Journal of Experimental Botany

Phloem sap and leaf {delta}13C, carbohydrates and amino acid concentrations in Eucalyptus globulus change systematically according to flooding and water deficit treatment.

Publication Date: 2010 Mar 8 PMID: 20211969
Authors: Merchant, A. - Peuke, A. D. - Keitel, C. - Macfarlane, C. - Warren, C. R. - Adams, M. A.
Journal: J Exp Bot

Phloem is a central conduit for the distribution of photoassimilate, nutrients, and signals among plant organs. A revised technique was used to collect phloem sap from small woody plants in order to assess changes in composition induced by water deficit and flooding. Bled phloem sap delta(13)C and sugar concentrations were compared to delta(13)C of bulk material, soluble carbon extracts, and the neutral sugar fraction from leaves. Amino acid composition and inorganic ions of the phloem sap was also analysed. Quantitative, systematic changes were detected in phloem sap composition and delta(13)C in response to altered water availability. Phloem sap delta(13)C was more sensitive to changes of water availability than the delta(13)C of bulk leaf, the soluble carbon fraction, and the neutral soluble fraction of leaves. Changes in water availability also resulted in significant changes in phloem sugar (sucrose and raffinose), inorganic nutrient (potassium), and amino acid (phenylalanine) concentrations with important implications for the maintenance of phloem function and biomass partitioning. The differences in carbohydrate and amino acid composition as well as the delta(13)C in the phloem, along with a new model system for phloem research, offer an improved understanding of the phloem-mediated signal, nutrient, and photoassimilate transduction in relation to water availability.

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Regulation of two germin-like protein genes during plum fruit development.

Publication Date: 2010 Mar 4 PMID: 20202999
Authors: El-Sharkawy, I. - Mila, I. - Bouzayen, M. - Jayasankar, S.
Journal: J Exp Bot

Germin-like proteins (GLPs) have several proposed roles in plant development and defence. Two novel genes (Ps-GLP1 and 2) encoding germin-like protein were isolated from plum (Prunus salicina). Their regulation was studied throughout fruit development and during ripening of early and late cultivars. These two genes exhibited similar expression patterns throughout the various stages of fruit development excluding two important stages, pit hardening (S2) and fruit ripening (S4). During fruit development until the ripening phase, the accumulation of both Ps-GLPs is related to the evolution of auxin. However, during the S2 stage only Ps-GLP1 is induced and this could putatively be in a H(2)O(2)-dependent manner. On the other hand, the diversity in the Ps-GLPs accumulation profile during the ripening process seems to be putatively due to the variability of endogenous auxin levels among the two plum cultivars, which consequently change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating Ps-GLPs transcripts was also investigated. These data, supported by their localization in the extracellular matrix, suggest that auxin is somehow involved in the regulation of both transcripts throughout fruit development and ripening.

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Terrestrial plant production and climate change.

Publication Date: 2010 Mar 4 PMID: 20202998
Authors: Friend, A. D.
Journal: J Exp Bot

The likely future increase in atmospheric CO(2) and associated changes in climate will affect global patterns of plant production. Models integrate understanding of the influence of the environment on plant physiological processes and so enable estimates of future changes to be made. Moreover, they allow us to assess the consequences of different assumptions for predictions and so stimulate further research. This paper is a review of the sensitivities of one such model, Hybrid6.5, a detailed mechanistic model of terrestrial primary production. This model is typical of its type, and the sensitivities of the global distribution of predicted production to model assumptions and possible future CO(2) levels and climate are assessed. Sensitivity tests show that leaf phenology has large effects on mean C(3) crop and needleleaved cold deciduous tree production, reducing potential net primary production (NPP) from that obtained using constant maximum annual leaf area index by 32.9% and 41.6%, respectively. Generalized Plant Type (GPT) specific parameterizations, particularly photosynthetic capacity per unit leaf N, affect mean predicted NPP of higher C(3) plants by -22.3% to 27.9%, depending on the GPT, compared to NPP predictions obtained using mean parameter values. An increase in atmospheric CO(2) concentrations from current values to 720 ppm by the end of this century, with associated effects on climate from a typical climate model, is predicted to increase global NPP by 37.3%. Mean increases range from 43.9-52.9% across different C(3) GPTs, whereas the mean NPP of C(4) grass and crop increases by 5.9%. Significant uncertainties concern the extent to which acclimative processes may reduce any potential future increase in primary production and the degree to which any gains are transferred to durable, and especially edible, biomass. Experimentalists and modellers need to work closely together to reduce these uncertainties. A number of research priorities are suggested.'The green leaf or, to be more precise, the microscopic green grain of chlorophyll, is the focus, the point in the world to which solar energy flows on one side while all the manifestations of life on earth take their source on the other side.' Kliment Arkadievich Timiryazev The conclusions of a century of plant physiology, speech at Moscow University, 12 January 1901.

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Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency.

Publication Date: 2010 Mar 4 PMID: 20202997
Authors: He, Y. - Jones, H. D. - Chen, S. - Chen, X. M. - Wang, D. W. - Li, K. X. - Wang, D. S. - Xia, L. Q.
Journal: J Exp Bot

An efficient Agrobacterium-mediated durum wheat transformation system has been developed for the production of 121 independent transgenic lines. This improved system used Agrobacterium strain AGL1 containing the superbinary pGreen/pSoup vector system and durum wheat cv Stewart as the recipient plant. Acetosyringone at 400 muM was added to both the inoculation and cultivation medium, and picloram at 10 mg l(-1) and 2 mg l(-1) was used in the cultivation and induction medium, respectively. Compared with 200 muM in the inoculation and cultivation media, the increased acetosyringone concentration led to significantly higher GUS (beta-glucuronidase) transient expression and T-DNA delivery efficiency. However, no evident effects of acetosyringone concentration on regeneration frequency were observed. The higher acetosyringone concentration led to an improvement in average final transformation efficiency from 4.7% to 6.3%. Furthermore, the concentration of picloram in the co-cultivation medium had significant effects on callus induction and regeneration. Compared with 2 mg l(-1) picloram in the co-cultivation medium, increasing the concentration to 10 mg l(-1) picloram resulted in improved final transformation frequency from 2.8% to 6.3%, with the highest frequency of 12.3% reached in one particular experiment, although statistical analysis showed that this difference in final transformation efficiency had a low level of significance. Stable integration of foreign genes, their expression, and inheritance were confirmed by Southern blot analyses, GUS assay, and genetic analysis. Analysis of T(1) progeny showed that, of the 31 transgenic lines randomly selected, nearly one-third had a segregation ratio of 3:1, while the remainder had ratios typical of two or three independently segregating loci.

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Metabolic networking in Brunfelsia calycina petals after flower opening.

Publication Date: 2010 Mar 4 PMID: 20202996
Authors: Bar-Akiva, A. - Ovadia, R. - Rogachev, I. - Bar-Or, C. - Bar, E. - Freiman, Z. - Nissim-Levi, A. - Gollop, N. - Lewinsohn, E. - Aharoni, A. - Weiss, D. - Koltai, H. - Oren-Shamir, M.
Journal: J Exp Bot

Brunfelsia calycina flowers change colour from purple to white due to anthocyanin degradation, parallel to an increase in fragrance and petal size. Here it was tested whether the production of the fragrant benzenoids is dependent on induction of the shikimate pathway, or if they are formed from the anthocyanin degradation products. An extensive characterization of the events taking place in Brunfelsia flowers is presented. Anthocyanin characterization was performed using ultraperfomance liquid chromatography-quadrupole time of flight-tandem mass specrometry (UPLC-QTOF-MS/MS). Volatiles emitted were identified by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Accumulated proteins were identified by 2D gel electrophoresis. Transcription profiles were characterized by cross-species hybridization of Brunfelsia cDNAs to potato cDNA microarrays. Identification of accumulated metabolites was performed by UPLC-QTOF-MS non-targeted metabolite analysis. The results include characterization of the nine main anthocyanins in Brunfelsia flowers. In addition, 146 up-regulated genes, 19 volatiles, seven proteins, and 17 metabolites that increased during anthocyanin degradation were identified. A multilevel analysis suggests induction of the shikimate pathway. This pathway is the most probable source of the phenolic acids, which in turn are precursors of both the benzenoid and lignin production pathways. The knowledge obtained is valuable for future studies on degradation of anthocyanins, formation of volatiles, and the network of secondary metabolism in Brunfelsia and related species.

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Association between winter anthocyanin production and drought stress in angiosperm evergreen species.

Publication Date: 2010 Mar 4 PMID: 20202995
Authors: Hughes, N. M. - Reinhardt, K. - Feild, T. S. - Gerardi, A. R. - Smith, W. K.
Journal: J Exp Bot

Leaves of many evergreen angiosperm species turn red under high light during winter due to the production of anthocyanin pigments, while leaves of other species remain green. There is currently no explanation for why some evergreen species exhibit winter reddening while others do not. Conditions associated with low leaf water potentials (Psi) have been shown to induce reddening in many plant species. Because evergreen species differ in susceptibility to water stress during winter, it is hypothesized that species which undergo winter colour change correspond with those that experience/tolerate the most severe daily declines in leaf Psi during winter. Six angiosperm evergreen species which synthesize anthocyanin in leaves under high light during winter and five species which do not were studied. Field Psi, pressure/volume curves, and gas exchange measurements were derived in summer (before leaf colour change had occurred) and winter. Consistent with the hypothesis, red-leafed species as a group had significantly lower midday Psi in winter than green-leafed species, but not during the summer when all the leaves were green. However, some red-leafed species showed midday declines similar to those of green-leafed species, suggesting that low Psi alone may not induce reddening. Pressure-volume curves also provided some evidence of acclimation to more negative water potentials by red-leafed species during winter (e.g. greater osmotic adjustment and cell wall hardening on average). However, much overlap in these physiological parameters was observed as well between red and green-leafed species, and some of the least drought-acclimated species were red-leafed. No difference was observed in transpiration (E) during winter between red and green-leaved species. When data were combined, only three of the six red-leafed species examined appeared physiologically acclimated to prolonged drought stress, compared to one of the five green-leafed species. This suggests that drought stress alone is not sufficient to explain winter reddening in evergreen angiosperms.

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An artificial solar spectrum substantially alters plant development compared with usual climate room irradiance spectra.

Publication Date: 2010 Mar 4 PMID: 20202994
Authors: Hogewoning, S. W. - Douwstra, P. - Trouwborst, G. - van Ieperen, W. - Harbinson, J.
Journal: J Exp Bot

Plant responses to the light spectrum under which plants are grown affect their developmental characteristics in a complicated manner. Lamps widely used to provide growth irradiance emit spectra which are very different from natural daylight spectra. Whereas specific responses of plants to a spectrum differing from natural daylight may sometimes be predictable, the overall plant response is generally difficult to predict due to the complicated interaction of the many different responses. So far studies on plant responses to spectra either use no daylight control or, if a natural daylight control is used, it will fluctuate in intensity and spectrum. An artificial solar (AS) spectrum which closely resembles a sunlight spectrum has been engineered, and growth, morphogenesis, and photosynthetic characteristics of cucumber plants grown for 13 d under this spectrum have been compared with their performance under fluorescent tubes (FTs) and a high pressure sodium lamp (HPS). The total dry weight of the AS-grown plants was 2.3 and 1.6 times greater than that of the FT and HPS plants, respectively, and the height of the AS plants was 4-5 times greater. This striking difference appeared to be related to a more efficient light interception by the AS plants, characterized by longer petioles, a greater leaf unfolding rate, and a lower investment in leaf mass relative to leaf area. Photosynthesis per leaf area was not greater for the AS plants. The extreme differences in plant response to the AS spectrum compared with the widely used protected cultivation light sources tested highlights the importance of a more natural spectrum, such as the AS spectrum, if the aim is to produce plants representative of field conditions.

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The regulation of MADS-box gene expression during ripening of banana and their regulatory interaction with ethylene.

Publication Date: 2010 Mar 3 PMID: 20200120
Authors: Elitzur, T. - Vrebalov, J. - Giovannoni, J. J. - Goldschmidt, E. E. - Friedman, H.
Journal: J Exp Bot

Six MaMADS-box genes have been cloned from the banana fruit cultivar Grand Nain. The similarity of these genes to tomato LeRIN is low and neither MaMADS2 nor MaMADS1 complement the tomato rin mutation. Nevertheless, the expression patterns, specifically in fruit and the induction during ripening and in response to ethylene and 1-MCP, suggest that some of these genes may participate in ripening. MaMADS1, 2, and 3, are highly expressed in fruit only, while the others are expressed in fruit as well as in other organs. Moreover, the suites of MaMADS-box genes and their temporal expression differ in peel and pulp during ripening. In the pulp, the increase in MaMADS2, 3, 4, and 5 expression preceded an increase in ethylene production, but coincides with the CO(2) peak. However, MaMADS1 expression in pulp coincided with ethylene production, but a massive increase in its expression occurred late during ripening, together with a second wave in the expression of MaMADS2, 3, and 4. In the peel, on the other hand, an increase in expression of MaMADS1, 3, and to a lesser degree also of MaMADS4 and 2 coincided with an increase in ethylene production. Except MaMADS3, which was induced by ethylene in pulp and peel, only MaMADS4, and 5 in pulp and MaMADS1 in peel were induced by ethylene. 1-MCP applied at the onset of the increase in ethylene production, increased the levels of MaMADS4 and MaMADS1 in pulp, while it decreased MaMADS1, 3, 4, and 5 in peel, suggesting that MaMADS4 and MaMADS1 are negatively controlled by ethylene at the onset of ethylene production only in pulp. Only MaMADS2 is neither induced by ethylene nor by 1-MCP, and it is expressed mainly in pulp. Our results suggest that two independent ripening programs are employed in pulp and peel which involve the activation of mainly MaMADS2, 4, and 5 and later on also MaMADS1 in pulp, and mainly MaMADS1, and 3 in peel. Hence, our results are consistent with MaMADS2, a SEP3 homologue, acting in the pulp upstream of the increase in ethylene production similarly to LeMADS-RIN.

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Modelling temperature-compensated physiological rates, based on the coordination of responses to temperature of developmental processes.

Publication Date: 2010 Mar 1 PMID: 20194927
Authors: Parent, B. - Turc, O. - Gibon, Y. - Stitt, M. - Tardieu, F.
Journal: J Exp Bot

Temperature fluctuates rapidly and affects all developmental and metabolic processes. This often obscures the effects of developmental trends or of other environmental conditions when temperature fluctuates naturally. A method is proposed for modelling temperature-compensated rates, based on the coordination of temperature responses of developmental processes. In a data set comprising 41 experiments in the greenhouse, growth chamber, or the field, the temperature responses in the range of 6-36 degrees C for different processes were compared in three species, maize, rice, and Arabidopsis thaliana. Germination, cell division, expansive growth rate, leaf initiation, and phenology showed coordinated temperature responses and followed common laws within each species. The activities of 10 enzymes involved in carbon metabolism exhibited monotonous exponential responses across the whole range 10-40 degrees C. Hence, the temperature dependence of developmental processes is not explained by a simple relationship to central metabolism. Temperature-compensated rates of development were calculated from the equations of response curve, by expressing rates per unit equivalent time at 20 degrees C. This resulted in stable rates when temperatures fluctuated over a large range (for which classical thermal time was inefficient), and in time courses of leaf development which were common to several experiments with different temperature scenarios.

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cpSecA, a thylakoid protein translocase subunit, is essential for photosynthetic development in Arabidopsis.

Publication Date: 2010 Mar 1 PMID: 20194926
Authors: Liu, D. - Gong, Q. - Ma, Y. - Li, P. - Li, J. - Yang, S. - Yuan, L. - Yu, Y. - Pan, D. - Xu, F. - Wang, N. N.
Journal: J Exp Bot

The endosymbiont-derived Sec-dependent protein sorting pathway is essential for protein import into the thylakoid lumen and is important for the proper functioning of the chloroplast. Two loss-of-function mutants of cpSecA, the ATPase subunit of the chloroplast Sec translocation machinery, were analysed in Arabidopsis. The homozygous mutants were albino and seedling lethal under autotrophic conditions and remained dwarf and infertile with an exogenous carbon supply. They were subject to oxidative stress and accumulated superoxide under normal lighting conditions. Electron microscopy revealed that the chloroplast of the mutants had underdeveloped thylakoid structures. Histochemical GUS assay of the AtcpSecA::GUS transgenic plants confirmed that AtcpSecA was expressed in green organs in a light-inducible way. Real-time RT-PCR and microarray analysis revealed repressed transcription of nucleus- and chloroplast- encoded subunits of photosynthetic complexes, and induced transcription of chloroplast protein translocation machinery and mitochondrion-encoded respiratory complexes in the mutants. It is inferred that AtcpSecA plays an essential role in chloroplast biogenesis, the absence of which triggered a retrograde signal, eventually leading to a reprogramming of chloroplast and mitochondrial gene expression.

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A model for gas transport in pear fruit at multiple scales.

Publication Date: 2010 Mar 1 PMID: 20194925
Authors: Ho, Q. T. - Verboven, P. - Verlinden, B. E. - Nicolai, B. M.
Journal: J Exp Bot

A two-dimensional multiscale gas exchange model was developed to evaluate the effect of ambient conditions, fruit size, and maturity on intracellular O(2) and CO(2) concentrations in pear fruit via computational analysis. The model consists of interconnected submodels that describe the gas exchange at the macroscopic scale of the fruit and the microscopic scale of the cells. The multiscale model resulted in a comprehensive description of gas exchange at different scales. The macroscale model was used to describe the gas exchange of the fruit under controlled atmosphere conditions while corresponding intracellular concentrations of microstructure tissue were computed from the microscale. Ripening of the fruit increased the risk of physiological disorders, since increased respiration resulted in anoxia in the fruit centre even under typical storage conditions.

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A tomato strigolactone-impaired mutant displays aberrant shoot morphology and plant interactions.

Publication Date: 2010 Mar 1 PMID: 20194924
Authors: Koltai, H. - Lekkala, S. P. - Bhattacharya, C. - Mayzlish-Gati, E. - Resnick, N. - Wininger, S. - Dor, E. - Yoneyama, K. - Yoneyama, K. - Hershenhorn, J. - Joel, D. M. - Kapulnik, Y.
Journal: J Exp Bot

Strigolactones are considered a new group of plant hormones. Their role as modulators of plant growth and signalling molecules for plant interactions first became evident in Arabidopsis, pea, and rice mutants that were flawed in strigolactone production, release, or perception. The first evidence in tomato (Solanum lycopersicon) of strigolactone deficiency is presented here. Sl-ORT1, previously identified as resistant to the parasitic plant Orobanche, had lower levels of arbuscular mycorrhizal fungus (Glomus intraradices) colonization, possibly as a result of its reduced ability to induce mycorrhizal hyphal branching. Biochemical analysis of mutant root extracts suggested that it produces only minute amounts of two of the tomato strigolactones: solanacol and didehydro-orobanchol. Accordingly, the transcription level of a key enzyme (CCD7) putatively involved in strigolactone synthesis in tomato was reduced in Sl-ORT1 compared with the wild type (WT). Sl-ORT1 shoots exhibited increased lateral shoot branching, whereas exogenous application of the synthetic strigolactone GR24 to the mutant restored the WT phenotype by reducing the number of lateral branches. Reduced lateral shoot branching was also evident in grafted plants which included a WT interstock, which was grafted between the mutant rootstock and the scion. In roots of these grafted plants, the CCD7 transcription level was not significantly induced, nor was mycorrhizal sensitivity restored. Hence, WT-interstock grafting, which restores mutant shoot morphology to WT, does not restore mutant root properties to WT. Characterization of the first tomato strigolactone-deficient mutant supports the putative general role of strigolactones as messengers of suppression of lateral shoot branching in a diversity of plant species.

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Synergic effect of salinity and CO2 enrichment on growth and photosynthetic responses of the invasive cordgrass Spartina densiflora.

Publication Date: 2010 Mar 1 PMID: 20194923
Authors: Mateos-Naranjo, E. - Redondo-Gomez, S. - Alvarez, R. - Cambrolle, J. - Gandullo, J. - Figueroa, M. E.
Journal: J Exp Bot

Spartina densiflora is a C(4) halophytic species that has proved to have a high invasive potential which derives from its clonal growth and its physiological plasticity to environmental factors, such as salinity. A greenhouse experiment was designed to investigate the synergic effect of 380 and 700 ppm CO(2) at 0, 171, and 510 mM NaCl on the growth and the photosynthetic apparatus of S. densiflora by measuring chlorophyll fluorescence parameters, gas exchange and photosynthetic pigment concentrations. PEPC activity and total ash, sodium, potassium, calcium, magnesium, and zinc concentrations were determined, as well as the C/N ratio. Elevated CO(2) stimulated growth of S. densiflora at 0 and 171 mM NaCl external salinity after 90 d of treatment. This growth enhancement was associated with a greater leaf area and improved leaf water relations rather than with variations in net photosynthetic rate (A). Despite the fact that stomatal conductance decreased in response to 700 ppm CO(2) after 30 d of treatment, A was not affected. This response of A to elevated CO(2) concentration might be explained by an enhanced PEPC carboxylation capacity. On the whole, plant nutrient concentrations declined under elevated CO(2), which can be ascribed to the dilution effect caused by an increase in biomass and the higher water content found at 700 ppm CO(2). Finally, CO(2) and salinity had a marked overall effect on the photochemical (PSII) apparatus and the synthesis of photosynthetic pigments.

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Glyoxylate rather than ascorbate is an efficient precursor for oxalate biosynthesis in rice.

Publication Date: 2010 Mar 1 PMID: 20194922
Authors: Yu, L. - Jiang, J. - Zhang, C. - Jiang, L. - Ye, N. - Lu, Y. - Yang, G. - Liu, E. - Peng, C. - He, Z. - Peng, X.
Journal: J Exp Bot

Oxalate is widely distributed in the plant kingdom. While excess oxalate in food crops is detrimental to animal and human health, it may play various functional roles in plants, particularly for coping with environmental stresses. Understanding its biosynthetic mechanism in plants, therefore, becomes increasingly important both theoretically and practically. However, it is still a matter of debate as to what precursor and pathway are ultimately used for oxalate biosynthesis in plants. In this study, both physiological and molecular approaches were applied to address these questions. First, it was observed that when glycolate or glyoxylate was fed into detached leaves, both organic acids were equally effective in stimulating oxalate accumulation. In addition, the stimulation could be completely inhibited by cysteine, a glyoxylate scavenger that forms cysteine-glyoxylate adducts. To verify the role of glyoxylate further, various transgenic plants were generated, in which several genes involved in glyoxylate metabolism [i.e. SGAT (serine-glyoxylate aminotransferase), GGAT (glutamate-glyoxylate aminotransferase), HPR (hydroxypyruvate reductase), ICL (isocitrate lyase)], were transcriptionally regulated through RNAi or over-expression. Analyses on these transgenic plants consistently revealed that glyoxylate acted as an efficient precursor for oxalate biosynthesis in rice. Unexpectedly, it was found that oxalate accumulation was not correlated with photorespiration, even though this pathway is known to be a major source of glyoxylate. Further, when GLDH (L-galactono-1,4-lactone dehydrogenase), a key enzyme gene for ascorbate biosynthesis, was down-regulated, the oxalate abundance remained constant, despite ascorbate having been largely reduced as expected in these transgenic plants. Taken together, our results strongly suggest that glyoxylate rather than ascorbate is an efficient precursor for oxalate biosynthesis, and that oxalate accumulation and regulation do not necessarily depend on photorespiration, possibly due to the occurrence of the anaplerotic reaction that may compensate for glyoxylate formation in rice.

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PtAAP11, a high affinity amino acid transporter specifically expressed in differentiating xylem cells of poplar.

Publication Date: 2010 Feb 26 PMID: 20190041
Authors: Couturier, J. - de Fay, E. - Fitz, M. - Wipf, D. - Blaudez, D. - Chalot, M.
Journal: J Exp Bot

Amino acids are the currency of nitrogen exchange between source and sink tissues in plants and constitute a major source of the components used for cellular growth and differentiation. The characterization of a new amino acid transporter belonging to the amino acid permease (AAP) family, AAP11, expressed in the perennial species Populus trichocarpa is reported here. PtAAP11 expression analysis was performed by semi-quantitative RT-PCR and GUS activity after poplar transformation. PtAAP11 function was studied in detail by heterologous expression in yeast. The poplar genome contains 14 putative AAPs which is quite similar to other species analysed except Arabidopsis. PtAAP11 was mostly expressed in differentiating xylem cells in different organs. Functional characterization demonstrated that PtAAP11 was a high affinity amino acid transporter, more particularly for proline. Compared with other plant amino acid transporters, PtAAP11 represents a novel high-affinity system for proline. Thus, the functional characterization and expression studies suggest that PtAAP11 may play a major role in xylogenesis by providing proline required for xylem cell wall proteins. The present study provides important information highlighting the role of a specific amino acid transporter in xylogenesis in poplar.

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A transcriptomic study of grapevine (Vitis vinifera cv. Cabernet-Sauvignon) interaction with the vascular ascomycete fungus Eutypa lata.

Publication Date: 2010 Feb 26 PMID: 20190040
Authors: Camps, C. - Kappel, C. - Lecomte, P. - Leon, C. - Gomes, E. - Coutos-Thevenot, P. - Delrot, S.
Journal: J Exp Bot

Eutypa dieback is a vascular disease that may severely affect vineyards throughout the world. In the present work, microarrays were made in order (i) to improve our knowledge of grapevine (Vitis vinifera cv. Cabernet-Sauvignon) responses to Eutypa lata, the causal agent of Eutypa dieback; and (ii) to identify genes that may prevent symptom development. Qiagen/Operon grapevine microarrays comprising 14 500 probes were used to compare, under three experimental conditions (in vitro, in the greenhouse, and in the vineyard), foliar material of infected symptomatic plants (S(+)R(+)), infected asymptomatic plants (S(-)R(+)), and healthy plants (S(-)R(-)). These plants were characterized by symptom notation after natural (vineyard) or experimental (in vitro and greenhouse) infection, re-isolation of the fungus located in the lignified parts, and the formal identification of E. lata mycelium by PCR. Semi-quantitative real-time PCR experiments were run to confirm the expression of some genes of interest in response to E. lata. Their expression profiles were also studied in response to other grapevine pathogens (Erysiphe necator, Plasmopara viticola, and Botrytis cinerea). (i) Five functional categories of genes, that is those involved in metabolism, defence reactions, interaction with the environment, transport, and transcription, were up-regulated in S(+)R(+) plants compared with S(-)R(-) plants. These genes, which cannot prevent infection and symptom development, are not specific since they were also up-regulated after infection by powdery mildew, downy mildew, and black rot. (ii) Most of the genes that may prevent symptom development are associated with the light phase of photosynthesis. This finding is discussed in the context of previous data on the mode of action of eutypin and the polypeptide fraction secreted by Eutypa.

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Temporal analysis of natural variation for the rate of leaf production and its relationship with flowering initiation in Arabidopsis thaliana.

Publication Date: 2010 Feb 26 PMID: 20190039
Authors: Mendez-Vigo, B. - Andres, M. T. - Ramiro, M. - Martinez-Zapater, J. M. - Alonso-Blanco, C.
Journal: J Exp Bot

Vegetative growth and flowering initiation are two crucial developmental processes in the life cycle of annual plants that are closely associated. The timing of both processes affects several presumed adaptive traits, such as flowering time (FT), total leaf number (TLN), or the rate of leaf production (RLP). However, the interactions among these complex processes and traits, and their mechanistic bases, remain largely unknown. To determine the genetic relationships between them, the natural genetic variation between A. thaliana accessions Fei-0 and Ler has been studied using a new population of 222 LerxFei-0 recombinant inbred lines. Temporal analysis of the parental development under a short day photoperiod distinguishes two vegetative phases differing in their RLP. QTL mapping of RLP in consecutive time intervals of vegetative development indicates that Ler/Fei-0 variation is caused by 10 loci whose small to moderate effects mainly display two different temporal patterns. Further comparative QTL analyses show that most of the genomic regions affecting FT or TLN also alter RLP. In addition, the partially independent genetic bases observed for FT and TLN appear determined by several genomic regions with two different patterns of phenotypic effects: regions with a larger effect on FT than TLN, and vice versa. The distinct temporal and pleiotropic patterns of QTL effects suggest that natural variation for flowering time is caused by different genetic mechanisms involved in vegetative and/or reproductive phase changes, most of them interacting with the control of leaf production rate. Thus, natural selection might contribute to maintain this genetic variation due to its phenotypic effects not only on the timing of flowering initiation but also on the rate of vegetative growth.

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Cloning and expression analysis of the Ccrboh gene encoding respiratory burst oxidase in Citrullus colocynthis and grafting onto Citrullus lanatus (watermelon).

Publication Date: 2010 Feb 24 PMID: 20181664
Authors: Si, Y. - Dane, F. - Rashotte, A. - Kang, K. - Singh, N. K.
Journal: J Exp Bot

A full-length drought-responsive gene Ccrboh, encoding the respiratory burst oxidase homologue (rboh), was cloned in Citrullus colocynthis, a very drought-tolerant cucurbit species. The robh protein, also named NADPH oxidase, is conserved in plants and animals, and functions in the production of reactive oxygen species (ROS). The Ccrboh gene accumulated in a tissue-specific pattern when C. colocynthis was treated with PEG, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), or NaCl, while the homologous rboh gene did not show any change in C. lanatus var. lanatus, cultivated watermelon, during drought. Grafting experiments were conducted using C. colocynthis or C. lanatus as the rootstock or scion. Results showed that the rootstock significantly affects gene expression in the scion, and some signals might be transported from the root to the shoot. Ccrboh in C. colocynthis was found to function early during plant development, reaching high mRNA transcript levels 3 d after germination. The subcellular location of Ccrboh was investigated by transient expression of the 35S::Ccrboh::GFP fusion construct in protoplasts. The result confirmed that Ccrboh is a transmembrane protein. Our data suggest that Ccrboh might be functionally important during the acclimation of plants to stress and also in plant development. It holds great promise for improving drought tolerance of other cucurbit species.

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Pollen-pistil interactions regulating successful fertilization in the Brassicaceae.

Publication Date: 2010 Feb 24 PMID: 20181663
Authors: Chapman, L. A. - Goring, D. R.
Journal: J Exp Bot

In the Brassicaceae, the acceptance of compatible pollen and the rejection of self-incompatible pollen by the pistil involves complex molecular communication systems between the pollen grain and the female reproductive structures. Preference towards species related-pollen combined with self-recognition systems, function to select the most desirable pollen; and thus, increase the plant's chances for the maximum number of successful fertilizations and vigorous offspring. The Brassicaceae is an ideal group for studying pollen-pistil interactions as this family includes a diverse group of agriculturally relevant crops as well as several excellent model organisms for studying both compatible and self-incompatible pollinations. This review will describe the cellular systems in the pistil that guide the post-pollination events, from pollen capture on the stigmatic papillae to pollen tube guidance to the ovule, with the final release of the sperm cells to effect fertilization. The interplay of other recognition systems, such as the self-incompatibility response and interspecific interactions, on regulating post-pollination events and selecting for compatible pollen-pistil interactions will also be explored.

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Elevation of night-time temperature increases terpenoid emissions from Betula pendula and Populus tremula.

Publication Date: 2010 Feb 24 PMID: 20181662
Authors: Ibrahim, M. A. - Maenpaa, M. - Hassinen, V. - Kontunen-Soppela, S. - Malec, L. - Rousi, M. - Pietikainen, L. - Tervahauta, A. - Karenlampi, S. - Holopainen, J. K. - Oksanen, E. J.
Journal: J Exp Bot

Volatile organic compounds (VOCs) are expected to have an important role in plant adaptation to high temperatures. The impacts of increasing night-time temperature on daytime terpenoid emissions and related gene expression in silver birch (Betula pendula) and European aspen (Populus tremula) clones were studied. The plants were grown under five different night-time temperatures (6, 10, 14, 18, and 22 degrees C) while daytime temperature was kept at a constant 22 degrees C. VOC emissions were collected during the daytime and analysed by gas chromatography-mass spectrometry (GC-MS). In birch, emissions per leaf area of the C11 homoterpene 4,8-dimethy1-nona-1,3,7-triene (DMNT) and several sesquiterpenes were consistently increased with increasing night-time temperature. Total sesquiterpene (SQT) emissions showed an increase at higher temperatures. In aspen, emissions of DMNT and beta-ocimene increased from 6 degrees C to 14 degrees C, while several other monoterpenes and the SQTs (Z,E)-alpha-farnesene and (E,E)-alpha-farnesene increased up to 18 degrees C. Total monoterpene and sesquiterpene emission peaked at 18 degrees C, whereas isoprene emissions decreased at 22 degrees C. Leaf area increased across the temperature range of 6-22 degrees C by 32% in birch and by 59% in aspen. Specific leaf area (SLA) was also increased in both species. The genetic regulation of VOC emissions seems to be very complex, as indicated by several inverse relationships between emission profiles and expression of several regulatory genes (DXR, DXS, and IPP). The study indicates that increasing night temperature may strongly affect the quantity and quality of daytime VOC emissions of northern deciduous trees.

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Accumulation of xylem transported protein at pit membranes and associated reductions in hydraulic conductance.

Publication Date: 2010 Feb 24 PMID: 20181661
Authors: Neumann, P. M. - Weissman, R. - Stefano, G. - Mancuso, S.
Journal: J Exp Bot

Proteins and traces of polysaccharide are the only polymeric colloids consistently transported in the xylem sap of plants. The hypothesis that such proteins could have physical inhibitory effects on xylem water transport was investigated. Ovalbumin, with a molecular weight of 45 kDa and a molecular diameter of 5.4 nm, is an inert, water-soluble protein that is midway along the size range of endogenous xylem sap proteins. Solutions of ovalbumin conjugated to a fluorescent marker and supplied to transpiring shoot explants of tobacco (Nicotiana tabacum L.) and olive (Olea europaea L.) were shown by confocal laser scanning microscopy to accumulate specifically at wall-based pit membranes that connect neighbouring xylem conduits. In addition, pressure-induced perfusion of micro-filtered ovalbumin solutions, at concentrations similar to those of endogenous xylem sap proteins, through the xylem of tobacco stem or olive twig segments resulted in the retention of c. 40% of the ovalbumin and reductions in the axial hydraulic conductance of the xylem. Smaller molecules such as Texas Red 3000 (MW 3 kDa) and Alexafluor 488-cadaverin conjugates (MW 0.64 kDa) did not show similar characteristics. The partial reduction in xylem hydraulic conductance appeared to be related to the accumulation of ovalbumin at xylem pit membranes and the consequent fouling of trans-membrane water-conducting pores with smaller diameters than those of the ovalbumin molecules. Potential implications of these novel findings for whole-plant water relations are considered.

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Characterization of plant sulfiredoxin and role of sulphinic form of 2-Cys peroxiredoxin.

Publication Date: 2010 Feb 22 PMID: 20176891
Authors: Iglesias-Baena, I. - Barranco-Medina, S. - Lazaro-Payo, A. - Lopez-Jaramillo, F. J. - Sevilla, F. - Lazaro, J. J.
Journal: J Exp Bot

The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent. In conditions of oxidative stress, the peroxidatic cysteine can be overoxidized to sulphinic acid inactivating the Prx. An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO(2)H). To explore the physiological functions of Srx in plants we have cloned, expressed and purified to homogeneity a Srx from Arabidopsis thaliana (AtSrx), as well as five variants by site-directed mutagenesis on amino acids involved in its activity. The activity of sulfiredoxin, determined by a new method, is dependent on the concentration of the sulphinic form of Prx and the conserved Srx is capable of regenerating the functionality of both pea and Arabidopsis Prx-SO(2)H. Molecular modelling of AtSrx and the facts that the R28Q variant shows a partial inactivation, that the activity of the E76A variant is equivalent to that of the native enzyme and that the double mutation R28Q/E76A abolishes the enzymatic activity suggests that the pair His100-Glu76 may be involved in the activation of C72 in the absence of R28. The knock-out mutant plants without Srx or 2-Cys Prx exhibited phenotypical differences under growth conditions of 16 h light, probably due to the signalling role of the sulphinic form of Prx. These mutants showed more susceptibility to oxidative stress than wild-type plants. This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function.

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Regulation of tissue-specific expression of SPATULA, a bHLH gene involved in carpel development, seedling germination, and lateral organ growth in Arabidopsis.

Publication Date: 2010 Feb 22 PMID: 20176890
Authors: Groszmann, M. - Bylstra, Y. - Lampugnani, E. R. - Smyth, D. R.
Journal: J Exp Bot

SPATULA is a bHLH transcription factor that promotes growth of tissues arising from the carpel margins, including the septum and transmitting tract. It is also involved in repressing germination of newly harvested seeds, and in inhibiting cotyledon, leaf, and petal expansion. Using a reporter gene construct, its expression profile was fully defined. Consistent with its known functions, SPT was expressed in developing carpel margin tissues, and in the hypocotyls and cotyledons of germinating seedlings, and in developing leaves and petals. It was also strongly expressed in tissues where no functions have been identified to date, including the dehiscence zone of fruits, developing anthers, embryos, and in the epidermal initials and new stele of root tips. The promoter region of SPT was dissected by truncation and deletion, and two main regions occupied by tissue-specific enhancers were identified. These were correlated with eight regions conserved between promoter regions of Arabidopsis, Brassica oleracea, and Brassica rapa. When transformed into Arabidopsis, the B. oleracea promoter drove expression in reproductive tissues mostly comparable to the equivalent Arabidopsis promoter. There is genetic evidence that SPT function in the gynoecium is associated with the perception of auxin. However, site-directed mutagenesis of three putative auxin-response elements had no detectable effect on SPT expression patterns. Even so, disruption of a putative E-box variant adjacent to one of these resulted in a loss of valve dehiscence zone expression. This expression was also specifically lost in mutants of another bHLH gene INDEHISCENT, indicating that IND may directly regulate SPT expression through this variant E-box.

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Air-coupled broadband ultrasonic spectroscopy as a new non-invasive and non-contact method for the determination of leaf water status.

Publication Date: 2010 Feb 22 PMID: 20176889
Authors: Sancho-Knapik, D. - Alvarez-Arenas, T. G. - Peguero-Pina, J. J. - Gil-Pelegrin, E.
Journal: J Exp Bot

The implementation of non-destructive methods for the study of water changes within plant tissues and/or organs has been a target for some time in plant physiology. Recent advances in air-coupled ultrasonic spectroscopy have enabled ultrasonic waves to be applied to the on-line and real-time assessment of the water content of different materials. In this study, this technique has been applied as a non-destructive, non-invasive, non-contact, and repeatable method for the determination of water status in Populusxeuramericana and Prunus laurocerasus leaves. Frequency spectra of the transmittance of ultrasounds through plant leaves reveal the presence of at least one resonance. At this resonant frequency, transmittance is at its maximum. This work demonstrates that changes in leaf relative water content (RWC) and water potential (Psi) for both species can be accurately monitored by the corresponding changes in resonant frequency. The differential response found between both species may be due to the contrasting leaf structural features and the differences found in the parameters derived from the P-V curves. The turgor loss point has been precisely defined by this new technique, as it is derived from the lack of significant differences between the relative water content at the turgor loss point (RWC(TLP)) obtained from P-V curves and ultrasonic measurements. The measurement of the turgor gradient between two different points of a naturally transpiring leaf is easily carried out with the method introduced here. Therefore, such a procedure can be an accurate tool for the study of all processes where changes in leaf water status are involved.

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HvALMT1 from barley is involved in the transport of organic anions.

Publication Date: 2010 Feb 22 PMID: 20176888
Authors: Gruber, B. D. - Ryan, P. R. - Richardson, A. E. - Tyerman, S. D. - Ramesh, S. - Hebb, D. M. - Howitt, S. M. - Delhaize, E.
Journal: J Exp Bot

Members of the ALMT gene family contribute to the Al(3+) resistance of several plant species by facilitating malate efflux from root cells. The first member of this family to be cloned and characterized, TaALMT1, is responsible for most of the natural variation of Al(3+) resistance in wheat. The current study describes the isolation and characterization of HvALMT1, the barley gene with the greatest sequence similarity to TaALMT1. HvALMT1 is located on chromosome 2H which has not been associated with Al(3+) resistance in barley. The relatively low levels of HvALMT1 expression detected in root and shoot tissues were independent of external aluminium or phosphorus supply. Transgenic barley plants transformed with the HvALMT1 promoter fused to the green fluorescent protein (GFP) indicated that expression of HvALMT1 was relatively high in stomatal guard cells and in root tissues containing expanding cells. GFP fused to the C-terminus of the full HvALMT1 protein localized to the plasma membrane and motile vesicles within the cytoplasm. HvALMT1 conferred both inward and outward currents when expressed in Xenopus laevis oocytes that were bathed in a range of anions including malate. Both malate uptake and efflux were confirmed in oocyte assays using [(14)C]malate as a radiotracer. It is suggested that HvALMT1 functions as an anion channel to facilitate organic anion transport in stomatal function and expanding cells.

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Development and verification of a water and sugar transport model using measured stem diameter variations.

Publication Date: 2010 Feb 22 PMID: 20176887
Authors: De Schepper, V. - Steppe, K.
Journal: J Exp Bot

In trees, water and sugars are transported by xylem and phloem conduits which are hydraulically linked. A simultaneous study of both flows is interesting, since they concurrently influence important processes such as stomatal regulation and growth. A few mathematical models have already been developed to investigate the influence of both hydraulically coupled flows. However, none of these models has so far been tested using real measured field data. In the present study, a comprehensive whole-tree model is developed that enables simulation of the stem diameter variations driven by both the water and sugar transport. Stem diameter variations are calculated as volume changes of both the xylem and the phloem tissue. These volume changes are dependent on: (i) water transport according to the cohesion-tension theory; (ii) sugar transport according to the Munch hypothesis; (iii) loading and unloading of sugars; and (iv) irreversible turgor-driven growth. The model considers three main compartments (crown, stem, and roots) and is verified by comparison with actual measured stem diameter variations and xylem sap flow rates. These measurements were performed on a young oak (Quercus robur L.) tree in controlled conditions and on an adult beech (Fagus sylvatica L.) tree in a natural forest. A good agreement was found between simulated and measured data. Hence, the model seemed to be a realistic representation of the processes observed in reality. Furthermore, the model is able to simulate several physiological variables which are relatively difficult to measure: phloem turgor, phloem osmotic pressure, and Munch's counterflow. Simulation of these variables revealed daily dynamics in their behaviour which were mainly induced by transpiration. Some of these dynamics are experimentally confirmed in the literature, while others are not.

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Molecular analysis of SCARECROW genes expressed in white lupin cluster roots.

Publication Date: 2010 Feb 18 PMID: 20167612
Authors: Sbabou, L. - Bucciarelli, B. - Miller, S. - Liu, J. - Berhada, F. - Filali-Maltouf, A. - Allan, D. - Vance, C.
Journal: J Exp Bot

The Scarecrow (SCR) transcription factor plays a crucial role in root cell radial patterning and is required for maintenance of the quiescent centre and differentiation of the endodermis. In response to phosphorus (P) deficiency, white lupin (Lupinus albus L.) root surface area increases some 50-fold to 70-fold due to the development of cluster (proteoid) roots. Previously it was reported that SCR-like expressed sequence tags (ESTs) were expressed during early cluster root development. Here the cloning of two white lupin SCR genes, LaSCR1 and LaSCR2, is reported. The predicted amino acid sequences of both LaSCR gene products are highly similar to AtSCR and contain C-terminal conserved GRAS family domains. LaSCR1 and LaSCR2 transcript accumulation localized to the endodermis of both normal and cluster roots as shown by in situ hybridization and gene promoter::reporter staining. Transcript analysis as evaluated by quantitative real-time-PCR (qRT-PCR) and RNA gel hybridization indicated that the two LaSCR genes are expressed predominantly in roots. Expression of LaSCR genes was not directly responsive to the P status of the plant but was a function of cluster root development. Suppression of LaSCR1 in transformed roots of lupin and Medicago via RNAi (RNA interference) delivered through Agrobacterium rhizogenes resulted in decreased root numbers, reflecting the potential role of LaSCR1 in maintaining root growth in these species. The results suggest that the functional orthologues of AtSCR have been characterized.

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Identification of a Mg-protoporphyrin IX monomethyl ester cyclase homologue, EaZIP, differentially expressed in variegated Epipremnum aureum 'Golden Pothos' is achieved through a unique method of comparative study using tissue regenerated plants.

Publication Date: 2010 Feb 18 PMID: 20167611
Authors: Hung, C. Y. - Sun, Y. H. - Chen, J. - Darlington, D. E. - Williams, A. L. - Burkey, K. O. - Xie, J.
Journal: J Exp Bot

Variegated plants provide a valuable tool for studying chloroplast biogenesis by allowing direct comparison between green and white/yellow sectors within the same leaf. While variegated plants are abundant in nature, the mechanism of leaf variegation remains largely unknown. Current studies are limited to a few mutants in model plant species, and are complicated by the potential for cross-contamination during dissection of leaf tissue into contrasting sectors. To overcome these obstacles, an alternative approach was explored using tissue-culture techniques to regenerate plantlets from unique sectors. Stable green and pale yellow plants were developed from a naturally variegated Epipremnum aureum 'Golden Pothos'. By comparing the gene expression between green and pale yellow plants using suppression subtractive hybridization in conjunction with homologous sequence search, nine down-regulated and 18 up-regulated genes were identified in pale yellow plants. Transcript abundance for EaZIP (Epipremnum aureum leucine zipper), a nuclear gene homologue of tobacco NTZIP and Arabidopsis CHL27, was reduced more than 4000-fold in qRT-PCR analysis. EaZIP encodes the Mg-protoporphyrin IX monomethyl ester cyclase, one of the key enzymes in the chlorophyll biosynthesis pathway. Examination of EaZIP expression in naturally variegated 'Golden Pothos' confirmed that EaZIP transcript levels were correlated with leaf chlorophyll contents, suggesting that this gene plays a major role in the loss of chlorophyll in the pale yellow sectors of E. aureum 'Golden Pothos'. This study further suggests that tissue-culture regeneration of plantlets from different coloured sectors of variegated leaves can be used to investigate the underlying mechanisms of variegation.

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Mechanisms of ozone tolerance in rice: characterization of two QTLs affecting leaf bronzing by gene expression profiling and biochemical analyses.

Publication Date: 2010 Feb 17 PMID: 20164144
Authors: Frei, M. - Tanaka, J. P. - Chen, C. P. - Wissuwa, M.
Journal: J Exp Bot

High surface ozone concentration is increasingly being recognized as a factor that negatively affects crop yields in Asia. However, little progress has been made in developing ozone-tolerant genotypes of rice-Asia's major staple crop. This study aimed to identify possible tolerance mechanisms by characterizing two quantitative trait loci (QTLs) that were previously shown to influence visible leaf symptoms under ozone exposure (120 nl l(-1), 7 h d(-1), 13 d). Two chromosome segment substitution lines (SL15 and SL41) that carried introgressions of the QTLs OzT3 and OzT9, respectively, were exposed to ozone at 120 nl l(-1) along with their parent Nipponbare. In accordance with the expected QTL effect, SL15 showed stronger visible symptoms of ozone damage than Nipponbare, whereas SL41 had fewer symptoms. Gene expression profiling by microarray hybridization yielded 470 probes that were differentially expressed in SL15 and 314 in SL41. Potential tolerance mechanisms were evaluated by investigating changes in gene expression in three general categories. (i) Processes involved in programmed cell death, in which a number of genes related to ethylene or jasmonic acid metabolism or general disease resistance were identified that were differentially regulated in one of the substitution lines. (ii) Biosynthesis of antioxidants. Testing this hypothesis did not reveal any genes differentially regulated between genotypes, and it was thus rejected. (iii) Turnover of antioxidants and enzymatic detoxification of radical oxygen species (ROS), in which a number of differentially regulated genes were also identified. Genes encoding antioxidant enzymes (catalase and peroxidases) tended to be more strongly expressed in SL15. A potential tolerance gene which encodes a putative ascorbate oxidase was identified within the QTL introgression in SL41. This gene showed consistently lower expression in SL41 under ozone exposure across different points in time within independent experiments. Its expression may be involved in mechanisms leading to enhanced ascorbic acid status in SL41 under ozone exposure, and may be linked to a higher concentration of total apoplastic ascorbic acid in SL41 that was observed in an independent experiment.

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Identification and localization of a caleosin in olive (Olea europaea L.) pollen during in vitro germination.

Publication Date: 2010 Feb 17 PMID: 20164143
Authors: Zienkiewicz, K. - Castro, A. J. - Alche, J. D. - Zienkiewicz, A. - Suarez, C. - Rodriguez-Garcia, M. I.
Journal: J Exp Bot

In plant organs and tissues, the neutral storage lipids are confined to discrete spherical organelles called oil bodies. Oil bodies from plant seeds contain 0.6-3% proteins, including oleosins, steroleosins, and caleosins. In this study, a caleosin isoform of approximately 30 kDa was identified in the olive pollen grain. The protein was mainly located at the boundaries of the oil bodies in the cytoplasm of the pollen grain and the pollen tube. In addition, caleosins were also visualized in the cytoplasm at the subapical zone, as well as in the tonoplast of vacuoles present in the pollen tube cytoplasm. The cellular behaviour of lipid bodies in the olive pollen was also monitored during in vitro germination. The number of oil bodies decreased 20-fold in the pollen grain during germination, whereas the opposite tendency occurred in the pollen tube, suggesting that oil bodies moved from one to the other. The data suggest that this pollen caleosin might have a role in the mobilization of oil bodies as well as in the reorganization of membrane compartments during pollen in vitro germination.

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Auxin response factor 2 (ARF2) plays a major role in regulating auxin-mediated leaf longevity.

Publication Date: 2010 Feb 17 PMID: 20164142
Authors: Lim, P. O. - Lee, I. C. - Kim, J. - Kim, H. J. - Ryu, J. S. - Woo, H. R. - Nam, H. G.
Journal: J Exp Bot

Auxin regulates a variety of physiological and developmental processes in plants. Although auxin acts as a suppressor of leaf senescence, its exact role in this respect has not been clearly defined, aside from circumstantial evidence. It was found here that ARF2 functions in the auxin-mediated control of Arabidopsis leaf longevity, as discovered by screening EMS mutant pools for a delayed leaf senescence phenotype. Two allelic mutations, ore14-1 and 14-2, caused a highly significant delay in all senescence parameters examined, including chlorophyll content, the photochemical efficiency of photosystem II, membrane ion leakage, and the expression of senescence-associated genes. A delay of senescence symptoms was also observed under various senescence-accelerating conditions, where detached leaves were treated with darkness, phytohormones, or oxidative stress. These results indicate that the gene defined by these mutations might be a key regulatory genetic component controlling functional leaf senescence. Map-based cloning of ORE14 revealed that it encodes ARF2, a member of the auxin response factor (ARF) protein family, which modulates early auxin-induced gene expression in plants. The ore14/arf2 mutation also conferred an increased sensitivity to exogenous auxin in hypocotyl growth inhibition, thereby demonstrating that ARF2 is a repressor of auxin signalling. Therefore, the ore14/arf2 lesion appears to cause reduced repression of auxin signalling with increased auxin sensitivity, leading to delayed senescence. Altogether, our data suggest that ARF2 positively regulates leaf senescence in Arabidopsis.

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Contribution of carbon fixed by Rubisco and PEPC to phloem export in the Crassulacean acid metabolism plant Kalanchoe daigremontiana.

Publication Date: 2010 Feb 16 PMID: 20159885
Authors: Wild, B. - Wanek, W. - Postl, W. - Richter, A.
Journal: J Exp Bot

Crassulacean acid metabolism (CAM) plants exhibit a complex interplay between CO(2) fixation by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), and carbon demand for CAM maintenance and growth. This study investigated the flux of carbon from PEPC and direct Rubisco fixation to different leaf carbon pools and to phloem sap over the diurnal cycle. Concentrations and carbon isotope compositions of starch, soluble sugars, and organic acids were determined in leaves and phloem exudates of Kalanchoe daigremontiana Hamet et Perr., and related to CO(2) fixation by PEPC and Rubisco. Three types of leaf carbon pools could be distinguished. (i) Starch and malate pools were dominant and showed a pattern of reciprocal mobilization and accumulation (85/54 and 13/48 mg C g(-1) DW, respective, at the beginning/end of phase I). The carbon isotope composition of these pools was compatible with predominant PEPC fixation (delta(13)C values of -13 and -11 per thousand for starch and malate compared to -11 per thousand of PEPC fixed carbon). (ii) Isotopic composition (-17 per thousand and -14 per thousand) and concentration of glucose and fructose (2 and 3 mg C g(-1) DW, respectively) were not affected by diurnal metabolism, suggesting a low turnover. (iii) Sucrose (1-3 mg C g(-1) DW), in contrast, exhibited large diurnal changes in delta(13)C values (from -17 per thousand in the evening to -12 per thousand in the morning), which were not matched by net changes in sucrose concentration. This suggests a high sucrose turnover, fed by nocturnal starch degradation and direct Rubisco fixation during the day. A detailed dissection of the carbon fixation and mobilization pattern in K. daigremontiana revealed that direct fixation of Rubisco during the light accounted for 30% of phloem sucrose, but only 15% of fixed carbon, indicating that carbon from direct Rubisco fixation was preferentially used for leaf export.

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Regulation of actin dynamics by actin-binding proteins in pollen.

Publication Date: 2010 Feb 16 PMID: 20159884
Authors: Staiger, C. J. - Poulter, N. S. - Henty, J. L. - Franklin-Tong, V. E. - Blanchoin, L.
Journal: J Exp Bot

A dynamic network of polymers, the actin cytoskeleton, co-ordinates numerous fundamental cellular processes. In pollen tubes, organelle movements and cytoplasmic streaming, organization of the tip zone, vesicle trafficking, and tip growth have all been linked to actin-based function. Further, during the self-incompatibility response of Papaver rhoeas, destruction of the cytoskeleton is a primary target implicated in the rapid cessation of pollen tube growth and alterations in actin dynamics are associated with the initiation of programmed cell death. Surprisingly, these diverse cellular processes are accomplished with only a small amount of filamentous actin and a huge pool of polymerizable monomers. These observations hint at incredibly fast and complex actin dynamics in pollen. To understand the molecular mechanisms regulating actin dynamics in plant cells, the abundant actin monomer-binding proteins, a major filament nucleator, a family of bundling and severing proteins, and a modulator of growth at the barbed-end of actin filaments have been characterized biochemically. The activities of these proteins are generally consistent with textbook models for actin turnover. For example, the three monomer-binding proteins, profilin, ADF, and CAP, are thought to function synergistically to enhance turnover and the exchange of subunits between monomer and polymer pools. How individual actin filaments behave in living cells, however, remains largely unexplored. Actin dynamics were examined using variable angle epifluorescence microscopy (VAEM) in expanding hypocotyl epidermal cells. Our observations of single filament behaviour are not consistent with filament turnover by treadmilling, but rather represent a novel property called stochastic dynamics. A new model for the dynamic control of actin filament turnover in plant cells is presented.

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Control of starch branching in barley defined through differential RNAi suppression of starch branching enzyme IIa and IIb.

Publication Date: 2010 Feb 15 PMID: 20156842
Authors: Regina, A. - Kosar-Hashemi, B. - Ling, S. - Li, Z. - Rahman, S. - Morell, M.
Journal: J Exp Bot

The roles of starch branching enzyme (SBE, EC 2.4.1.18) IIa and SBE IIb in defining the structure of amylose and amylopectin in barley (Hordeum vulgare) endosperm were examined. Barley lines with low expression of SBE IIa or SBE IIb, and with the low expression of both isoforms were generated through RNA-mediated silencing technology. These lines enabled the study of the role of each of these isoforms in determining the amylose content, the distribution of chain lengths, and the frequency of branching in both amylose and amylopectin. In lines where both SBE IIa and SBE IIb expression were reduced by >80%, a high amylose phenotype (>70%) was observed, while a reduction in the expression of either of these isoforms alone had minor impact on amylose content. The structure and properties of the high amylose starch resulting from the concomitant reduction in the expression of both isoforms of SBE II in barley were found to approximate changes seen in amylose extender mutants of maize, which result from lesions eliminating expression of the SBE IIb gene. Amylopectin chain length distribution analysis indicated that both SBE IIa and SBE IIb isoforms play distinct roles in determining the fine structure of amylopectin. A significant reduction in the frequency of branches in amylopectin was noticed only when both SBE IIa and SBE IIb were reduced, whereas there was a significant increase in the branching frequency of amylose when SBE IIb alone was reduced. Functional interactions between SBE isoforms are suggested, and a possible inhibitory role of SBE IIb on other SBE isoforms is discussed.

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Ion fluxes across the pitcher walls of three Bornean Nepenthes pitcher plant species: flux rates and gland distribution patterns reflect nitrogen sequestration strategies.

Publication Date: 2010 Feb 11 PMID: 20150519
Authors: Moran, J. A. - Hawkins, B. J. - Gowen, B. E. - Robbins, S. L.
Journal: J Exp Bot

Nepenthes pitcher plant species differ in their prey capture strategies, prey capture rates, and pitcher longevity. In this study, it is investigated whether or not interspecific differences in nutrient sequestration strategy are reflected in the physiology and microstructure of the pitchers themselves. Using a non-invasive technique (MIFE), ion fluxes in pitchers of Nepenthes ampullaria Jack, Nepenthes bicalcarata Hook.f., and Nepenthes rafflesiana Jack were measured. Scanning electron microscopy was also used to characterize the distribution of glandular and other structures on the inner pitcher walls. The results demonstrate that nutrient sequestration strategy is indeed mirrored in pitcher physiology and microstructure. Species producing long-lived pitchers with low prey capture rates (N. ampullaria, N. bicalcarata) showed lower rates of NH(4)(+) uptake than N. rafflesiana, a species producing short-lived pitchers with high capture rates. Crucially, species dependent upon aquatic commensals (N. ampullaria, N. bicalcarata) actively manipulated H(+) fluxes to maintain less acid pitcher fluid than found in 'typical' species; in addition, these species lacked the lunate cells and epicuticular waxes characteristic of 'typical' insectivorous congeners. An unexpected finding was that ion fluxes occurred in the wax-covered, non-glandular zones in N. rafflesiana. The only candidates for active transport of aqueous ions in these zones appear to be the epidermal cells lying beneath the lunate cells, as these are the only sites not visibly coated with epicuticular waxes.

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Metabolic profiling of Arabidopsis thaliana epidermal cells.

Publication Date: 2010 Feb 11 PMID: 20150518
Authors: Ebert, B. - Zoller, D. - Erban, A. - Fehrle, I. - Hartmann, J. - Niehl, A. - Kopka, J. - Fisahn, J.
Journal: J Exp Bot

Metabolic phenotyping at cellular resolution may be considered one of the challenges in current plant physiology. A method is described which enables the cell type-specific metabolic analysis of epidermal cell types in Arabidopsis thaliana pavement, basal, and trichome cells. To achieve the required high spatial resolution, single cell sampling using microcapillaries was combined with routine gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) based metabolite profiling. The identification and relative quantification of 117 mostly primary metabolites has been demonstrated. The majority, namely 90 compounds, were accessible without analytical background correction. Analyses were performed using cell type-specific pools of 200 microsampled individual cells. Moreover, among these identified metabolites, 38 exhibited differential pool sizes in trichomes, basal or pavement cells. The application of an independent component analysis confirmed the cell type-specific metabolic phenotypes. Significant pool size changes between individual cells were detectable within several classes of metabolites, namely amino acids, fatty acids and alcohols, alkanes, lipids, N-compounds, organic acids and polyhydroxy acids, polyols, sugars, sugar conjugates and phenylpropanoids. It is demonstrated here that the combination of microsampling and GC-MS based metabolite profiling provides a method to investigate the cellular metabolism of fully differentiated plant cell types in vivo.

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The proteome map of spinach leaf peroxisomes indicates partial compartmentalization of phylloquinone (vitamin K1) biosynthesis in plant peroxisomes.

Publication Date: 2010 Feb 11 PMID: 20150517
Authors: Babujee, L. - Wurtz, V. - Ma, C. - Lueder, F. - Soni, P. - van Dorsselaer, A. - Reumann, S.
Journal: J Exp Bot

Leaf peroxisomes are fragile, low-abundance plant cell organelles that are difficult to isolate from one of the few plant species whose nuclear genome has been sequenced. Leaf peroxisomes were enriched at high purity from spinach (Spinacia oleracea) and approximately 100 protein spots identified from 2-dimensional gels by a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and de novo sequencing. In addition to the predominant enzymes involved in photorespiration and detoxification, several minor enzymes were detected, underscoring the high sensitivity of the protein identification. The tryptic peptides of three unknown proteins shared high sequence similarity with Arabidopsis proteins that carry putative peroxisomal targeting signals type 1 or 2 (PTS1/2). The apparent Arabidopsis orthologues are a short-chain alcohol dehydrogenase (SDRa/IBR1, At4g05530, SRL>) and two enoyl-CoA hydratases/isomerases (ECHIa, At4g16210, SKL>; NS/ECHId, At1g60550, RLx(5)HL). The peroxisomal localization of the three proteins was confirmed in vivo by tagging with enhanced yellow fluorescent protein (EYFP), and the targeting signals were identified. The single Arabidopsis isoform of naphthoate synthase (NS) is orthologous to MenB from cyanobacteria, which catalyses an essential reaction in phylloquinone biosynthesis, a pathway previously assumed to be entirely compartmentalized in plastids in higher plants. In an extension of a previous study, the present in vivo targeting data furthermore demonstrate that the enzyme upstream of NS, chloroplastic acyl-CoA activating enzyme isoform 14 (AAE14, SSL>), is dually targeted to both plastids and peroxisomes. This proteomic study, extended by in vivo subcellular localization analyses, indicates a novel function for plant peroxisomes in phylloquinone biosynthesis.

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Agrobacterium tumefaciens-mediated transformation of Cleome gynandra L., a C4 dicotyledon that is closely related to Arabidopsis thaliana.

Publication Date: 2010 Feb 11 PMID: 20150516
Authors: Newell, C. A. - Brown, N. J. - Liu, Z. - Pflug, A. - Gowik, U. - Westhoff, P. - Hibberd, J. M.
Journal: J Exp Bot

In leaves of most C(4) plants, the biochemistry of photosynthesis is partitioned between mesophyll and bundle sheath cells. In addition, their cell biology and development also differs from that in C(3) plants. We have a poor understanding of the mechanisms that generate the cell-specific accumulation of proteins used in the C(4) pathway, and there are few genes that have been shown to be important for the cell biology and development of C(4) leaves. To facilitate functional analysis of C(4) photosynthesis, and to enable knowledge from Arabidopsis thaliana to be translated to C(4) species, an Agrobacterium tumefaciens-mediated transformation protocol was developed for the C(4) species Cleome gynandra. A. tumefaciens, harbouring the binary vector SLJ1006, was used to transfer the uidA gene under the control of the CaMV 35S promoter into C. gynandra. Co-incubation of hypocotyls or cotyledons with SLJ1006 allowed efficient transfer of DNA into C. gynandra, and media that allowed callus production and then shoot regeneration were identified. Stable transformants of C. gynandra with detectable amounts of beta-glucuronidase (GUS) were produced at an efficiency of 14%. When driven by the CaMV 35S promoter, GUS was visible in all leaf cells, whereas uidA translationally fused to a CgRbcS gene generated GUS accumulation specifically in bundle sheath cells. This transformation procedure is the first for an NAD-ME type C(4) plant and should significantly accelerate the analysis of mechanisms underlying C(4) photosynthesis.

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SRD1 is involved in the auxin-mediated initial thickening growth of storage root by enhancing proliferation of metaxylem and cambium cells in sweetpotato (Ipomoea batatas).

Publication Date: 2010 Feb 11 PMID: 20150515
Authors: Noh, S. A. - Lee, H. S. - Huh, E. J. - Huh, G. H. - Paek, K. H. - Shin, J. S. - Bae, J. M.
Journal: J Exp Bot

A sweetpotato (Ipomoea batatas cv. 'Jinhongmi') MADS-box protein cDNA (SRD1) has been isolated from an early stage storage root cDNA library. The role of the SRD1 gene in the formation of the storage root in sweetpotato was investigated by an expression pattern analysis and characterization of SRD1-overexpressing (ox) transgenic sweetpotato plants. Transcripts of SRD1 were detected only in root tissues, with the fibrous root having low levels of the transcript and the young storage root showing relatively higher transcript levels. SRD1 mRNA was mainly found in the actively dividing cells, including the vascular and cambium cells of the young storage root. The transcript level of SRD1 in the fibrous roots increased in response to 1000 muM indole-3-acetic acid (IAA) applied exogenously. During the early stage of storage root development, the endogenous IAA content and SRD1 transcript level increased concomitantly, suggesting an involvement of SRD1 during the early stage of the auxin-dependent development of the storage root. SRD1-ox sweetpotato plants cultured in vitro produced thicker and shorter fibrous roots than wild-type plants. The metaxylem and cambium cells of the fibrous roots of SRD1-ox plants showed markedly enhanced proliferation, resulting in the fibrous roots of these plants showing an earlier thickening growth than those of wild-type plants. Taken together, these results demonstrate that SRD1 plays a role in the formation of storage roots by activating the proliferation of cambium and metaxylem cells to induce the initial thickening growth of storage roots in an auxin-dependent manner.

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On how to disentangle the contribution of different organs and processes to the growth of whole plants.

Publication Date: 2010 Mar PMID: 20118495
Authors: Aphalo, P. J.
Journal: J Exp Bot

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Leaf lifetime photosynthetic rate and leaf demography in whole plants of Ipomoea pes-caprae growing with a low supply of calcium, a 'non-mobile' nutrient.

Publication Date: 2010 Mar PMID: 20080828
Authors: Suarez, N.
Journal: J Exp Bot

The adaptive significance of leaf longevity has been established in relation to restrictive nutrients that can be retranslocated within the plant. However, the effect of deficiencies in 'non-mobile' nutrients on leaf lifespan and photosynthetic carbon gain is uncertain. Calcium is frequently given as an example of an essential nutrient with low phloem mobility that may alter the leaf senescence process. This study has been designed to estimate leaf lifespan, leaf production (L(p)) and leaf death (L(d)) rates, the age structure of leaves, and the decline in maximum photosynthetic rate (A(max)) with age in plants of Ipomoea pes-caprae growing with a full supply of nutrients and with a low Ca supply. The Ca deficiency produced reductions in L(p) and leaf lifespan compared with control plants. In spite of the differences in the demographic parameters between treatments in control and low-Ca plants, the percentage of leaves of a given leaf age class is maintained in such a way that the number of leaves per plant continues to increase. No relationship was found between Ca supply and A(max). However, the decline in A(max) with leaf senescence was rather sudden in control plants compared with plants growing with a low Ca supply. The importance of simultaneously using the total leaf demographic census and the assimilation rate along with leaf lifespan data in order to understand the performance of whole plants under constrained conditions is discussed.

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Assembly and disassembly of plant microtubules: tubulin modifications and binding to MAPs.

Publication Date: 2010 Mar PMID: 20080825
Authors: Cai, G.
Journal: J Exp Bot

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Correlations in concentrations, xylem and phloem flows, and partitioning of elements and ions in intact plants. A summary and statistical re-evaluation of modelling experiments in Ricinus communis.

Publication Date: 2010 Mar PMID: 20032109
Authors: Peuke, A. D.
Journal: J Exp Bot

Within the last two decades, a series of papers have dealt with the effects of nutrition and nutrient deficiency, as well as salt stress, on the long-distance transport and partitioning of nutrients in castor bean. Flows in xylem and phloem were modelled according to an empirically-based modelling technique that permits additional quantification of the uptake and incorporation into plant organs. In the present paper these data were statistically re-evaluated, and new correlations are presented. Numerous relationships between different compartments and transport processes for single elements, but also between elements, were detected. These correlations revealed different selectivities for ions in bulk net transport. Generally, increasing chemical concentration gradients for mineral nutrients from the rhizosphere to the root and from the xylem to leaf tissue were observed, while such gradients decreased from root tissue to the xylem and from leaves to the phloem. These studies showed that, for the partitioning of nutrients within a plant, the correlated interactions of uptake, xylem and phloem flow, as well as loading and unloading of solutes from transport systems, are of central importance. For essential nutrients, tight correlations between uptake, xylem and phloem flow, and the resulting partitioning of elements, were observed, which allows the stating of general models. For non-essential ions like Na(+) or Cl(-), a statistically significant dependence of xylem transport on uptake was not detected. The central role of the phloem for adjusting, but also signalling, of nutrition status is discussed, since strong correlations between leaf nutrient concentrations and those in phloem saps were observed. In addition, negative correlations between phloem sap sugar concentration and net-photosynthesis, growth, and uptake of nutrients were demonstrated. The question remains whether this is only a consequence of an insufficient use of carbohydrates in plants or a ubiquitous signal for stress in plants. In general, high sugar concentrations in phloem saps indicate (nutritional) stress, and high nutrient concentrations in phloem saps indicate nutritional sufficiency of leaf tissues.

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Membrane-bound guaiacol peroxidases from maize (Zea mays L.) roots are regulated by methyl jasmonate, salicylic acid, and pathogen elicitors.

Publication Date: 2010 Mar PMID: 20032108
Authors: Mika, A. - Boenisch, M. J. - Hopff, D. - Luthje, S.
Journal: J Exp Bot

Plant peroxidases are involved in numerous cellular processes in plant development and stress responses. Four plasma membrane-bound peroxidases have been identified and characterized in maize (Zea mays L.) roots. In the present study, maize seedlings were treated with different stresses and signal compounds, and a functional analysis of these membrane-bound class III peroxidases (pmPOX1, pmPOX2a, pmPOX2b, and pmPOX3) was carried out. Total guaiacol peroxidase activities from soluble and microsomal fractions of maize roots were compared and showed weak changes. By contrast, total plasma membrane and washed plasma membrane peroxidase activities, representing peripheral and integral membrane proteins, revealed strong changes after all of the stresses applied. A proteomic approach using 2D-PAGE analysis showed that pmPOX3 was the most abundant class III peroxidase at plasma membranes of control plants, followed by pmPOX2a >pmPOX2b >pmPOX1. The molecular mass (63 kDa) and the isoelectric point (9.5) of the pmPOX2a monomer were identified for the first time. The protein levels of all four enzymes changed in response to multiple stresses. While pmPOX2b was the only membrane peroxidase down-regulated by wounding, all four enzymes were differentially but strongly stimulated by methyl jasmonate, salicylic acid, and elicitors (Fusarium graminearum and Fusarium culmorum extracts, and chitosan) indicating their function in pathogen defence. Oxidative stress applied as H(2)O(2) treatment up-regulated pmPOX2b >pmPOX2a, while pmPOX3 was down-regulated. Treatment with the phosphatase inhibitor chantharidin resulted in distinct responses.

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A major quantitative trait locus for increasing cadmium-specific concentration in rice grain is located on the short arm of chromosome 7.

Publication Date: 2010 Mar PMID: 20022924
Authors: Ishikawa, S. - Abe, T. - Kuramata, M. - Yamaguchi, M. - Ando, T. - Yamamoto, T. - Yano, M.
Journal: J Exp Bot

Large phenotypic variations in the cadmium (Cd) concentration of rice grains and shoots have been observed. However, the genetic control of Cd accumulation remains poorly understood. Quantitative trait loci (QTLs) determining the grain Cd concentration of rice grown in a Cd-polluted paddy field were identified. Using a mapping population consisting of 85 backcross inbred lines derived from a cross between the low-Cd-accumulating cultivar Sasanishiki (japonica) and high-Cd-accumulating cultivar Habataki (indica), two QTLs for increasing grain Cd concentration were found on chromosomes 2 and 7. A major-effect QTL, qGCd7 (QTL for grain Cd on chromosome 7), was detected on the short arm of chromosome 7. It accounted for 35.5% of all phenotypic variance in backcross inbred lines. qGCd7 was not genetically related to any QTLs for concentrations of essential trace metals (Cu, Fe, Mn, and Zn) or those for agronomic traits such as heading date, suggesting that this QTL is specific to Cd. Furthermore, the existence of qGCd7 was confirmed using chromosome segment substitution lines (CSSLs) and an F(2) population from a cross between the target CSSL and Sasanishiki grown in a Cd-polluted paddy soil. To our knowledge, qGCd7 is a novel QTL with major effects for increasing grain Cd concentrations.

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TaSnRK2.4, an SNF1-type serine/threonine protein kinase of wheat (Triticum aestivum L.), confers enhanced multistress tolerance in Arabidopsis.

Publication Date: 2010 Mar PMID: 20022921
Authors: Mao, X. - Zhang, H. - Tian, S. - Chang, X. - Jing, R.
Journal: J Exp Bot

Osmotic stresses such as drought, salinity, and cold are major environmental factors that limit agricultural productivity worldwide. Protein phosphorylation/dephosphorylation are major signalling events induced by osmotic stress in higher plants. Sucrose non-fermenting 1-related protein kinase2 family members play essential roles in response to hyperosmotic stresses in Arabidopsis, rice, and maize. In this study, the function of TaSnRK2.4 in drought, salt, and freezing stresses in Arabidopsis was characterized. A translational fusion protein of TaSnRK2.4 with green fluorescent protein showed subcellular localization in the cell membrane, cytoplasm, and nucleus. To examine the role of TaSnRK2.4 under various environmental stresses, transgenic Arabidopsis plants overexpressing wheat TaSnRK2.4 under control of the cauliflower mosaic virus 35S promoter were generated. Overexpression of TaSnRK2.4 resulted in delayed seedling establishment, longer primary roots, and higher yield under normal growing conditions. Transgenic Arabidopsis overexpressing TaSnRK2.4 had enhanced tolerance to drought, salt, and freezing stresses, which were simultaneously supported by physiological results, including decreased rate of water loss, enhanced higher relative water content, strengthened cell membrane stability, improved photosynthesis potential, and significantly increased osmotic potential. The results show that TaSnRK2.4 is involved in the regulation of enhanced osmotic potential, growth, and development under both normal and stress conditions, and imply that TaSnRK2.4 is a multifunctional regulatory factor in Arabidopsis. Since the overexpression of TaSnRK2.4 can significantly strengthen tolerance to drought, salt, and freezing stresses and does not retard the growth of transgenic Arabidopsis plants under well-watered conditions, TaSnRK2.4 could be utilized in transgenic breeding to improve abiotic stresses in crops.

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Induced production of antifungal naphthoquinones in the pitchers of the carnivorous plant Nepenthes khasiana.

Publication Date: 2010 Mar PMID: 20018905
Authors: Eilenberg, H. - Pnini-Cohen, S. - Rahamim, Y. - Sionov, E. - Segal, E. - Carmeli, S. - Zilberstein, A.
Journal: J Exp Bot

Nepenthes spp. are carnivorous plants that have developed insect capturing traps, evolved by specific modification of the leaf tips, and are able to utilize insect degradation products as nutritional precursors. A chitin-induced antifungal ability, based on the production and secretion to the trap liquid of droserone and 5-O-methyldroserone, is described here. Such specific secretion uniquely occurred when chitin injection was used as the eliciting agent and probably reflects a certain kind of defence mechanism that has been evolved for protecting the carnivory-based provision of nutritional precursors. The pitcher liquid containing droserone and 5-O-methyldroserone at 3:1 or 4:1 molar ratio, as well as the purified naphthoquinones, exerted an antifungal effect on a wide range of plant and human fungal pathogens. When tested against Candida and Aspergillus spp., the concentrations required for achieving inhibitory and fungicidal effects were significantly lower than those causing cytotoxicity in cells of the human embryonic kidney cell line, 293T. These naturally secreted 1,4-naphthoquinone derivatives, that are assumed to act via semiquinone enhancement of free radical production, may offer a new lead to develop alternative antifungal drugs with reduced selectable pressure for potentially evolved resistance.

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Supply of sulphur to S-deficient young barley seedlings restores their capability to cope with iron shortage.

Publication Date: 2010 Mar PMID: 20018904
Authors: Astolfi, S. - Zuchi, S. - Hubberten, H. M. - Pinton, R. - Hoefgen, R.
Journal: J Exp Bot

The effect of the S nutritional status on a plant's capability to cope with Fe shortage was studied in solution cultivation experiments in barley (Hordeum vulgare L. cv. Europa). Barley is a Strategy II plant and responds to Fe deficiency by secretion of chelating compounds, phytosiderophores (PS). All PS are derived from nicotianamine whose precursor is methionine. This suggests that a long-term supply of an inadequate amount of S could reduce a plant's capability to respond to Fe deficiency by limiting the rate of PS biosynthesis. The responses of barley (Hordeum vulgare L. cv. Europa) plants grown for 12 d on Fe-free nutrient solutions (NS) containing 0 or 1.2 mM SO(4)(2-), was examined after 24 h or 48 h from transfer to NS containing 1.2 mM SO(4)(2-). After the supply of S was restored to S-deprived plants, an increase in PS release in root exudates was evident after 24 h of growth in S-sufficient NS and the increment reached values up to 4-fold higher than the control 48 h after S resupply. When S was supplied to S-deficient plants, leaf ATPS (EC 2.7.7.4) and OASTL (EC 4.2.99.8) activities exhibited a progressive recovery. Furthermore, root HvST1 transcript abundance remained high for 48 h following S resupply and a significant increase in the level of root HvYS1 transcripts was also found after only 24 h of S resupply. Data support the idea that the extent to which the plant is able to cope with Fe starvation is strongly associated with its S nutritional status. In particular, our results are indicative that barley plants fully recover their capability to cope with Fe shortage after the supply of S is restored to S-deficient plants.

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Plant cell division is specifically affected by nitrotyrosine.

Publication Date: 2010 Mar PMID: 20018903
Authors: Jovanovic, A. M. - Durst, S. - Nick, P.
Journal: J Exp Bot

Virtually all eukaryotic alpha-tubulins harbour a C-terminal tyrosine that can be reversibly removed and religated, catalysed by a specific tubulin-tyrosine carboxypeptidase (TTC) and a specific tubulin-tyrosine ligase (TTL), respectively. The biological function of this post-translational modification has remained enigmatic. 3-nitro-L-tyrosine (nitrotyrosine, NO(2)Tyr), can be incorporated into detyrosinated alpha-tubulin instead of tyrosine, producing irreversibly nitrotyrosinated alpha-tubulin. To gain insight into the possible function of detyrosination, the effect of NO(2)Tyr has been assessed in two plant model organisms (rice and tobacco). NO(2)Tyr causes a specific, sensitive, and dose-dependent inhibition of cell division that becomes detectable from 1 h after treatment and which is not observed with non-nitrosylated tyrosine. These effects are most pronounced in cycling tobacco BY-2 cells, where the inhibition of cell division is accompanied by a stimulation of cell length, and a misorientation of cross walls. NO(2)Tyr reduces the abundance of the detyrosinated form of alpha-tubulin whereas the tyrosinated alpha-tubulin is not affected. These findings are discussed with respect to a model where NO(2)Tyr is accepted as substrate by TTL and subsequently blocks TTC activity. The irreversibly tyrosinated alpha-tubulin impairs microtubular functions that are relevant to cell division in general, and cell wall deposition in particular.

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Nicotiana tabacum EIL2 directly regulates expression of at least one tobacco gene induced by sulphur starvation.

Publication Date: 2010 Mar PMID: 20018902
Authors: Wawrzynska, A. - Lewandowska, M. - Sirko, A.
Journal: J Exp Bot

Sulphur deficiency severely affects plant growth and their agricultural productivity leading to diverse changes in development and metabolisms. Molecular mechanisms regulating gene expression under low sulphur conditions remain largely unknown. AtSLIM1, a member of the EIN3-like (EIL) family was reported to be a central transcriptional regulator of the plant sulphur response, however, no direct interaction of this protein with any sulphur-responsive promoters was demonstrated. The focus of this study was on the analysis of a promoter region of UP9C, a tobacco gene strongly induced by sulphur limitation. Cloning and subsequent examination of this promoter resulted in the identification of a 20-nt sequence (UPE-box), also present in the promoters of several Arabidopsis genes, including three out of four homologues of UP9C. The UPE-box, consisting of two parallel tebs sequences (TEIL binding site), proved to be necessary to bind the transcription factors belonging to the EIL family and of a 5-nt conserved sequence at the 3'-end. The yeast one-hybrid analysis resulted in the identification of one transcription factor (NtEIL2) capable of binding to the UPE-box. The interactions of NtEIL2, and its homologue from Arabidopsis, AtSLIM1, with DNA were affected by mutations within the UPE-box. Transient expression assays in Nicotiana benthamiana have further shown that both factors, NtEIL2 and AtSLIM1, activate the UP9C promoter. Interestingly, activation by NtEIL2, but not by AtSLIM1, was dependent on the sulphur-deficiency of the plants.

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Overexpression of OsRAN2 in rice and Arabidopsis renders transgenic plants hypersensitive to salinity and osmotic stress.

Publication Date: 2010 Mar PMID: 20018899
Authors: Zang, A. - Xu, X. - Neill, S. - Cai, W.
Journal: J Exp Bot

Nucleo-cytoplasmic partitioning of regulatory proteins is increasingly being recognized as a major control mechanism for the regulation of signalling in plants. Ras-related nuclear protein (Ran) GTPase is required for regulating transport of proteins and RNA across the nuclear envelope and also has roles in mitotic spindle assembly and nuclear envelope (NE) assembly. However, thus far little is known of any Ran functions in the signalling pathways in plants in response to changing environmental stimuli. The OsRAN2 gene, which has high homology (77% at the amino acid level) with its human counterpart, was isolated here. Subcellular localization results showed that OsRan2 is mainly localized in the nucleus, with some in the cytoplasm. Transcription of OsRAN2 was reduced by salt, osmotic, and exogenous abscisic acid (ABA) treatments, as determined by real-time PCR. Overexpression of OsRAN2 in rice resulted in enhanced sensitivity to salinity, osmotic stress, and ABA. Seedlings of transgenic Arabidopsis thaliana plants overexpressing OsRAN2 were overly sensitive to salinity stress and exogenous ABA treatment. Furthermore, three ABA- or stress-responsive genes, AtNCED3, AtPLC1, and AtMYB2, encoding a key enzyme in ABA synthesis, a phospholipase C homologue, and a putative transcriptional factor, respectively, were shown to have differentially induced expression under salinity and ABA treatments in transgenic and wild-type Arabidopsis plants. OsRAN2 overexpression in tobacco epidermal leaf cells disturbed the nuclear import of a maize (Zea mays L.) leaf colour transcription factor (Lc). In addition, gene-silenced rice plants generated via RNA interference (RNAi) displayed pleiotropic developmental abnormalities and were male sterile.

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Genetic evidence that two independent S-loci control RNase-based self-incompatibility in diploid strawberry.

Publication Date: 2010 Mar PMID: 20008462
Authors: Boskovic, R. I. - Sargent, D. J. - Tobutt, K. R.
Journal: J Exp Bot

The self-incompatibility mechanism that reduces inbreeding in many plants of the Rosaceae is attributed to a multi-allelic S locus which, in the Prunoideae and Maloideae subfamilies, comprises two complementary genes, a stylar-expressed S-RNase and a pollen-expressed SFB. To elucidate incompatibility in the subfamily Rosoideae, stylar-specific RNases and self-(in)compatibility status were analysed in various diploid strawberries, especially Fragaria nubicola and F. viridis, both self-incompatible, and F. vesca, self-compatible, and in various progenies derived from them. Unexpectedly, two unlinked RNase loci, S and T, were found, encoding peptides distinct from Prunoideae and Maloideae S-RNases; the presence of a single active allele at either is sufficient to confer self-incompatibility. By contrast, in diploid Maloideae and Prunoideae a single locus encodes S-RNases that share several conserved regions and two active alleles are required for self-incompatibility. Our evidence implicates the S locus in unilateral inter-specific incompatibility and shows that S and T RNases can, remarkably, confer not only allele-specific rejection of cognate pollen but also unspecific rejection of Sn Tn pollen, where n indicates a null allele, consistent with the the presence of the pollen component, SFB, activating the cognitive function of these RNases. Comparison of relevant linkage groups between Fragaria and Prunus suggests that Prunus S-RNases, unique in having two introns, may have resulted from gene conversion in an ancestor of Prunus. In addition, it is shown that there is a non-S locus that is essential for self-incompatibility in diploid Fragaria.

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Expression of the ethylene biosynthetic machinery in maize roots is regulated in response to hypoxia.

Publication Date: 2010 Mar PMID: 20008461
Authors: Geisler-Lee, J. - Caldwell, C. - Gallie, D. R.
Journal: J Exp Bot

Ethylene regulates plant growth in response to many adverse environmental conditions, including the induction of aerenchyma, i.e. the formation of air spaces, in flooded roots in an effort to maintain oxygen levels. In this work, quantitative RT-PCR and in situ RNA hybridization were used to determine how the expression of the ethylene biosynthetic machinery in maize roots is spatially and temporally regulated following exposure to 4% oxygen (i.e. hypoxia) for up to 24 h, conditions that induced aerenchyma formation in the fully-expanded region of the root and reduced cytoplasmic density throughout the root. Expression of ACC oxidase, the ethylene forming enzyme, was observed in the root cap, protophloem sieve elements, and companion cells associated with metaphloem sieve elements. Exposure to 4% oxygen induced ACC oxidase expression in these cell types as well as in the root cortex. ACC synthase, which generates the ethylene precursor, was expressed in the root cap and the cortex and its expression was induced in cortical cells following low oxygen treatment. The induction of expression of the ethylene biosynthetic machinery was accompanied by an induction of ethylene evolution and a reduced rate of root growth. These results suggest that maize roots respond to conditions of hypoxia by inducing the spatially restricted expression of the ethylene biosynthetic machinery, resulting in increased ethylene production.

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Identification and functional analysis of PCNA1 and PCNA-like1 genes of Phaseolus coccineus.

Publication Date: 2010 Mar PMID: 20007687
Authors: Strzalka, W. - Kaczmarek, A. - Naganowska, B. - Ziemienowicz, A.
Journal: J Exp Bot

Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication and in many other processes in eukaryotic cells. Genetic analysis of Phaseolus coccineus showed the presence of at least two PCNA-like genes in the runner bean genome. Two PCNA genes have previously been found in a few plant species including Arabidopsis, tobacco, and maize. In these species, genes were nearly identical. Two cDNAs of P. coccineus PCNA (PcPCNA1 and PcPCNA-like1) have been identified that differ distinctly from each other. Interestingly, both the genetic organization of PcPCNA1 and PcPCNA-like1 genes and their expression patterns were similar, but these were the only similarities between these genes and their products. The identity between PcPCNA1 and PcPCNA-like1 at the amino acid level was only 54%, with PcPCNA-like1 lacking motifs that are crucial for the activity typical of PCNA. Consequently, these two proteins showed different properties. PcPCNA1 behaved like a typical PCNA protein: it formed a homotrimer and stimulated the activity of human DNA polymerase delta. In addition, PcPCNA1 interacted with a p21 peptide and was recognized by an anti-human PCNA monoclonal antibody PC10. By contrast, PcPCNA-like1 was detected as a monomer and was unable to stimulate the DNA polymerase delta activity. PcPCNA-like1 also could not interact with p21 and was not recognized by the PC10 antibody. Our results suggest that PcPCNA-like1 either is unable to function alone and therefore might be a component of the heterotrimeric PCNA ring or may have other, yet unknown functions. Alternatively, the PcPCNA-like1 gene may represent a pseudogene.

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Silymarin secretion and its elicitation by methyl jasmonate in cell cultures of Silybum marianum is mediated by phospholipase D-phosphatidic acid.

Publication Date: 2010 Mar PMID: 20007197
Authors: Madrid, E. - Corchete, P.
Journal: J Exp Bot

The flavonolignan silymarin is released to the extracellular medium of Silybum marianum cultures and its production can be stimulated by the elicitor methyljasmonate (MeJA). The sequence of the signalling processes leading to this response is unknown at present. It is reported in this work that MeJA increased the activity of the enzyme phospholipase D (PLD). Treatment with mastoparan (Mst), a PLD activity stimulator, also enhanced PLD and caused a substantial increase in silymarin production. The application of the product of PLD activity, phosphatidic acid (PA) promoted silymarin accumulation. Altering PLD activity by introducing in cultures n-butanol (nBuOH), which inhibits PA production by PLD, prevented silymarin elicitation by MeJA or Mst and also impeded its release in non-elicited cultures. Treatment with iso-, sec- or tert- butanol had no effect on silymarin production. The exogenous addition of PA reversed the inhibitory action of nBuOH, both in control and MeJA-treated cultures. These results suggest that the enzyme PLD and its product PA mediate silymarin secretion to the medium of S. marianum cultures.

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Heteroplasmy and stoichiometric complexity of plant mitochondrial genomes--though this be madness, yet there's method in't.

Publication Date: 2010 Mar PMID: 19995826
Authors: Woloszynska, M.
Journal: J Exp Bot

Mitochondrial heteroplasmy is defined as the coexistence of divergent mitochondrial genotypes in a cell. The ratio of the alternative genomes may be variable, but in plants, the usually prevalent main genome is accompanied by sublimons--substoichiometric mitochondrial DNA (mtDNA) molecules. Plant mitochondrial heteroplasmy was originally viewed as being associated with pathological mutations or was found in non-natural plant populations. Currently, it is considered to be a common situation in plants. Recent years have changed the previous view on the role of homologous recombination, small-scale mutations, and paternal leakage of mtDNA in the generation of heteroplasmy. Newly developed sensitive techniques have allowed the precise estimation of mtDNA stoichiometry. Mechanisms of maintenance and transmission of heteroplasmic genomes, including DNA recombination and replication, as well as mitochondrial fusion and fission, have been studied. This review describes the high level of plant mitochondrial genome complication--the 'madness' resulting from the heteroplasmic state and explains the method hidden in this madness. Heteroplasmy is described as the evolutionary strategy of uniparentally inherited plant mitochondrial genomes which do not undergo sexual recombination. In order to compensate for this deficiency, alternative types of mtDNA are substoichiometrically accumulated as a reservoir of genetic variability and may undergo accelerated evolution. Occasionally, sublimons are selected and amplified in the process called substoichiometric shifting, to take over the role of the main genome. Alternative mitochondrial genomes may recombine, yielding new mtDNA variants, or segregate during plant growth resulting in plants with mosaic phenotypes. Two opposite roles of mitochondrial heteroplasmy with respect to acceleration or counteracting of mutation accumulation are also discussed. Finally, nuclear control of heteroplasmy and substoichiometric shifting is described.

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Transpiration response of 'slow-wilting' and commercial soybean (Glycine max (L.) Merr.) genotypes to three aquaporin inhibitors.

Publication Date: 2010 Mar PMID: 19969533
Authors: Sadok, W. - Sinclair, T. R.
Journal: J Exp Bot

The slow-wilting soybean [Glycine max (L.) Merr.] genotype, PI 416937, exhibits a limiting leaf hydraulic conductance for transpiration rate (TR) under high vapour pressure deficit (VPD). This genotype has a constant TR at VPD greater than 2 kPa, which may be responsible for its drought tolerance as a result of soil water conservation. However, the exact source of the hydraulic limitation between symplastic and apoplastic water flow in the leaf under high VPD conditions are not known for PI 416937. A comparison was made in the TR response to aquaporin (AQP) inhibitors between PI 416937 and N01-11136, a commercial genotype that has a linear TR response to VPD in the 1-3.5 kPa range. Three AQP inhibitors were tested: cycloheximide (CHX, a de novo synthesis inhibitor), HgCl(2), and AgNO(3). Dose-response curves for the decrease in TR following exposure to each inhibitor were developed. Decreases in TR of N01-11136 following treatment with inhibitors were up to 60% for CHX, 82% for HgCl(2), and 42% for AgNO(3). These results indicate that the symplastic pathway terminating in the guard cells of these soybean leaves may be at least as important as the apoplastic pathway for water flow in the leaf under high VPD. While the decrease in TR for PI 416937 was similar to that of N01-11136 following exposure to CHX and HgCl(2), TR of PI 416937 was insensitive to AgNO(3) exposure. These results indicate the possibility of a lack of a Ag-sensitive leaf AQP population in the slow-wilting line, PI 416937, and the presence of such a population in the commercial line, N01-11136.

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Overexpression of serine acetlytransferase produced large increases in O-acetylserine and free cysteine in developing seeds of a grain legume.

Publication Date: 2010 Mar PMID: 19939888
Authors: Tabe, L. - Wirtz, M. - Molvig, L. - Droux, M. - Hell, R.
Journal: J Exp Bot

There have been many attempts to increase concentrations of the nutritionally essential sulphur amino acids by modifying their biosynthetic pathway in leaves of transgenic plants. This report describes the first modification of cysteine biosynthesis in developing seeds; those of the grain legume, narrow leaf lupin (Lupinus angustifolius, L.). Expression in developing lupin embryos of a serine acetyltransferase (SAT) from Arabidopsis thaliana (AtSAT1 or AtSerat 2;1) was associated with increases of up to 5-fold in the concentrations of O-acetylserine (OAS), the immediate product of SAT, and up to 26-fold in free cysteine, resulting in some of the highest in vivo concentrations of these metabolites yet reported. Despite the dramatic changes in free cysteine in developing embryos of SAT overexpressers, concentrations of free methionine in developing embryos, and the total cysteine and methionine concentrations in mature seeds were not significantly altered. Pooled F(2) seeds segregating for the SAT transgene and for a transgene encoding a methionine- and cysteine-rich sunflower seed storage protein also had increased OAS and free cysteine, but not free methionine, during development, and no increase in mature seed total sulphur amino acids compared with controls lacking SAT overexpression. The data support the view that the cysteine biosynthetic pathway is active in developing seeds, and indicate that SAT activity limits cysteine biosynthesis, but that cysteine supply is not limiting for methionine biosynthesis or for storage protein synthesis in maturing lupin embryos in conditions of adequate sulphur nutrition. OAS and free methionine, but not free cysteine, were implicated as signalling metabolites controlling expression of a gene for a cysteine-rich seed storage protein.

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Intraspecific variation in herbivore community composition and transcriptional profiles in field-grown Brassica oleracea cultivars.

Publication Date: 2010 Mar PMID: 19934173
Authors: Broekgaarden, C. - Poelman, E. H. - Voorrips, R. E. - Dicke, M. - Vosman, B.
Journal: J Exp Bot

Intraspecific differences in plant defence traits are often correlated with variation in transcriptional profiles and can affect the composition of herbivore communities on field-grown plants. However, most studies on transcriptional profiling of plant-herbivore interactions have been carried out under controlled conditions in the laboratory or greenhouse and only a few examine intraspecific transcriptional variation. Here, intraspecific variation in herbivore community composition and transcriptional profiles between two Brassica oleracea cultivars grown in the field is addressed. Early in the season, no differences in community composition were found for naturally occurring herbivores, whereas cultivars differed greatly in abundance, species richness, and herbivore community later in the season. Genome-wide transcriptomic analysis using an Arabidopsis thaliana oligonucleotide microarray showed clear differences for the expression levels of 26 genes between the two cultivars later in the season. Several defence-related genes showed higher levels of expression in the cultivar that harboured the lowest numbers of herbivores. Our study shows that herbivore community composition develops differentially throughout the season on the two B. oleracea cultivars grown in the field. The correlation between the differences in herbivore communities and differential expression of particular defence-related genes is discussed.

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Effects of drought stress and subsequent rewatering on photosynthetic and respiratory pathways in Nicotiana sylvestris wild type and the mitochondrial complex I-deficient CMSII mutant.

Publication Date: 2010 Mar PMID: 19933320
Authors: Galle, A. - Florez-Sarasa, I. - Thameur, A. - de Paepe, R. - Flexas, J. - Ribas-Carbo, M.
Journal: J Exp Bot

The interaction of photosynthesis and respiration has been studied in vivo under conditions of limited water supply and after consecutive rewatering. The role of the alternative (v(alt)) and cytochrome (v(cyt)) pathways on drought stress-induced suppression of photosynthesis and during photosynthetic recovery was examined in the Nicotiana sylvestris wild type (WT) and the complex I-deficient CMSII mutant. Although photosynthetic traits, including net photosynthesis (A(N)), stomatal (g(s)) and mesophyll conductances (g(m)), as well as respiration (v(cyt) and v(alt)) differed between well-watered CMSII and WT, similar reductions of A(N), g(s), and g(m) were observed during severe drought stress. However, total respiration (V(t)) remained slightly higher in CMSII due to the still increased v(cyt) (to match ATP demand). v(alt) and maximum carboxylation rates remained almost unaltered in both genotypes, while in CMSII, changes in photosynthetic light harvesting (i.e. Chl a/b ratio) were detected. In both genotypes, photosynthesis and respiration were restored after 2 d of rewatering, predominantly limited by a delayed stomatal response. Despite complex I dysfunction and hence altered redox balance, the CMSII mutant seems to be able to adjust its photosynthetic machinery during and after drought stress to reduce photo-oxidation and to maintain the cell redox state and the ATP level.

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Carotenoid composition and carotenogenic gene expression during Ipomoea petal development.

Publication Date: 2010 Mar PMID: 19933319
Authors: Yamamizo, C. - Kishimoto, S. - Ohmiya, A.
Journal: J Exp Bot

Japanese morning glory (Ipomoea nil) is a representative plant lacking a yellow-flowered cultivar, although a few wild Ipomoea species contain carotenoids in their petals such as Ipomoea sp. (yellow petals) and I. obscura (pale-yellow petals). In the present study, carotenoid composition and the expression patterns of carotenogenic genes during petal development were compared among I. nil, I. obscura, and Ipomoea sp. to identify the factors regulating carotenoid accumulation in Ipomoea plant petals. In the early stage, the carotenoid composition in petals of all the Ipomoea plants tested was the same as in the leaves mainly showing lutein, violaxanthin, and beta-carotene (chloroplast-type carotenoids). However, in fully opened flowers, chloroplast-type carotenoids were entirely absent in I. nil, whereas they were present in trace amounts in the free form in I. obscura. At the late stage of petal development in Ipomoea sp., the majority of carotenoids were beta-cryptoxanthin, zeaxanthin, and beta-carotene (chromoplast-type carotenoids). In addition, most of them were present in the esterified form. Carotenogenic gene expression was notably lower in I. nil than in Ipomoea sp. In particular, beta-ring hydroxylase (CHYB) was considerably suppressed in petals of both I. nil and I. obscura. The CHYB expression was found to be significantly high in the petals of Ipomoea sp. during the synthesis of chromoplast-type carotenoids. The expression levels of carotenoid cleavage genes (CCD1 and CCD4) were not correlated with the amount of carotenoids in petals. These results suggest that both I. obscura and I. nil lack the ability to synthesize chromoplast-type carotenoids because of the transcriptional down-regulation of carotenogenic genes. CHYB, an enzyme that catalyses the addition of a hydroxyl residue required for esterification, was found to be a key enzyme for the accumulation of chromoplast-type carotenoids in petals.

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The syncytium-specific expression of the Orysa;KRP3 CDK inhibitor: implication of its involvement in the cell cycle control in the rice (Oryza sativa L.) syncytial endosperm.

Publication Date: 2010 Mar PMID: 19933315
Authors: Mizutani, M. - Naganuma, T. - Tsutsumi, K. - Saitoh, Y.
Journal: J Exp Bot

During rice (Oryza sativa L.) seed development, the primary endosperm nucleus undergoes a series of divisions without cytokinesis, producing a multinucleate cell, known as a syncytium. After several rounds of rapid nuclear proliferation, the syncytium ceases to undergo mitosis; thereafter, the syncytium is partitioned into individual cells by a specific type of cytokinesis called cellularization. The transition between syncytium and cellularization is important in determining the final seed size and is a model for studying the cell cycle and cytokinesis. The involvement of cyclin-dependent kinase (CDK) inhibitors (CKIs) in cell cycle control was investigated here during the transition between syncytium and cellularization. It was found that one of the rice CKIs, Orysa;KRP3, is strongly expressed in the caryopsis at 2 d after flowering (DAF), and its expression is significantly reduced at 3 DAF. The other CKI transcripts did not show such a shift at 2 DAF. In situ hybridization analysis revealed that Orysa;KRP3 is expressed in multinucleate syncytial endosperm at 2 DAF, but not in cellularized endosperm at 3 DAF. Two-hybrid assays showed that Orysa;KRP3 binds Orysa;CDKA;1, Orysa;CDKA;2, Orysa;CycA1;1, and Orysa;CycD2;2. By contrast, Orysa;CDKB2;1 and Orysa;CycB2;2 do not show binding to Orysa;KRP3. Orysa;KRP3 was able to rescue yeast premature cell division due to the dominant positive expression of mutant rice CDKA;1 indicating that Orysa;KRP3 inhibited rice CDK. These data suggest that Orysa;KRP3 is involved in cell cycle control of syncytial endosperm.

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Sporophytic control of pollen tube growth and guidance in maize.

Publication Date: 2010 Mar PMID: 19926683
Authors: Lausser, A. - Kliwer, I. - Srilunchang, K. O. - Dresselhaus, T.
Journal: J Exp Bot

Pollen tube germination, growth, and guidance (progamic phase) culminating in sperm discharge is a multi-stage process including complex interactions between the male gametophyte as well as sporophytic tissues and the female gametophyte (embryo sac), respectively. Inter- and intra-specific crossing barriers in maize and Tripsacum have been studied and a precise description of progamic pollen tube development in maize is reported here. It was found that pollen germination and initial tube growth are rather unspecific, but an early, first crossing barrier was detected before arrival at the transmitting tract. Pollination of maize silks with Tripsacum pollen and incompatible pollination of Ga1s/Ga1s-maize silks with ga1-maize pollen revealed another two incompatibility barriers, namely transmitting tract mistargeting and insufficient growth support. Attraction and growth support by the transmitting tract seem to play key roles for progamic pollen tube growth. After leaving transmitting tracts, pollen tubes have to navigate across the ovule in the ovular cavity. Pollination of an embryo sac-less maize RNAi-line allowed the role of the female gametophyte for pollen tube guidance to be determined in maize. It was found that female gametophyte controlled guidance is restricted to a small region around the micropyle, approximately 50-100 microm in diameter. This area is comparable to the area of influence of previously described ZmEA1-based short-range female gametophyte signalling. In conclusion, the progamic phase is almost completely under sporophytic control in maize.

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Phylogenetically-based variation in the regulation of the Calvin cycle enzymes, phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase, in algae.

Publication Date: 2010 Mar PMID: 19926682
Authors: Maberly, S. C. - Courcelle, C. - Groben, R. - Gontero, B.
Journal: J Exp Bot

Aquatic photosynthesis is responsible for about half of the global production and is undertaken by a huge phylogenetic diversity of algae that are poorly studied. The diversity of redox-regulation of phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was investigated in a wide range of algal groups under standard conditions. Redox-regulation of PRK was greatest in chlorophytes, low or absent in a red alga and most chromalveolates, and linked to the number of amino acids between two regulatory cysteine residues. GAPDH regulation was not strongly-related to the different forms of this enzyme and was less variable than for PRK. Addition of recombinant CP12, a protein that forms a complex with PRK and GAPDH, to crude extracts inhibited GAPDH and PRK inversely in the Plantae, but in most chromalveolates had little effect on GAPDH and inhibited or stimulated PRK depending on the species. Patterns of enzyme regulation were used to produce a phylogenetic tree in which cryptophytes and haptophytes, at the base of the chromalveolates, formed a distinct clade. A second clade comprised only chromalveolates. A third clade comprised a mixture of Plantae, an excavate and three chromalveolates: a marine diatom and two others (a xanthophyte and eustigmatophyte) that are distinguished by a low content of chlorophyll c and a lack of fucoxanthin. Regulation of both enzymes was greater in freshwater than in marine taxa, possibly because most freshwaters are more dynamic than oceans. This work highlights the importance of understanding enzyme regulation in diverse algae if their ecology and productivity is to be understood.

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Determinants of ozone fluxes and metrics for ozone risk assessment in plants.

Publication Date: 2010 Mar PMID: 19923198
Authors: Fares, S. - Goldstein, A. - Loreto, F.
Journal: J Exp Bot

Tropospheric ozone concentration is increasing and represents a threat to single plants and whole ecosystems. The deleterious ozone effects mainly occur when (i) ozone concentration in the air builds up; (ii) the pollutant enters the leaf through stomatal uptake, and (iii) ozone-produced reactive oxygen species are not efficiently scavenged by leaf antioxidants and then oxidize leaf tissues. The sensitivity of plants to ozone is species-specific, and a correct risk assessment should be based on a metric that correctly takes into account the ambient concentration of ozone, the physiological control on stomatal apertures, and the efficiency of leaf antioxidant system. Current methodologies have been analysed to evaluate ozone risk assessment, and, by phasing-in and phasing out sources and sinks of ozone, elements of improvements for the current metrics have been suggested.

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Silencing Sl-EBF1 and Sl-EBF2 expression causes constitutive ethylene response phenotype, accelerated plant senescence, and fruit ripening in tomato.

Publication Date: 2010 Mar PMID: 19903730
Authors: Yang, Y. - Wu, Y. - Pirrello, J. - Regad, F. - Bouzayen, M. - Deng, W. - Li, Z.
Journal: J Exp Bot

The hormone ethylene regulates a wide range of plant developmental processes and EBF (EIN3-binding F-box) proteins were shown to negatively regulate the ethylene signalling pathway via mediating the degradation of EIN3/EIL proteins. The present study reports on the identification of two tomato F-box genes, Sl-EBF1 and Sl-EBF2 from the EBF subfamily. The two genes display contrasting expression patterns in reproductive and vegetative tissues and in response to ethylene and auxin treatment. Sl-EBF1 and Sl-EBF2 genes are actively regulated at crucial stages in the development of the reproductive organs. Their dynamic expression in flowers during bud-to-anthesis and anthesis-to-post-anthesis transitions, and at the onset of fruit ripening, suggests their role in situations where ethylene is required for stimulating flower opening and triggering fruit ripening. VIGS-mediated silencing of a single tomato EBF gene uncovered a compensation mechanism that tends to maintain a threshold level of Sl-EBF expression via enhancing the expression of the second Sl-EBF gene. In line with this compensation, tomato plants silenced for either of the Sl-EBF genes were indistinguishable from control plants, indicating functional redundancy among Sl-EBF genes. By contrast, co-silencing of both Sl-EBFs resulted in ethylene-associated phenotypes. While reports on EBF genes to date have focused on their role in modulating ethylene responses in Arabidopsis, the present study uncovered their role in regulating crucial stages of flower and fruit development in tomato. The data support the hypothesis that protein degradation via the ubiquitin/26S proteasome pathway is a control point of fruit ripening and open new leads for engineering fruit quality.

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