Planta

Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L.

Publication Date: 2012 May 10 PMID: 22572777
Authors: Shang, Q. M. - Li, L. - Dong, C. J.
Journal: Planta

Phenylalanine ammonia-lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway, and therefore plays a key role in both plant development and stress defense. In many plants, PAL is encoded by a multi-gene family, and each member is differentially regulated in response to environmental stimuli. In the present study, we report that PAL in cucumber (Cucumis sativus L.) is encoded for by a family of seven genes (designated as CsPAL1-7). All seven CsPALs are arranged in tandem in two duplication blocks, which are located on chromosomes 4 and 6, respectively. The cDNA and protein sequences of the CsPALs share an overall high identity to each other. Homology modeling reveals similarities in their protein structures, besides several slight differences, implying the different activities in conversion of phenylalanine. Phylogenic analysis places CsPAL1-7 in a separate cluster rather than clustering with other plant PALs. Analyses of expression profiles in different cucumber tissues or in response to various stress or plant hormone treatments indicate that CsPAL1-7 play redundant, but divergent roles in cucumber development and stress response. This is consistent with our finding that CsPALs possess overlapping but different cis-elements in their promoter regions. Finally, several duplication events are discussed to explain the evolution of the cucumber PAL genes.

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Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response.

Publication Date: 2012 May 9 PMID: 22569921
Authors: Kuzuoglu-Ozturk, D. - Cebeci Yalcinkaya, O. - Akpinar, B. A. - Mitou, G. - Korkmaz, G. - Gozuacik, D. - Budak, H.
Journal: Planta

An autophagy-related gene Atg8 was cloned for the first time from wild emmer wheat, named as TdAtg8, and its role on autophagy under abiotic stress conditions was investigated. Examination of TdAtg8 expression patterns indicated that Atg8 expression was strongly upregulated under drought stress, especially in the roots when compared to leaves. LysoTracker((R)) red marker, utilized to observe autophagosomes, revealed that autophagy is constitutively active in Triticum dicoccoides. Moreover, autophagy was determined to be induced in plants exposed to osmotic stress when compared to plants grown under normal conditions. Functional studies were executed in yeast to confirm that the TdATG8 protein is functional, and showed that the TdAtg8 gene complements the atg8::kan MX yeast mutant strain grown under nitrogen deficiency. For further functional analysis, TdATG8 protein was expressed in yeast and analyzed using Western immunoblotting. Atg8-silenced plants were exposed to drought stress and chlorophyll and malondialdehyde (MDA) content measurements demonstrated that Atg8 plays a key role on drought stress tolerance. In addition, Atg8-silenced plants exposed to osmotic stress were found to have decreased Atg8 expression level in comparison to controls. Hence, Atg8 is a positive regulator in osmotic and drought stress response.

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Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions.

Publication Date: 2012 May 9 PMID: 22569920
Authors: Azuma, A. - Yakushiji, H. - Koshita, Y. - Kobayashi, S.
Journal: Planta

Temperature and light are important environmental factors that affect flavonoid biosynthesis in grape berry skin. However, the interrelationships between temperature and light effects on flavonoid biosynthesis have not been fully elucidated at the molecular level. Here, we investigated the effects of temperature and light conditions on the biosynthesis of flavonoids (anthocyanins and flavonols) and the expression levels of related genes in an in vitro environmental experiment using detached grape berries. Sufficient anthocyanin accumulation in the grape skin was observed under a low temperature (15 degrees C) plus light treatment, whereas high temperature (35 degrees C) or dark treatment severely suppressed anthocyanin accumulation. This indicates that the accumulation of anthocyanins is dependent on both low temperature and light. qRT-PCR analysis showed that the responses of three MYB-related genes (VlMYBA1-3, VlMYBA1-2, and VlMYBA2) to temperature and light differed greatly even though the products of all three genes had the ability to regulate anthocyanin biosynthesis pathway genes. Furthermore, the expression levels of other MYB-related genes and many flavonoid biosynthesis pathway genes were regulated independently by temperature and light. We also found that temperature and light conditions affected the anthocyanin composition in the skin through the regulation of flavonoid biosynthesis pathway genes. Our results suggest that low temperature and light have a synergistic effect on the expression of genes in the flavonoid biosynthesis pathway. These findings provide new information about the relationships between environmental factors and flavonoid accumulation in grape berry skin.

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Expression of flavonoid genes in the red grape berry of 'Alicante Bouschet' varies with the histological distribution of anthocyanins and their chemical composition.

Publication Date: 2012 May 3 PMID: 22552639
Authors: Falginella, L. - Di Gaspero, G. - Castellarin, S. D.
Journal: Planta

The mature berry of Vitis vinifera 'Alicante Bouschet' is entirely red, but anthocyanin metabolism discloses elements of histological discontinuity. This provides an experimental system amenable to studies of compartmentalised secondary metabolism in a fleshly fruit. We compared microscopy of fixed berry sections and chemical composition of anthocyanin extracts with the expression of 41 flavonoid genes in three berry tissues. In the pericarp, anthocyanins formed membrane-encased spherical coalescences that gradually enlarged and were shuttled into the vacuolar system. The size and the intensity of in situ pigmentation and of colour extracts of anthocyanin vesicles all decreased with depth beneath the epidermis. Shades of red colour, and the quantity and types of anthocyanins in skin, flesh, and seed extracts were correlated with differences in the expression of flavonoid 3',5'-hydroxylases and anthocyanin genes encoding transcription factors, enzymatic proteins, and transporters. Fine adjustments in the global transcriptional modulation of the pathway occurred distinctively in each tissue, within four groups of co-expressed genes that were more associated with either the pericarp or the seed, and with either early or late-ripening stages. All structural genes controlling early steps of the flavonoid pathway exist in the grapevine genome in multiple copies that were recruited by antagonistic branches of the pathway in the 'Alicante Bouschet' berry. Expression patterns of individual paralogs were spatiotemporally distinct from one another, in step with either anthocyanin genes or proanthocyanidin genes.

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Role of trichomes in defense against herbivores: comparison of herbivore response to woolly and hairless trichome mutants in tomato (Solanum lycopersicum).

Publication Date: 2012 May 3 PMID: 22552638
Authors: Tian, D. - Tooker, J. - Peiffer, M. - Chung, S. H. - Felton, G. W.
Journal: Planta

Trichomes contribute to plant resistance against herbivory by physical and chemical deterrents. To better understand their role in plant defense, we systemically studied trichome morphology, chemical composition and the response of the insect herbivores Helicoverpa zea and Leptinotarsa decemlineata (Colorado potato beetle = CPB) on the tomato hairless (hl), hairy (woolly) mutants and wild-type Rutgers (RU) and Alisa Craig (AC) plants. Hairless mutants showed reduced number of twisted glandular trichomes (types I, IV, VI and VII) on leaf and stem compared to wild-type Rutgers (RU), while woolly mutants showed high density of non-glandular trichomes (types II, III and V) but only on the leaf. In both mutants, trichome numbers were increased by methyl jasmonate (MeJA), but the types of trichomes present were not affected by MeJA treatment. Glandular trichomes contained high levels of monoterpenes and sesquiterpenes. A similar pattern of transcript accumulation was observed for monoterpene MTS1 (=TPS5) and sesquiterpene synthase SST1 (=TPS9) genes in trichomes. While high density of non-glandular trichome on leaves negatively influenced CPB feeding behavior and growth, it stimulated H. zea growth. High glandular trichome density impaired H. zea growth, but had no effect on CPB. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that glandular trichomes highly express protein inhibitors (PIN2), polyphenol oxidase (PPOF) and hydroperoxide lyase (HPL) when compared to non-glandular trichomes. The SlCycB2 gene, which participates in woolly trichome formation, was highly expressed in the woolly mutant trichomes. PIN2 in trichomes was highly induced by insect feeding in both mutant and wild-type plants. Thus, both the densities of trichomes and the chemical defenses residing in the trichomes are inducible.

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Overexpression of Arabidopsis miR157b induces bushy architecture and delayed phase transition in Torenia fournieri.

Publication Date: 2012 May 3 PMID: 22552637
Authors: Shikata, M. - Yamaguchi, H. - Sasaki, K. - Ohtsubo, N.
Journal: Planta

miR156/157 is a small RNA molecule that is highly conserved among various plant species. Overexpression of miR156/157 has been reported to induce bushy architecture and delayed phase transition in several plant species. To investigate the effect of miR157 overexpression in a horticultural plant, and to explore the applicability of miRNA to molecular breeding, we introduced Arabidopsis MIR157b (AtMIR157b) into torenia (Torenia fournieri). The resulting 35S:AtMIR157b plants showed a high degree of branching along with small leaves, which resembled miR156/157-overexpressing plants of other species. We also isolated torenia SBP-box genes with target miR156/157 sequences and confirmed that their expression was selectively downregulated in 35S:AtMIR157b plants. The reduced accumulation of mRNA was probably due to sequence specificity. Moreover, expression of torenia homologs of the SBP-box protein-regulated genes TfLFY and TfMIR172 was also reduced by AtmiR157 overexpression. These findings suggest that the molecular mechanisms of miR156/157 regulation are conserved between Arabidopsis and torenia. The bushy architecture and small leaves of 35S:AtMIR157b torenia plants could be applied in molecular breeding of various horticultural plants as well as for increasing biomass and crop production.

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Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae).

Publication Date: 2012 May 1 PMID: 22547029
Authors: Terauchi, M. - Nagasato, C. - Kajimura, N. - Mineyuki, Y. - Okuda, K. - Katsaros, C. - Motomura, T.
Journal: Planta

Plasmodesmata are intercellular bridges that directly connect the cytoplasm of neighboring cells and play a crucial role in cell-to-cell communication and cell development in multicellular plants. Although brown algae (Phaeophyceae, Heterokontophyta) are phylogenetically distant to land plants, they nevertheless possess a complex multicellular organization that includes plasmodesmata. In this study, the ultrastructure and formation of plasmodesmata in the brown alga Dictyota dichotoma were studied using transmission electron microscopy and electron tomography with rapid freezing and freeze substitution. D. dichotoma possesses plasma membrane-lined, simple plasmodesmata without internal endoplasmic reticulum (desmotubule). This structure differs from those in land plants. Plasmodesmata were clustered in regions with thin cell walls and formed pit fields. Fine proteinaceous "internal bridges" were observed in the cavity. Ultrastructural observations of cytokinesis in D. dichotoma showed that plasmodesmata formation began at an early stage of cell division with the formation of tubular pre-plasmodesmata within membranous sacs of the cytokinetic diaphragm. Clusters of pre-plasmodesmata formed the future pit field. As cytokinesis proceeded, electron-dense material extended from the outer surface of the mid region of the pre-plasmodesmata and finally formed the nascent cell wall. From these results, we suggest that pre-plasmodesmata are associated with cell wall development during cytokinesis in D. dichotoma.

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Transcriptional response of Medicago truncatula sulphate transporters to arbuscular mycorrhizal symbiosis with and without sulphur stress.

Publication Date: 2012 Apr 26 PMID: 22535379
Authors: Casieri, L. - Gallardo, K. - Wipf, D.
Journal: Planta

Sulphur is an essential macronutrient for plant growth, development and response to various abiotic and biotic stresses due to its key role in the biosynthesis of many S-containing compounds. Sulphate represents a very small portion of soil S pull and it is the only form that plant roots can uptake and mobilize through H(+)-dependent co-transport processes implying sulphate transporters. Unlike the other organically bound forms of S, sulphate is normally leached from soils due to its solubility in water, thus reducing its availability to plants. Although our knowledge of plant sulphate transporters has been growing significantly in the past decades, little is still known about the effect of the arbuscular mycorrhiza interaction on sulphur uptake. Carbon, nitrogen and sulphur measurements in plant parts and expression analysis of genes encoding putative Medicago sulphate transporters (MtSULTRs) were performed to better understand the beneficial effects of mycorrhizal interaction on Medicago truncatula plants colonized by Glomus intraradices at different sulphate concentrations. Mycorrhization significantly promoted plant growth and sulphur content, suggesting increased sulphate absorption. In silico analyses allowed identifying eight putative MtSULTRs phylogenetically distributed over the four sulphate transporter groups. Some putative MtSULTRs were transcribed differentially in roots and leaves and affected by sulphate concentration, while others were more constitutively transcribed. Mycorrhizal-inducible and -repressed MtSULTRs transcripts were identified allowing to shed light on the role of mycorrhizal interaction in sulphate uptake.

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A possible involvement of autophagy in amyloplast degradation in columella cells during hydrotropic response of Arabidopsis roots.

Publication Date: 2012 Apr 25 PMID: 22532286
Authors: Nakayama, M. - Kaneko, Y. - Miyazawa, Y. - Fujii, N. - Higashitani, N. - Wada, S. - Ishida, H. - Yoshimoto, K. - Shirasu, K. - Yamada, K. - Nishimura, M. - Takahashi, H.
Journal: Planta

Seedling roots display not only gravitropism but also hydrotropism, and the two tropisms interfere with one another. In Arabidopsis (Arabidopsis thaliana) roots, amyloplasts in columella cells are rapidly degraded during the hydrotropic response. Degradation of amyloplasts involved in gravisensing enhances the hydrotropic response by reducing the gravitropic response. However, the mechanism by which amyloplasts are degraded in hydrotropically responding roots remains unknown. In this study, the mechanistic aspects of the degradation of amyloplasts in columella cells during hydrotropic response were investigated by analyzing organellar morphology, cell polarity and changes in gene expression. The results showed that hydrotropic stimulation or systemic water stress caused dramatic changes in organellar form and positioning in columella cells. Specifically, the columella cells of hydrotropically responding or water-stressed roots lost polarity in the distribution of the endoplasmic reticulum (ER), and showed accelerated vacuolization and nuclear movement. Analysis of ER-localized GFP showed that ER redistributed around the developed vacuoles. Cells often showed decomposing amyloplasts in autophagosome-like structures. Both hydrotropic stimulation and water stress upregulated the expression of AtATG18a, which is required for autophagosome formation. Furthermore, analysis with GFP-AtATG8a revealed that both hydrotropic stimulation and water stress induced the formation of autophagosomes in the columella cells. In addition, expression of plastid marker, pt-GFP, in the columella cells dramatically decreased in response to both hydrotropic stimulation and water stress, but its decrease was much less in the autophagy mutant atg5. These results suggest that hydrotropic stimulation confers water stress in the roots, which triggers an autophagic response responsible for the degradation of amyloplasts in columella cells of Arabidopsis roots.

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Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants.

Publication Date: 2012 Apr 22 PMID: 22526507
Authors: Huang, J. - Zhong, Y. - Sandmann, G. - Liu, J. - Chen, F.
Journal: Planta

beta-Carotene ketolase (BKT) catalyzes the rate-limiting steps for the biosynthesis of astaxanthin. Several bkt genes have been isolated and explored to modify plant carotenoids to astaxanthin with limited success. In this study, five algal BKT cDNAs were isolated and characterized for the engineering of high-yield astaxanthin in plants. The products of the cDNAs showed high similarity in sequence and enzymatic activity of converting beta-carotene into canthaxanthin. However, the enzymes exhibited extremely different activities in converting zeaxanthin into astaxanthin. Chlamydomonas reinhardtii BKT showed the highest conversion rate (ca 85 %), whereas, Neochloris wimmeri BKT exhibited very poor activity of ketolating zeaxanthin. Expression of C. reinhardtii BKT in tobacco led to a twofold increase of total carotenoids in the leaves with astaxanthin being the predominant. The bkt genes described here provide a valuable resource for metabolic engineering of plants as cell factories for astaxanthin production.

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Substituent-specific antibody against glucuronoxylan reveals close association of glucuronic acid and acetyl substituents and distinct labeling patterns in tree species.

Publication Date: 2012 Apr 24 PMID: 22526506
Authors: Koutaniemi, S. - Guillon, F. - Tranquet, O. - Bouchet, B. - Tuomainen, P. - Virkki, L. - Petersen, H. L. - Willats, W. G. - Saulnier, L. - Tenkanen, M.
Journal: Planta

Immunolabeling can be used to locate plant cell wall carbohydrates or other components to specific cell types or to specific regions of the wall. Some antibodies against xylans exist; however, many partly react with the xylan backbone and thus provide limited information on the type of substituents present in various xylans. We have produced a monoclonal antibody which specifically recognizes glucopyranosyl uronic acid (GlcA), or its 4-O-methyl ether (meGlcA), substituents in xylan and has no cross-reactivity with linear or arabinofuranosyl-substituted xylans. The UX1 antibody binds most strongly to (me)GlcA substitutions at the non-reducing ends of xylan chains, but has a low cross-reactivity with internal substitutions as well, at least on oligosaccharides. The antibody labeled plant cell walls from both mono- and dicotyledons, but in most tissues an alkaline pretreatment was needed for antibody binding. The treatment removed acetyl groups from xylan, indicating that the vicinity of glucuronic acid substituents is also acetylated. The novel labeling patterns observed in the xylem of tree species suggested that differences within the cell wall exist both in acetylation degree and in glucuronic acid content.

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Cell wall polysaccharides are involved in P-deficiency-induced Cd exclusion in Arabidopsis thaliana.

Publication Date: 2012 Apr 25 PMID: 22526505
Authors: Zhu, X. F. - Lei, G. J. - Jiang, T. - Liu, Y. - Li, G. X. - Zheng, S. J.
Journal: Planta

The physiological and molecular mechanisms leading to the competitive interactions between phosphorus (P) and metal elements are a matter of debate. In this study, we found that P deficiency can alleviate cadmium (Cd) toxicity in Arabidopsis thaliana (Col-0). Under P deficiency (-P), less Cd was accumulated in the plants and the root cell walls, indicating the operation of a P-deficiency-induced Cd exclusion mechanism. However, organic acid efflux was similar under -P+Cd and +Cd treatments, suggesting that organic acid efflux is not responsible for the Cd exclusion. Interestingly, P deficiency significantly decreased cell wall polysaccharides (pectin and hemicellulose) contents and pectin methylesterase activity, and decreased the Cd retained by the extracted root cell wall. Therefore, we conclude that the modification of cell wall composition is responsible for the Cd exclusion of the root under P deficiency.

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Root metabolic response of rice (Oryza sativa L.) genotypes with contrasting tolerance to zinc deficiency and bicarbonate excess.

Publication Date: 2012 Apr 24 PMID: 22526504
Authors: Rose, M. T. - Rose, T. J. - Pariasca-Tanaka, J. - Yoshihashi, T. - Neuweger, H. - Goesmann, A. - Frei, M. - Wissuwa, M.
Journal: Planta

Plants are routinely subjected to multiple environmental stresses that constrain growth. Zinc (Zn) deficiency and high bicarbonate are two examples that co-occur in many soils used for rice production. Here, the utility of metabolomics in diagnosing the effect of each stress alone and in combination on rice root function is demonstrated, with potential stress tolerance indicators identified through the use of contrasting genotypes. Responses to the dual stress of combined Zn deficiency and bicarbonate excess included greater root solute leakage, reduced dry matter production, lower monosaccharide accumulation and increased concentrations of hydrogen peroxide, phenolics, peroxidase and N-rich metabolites in roots. Both hydrogen peroxide concentration and root solute leakage were correlated with higher levels of citrate, allantoin and stigmasterol. Zn stress resulted in lower levels of the tricarboxylic acid (TCA) cycle intermediate succinate and the aromatic amino acid tyrosine. Bicarbonate stress reduced shoot iron (Fe) concentrations, which was reflected by lower Fe-dependent ascorbate peroxidase activity. Bicarbonate stress also favoured the accumulation of the TCA cycle intermediates malate, fumarate and succinate, along with the non-polar amino acid tyrosine. Genotypic differentiation revealed constitutively higher levels of D: -gluconate, 2-oxoglutarate and two unidentified compounds in the Zn-efficient line RIL46 than the Zn-inefficient cultivar IR74, suggesting a possible role for these metabolites in overcoming oxidative stress or improving metal re-distribution.

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Damage to photosystem II due to heat stress without light-driven electron flow: involvement of enhanced introduction of reducing power into thylakoid membranes.

Publication Date: 2012 Apr 24 PMID: 22526503
Authors: Marutani, Y. - Yamauchi, Y. - Kimura, Y. - Mizutani, M. - Sugimoto, Y.
Journal: Planta

Under a moderately heat-stressed condition, the photosystems of higher plants are damaged in the dark more easily than they are in the presence of light. To obtain a better understanding of this heat-derived damage mechanism that occurs in the dark, we focused on the involvement of the light-independent electron flow that occurs at 40 degrees C during the damage. In various plant species, the maximal photochemical quantum yield of photosystem (PS) II (F (v)/F (m)) decreased as a result of heat treatment in the dark. In the case of wheat, the most sensitive plant species tested, both F (v)/F (m) and oxygen evolution rapidly decreased by heat treatment at 40 degrees C for 30 min in the dark. In the damage, specific degradation of D1 protein was involved, as shown by immunochemical analysis of major proteins in the photosystem. Because light canceled the damage to PSII, the light-driven electron flow may play a protective role against PSII damage without light. Light-independent incorporation of reducing power from stroma was enhanced at 40 degrees C but not below 35 degrees C. Arabidopsis mutants that have a deficit of enzymes which mediate the incorporation of stromal reducing power into thylakoid membranes were tolerant against heat treatment at 40 degrees C in the dark, suggesting that the reduction of the plastoquinone pool may be involved in the damage. In conclusion, the enhanced introduction of reducing power from stroma into thylakoid membranes that occurs around 40 degrees C causes over-reduction of plastoquinone, resulting in the damage to D1 protein under heat stress without linear electron flow.

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Iron and reactive oxygen responses in Pinus sylvestris root cortical cells infected with different species of Heterobasidion annosum sensu lato.

Publication Date: 2012 Apr 24 PMID: 22526502
Authors: Mucha, J. - Guzicka, M. - Lakomy, P. - Zadworny, M.
Journal: Planta

Defence mechanisms in trees are not well understood. We assessed whether distribution of iron ions and their co-localisation with reactive oxygen species in Pinus sylvestris root cells reflect differential preferences of the pathogens Heterobasidion annosum sensu stricto, H. parviporum and H. abietinum to the host. Strains of H. annosum s.s. characterised by a greater preference for P. sylvestris induced accumulation of superoxide (O(2) (-)) in host cells 6 h after inoculation, whereas two peaks in accumulation of O(2) (-) (after 4 and 48 h) were observed after infection with strains of the pathogens H. parviporum and H. abietinum, which have a lower preference for P. sylvestris. Moreover, strains of H. annosum s.s. caused increased production of hydrogen peroxide (H(2)O(2)) in P. sylvestris cells, in contrast with strains of the other two species (H. parviporum and H. abietinum). Following inoculation with H. annosum s.s. strains, H(2)O(2) was correlated negatively with O(2) (-) and correlated positively with ferrous iron (Fe(2+)). Co-localisation of Fe(3+) with H(2)O(2) may suggest that they are involved in inducing hypersensitive responses and eventually cell death in roots inoculated with H. annosum s.s. strains, in contrast with H. parviporum, in which other mechanisms operate when the host is parasitised.

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The response to daylight or continuous ozone of phenylpropanoid and lignin biosynthesis pathways in poplar differs between leaves and wood.

Publication Date: 2012 Apr 24 PMID: 22526501
Authors: Richet, N. - Tozo, K. - Afif, D. - Banvoy, J. - Legay, S. - Dizengremel, P. - Cabane, M.
Journal: Planta

Ozone induces a stimulation of the phenylpropanoid and lignin biosynthesis pathways in leaves but the response of wood, the main lignin-producing tissue, is not well documented. The purpose of this study was to compare the responses of phenylpropanoid and lignin pathways in leaves and stem wood by a simultaneous analysis of both organs. Young poplars (Populus tremula x alba) were subjected either to daylight ozone (200 nL L(-1) during light period) or continuous ozone (200 nL L(-1) during light and dark periods) in controlled chambers. The trees were tilted so as to limit the formation of tension wood to the upper side of the stem and that of opposite wood to the lower side. Continuous ozone fumigation induced more pronounced effects in leaves than daylight ozone. Tension wood and opposite wood displayed similar responses to ozone. Enzyme activities involved in phenylpropanoid and lignin biosynthesis increased in the leaves of ozone-treated poplars and decreased in the wood. All steps involved in phenylpropanoid and monolignol synthesis in leaves and stem wood, were also altered at the transcript level (except coniferyl aldehyde 5-hydroxylase in leaves) suggesting that the responses were tightly coordinated. The response occurred rapidly in the leaves and much later in the wood. Phenylpropanoid and lignin biosynthesis is probably first involved in a defensive role against ozone in the leaves, which would lead to considerable rerouting of the carbon skeletons. The later response of phenylpropanoid and lignin metabolism in wood seemed to result from readjustment to the reduced carbon supply.

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Translational fusion and redirection to thylakoid lumen as strategies to improve the accumulation of a camelid antibody fragment in transplastomic tobacco.

Publication Date: 2012 Apr 24 PMID: 22526499
Authors: Lentz, E. M. - Garaicoechea, L. - Alfano, E. F. - Parreno, V. - Wigdorovitz, A. - Bravo-Almonacid, F. F.
Journal: Planta

Fragments from camelid single-chain antibodies known as VHHs or nanobodies represent a valuable tool in diagnostics, investigation and passive immunity therapy. Here, we explored different strategies to improve the accumulation of a neutralizing VHH antibody against rotavirus in tobacco transplastomic plants. First, we attempted to express the VHH in the chloroplast stroma and then two alternative strategies were carried out to improve the expression levels: expression as a translational fusion to the beta-glucuronidase enzyme (GUS-E-VHH), and redirection of the VHH into the thylakoid lumen (pep-VHH). Every attempt to produce transplastomic plants expressing the VHH in the stroma was futile. The transgene turned out to be unstable and the presence of the VHH protein was almost undetectable. Although pep-VHH plants also presented some of the aforementioned problems, higher accumulation of the nanobody was observed (2-3 % of the total soluble proteins). The use of beta-glucuronidase as a partner protein turned out to be a successful strategy and expression levels reached 3 % of the total soluble proteins. The functionality of the VHHs produced by pep-VHH and GUS-E-VHH plants was studied and compared with that of the antibody produced in Escherichia coli. This work contributes to optimizing the expression of VHH in transplastomic plants. Recombinant proteins could be obtained either by accumulation in the thylakoid lumen or as a fusion protein with beta-glucuronidase, and both strategies allow for further optimization.

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Physiological and molecular changes in plants grown at low temperatures.

Publication Date: 2012 Apr 20 PMID: 22526498
Authors: Theocharis, A. - Clement, C. - Barka, E. A.
Journal: Planta

Apart from water availability, low temperature is the most important environmental factor limiting the productivity and geographical distribution of plants across the world. To cope with cold stress, plant species have evolved several physiological and molecular adaptations to maximize cold tolerance by adjusting their metabolism. The regulation of some gene products represents an additional mechanism of cold tolerance. A consequence of these mechanisms is that plants are able to survive exposure to low temperature via a process known as cold acclimation. In this review, we briefly summarize recent progress in research and hypotheses on how sensitive plants perceive cold. We also explore how this perception is translated into changes within plants following exposure to low temperatures. Particular emphasis is placed on physiological parameters as well as transcriptional, post-transcriptional and post-translational regulation of cold-induced gene products that occur after exposure to low temperatures, leading to cold acclimation.

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The perichromatin region of the plant cell nucleus is the area with the strongest co-localisation of snRNA and SR proteins.

Publication Date: 2012 Apr 24 PMID: 22526497
Authors: Niedojadlo, J. - Mikulski, Z. - Delenko, K. - Szmidt-Jaworska, A. - Smolinski, D. J. - Epstein, A. L.
Journal: Planta

The spatial organisation of the splicing system in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus) nuclei was studied by immunolabelling of SR proteins, snRNA, and the PANA antigen, known markers for interchromatin granule clusters in mammalian cells. Electron microscope results allowed us to determine the distribution of these molecules within the structural domains of the nucleus. Similar to animal cells, in both plant species SR proteins were localised in interchromatin granules, but contrary to animal cells contained very small amounts of snRNA. The area with the strongest snRNA and SR protein co-localisation was the perichromatin region, which may be the location of pre-mRNA splicing in the plant cell nuclei. The only observable differences in the organisation of reticular and chromocentric nuclei were the size of the speckles and the number of snRNA pools in the condensed chromatin. We conclude that, despite remarkable changes in the nuclear architecture, the organisation of the splicing system is remarkably similar in both types of plant cell nuclei.

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Rice PLASTOCHRON genes regulate leaf maturation downstream of the gibberellin signal transduction pathway.

Publication Date: 2012 May PMID: 22476293
Authors: Mimura, M. - Nagato, Y. - Itoh, J.
Journal: Planta

Rice PLASTOCHRON 1 (PLA1) and PLA2 genes regulate leaf maturation and plastochron, and their loss-of-function mutants exhibit small organs and rapid leaf emergence. They encode a cytochrome P450 protein CYP78A11 and an RNA-binding protein, respectively. Their homologs in Arabidopsis and maize are also associated with plant development/organ size. Despite the importance of PLA genes in plant development, their molecular functions remain unknown. Here, we investigated how PLA1 and PLA2 genes are related to phytohormones. We found that gibberellin (GA) is the major phytohormone that promotes PLA1 and PLA2 expression. GA induced PLA1 and PLA2 expression, and conversely the GA-inhibitor uniconazole suppressed PLA1 and PLA2 expression. In pla1-4 and pla2-1 seedlings, expression levels of GA biosynthesis genes and the signal transduction gene were similar to those in wild-type seedlings. GA treatment slightly down-regulated the GA biosynthesis gene GA20ox2 and up-regulated the GA-catabolizing gene GA2ox4, whereas the GA biosynthesis inhibitor uniconazole up-regulated GA20ox2 and down-regulated GA2ox4 both in wild-type and pla mutants, suggesting that the GA feedback mechanism is not impaired in pla1 and pla2. To reveal how GA signal transduction affects the expression of PLA1 and PLA2, PLA expression in GA-signaling mutants was examined. In GA-insensitive mutant, gid1 and less-sensitive mutant, Slr1-d1, PLA1 and PLA2 expression was down-regulated. On the other hand, the expression levels of PLA1 and PLA2 were highly enhanced in a GA-constitutive-active mutant, slr1-1, causing ectopic overexpression. These results indicate that both PLA1 and PLA2 act downstream of the GA signal transduction pathway to regulate leaf development.

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dHPLC efficiency for semi-automated cDNA-AFLP analyses and fragment collection in the apple scab-resistance gene model.

Publication Date: 2012 May PMID: 22270558
Authors: Paris, R. - Dondini, L. - Zannini, G. - Bastia, D. - Marasco, E. - Gualdi, V. - Rizzi, V. - Piffanelli, P. - Mantovani, V. - Tartarini, S.
Journal: Planta

cDNA-AFLP analysis for transcript profiling has been successfully applied to study many plant biological systems, particularly plant-microbe interactions. However, the separation of cDNA-AFLP fragments by gel electrophoresis is reported to be labor-intensive with only limited potential for automation, and the collection of differential bands for gene identification is even more cumbersome. In this work, we present the use of dHPLC (denaturing high performance liquid chromatography) and automated DNA fragment collection using the WAVE((R)) System to analyze and recover cDNA-AFLP fragments. The method is successfully applied to the Malus-Venturia inaequalis interaction, making it possible to collect 66 different transcript-derived fragments for apple genes putatively involved in the defense response activated by the HcrVf2 resistance gene. The results, validated by real time quantitative RT-PCR, were consistent with the plant-pathogen interaction under investigation and this further supports the suitability of dHPLC for cDNA-AFLP transcript profiling. Features and advantages of this new approach are discussed, evincing that it is an almost fully automatable and cost-effective solution for processing large numbers of samples and collecting differential genes involved in other biological processes and non-model plants.

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High biological variability of plastids, photosynthetic pigments and pigment forms of leaf primordia in buds.

Publication Date: 2012 May PMID: 22160501
Authors: Solymosi, K. - Morandi, D. - Boka, K. - Boddi, B. - Schoefs, B.
Journal: Planta

To study the formation of the photosynthetic apparatus in nature, the carotenoid and chlorophyllous pigment compositions of differently developed leaf primordia in closed and opening buds of common ash (Fraxinus excelsior L.) and horse chestnut (Aesculus hippocastanum L.) as well as in closed buds of tree of heaven (Ailanthus altissima P. Mill.) were analyzed with HPLC. The native organization of the chlorophyllous pigments was studied using 77 K fluorescence spectroscopy, and plastid ultrastructure was investigated with electron microscopy. Complete etiolation, i.e., accumulation of protochlorophyllide, and absence of chlorophylls occurred in the innermost leaf primordia of common ash buds. The other leaf primordia were partially etiolated in the buds and contained protochlorophyllide (0.5-1 mug g(-1) fresh mass), chlorophyllides (0.2-27 mug g(-1) fresh mass) and chlorophylls (0.9-643 mug g(-1) fresh mass). Etio-chloroplasts with prolamellar bodies and either regular or only low grana were found in leaves having high or low amounts of chlorophyll a and b, respectively. After bud break, etioplast-chloroplast conversion proceeded and the pigment contents increased in the leaves, similarly to the greening processes observed in illuminated etiolated seedlings under laboratory conditions. The pigment contents and the ratio of the different spectral forms had a high biological variability that could be attributed to (i) various light conditions due to light filtering in the buds resulting in differently etiolated leaf primordia, (ii) to differences in the light-exposed and inner regions of the same primordia in opening buds due to various leaf folding, and (iii) to tissue-specific slight variations of plastid ultrastructure.

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Sesuvium portulacastrum (L.) L.: a potential halophyte for the degradation of toxic textile dye, Green HE4B.

Publication Date: 2012 May PMID: 22160500
Authors: Patil, A. V. - Lokhande, V. H. - Suprasanna, P. - Bapat, V. A. - Jadhav, J. P.
Journal: Planta

Sesuvium portulacastrum is a common halophyte growing well in adverse surroundings and is exploited mainly for the environmental protection including phytoremediation, desalination and stabilization of contaminated soil. In the present investigation, attempts have been made on the decolorization of a toxic textile dye Green HE4B (GHE4B) using in vitro grown Sesuvium plantlets. The plantlets exhibited significant (70%) decolorization of GHE4B (50 mg l(-1)) that sustain 200 mM sodium chloride (NaCl) within 5 days of incubation. The enzymatic analysis performed on the root and shoot tissues of the in vitro plantlets subjected to GHE4B decolorization in the presence of 200 mM NaCl showed a noteworthy induction of tyrosinase, lignin peroxidase and NADH-DCIP reductase activities, indicating the involvement of these enzymes in the metabolism of the dye GHE4B. The UV-visible spectrophotometer, HPLC and Fourier Transform Infrared Spectroscopy (FTIR) analyses of the samples before and after decolorization of the dye confirmed the efficient phytotransformation of GHE4B in the presence of 200 mM NaCl. Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of the products revealed the formation of three metabolites such as p -amino benzene, p -amino toluene and 1, 2, 7-amino naphthalene after phytotransformation of GHE4B. Based on the FTIR and GC-MS results, the possible pathway for the biodegradation of GHE4B in the presence of 200 mM NaCl has been proposed. The phytotoxicity experiments confirmed the non-toxicity of the degraded products. The present study demonstrates for the first time the potential of Sesuvium for the efficient degradation of textile dyes and its efficacy on saline soils contaminated with toxic compounds.

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Fluorescence kinetic parameters and cyclic electron transport in guard cell chloroplasts of chlorophyll-deficient leaf tissues from variegated weeping fig (Ficus benjamina L.).

Publication Date: 2012 May PMID: 22134781
Authors: Lysenko, V.
Journal: Planta

Residual chlorophyll in chlorophyll-deficient (albino) areas of variegated leaves of Ficus benjamina originates from guard cell chloroplasts. Photosynthetic features of green and albino sectors of F. benjamina were studied by imaging the distribution of the fluorescence decrease ratio Rfd within a leaf calculated from maximum (Fm) and steady-state leaf chlorophyll fluorescence (Fs) at 690 and 740 nm. Local areas of albino sectors demonstrated an abnormally high Rfd(740)/Rfd(690) ratio. Fluorescence transients excited in albino sectors at red (640 and 690 nm) wavelengths showed an abrupt decrease of the Rfd values (0.4 and 0.1, correspondingly) as compared with those excited at blue wavelengths (1.7-2.4). This "Red Drop" was not observed for green sectors. Normal and chlorophyll-deficient leaf sectors of F. benjamina were also tested for linear and cyclic electron transport in thylakoids. The tests have been performed studying fluorescence at a steady-state phase with CO(2)-excess impulse feeding, photoacoustic signal generated by pulse light source at wavelengths selectively exciting PSI, fluorescence kinetics under anaerobiosis and fluorescence changes observed by dual-wavelength excitation method. The data obtained for albino sectors strongly suggest the possibility of a cyclic electron transport simultaneously occurring in guard cell thylakoids around photosystems I and II under blue light, whereas linear electron transport is absent or insufficient.

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The heterologous expression in Arabidopsis of a chrysanthemum Cys2/His2 zinc finger protein gene confers salinity and drought tolerance.

Publication Date: 2012 May PMID: 22127738
Authors: Gao, H. - Song, A. - Zhu, X. - Chen, F. - Jiang, J. - Chen, Y. - Sun, Y. - Shan, H. - Gu, C. - Li, P. - Chen, S.
Journal: Planta

Zinc finger proteins (ZFPs) play an important role in plant growth and development. Here, we describe the isolation of CgZFP1, a Cys2/His2 (C(2)H(2)) ZFP gene, using RACE PCR from chrysanthemum, and the investigation of its function with ectopic expression in Arabidopsis. CgZFP1 contains two conserved C(2)H(2) regions, a nuclear localization domain (B box), a Leu-rich domain (L box) and a conserved DLN sequence (DLN box) close to its C-terminus. Its expression in the chrysanthemum leaf was strongly induced by salinity or drought, but not by ABA. Subcellular localization assay indicated that CgZFP1 protein is localized in nucleus in vivo. Yeast-one hybrid assay showed that CgZFP1 possesses transcriptional activation ability, heterologous expression of CgZFP1 conferred tolerance of transgenic Arabidopsis plants to both salinity and drought stresses. Under salinity stress, genes involved in osmotic adjustment, ROS scavenging, and ion homeostasis: Atlea3, AtP5CS2, AtProT1, and AtMnSOD, AtPOD, AtAPX1, and AtSOS1, AtSOS2, AtSOS3, AtNHX1 were enhanced in CgZFP1 transgenic Arabidopsis plants. Moreover, genes involved in the osmotic adjustment and oxidative stress responses: Atlea3, AtP5CS2, AtProT1, the aquaporin AtPIP2A, and AtMnSOD, AtPOD, AtAPX1 were induced in CgZFP1 transgenic Arabidopsis under drought stress. These results indicate CgZFP1 is an important regulator involved in the salt and drought stress response in plants.

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Role of asparaginase variable loop at the carboxyl terminal of the alpha subunit in the determination of substrate preference in plants.

Publication Date: 2012 May PMID: 22127737
Authors: Gabriel, M. - Telmer, P. G. - Marsolais, F.
Journal: Planta

Structural determinants responsible for the substrate preference of the potassium-independent (ASPGA1) and -dependent (ASPGB1) asparaginases from Arabidopsis thaliana have been investigated. Like ASPGA1, ASPGB1 was found to be catalytically active with both L: -Asn and beta-Asp-His as substrates, contrary to a previous report. However, ASPGB1 had a 45-fold higher specific activity with Asn as substrate than ASPGA1. A divergent sequence between the two enzymes forms a variable loop at the C-terminal of the alpha subunit. The results of dynamic simulations have previously implicated a movement of the C-terminus in the allosteric transduction of K(+)-binding at the surface of LjNSE1 asparaginase. In the crystal structure of Lupinus luteus asparaginase, most residues in this segment cannot be visualized due to a weak electron density. Exchanging the variable loop in ASPGA1 with that from ASPGB1 increased the affinity for Asn, with a 320-fold reduction in K (m) value. Homology modeling identified a residue specific to ASPGB1, Phe(162), preceding the variable loop, whose side chain is located in proximity to the beta-carboxylate group of the product aspartate, and to Gly(246), a residue participating in an oxyanion hole which stabilizes a negative charge forming on the side chain oxygen of asparagine during catalysis. Replacement with the corresponding leucine from ASPGA1 specifically lowered the V (max) value with Asn as substrate by 8.4-fold.

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From callus to embryo: a proteomic view on the development and maturation of somatic embryos in Cyclamen persicum.

Publication Date: 2012 May PMID: 22127736
Authors: Rode, C. - Lindhorst, K. - Braun, H. P. - Winkelmann, T.
Journal: Planta

In this study, the proteome structures following the pathway in somatic embryogenesis of Cyclamen persicum were analysed via high-resolution 2D-SDS-PAGE with two objectives: (1) to identify the significant physiological processes during somatic embryogenesis in Cyclamen and (2) to improve the maturation of somatic embryos. Therefore, the effects of maturation-promoting plant growth regulator abscisic acid (ABA) and high sucrose levels on torpedo-shaped embryos were investigated. In total, 108 proteins of differential abundance were identified using a combination of tandem mass spectrometry and a digital proteome reference map. In callus, enzymes related to energy supply were especially distinct, most likely due to energy demand caused by fast growth and cell division. The switch from callus to globular embryo as well as from globular to torpedo-shaped embryo was associated with controlled proteolysis via the ubiquitin-26S proteasome pathway. Storage compound accumulation was first detected 21 days after transfer to plant growth regulator (PGR)-free medium in early torpedo-shaped embryos. Increase in abundance of auxin-amidohydrolase during embryogenesis suggests a possible increase in auxin release in the late embryo stages of Cyclamen. A development-specific isoelectric point switch of catalases has been reported for the first time for somatic embryogenesis. Several proteins were identified to represent markers for the different developmental stages analysed. High sucrose levels and ABA treatment promoted the accumulation of storage compounds in torpedo-shaped embryos. Additionally, proteins of the primary metabolic pathways were decreased in the proteomes of ABA-treated embryos. Thus, ABA and high sucrose concentration in the culture medium improved maturation and consequently the quality of somatic embryos in C. persicum.

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Fumonisin B1, a toxin produced by Fusarium verticillioides, modulates maize beta-1,3-glucanase activities involved in defense response.

Publication Date: 2012 May PMID: 22120123
Authors: Sanchez-Rangel, D. - Sanchez-Nieto, S. - Plasencia, J.
Journal: Planta

Fusarium verticillioides is an important pathogen in maize that causes various diseases affecting all stages of plant development worldwide. The fungal pathogen could be seed borne or survive in soil and penetrate the germinating seed. Most F. verticillioides strains produce fumonisins, which are of concern because of their toxicity to animals and possibly humans, and because they enhance virulence against seedlings of some maize genotypes. In this work, we studied the action of fumonisin B1 (FB1) on the activity of maize beta-1,3-glucanases involved in plant defense response. In maize embryos, FB1 induced an acidic isoform while suppressing the activity of two basic isoforms. This acidic isoform was induced also with 2,6-dichloroisonicotinic acid, an analog of salicylic acid. Repression of the basic isoforms suggested a direct interaction of the enzymes with the mycotoxin as in vitro experiments showed that pure FB1 inhibited the basic beta-1,3-glucanases with an IC(50) of 53 muM. When germinating maize embryos were inoculated with F. verticillioides the same dual effect on beta-1,3-glucanase activities that we observed with the pure toxin was reproduced. Similar levels of FB1 were recovered at 24 h germination in maize tissue when they were treated with pure FB1 or inoculated with an FB1-producing strain. These results suggest that beta-1,3-glucanases are a relevant physiological target and their modulation by FB1 might contribute to F. verticillioides colonization.

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Sucrose synthase in unicellular cyanobacteria and its relationship with salt and hypoxic stress.

Publication Date: 2012 May PMID: 22113826
Authors: Kolman, M. A. - Torres, L. L. - Martin, M. L. - Salerno, G. L.
Journal: Planta

Higher plants and cyanobacteria metabolize sucrose (Suc) by a similar set of enzymes. Suc synthase (SuS, A/UDP-glucose: D: -fructose 2-alpha-D: -glucosyl transferase) catalyzes a reversible reaction. However, it is in the cleavage of Suc that this enzyme plays an important role in vivo, providing sugar nucleotides for polysaccharide biosynthesis. In cyanobacteria, SuS occurrence has been reported in heterocyst-forming strains, where it was shown to be involved also in nitrogen fixation. We investigated the presence of sequences homologous to SuS-encoding genes (sus) in recently sequenced cyanobacterial genomes. In this work, we show for the first time the presence of SuS in unicellular cyanobacterium strains (Microcystis aeruginosa PCC 7806, Gloebacter violaceus PCC 7421, and Thermosynechococcus elongatus BP-1). After functional characterization of SuS encoding genes, we demonstrated an increase in their transcript levels after a salt treatment or hypoxic stress in M. aeruginosa and G. violaceus cells. Based on phylogenetic analysis and on the presence of sus homologs in the most recently radiated cyanobacterium strains, we propose that sus genes in unicellular cyanobacteria may have been acquired through horizontal gene transfer. Taken together, our data indicate that SuS acquisition by cyanobacteria might be related to open up new ecological niches.

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Activation of a flavin monooxygenase gene YUCCA7 enhances drought resistance in Arabidopsis.

Publication Date: 2012 May PMID: 22109847
Authors: Lee, M. - Jung, J. H. - Han, D. Y. - Seo, P. J. - Park, W. J. - Park, C. M.
Journal: Planta

Auxin regulates diverse molecular and physiological events at the cellular and organismal levels during plant growth and development in response to environmental stimuli. It acts either through distinct signaling pathways or in concert with other growth hormones. Its biological functions are adjusted by modulating biosynthesis, conjugate formation, and polar transport and distribution. Several tryptophan-dependent and -independent auxin biosynthetic pathways have been proposed. Recent studies have shown that a few flavin monooxygenase enzymes contribute to the tryptophan-dependent auxin biosynthesis. Here, we show that activation of a flavin monooxygenase gene YUCCA7 (YUC7), which belongs to the tryptophan-dependent auxin biosynthetic pathway, enhances drought resistance. An Arabidopsis activation-tagged mutant yuc7-1D exhibited phenotypic changes similar to those observed in auxin-overproducing mutants, such as tall, slender stems and curled, narrow leaves. Accordingly, endogenous levels of total auxin were elevated in the mutant. The YUC7 gene was induced by drought, primarily in the roots, in an abscisic acid (ABA)-dependent manner. The yuc7-1D mutant was resistant to drought, and drought-responsive genes, such as RESPONSIVE TO DESSICATION 29A (RD29A) and COLD-REGULATED 15A (COR15A), were up-regulated in the mutant. Interestingly, whereas stomatal aperture and production of osmoprotectants were not discernibly altered, lateral root growth was significantly promoted in the yuc7-1D mutant when grown under drought conditions. These observations support that elevation of auxin levels in the roots enhances drought resistance possibly by promoting root growth.

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Experimental sink removal induces stress responses, including shifts in amino acid and phenylpropanoid metabolism, in soybean leaves.

Publication Date: 2012 May PMID: 22109846
Authors: Turner, G. W. - Cuthbertson, D. J. - Voo, S. S. - Settles, M. L. - Grimes, H. D. - Lange, B. M.
Journal: Planta

The repeated removal of flower, fruit, or vegetative buds is a common treatment to simulate sink limitation. These experiments usually lead to the accumulation of specific proteins, which are degraded during later stages of seed development, and have thus been designated as vegetative storage proteins. We used oligonucleotide microarrays to assess global effects of sink removal on gene expression patterns in soybean leaves and found an induction of the transcript levels of hundreds of genes with putative roles in the responses to biotic and abiotic stresses. In addition, these data sets indicated potential changes in amino acid and phenylpropanoid metabolism. As a response to sink removal we detected an induced accumulation of gamma-aminobutyric acid, while proteinogenic amino acid levels decreased. We also observed a shift in phenylpropanoid metabolism with an increase in isoflavone levels, concomitant with a decrease in flavones and flavonols. Taken together, we provide evidence that sink removal leads to an up-regulation of stress responses in distant leaves, which needs to be considered as an unintended consequence of this experimental treatment.

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The chloroplast ribosomal protein L21 gene is essential for plastid development and embryogenesis in Arabidopsis.

Publication Date: 2012 May PMID: 22105802
Authors: Yin, T. - Pan, G. - Liu, H. - Wu, J. - Li, Y. - Zhao, Z. - Fu, T. - Zhou, Y.
Journal: Planta

Embryogenesis in higher plants is controlled by a complex gene network. Identification and characterization of genes essential for embryogenesis will provide insights into the early events in embryo development. In this study, a novel mutant with aborted seed development (asd) was identified in Arabidopsis. The asd mutant produced about 25% of albino seeds at the early stage of silique development. The segregation of normal and albino seeds was inherited as a single recessive embryo-lethal trait. The gene disrupted in the asd mutant was isolated through map-based cloning. The mutated gene contains a single base change (A to C) in the coding region of RPL21C (At1g35680) that is predicted to encode the chloroplast 50S ribosomal protein L21. Allele test with other two T-DNA insertion lines in RPL21C and a complementation test demonstrated that the mutation in RPL21C was responsible for the asd phenotype. RPL21C exhibits higher expression in leaves and flowers compared with expression levels in roots and developing seeds. The RPL21C-GFP fusion protein was localized in chloroplasts. Cytological observations showed that the asd embryo development was arrested at the globular stage. There were no plastids with normal thylakoids and as a result no normal chloroplasts formed in mutant cells, indicating an indispensable role of the ASD gene in chloroplasts biogenesis. Our studies suggest that the chloroplast ribosomal protein L21 gene is required for chloroplast development and embryogenesis in Arabidopsis.

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Expression of ethylene response genes during persimmon fruit astringency removal.

Publication Date: 2012 May PMID: 22101946
Authors: Yin, X. R. - Shi, Y. N. - Min, T. - Luo, Z. R. - Yao, Y. C. - Xu, Q. - Ferguson, I. - Chen, K. S.
Journal: Planta

Thirteen ethylene signaling related genes were isolated and studied during ripening of non-astringent 'Yangfeng' and astringent 'Mopan' persimmon fruit. Some of these genes were characterized as ethylene responsive. Treatments, including ethylene and CO(2), had different effects on persimmon ripening, but overlapping roles in astringency removal, such as increasing the reduction in levels of soluble tannins. DkERS1, DkETR2, and DkERF8, may participate in persimmon fruit ripening and softening. The expression patterns of DkETR2, DkERF4, and DkERF5 had significant correlations with decreases in soluble tannins in 'Mopan' persimmon fruit, suggesting that these genes might be key components in persimmon fruit astringency removal and be the linkage between different treatments, while DkERF1 and DkERF6 may be specifically involved in CO(2) induced astringency removal. The possible roles of ethylene signaling genes in persimmon fruit astringency removal are discussed.

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Morphological and photosynthetic variations in the process of spermatia formation from vegetative cells in Porphyra yezoensis Ueda (Bangiales, Rhodophyta) and their responses to desiccation.

Publication Date: 2012 May PMID: 22101945
Authors: Yang, R. L. - Zhou, W. - Shen, S. D. - Wang, G. C. - He, L. W. - Pan, G. H.
Journal: Planta

Porphyra yezoensis has a macroscopic foliage gametophyte phase with only a single cell layer, and is ideally suited for the study of the sexual differentiation process, from the vegetative cell to the spermatia. Firstly, we compared variations in the responses of the vegetative and male sectors to desiccation. Later, cell tracking experiments were carried out during the formation of spermatia from vegetative cells. The two sectors showed similar tolerance to desiccation, and the formation of spermatia from vegetative cells was independent of the degree of desiccation. Both light and scanning electron microscopy (SEM) observations of the differentiation process showed that the formation of spermatia could be divided into six phases: the one-cell, two-cell, four-cell, eight-cell, pre-release and spermatia phases. Photomicrographs of Fluorescent Brightener staining showed that the released spermatia had no cell walls. Photosynthetic data showed that there was a significant rise in Y(II) in the four-cell phase, indicating an increase in photosynthetic efficiency of PSII during this phase. We propose that this photosynthetic rise may be substantial and provide the increased energy needed for the formation and release of spermatia in P. yezoensis.

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Genome-wide profiling of novel and conserved Populus microRNAs involved in pathogen stress response by deep sequencing.

Publication Date: 2012 May PMID: 22101925
Authors: Chen, L. - Ren, Y. - Zhang, Y. - Xu, J. - Zhang, Z. - Wang, Y.
Journal: Planta

MicroRNAs (miRNAs) are small RNAs, generally of 20-23 nt, that down-regulate target gene expression during development, differentiation, growth, and metabolism. In Populus, extensive studies of miRNAs involved in cold, heat, dehydration, salinity, and mechanical stresses have been performed; however, there are few reports profiling the miRNA expression patterns during pathogen stress. We obtained almost 38 million raw reads through Solexa sequencing of two libraries from Populus inoculated and uninoculated with canker disease pathogen. Sequence analyses identified 74 conserved miRNA sequences belonging to 37 miRNA families from 154 loci in the Populus genome and 27 novel miRNA sequences from 35 loci, including their complementary miRNA* strands. Intriguingly, the miRNA* of three conserved miRNAs were more abundant than their corresponding miRNAs. The overall expression levels of conserved miRNAs increased when subjected to pathogen stress, and expression levels of 33 miRNA sequences markedly changed. The expression trends determined by sequencing and by qRT-PCR were similar. Finally, nine target genes for three conserved miRNAs and 63 target genes for novel miRNAs were predicted using computational analysis, and their functions were annotated. Deep sequencing provides an opportunity to identify pathogen-regulated miRNAs in trees, which will help in understanding the regulatory mechanisms of plant defense responses during pathogen infection.

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