Plant and Cell Physiology

Identification and Localization of the CupB Protein Involved in Constitutive CO2-uptake in the Cyanobacterium, Synechocystis sp. Strain PCC 6803.

Publication Date: 2008 May 7 PMID: 18467341
Authors: Xu, M. - Ogawa, T. - Pakrasi, H. B. - Mi, H.
Journal: Plant Cell Physiol

Antibody against cMyc cross-reacted strongly with the CupB protein tagged with His6-cMyc (HM) in thylakoid membrane of Synechocystis sp. strain PCC 6803 but only faintly with cytoplasmic membrane fraction. The protein was not detected in the membranes of the triangle upndhD4 and triangle upndhF4 mutants in which CupB was tagged with HM. We concluded that a CupB complex containing NdhD4 and NdhF4 is largely, if not exclusively, confined to the thylakoid membrane. Both CupB and NdhH were detected in a fraction containing protein complexes of > 450 kDa, obtained after nickel column and gel filtration chromatography of the membranes solubilized with n-dodecyl-beta-maltoside.

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Antagonistic Jacalin-Related Lectins Regulate the Size of ER-Body-Type {beta}-Glucosidase Complexes in Arabidopsis thaliana.

Publication Date: 2008 May 8 PMID: 18467340
Authors: Nagano, A. J. - Fukao, Y. - Fujiwara, M. - Nishimura, M. - Hara-Nishimura, I.
Journal: Plant Cell Physiol

PYK10/BGLU23 is a beta-glucosidase that is a major protein of ER bodies, which are endoplasmic reticulum (ER)-derived organelles that may be involved in defense systems. PYK10 has active and inactive forms. Active PYK10 molecules form large complexes with diameters ranging from 0.65 mum to over 70 mum. We identified three beta-glucosidases (PYK10, BGLU21 and BGLU22), five jacalin-related lectins (JALs) and a GDSL lipase-like protein (GLL) in the purified PYK10 complex. Expression levels of JALs and GLLs were lower in the nai1-1 mutant, which has no ER bodies, than in Col-0. The subcellular localization of PYK10 is predicted to be different from the localizations of JALs and GLLs. This suggests that PYK10 interacts with its partners (JALs and GLLs) when subcellular structure is destroyed by pathogens. The PYK10 complex was found to be larger in the pbp1-1 and jal22-1 mutants than in Col-0, while it was smaller in the jal23-1, jal31-1 and jal31-2 mutants than that in Col-0. These results show that two types of JALs having opposite roles regulate the size of the PYK10 complex antagonistically. We define the two types of lectins as a "polymerizer-type lectin" and an "inhibitor-type lectin". Interestingly, the closest homologues of polymerizer-type lectins (JAL31 and JAL23) were inhibitor-type lectins (PBP1/JAL30 and JAL22). The pairs of polymerizer-type and inhibitor-type lectins reported here are good examples of genes that have evolved new functions after gene duplication (neofunctionalization).

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The bHLH Protein, MUTE, Controls Differentiation of Stomata and the Hydathode Pore in Arabidopsis.

Publication Date: 2008 May 1 PMID: 18450784
Authors: Pillitteri, L. J. - Bogenschutz, N. L. - Torii, K. U.
Journal: Plant Cell Physiol

Stomata are turgor-driven epidermal valves on the surface of plants that allow for efficient gas and water exchange between the plant and its environment. The Arabidopsis thaliana basic helix-loop-helix (bHLH) protein, MUTE, is a master regulator of stomatal differentiation where it is required for progression through the stomatal lineage and the differentiation of stomata. The genetic control of stomatal spacing across the epidermal surface is variable in different organs. For instance, a distinct suite of genes from those in leaves regulates stomatal patterning in hypocotyls. Here we report that regardless of organ type MUTE controls downstream events directing stomatal differentiation, specifically the transition from meristemoid to guard mother cell. Ectopic MUTE expression is sufficient to override cell-fate specification in cell types that do not normally differentiate stomata. Furthermore, MUTE is required for the production of the structure evolutionarily related to stomata, the hydathode pore. Consistently, MUTE displays expression at the tip of cotyledons and leaves, thus co-localizing with the auxin maxima. However, MUTE itself was not regulated by the auxin, and the absence of hydathode pores in mute did not affect the auxin maxima. Surprisingly, our analysis revealed that the requirement for MUTE could be partially circumvented under conditions of compromised inhibitory signaling.

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Cryobehavior of the plasma membrane in protoplasts isolated from cold-acclimated Arabidopsis leaves is related to surface area regulation.

Publication Date: 2008 May 1 PMID: 18450783
Authors: Yamazaki, T. - Kawamura, Y. - Uemura, M.
Journal: Plant Cell Physiol

Extracellular freezing in plants results in dehydration and mechanical stresses upon the plasma membrane. Plants that acquire enhanced freezing tolerance after cold acclimation can withstand these two physical stresses. To understand the tolerance to freeze-induced physical stresses, the cryobehavior of the plasma membrane was observed using protoplasts isolated from cold-acclimated Arabidopsis thaliana leaves with the combination of a lipophilic fluorescent dye FM 1-43 and cryomicroscopy. We found that many vesicular structures appeared in the cytoplasmic region near the plasma membrane just after extracellular freezing occurred. These structures, referred to as freeze-induced vesicular structures (FIVs), then developed horizontally near the plasma membrane during freezing. There was a strong correlation between the increase in individual FIV size and the decrease in the surface area of the protoplasts during freezing. Some FIVs fused with their neighbors as the temperature decreased. Occasionally, FIVs fused with the plasma membrane, which may be necessary to relax the stress upon the plasma membrane during freezing. Vesicular structures resembling FIVs were also induced when protoplasts were mechanically pressed between a coverslip and slide glass. Fewer FIVs formed when protoplasts were subjected to hyperosmotic solution, suggesting that FIV formation is associated with mechanical stress rather than dehydration. Collectively, these results suggest that cold-acclimated plant cells may balance membrane tension in the plasma membrane by regulating surface area. This enables plant cells to withstand the direct mechanical stress imposed by extracellular freezing.

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Occurrence of plastids in the sperm cells of Caprifoliaceae: biparental plastid inheritance in angiosperms is unilaterally derived from maternal inheritance.

Publication Date: 2008 Apr 29 PMID: 18448473
Authors: Hu, Y. - Zhang, Q. - Rao, G. - Sodmergen
Journal: Plant Cell Physiol

It is widely held that organelles inherit from the maternal lineage. However, the plastid genome in quite a few angiosperms appears to be biparentally transmitted. It is unclear how and why biparental inheritance of the genome became aroused. Here, we detected widespread occurrence of plastids in the sperm cells (a cellular prerequisite for biparental inheritance) of traditional Caprifoliaceae. Of the 12 genera sampled, the sperm cells of Abelia, Dipelta, Heptacodium, Kolkwitzia, Leycesteria, Linnaea, Lonicera, Symphoricarpos, Triosteum, and Weigela possessed inheritable plastids. The other genera, Sambucus and Viburnum, lacked plastids in sperm cells. Interestingly, such exclusion of plastids in the sperm cells of some Caprifoliaceae appeared to be associated with the divergence of Dipsacales phylogeny. Closer examination of Weigela florida revealed that both plastids and plastid DNA were highly duplicated in the generative cells. This implies that the appearance of plastids in sperm cells involved cellular mechanisms. Because such mechanisms must enhance the strength of plastid transmission through the paternal lineage and appear ubiquitous in species exhibiting biparental or potential biparental plastid inheritance, we presume that biparental plastid genetics may be a derived trait in angiosperms. This is consistent with our extended phylogenetic analysis using species with recently discovered modes of potential plastid inheritance. The results show that basal and early angiosperms have maternal plastid transmission, whereas all potential biparental transmission occurs at terminal branches of the tree. Thus, unlike previous studies, we suggest that biparental plastid inheritance in angiosperms was unilaterally converted from the maternal transmission mode during late angiosperm evolution.

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Functional characterization of Arabidopsis Calreticulin1a: a key alleviator of endoplasmic reticulum stress.

Publication Date: 2008 Apr 23 PMID: 18436549
Authors: Christensen, A. - Svensson, K. - Persson, S. - Jung, J. - Michalak, M. - Widell, S. - Sommarin, M.
Journal: Plant Cell Physiol

The chaperone calreticulin plays important roles in a variety of processes in the endoplasmic reticulum of animal cells, such as Ca(2+) signaling and protein folding. Although the functions of calreticulin are well characterized in animals only indirect evidences are available for plants. To increase our understanding of plant calreticulins we introduced one of the Arabidopsis isoforms, AtCRT1a, into calreticulin deficient (crt(-/-)) mouse embryonic fibroblasts. As a result of calreticulin deficiency the mouse crt(-/-) fibroblasts have decreased levels of Ca(2+ )in the endoplasmic reticulum and impaired protein folding abilities. Expression of the AtCRT1a in mouse crt(-/-) fibroblasts rescued these phenotypes, that is AtCRT1a restored the Ca(2+)-holding capacity and chaperone functions in the endoplasmic reticulum of the mouse crt(-/-) fibroblasts, demonstrating that the animal sorting machinery also was functional for a plant protein, and that basic calreticulin functions are conserved across the Kingdoms. Expression analyses using a GUS- AtCRT1a promoter construct revealed high expression of CRT1a in root tips, floral tissues and in association with vascular bundles. To assess the impact of AtCRT1a in planta, we generated Atcrt1a mutant plants. The Atcrt1a mutants exhibited increased sensitivity to the drug tunicamycin, an inducer of unfolded protein response. We therefore conclude that AtCRT1a is an alleviator of the tunicamycin-induced unfolded protein response, and propose that the use of the mouse crt(-/-) fibroblasts as a calreticulin expression system may prove useful to assess functionalities of calreticulins from different species.

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Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit-chains of amylopectin in rice.

Publication Date: 2008 Apr 22 PMID: 18430767
Authors: Hanashiro, I. - Itoh, K. - Kuratomi, Y. - Yamazaki, M. - Igarashi, T. - Matsugasako, J. I. - Takeda, Y.
Journal: Plant Cell Physiol

A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice (Plant Cell Physiol., 44: 473-480 (2003)) and its effect on endosperm starches was examined. Apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit-chains (ELC) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELC, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELC isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from cv. Labelle (Wx(a)) in terms of higher degree of branching and size distribution. Amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown, and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELC. Structural analysis of Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELC in cultivars carrying the Wx(a).

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Separation of Light-induced Linear, Cyclic and Stroma-sourced Electron Fluxes to P700+ in Cucumber Leaf Discs after Pre-illumination at a Chilling Temperature.

Publication Date: 2008 Apr 20 PMID: 18426807
Authors: Fan, D. Y. - Hope, A. B. - Jia, H. - Chow, W. S.
Journal: Plant Cell Physiol

Pre-illumination of cucumber leaf discs at 4 degrees C with low-irradiance white light (1) led to a marked decrease in the extent of photo-oxidation of P700 [the special chlorophyll pair in the photosystem I (PSI) reaction centre] in actinic light at room temperature and (2) hastened the post-illumination re-reduction of P700(+). Quantifying the linear, cyclic and stroma-sourced electron fluxes to P700(+ )in two actinic-light regimes, we found that there was no increase in cyclic or linear electron fluxes to account for these changes. Rather, we observed a decrease in the maximum extent of P700 photo-oxidation assayed by a strong flash superimposed on continuous, background light of wavelength 723 nm, which we interpret to represent a loss of stable charge separation in PSI due to enhanced charge recombination as a result of the pre-illumination treatment. The funnelling of electrons towards fewer non-damaged PSI complexes could explain the hastened post-illumination re-reduction of P700(+), aided by a slight increase in a stroma-sourced electron flux after prolonged pre-illumination at 4 degrees C. Quantifying the separate fluxes to P700(+) helps to elucidate the effects of chilling of cucumber leaf discs in the light and the reasons for the hastened post-illumination re-reduction of P700(+).

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Cytokinin inhibits the proteasome-mediated degradation of carbonylated proteins in Arabidopsis leaves.

Publication Date: 2008 May PMID: 18420596
Authors: Jain, V. - Kaiser, W. - Huber, S. C.
Journal: Plant Cell Physiol

Under normal conditions, plants contain numerous carbonylated proteins, which are thought to be indicative of oxidative stress damage. Conditions that promote formation of reactive oxygen species (ROS) enhance protein carbonylation, and protein degradation is required to reverse the damage. However, it is not clear how the degradation of carbonylated proteins is controlled in planta. In this report, we show that detached Arabidopsis leaves rapidly and selectively degrade carbonylated proteins when kept in the dark. The loss of carbonylated proteins corresponded to a loss of soluble protein and accumulation of free amino acids. Degradation of carbonylated proteins and the loss of soluble protein was blocked by MG132 but not 3-methyladenine, suggesting that the 26S proteasome pathway rather than the autophagic pathway was involved. Consistent with this, rpn10 and rpn12 mutants, which are defective in proteasome function, had increased (rather than decreased) levels of carbonylated proteins when detached in the dark. Feeding metabolites (amino acids and sucrose) to detached leaves of wild-type Arabidopsis in the dark had little or no effect on the loss of carbonylated proteins, whereas providing soybean xylem sap via the transpiration stream effectively prevented degradation. The effect of xylem sap was mimicked by feeding 10 muM kinetin. We postulate that disruption of cytokinin flux to detached leaves triggers the selective degradation of carbonylated proteins via the proteasome pathway. The results may have implications for the control of protein mobilization in response to changes in N availability.

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Overexpression of a new rice vacuolar antiporter regulating protein OsARP improves salt tolerance in tobacco.

Publication Date: 2008 Apr 17 PMID: 18420595
Authors: Uddin, M. I. - Qi, Y. - Yamada, S. - Shibuya, I. - Deng, X. P. - Kwak, S. S. - Kaminaka, H. - Tanaka, K.
Journal: Plant Cell Physiol

We examined function of the rice (Oryza sativa L.) antiporter regulating protein OsARP by overexpressing in tobacco (Nicotiana tabacum L.). In public databases, this protein was annotated as putative Os02g0465900 protein of rice. The OsARP gene was introduced into tobacco under the control of cauliflower mosaic virus 35S promoter. The transformants were selected for their ability to grow on medium containing kanamycin. Incorporation of the transgene in the genome of tobacco was confirmed by polymerase chain reaction, and its expression was confirmed by Western-blot analysis. Transgenic plants had better growth and vigor than that of non-transgenic under salt stress in vitro. Overexpression of OsARP in transgenic tobacco plants resulted in salt tolerance had higher photosynthesis and effective PSII photon yield when compared with the wild type. The OsARP protein was localized in tonoplast of rice plants. Transgenic plants accumulated more Na(+) in their leaf tissue than those of wild type plants. It is conceivable that the toxic effect of Na(+) in cytosol might be reduced by sequestration into vacuoles. The rate of water loss was higher in wild type than that of transgenic under salt stress. Increased vacuolar solute accumulation and water retention could confer salt tolerance in transgenic plants. Tonoplast vesicles isolated from OsARP transgenic plants showed Na(+)/H(+) exchange rates three-fold higher than those of wild type plants. These results suggest that OsARP on the tonoplasts plays important role in compartmentation of Na(+) into vacuoles. We suggest OsARP is a new type protein participating in Na(+) uptake in vacuoles.

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The Gravity-Regulated Growth of Axillary Buds is Mediated by a Mechanism Different from Decapitation-Induced Release.

Publication Date: 2008 Apr 17 PMID: 18420594
Authors: Kitazawa, D. - Miyazawa, Y. - Fujii, N. - Hoshino, A. - Iida, S. - Nitasaka, E. - Takahashi, H.
Journal: Plant Cell Physiol

When the upper part of the main shoot of the Japanese morning glory (Pharbitis nil or Ipomoea nil) is bent down, the axillary bud situated on the uppermost node of the bending region is released from apical dominance and elongates. Here, we demonstrate that this release of axillary buds from apical dominance is gravity-regulated. We utilized two agravitropic mutants of morning glory defective in gravisensing cell differentiation, weeping (we) and weeping2 (we2). Bending the main shoots of either we or we2 plants resulted in minimal elongation of their axillary buds. This aberration was genetically linked to the agravitropism phenotype of the weeping mutants, which implied that shoot bending-induced release from apical dominance required gravisensing cells. Previous studies have shown that basipetal translocation of auxin from the apical bud inhibits axillary bud growth, whereas cytokinin promotes axillary bud outgrowth. We therefore compared the roles of auxin and cytokinin in bending- or decapitation-induced axillary bud growth. In the wild-type and we plants, decapitation increased cytokinin levels and reduced auxin response. In contrast, shoot bending did not cause significant changes in either cytokinin level or auxin response, suggesting that the mechanisms underlying gravity- and decapitation-regulated release from apical dominance are distinct and unique.

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