Plant and Cell Physiology - Advance Access

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

Nagano, A. J., Fukao, Y., Fujiwara, M., Nishimura, M., Hara-Nishimura, I. — 2008-05-08

PYK10/BGLU23 is a β-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 µm to over 70 µm. We identified three β-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).

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

Xu, M., Ogawa, T., Pakrasi, H. B., Mi, H. — 2008-05-07

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 ndhD4 and ndhF4 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-β-maltoside.

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

Yamazaki, T., Kawamura, Y., Uemura, M. — 2008-05-01

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.

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

Pillitteri, L. J., Bogenschutz, N. L., Torii, K. U. — 2008-05-01

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.

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

Hu, Y., Zhang, Q., Rao, G., Sodmergen, — 2008-04-29

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.

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

2008-04-23

The chaperone calreticulin plays important roles in a variety of processes in the endoplasmic reticulum of animal cells, such as Ca²⁺ 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 Ca²⁺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 Ca²⁺-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.

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

2008-04-22

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.

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

Fan, D.-Y., Hope, A. B., Jia, H., Chow, W. S. — 2008-04-20

Pre-illumination of cucumber leaf discs at 4°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°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⁺.

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

2008-04-17

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.

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

2008-04-17

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.

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

Jain, V., Kaiser, W., Huber, S. C. — 2008-04-16

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 µM 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.

A Comprehensive Transcriptional Profiling of the WRKY Gene Family in Rice under Various Abiotic and Phytohormone Treatments

Ramamoorthy, R., Jiang, S.-Y., Kumar, N., Venkatesh, P. N., Ramachandran, S. — 2008-04-15

WRKY transcription factors play important roles in the regulation of various biological processes. We have analyzed the rice genome sequence databases publicly available and predicted 103 genes encoding WRKY transcription factors. Among them, the majority of rice WRKY genes (77.7%) were located in duplicated regions; 45.6% of WRKY genes were fragmentally duplicated and 35% of them were tandemly duplicated. These results suggested that genome duplications might be regarded as a major mechanism for expansion of this family in rice genome. Subsequently, we analyzed their expression profiles under normal and abiotic stress as well as various hormone treatments. Under normal growth conditions, 65 WRKY genes were expressed differentially either in their transcript abundance or in their expression patterns. Under abiotic (cold, drought and salinity) stresses and various phytohormone treatments, 54 WRKY genes exhibited significant differences in their transcript abundance, among them 3 genes were expressed only in stressed conditions. Among the stress-inducible genes, 13 genes were regulated only by abiotic stresses, another set of 13 genes were responsive to only phytohormone treatments and the remaining 28 genes were regulated by both factors, suggesting an interaction between abiotic stress and hormone signaling. On the other hand, we have also surveyed expression divergence of duplicated genes under normal or stressed conditions and the results showed that high expression divergence has occurred not only among fragmentally but also among tandemly duplicated genes. These results suggested that the high expression divergence could be one of mechanisms for the retention of these duplicated WRKY genes.

Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid

2008-04-04

In our previous studies, we identified four DEF-like genes and one GLO-like gene involved in floral organ development in Phalaenopsis equestris. Revealing the DNA-binding properties and protein-protein interactions of these floral homeotic MADS-box protein complexes (PeMADS) in orchids is crucial to elucidate the unique orchid floral morphogenesis. In this study, the interactome of B-class PeMADS proteins was assayed by yeast two-hybrid system (Y2H) and GST pull-down assays. Furthermore, the DNA binding activities of these proteins were assessed by using electrophoretic mobility shift assay (EMSA). All four DEF-like PeMADS proteins interacted individually with the GLO-like PeMADS6 under Y2H assay, yet with differential interaction strength. Generally, the PeMADS3/PeMADS4 lineage interacted stronger with PeMADS6 than the PeMADS2/PeMADS5 lineage did. In addition, independent homodimer formation for both PeMADS4 (DEF-like) and PeMADS6 (GLO-like) were detected. The protein-protein interactions between pairs of PeMADS proteins were further confirmed by using GST pull-down assay. Furthermore, both the PeMADS4 homodimer and the PeMADS6 homodimer/homomultimer per se were able to bind to the MADS-box protein binding motif CArG. The heterodimeric complexes of PeMADS2/PeMADS6, PeMADS4/PeMADS6 and PeMADS5/PeMADS6 showed the CArG-binding activity. Taken together, these results suggest that various complexes formed among different combinations of the five B-class PeMADS proteins may increase the complexity of their regulatory functions and thus specify the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in orchids.

CRR23/NdhL is a subunit of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

Shimizu, H., Peng, L., Myouga, F., Motohashi, R., Shinozaki, K., Shikanai, T. — 2008-04-03

The chloroplast NAD(P)H dehydrogenase (NDH) complex functions in photosystem I cyclic and chlororespiratory electron transport in higher plants. Eleven plastid-encoded and three nuclear-encoded subunits have been identified so far, but the entire subunit composition, especially of the putative electron-donor-binding module, is unclear. We isolated Arabidopsis thaliana crr23 (chlororespiratory reduction) mutants lacking NDH activity according to the absence of a transient increase in Chl fluorescence after actinic light illumination. Although CRR23 shows similarity to the NdhL subunit of cyanobacterial NDH-1, it has three transmembrane domains rather than the two in cyanobacterial NdhL. Unlike cyanobacterial NdhL, CRR23 is essential for stabilizing the NDH complex, which in turn is required for the accumulation of CRR23. Furthermore, CRR23 and NdhH, a subunit of chloroplast NDH, co-localized in blue-native gel. All the results indicate that CRR23 is an ortholog of cyanobacterial ndhL in Arabidopsis, despite its diversity of structure and function.

Characterization of factors affecting the activity of photosystem I cyclic electron transport in chloroplasts

Okegawa, Y., Kagawa, Y., Kobayashi, Y., Shikanai, T. — 2008-04-03

PSI cyclic electron transport is essential for photosynthesis and photoprotection. In higher plants, antimycin A-sensitive pathway is the main route of electrons in PSI cyclic electron transport. Although a small thylakoid protein, PGR5 (PROTON GRADIENT REGULATION 5), is essential for this pathway, its function is still unclear, and there are numerous debates on the rate of electron transport in vivo and its regulation. To assess how PGR5-dependent PSI cyclic electron transport is regulated in vivo, we characterized its activity in ruptured chloroplasts isolated from Arabidopsis thaliana. The activity of ferredoxin (Fd)-dependent plastoquinone (PQ) reduction in the dark is impaired in the pgr5 mutant. Alkalinization of the reaction medium enhanced the activity of Fd-dependent PQ reduction in the wild type. Even weak actinic light (AL) illumination also markedly activated PGR5-dependent PSI cyclic electron transport in ruptured chloroplasts. And even in the presence of linear electron transport (11 µmol O₂ mg Chl^-1 h^-1), PGR5-dependent PSI electron transport was detected as a difference in Chl fluorescence levels in ruptured chloroplasts. In the wild type, PGR5-dependent PSI cyclic electron transport competed with NADP⁺ photoreduction. These results suggest that the rate of PGR5-dependent PSI cyclic electron transport is high enough to balance the production ratio of ATP and NADPH during steady-state photosynthesis, consistently with the pgr5 mutant phenotype. Our results also suggest that the activity of PGR5-dependent PSI cyclic electron transport is regulated by the redox state of the NADPH pool.

Unequal Genetic Redundancy of Rice PISTILLATA Orthologs, OsMADS2 and OsMADS4, in Lodicule and Stamen Development

Yao, S.-G., Ohmori, S., Kimizu, M., Yoshida, H. — 2008-04-03

Although two homologs of PISTILLATA, OsMADS2 and OsMADS4, have been identified in the rice genome, their roles in floral organ development are controversial. Here, we demonstrate that the two genes show unequal redundancy of class-B function. Although OsMADS2 plays the important role in lodicule development, OsMADS4 also supports specification of lodicule identity. On the other hand, the two genes are almost equally important in stamen development. Consistent with their redundant function, both OsMADS2 and OsMADS4 interact with SPW1, the unique rice AP3 ortholog.

Drought Stress Alters Water relations and Expression of PIP-type Aquaporin Genes in Nicotiana tabacum Plants

Najafabadi, M. M., Mostajeran, A., Horie, T., Katsuhara, M. — 2008-04-01

Plasma membrane intrinsic proteins (PIPs), a type of aquaporins, mediate water transport in many plant species. In this study, we investigated the relation between the functions of PIP-type water channels and water relations of tobacco plants (Nicotiana tabacum cv. Samsun) under drought stress. Drought stress treatments have led to reductions in the stomatal conductance, transpiration, water potential, and turgor pressure in leaves, and also the sap flow rate and osmotic hydraulic conductance in roots. In contrast, leaf osmotic pressure was increased in response to drought stress. Interestingly, the accumulation of NtPIP1;1 and NtPIP2;1 transcripts was significantly decreased, but only that of the NtAQP1 transcript was increased under drought stress. Functional analysis using Xenopus laevis oocytes revealed that NtPIP2;1 shows marked water transport activity, but those of NtAQP1 and NtPIP1;1 are weak or almost negligible, respectively, when solely expressed. However, co-expression of NtPIP1;1 with NtPIP2;1 significantly enhanced water transport activity compared to that of NtPIP1;1- or NtPIP2;1-expressing oocytes, suggesting that these two aquaporins may function as a water channel forming a heterotetramer. Heteromerization of NtPIP1;1 and NtPIP2;1 was also suggested by co-expression analyses of NtPIP1;1-GFP and NtPIP2;1 in Xenopus oocytes. Re-watering treatments recovered water relation parameters and the accumulation of the three NtPIP transcripts to similar levels to control conditions. These results suggest that NtPIP1;1 and NtPIP2;1 play an important role in water transport in roots, and that expressions of NtPIP1;1 and NtPIP2;1 are down-regulated in order to reduce osmotic hydraulic conductance in the roots of tobacco plants under drought stress.

ACTIN-RELATED RROTEIN8 encodes an F-box protein localized to the nucleolus in Arabidopsis

Kandasamy, M. K., McKinney, E. C., Meagher, R. B. — 2008-04-01

Arabidopsis encodes six nuclear actin related proteins (ARPs), among them ARP8 is unique in having a F-box domain and an actin-homology domain. Analysis of ARP8 promoter-GUS fusion suggests that ARP8 is ubiquitously expressed in all organs and cell types. Immunocytochemical analysis with ARP8-specific monoclonal antibodies revealed that ARP8 protein is localized to the nucleolus in interphase cells and dispersed in the cytoplasm in mitotic cells. The cell cycle-dependent subcellular patterns of distribution of ARP8 are conserved in other members of Brassicaceae. Our findings provide the first insight into the possible contributions of plant ARP8 to the nucleolar functions.

Tomato Rab11a characterization evidenced a difference between SYP121 dependent and SYP122 dependent exocytosis

2008-04-01

The regulatory functions of Rab proteins in membrane trafficking lie in their ability to perform as molecular switches that oscillate between a GTP- and a GDP- bound conformation. The role of tomato LeRab11a in secretion was analysed in tobacco protoplasts. GFP/RFP-tagged LeRab11a was localized at the TGN in vivo. Two serines in the GTP-binding site of the protein were mutagenised, giving rise to the three mutants Rab11S22N, Rab11S27N, and Rab11S22/27N. The double mutation reduced secretion of a marker protein secRGUS, by half, whereas each of the single mutations alone had a much smaller effect, showing that both serines have to be mutated to obtain a dominant negative effect on LeRab11a function. The dominant negative mutant was used to determine whether Rab11 is involved in the pathway(s) regulated by the plasma membrane syntaxins SYP121 and SYP122. Co-expression of either of these GFP-tagged syntaxins with the dominant negative Rab11S22/27N mutant led to the appearance of endosomes but co-expression of GFP-tagged SYP122 also labelled the endoplasmic reticulum and dotted structures. However, co-expression of Rab11S22/27N with SYP121 dominant negative mutants decreased secretion of secRGUS further compared to the expression of Rab11S22/27N alone, whereas co-expression of Rab11S22/27N with SYP122 had no synergistic effect. With the same essay the difference between SYP121 and SYP122 dependent secretion was then evidenced. The results suggest that Rab11 regulates anterograde transport from the TGN to the PM and strongly implicate SYP122, rather than SYP121. LeRab11a differential effect supports the possibility that SYP121 and SYP122 drive independent secretory events.

Apical wilting and petiole xylem vessel diameter of the rms2 branching mutant of pea are shoot controlled and independent of a long-distance signal regulating branching

Dodd, I. C., Ferguson, B. J., Beveridge, C. A. — 2008-03-31

RMS2 (RAMOSUS2) affects the level or transport of a graft-transmissible signal produced in the shoot and root that controls axillary bud outgrowth in pea (Pisum sativum L.). The shoot apex of rms2 transiently wilts under high evaporative demand. The origin of this phenotype was investigated to determine whether it was involved in the regulation of branching. Wild-type (WT) and rms2 leaves showed a similar stomatal conductance at both low and high evaporative demand in vivo, indicating normal stomatal function. Leaves of both genotypes had similar abscisic acid (ABA) content and response to ABA. Although root hydraulic conductance (determined by pressure-induced flow) of rms2 plants was normal, more xylem vessels per vascular bundle were identified in cross-sections of fully-expanded rms2 petioles compared with those of the WT. However, the diameter of these vessels was nearly half that of the WT. Since the conductance of each vessel is proportional to the fourth power of vessel radius (according to the Hagen-Poiseulle Law), theoretical (calculated) petiole hydraulic conductance of rms2 was greatly decreased compared to WT plants. Under high evaporative demand, this would cause a temporary imbalance between water supply to, and demand from, rms2 shoots, directly resulting in the wilting phenotype of the mutant. Reciprocal grafting showed that xylem vessel development in rms2 shoots is strictly shoot controlled, likely via elevated auxin levels. This altered xylem vessel development, though causing wilting in rms2 shoot tips, does not appear to affect shoot branching.

Nano Scale Proteomics Revealed the Presence of Regulatory Proteins Including Three FT-like proteins in Phloem and Xylem Saps from Rice

Aki, T., Shigyo, M., Nakano, R., Yoneyama, T., Yanagisawa, S. — 2008-03-27

The main physiological roles of phloem and xylem in higher plants involve the transport of water, nutrients and metabolites. They are also involved, however, in whole plant events including stress responses and long-distance signaling. Phloem and xylem saps therefore include a variety of proteins. In this study, we have performed a shotgun analysis of the proteome of phloem and xylem saps from rice, taking advantage of the complete and available genomic information for this plant. Xylem sap was prepared using the root pressure method, whereas phloem sap was prepared with a unique method with the assistance of planthoppers to ensure the robustness of the detected proteins. The technical difficulties caused by the very limited availability of rice samples were overcome by the use of nano-flow liquid chromatography linked to a mass spectrometer. We identified 118 different proteins and eight different peptides in xylem sap and 107 different proteins and five different peptides in phloem sap. Signal transduction proteins, putative transcription factors and stress response factors as well as metabolic enzymes were identified in these saps. Interestingly, we found the presence of three TERMINAL FLOWER 1/FLOWERING LOCUS T (FT)-like proteins in phloem sap. The detected FT-like proteins were not rice Hd3a (OsFTL2) itself that acted as a non-cell-autonomous signal for flowering control, but they were members of distinct subfamilies of the FT family with differential expression patterns. These results imply that proteomics on a nano scale is a potent tool for investigation of biological processes in plants.

Biochemical and Genetic Analysis of Carbohydrate Accumulation in Allium cepa L.

2008-03-27

Onion and shallot (Allium cepa L.) exhibit wide variation in bulb fructan content and the Frc locus on chromosome 8 conditions much of this variation. To understand the biochemical basis of Frc we conducted biochemical and genetic analyses of Allium fistulosum (FF) - shallot (A. cepa Aggregatum group) alien monosomic addition lines (AALs; FF+1A-FF+8A) and onion mapping populations. Sucrose and fructan levels in leaves of FF+2A were significantly lower than FF throughout the year and springtime activity of acid invertase was also lower. FF+8A showed significantly higher winter sucrose accumulation and sucrose phosphate synthase (SPS) activity. Inbred high fructan (Frc_) lines from the ‘W202A x Texas Grano 438' (WxT) onion population exhibited significantly higher sucrose levels prior to bulbing than low fructan (frcfrc) lines. SuSy activity in these lines was correlated with leaf hexose content but not with Frc phenotype. Markers for additional candidate genes for sucrose metabolism were obtained by cloning a major SPS expressed in onion leaf and exhaustively mining onion EST resources. SPS and SuSy loci were assigned to chromosome 8 and 6 respectively using AALs and linkage mapping. Further loci were assigned, using AALs, to chromosomes 1 (sucrose phosphate phosphatase), 2 (SuSy and 3 invertases) and 8 (neutral invertase). The concordance between chromosome 8 localization of SPS and elevated leaf sucrose levels conditioned by high fructan alleles at the Frc locus in bulb onion or alien monosomic additions of chromosome 8 in A. fistulosum suggest that the Frc locus may condition variation in SPS activity.

An analysis of long-distance water transport in the soybean stem using H215O

2008-03-27

The lateral water movement in the intact stem of a transpiring soybean plant was analyzed quantitatively by a real-time measurement system utilizing labeled water, H₂¹⁵O, and gamma ray detectors. A large volume of water escaping from xylem vessels during its transport was detected. The escape of water was not influenced by evaporation from the stem surface or mass flow in the sieve tubes. It was assumed that the total amount of water transported through xylem vessels was kept almost completely constant along the internode. As a result, most of the escaped water was found to re-enter the xylem vessels, i.e. water exchange occurred. The analysis of radiographs of THO suggested that the self-diffusion effect of water was strong for lateral water movement though another driving force besides thermal motion was included in the process, and that the process was also affected by the water permeability of plasma membrane. An analysis based on a mathematical model showed that the net volume of water escaped from xylem vessels was not dependent on the transpiration rate of the plant.

Functional Analysis of Three Lily (Lilium longiflorum) APETALA1-like MADS Box Genes in Regulating Floral Transition and Formation

Chen, M.-K., Lin, I-C., Yang, C.-H. — 2008-03-26

Three cDNAs showed high homology to the SQUA subfamily of MADS box genes were isolated and characterized from the lily (Lilium longiflorum). Lily MADS Box Gene 5 (LMADS5) showed high sequence identity to oil palm (Elaeis guineensis) SQUAMOSA3 (EgSQUA3). LMADS6 is closely related to LMADS5 whereas LMADS7 is more related to DOMADS2, an orchid (Dendrobium) gene in the SQUA subfamily. The expression pattern for these three genes was similar and their RNA was detected in vegetative stem and inflorescence meristem. LMADS5, 6 were highly expressed in vegetative leaves and carpel whereas the LMADS7 expression was absent. Ectopic expression of LMADS5, 6 or 7 in transgenic Arabidopsis plants showed novel phenotypes by flowering early and producing terminal flowers. Homeotic conversion of sepals to carpelloid and petal to stamen-like structures were also observed in 35S::LMADS5, 6 or 7 flowers. Ectopic expression of LMADS6 or LMADS7 was able to complement the ap1 flower defect in transgenic Arabidopsis ap1 mutant plants. These results strongly indicated that the function of these three lily genes was involved in flower formation as well as in floral induction. Furthermore, the ability for lily LMADS 6 and 7 to complement the Arabidopsis ap1 mutant provided further evidence to show that the conserved motifs (paleoAP1 or euAP1) in C terminus of SQUA/AP1 subfamily of MADS box genes is not strictly necessary for their function.

ACC synthase genes are polymorphic in watermelon (Citrullus spp.) and differentially expressed in flowers and in response to auxin and gibberellin

Salman-Minkov, A., Levi, A., Wolf, S., Trebitsh, T. — 2008-03-26

The flowering pattern of watermelon species (Citrullus spp.) is either monoecious or andromonoecious. Ethylene is known to play a critical role in floral sex determination of Cucurbit species. In contrast to its feminizing effect in cucumber and melon, in watermelon ethylene promotes male flower development. In cucumber the rate-limiting enzyme of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), regulates unisexual flower development. To investigate the role of ethylene in flower development we isolated four genomic sequences of ACS from watermelon (CitACS1-4). Both CitACS1 and CitACS3 are expressed in floral tissue. CitACS1 is also expressed in vegetative tissue and it may be involved in cell growth processes. Expression of CitACS1 is up-regulated by exogenous treatment with auxin, gibberellin or ACC, the immediate precursor of ethylene. No discernable differential floral sex-dependent expression pattern was observed for this gene. The CitACS3 gene is expressed in open flowers and in young staminate floral buds (male or hermaphrodite) but not in female flowers. CitACS3 is also up-regulated by ACC, and is likely to be involved in ethylene-regulated anther development. The expression of CitACS2 was not detected in vegetative or reproductive organs but was up-regulated by auxin. CitACS4 transcript was not detected under our experimental conditions. Restriction fragment length polymorphism (RFLP) and sequence tagged site (STS) marker analyses of the CitACS genes showed polymorphism among and within the different Citrullus groups, including watermelon cultivars, Citrullus lanatus var. lanatus, the central subspecies Citrullus lanatus var. citroides, and the desert species Citrullus colocynthis (L).

Metabolite profiling reveals distinct changes in carbon and nitrogen metabolism in phosphate-deficient barley plants (Hordeum vulgare L.)

2008-03-15

Plants modify metabolic processes for adaptation to low phosphate (P) conditions. Whilst transcriptomic analyses show that P deficiency changes hundreds of genes related to various metabolic processes, there is limited information available for global metabolite changes of P-deficient plants, especially for cereals. As changes in metabolites are the ultimate "readout" of changes in gene expression, we profiled polar metabolites from both shoots and roots of P-deficient barley (Hordeum vulgare) using GC-MS. Results showed that mildly P-deficient plants accumulated di- and tri-saccharides (sucrose, maltose, raffinose and 6-kestose), especially in shoots. Severe P-deficiency increased the levels of metabolites related to ammonium metabolism in addition to di- and tri-saccharides, but reduced the levels of phosphorylated intermediates (glucose-6-P, fructose-6-P, inositol-1-P, and glycerol-3-P) and organic acids (-ketoglutarate, succinate, fumarate and malate). The results revealed that P-deficient plants modify carbohydrate metabolism initially to reduce P consumption, and salvage P from small P-containing metabolites when P deficiency is severe, which consequently reduced levels of organic acids in the TCA cycle. The extent of the effect of severe P deficiency on ammonium metabolism was also revealed by LC-MS quantitative analysis of free amino acids. A sharp increase in the concentrations of glutamine and asparagine was observed in both shoots and roots of severely P-deficient plants. Based on these data a strategy for improving the ability of cereals to adapt to low P environments is proposed that involves alteration in partitioning of carbohydrates into organic acids and amino acids to enable more efficient utilization of carbon in P-deficient plants.

GIBBERELLIN HOMEOSTASIS IN TOBACCO IS REGULATED BY GA METABOLISM GENES WITH DIFFERENT GA SENSITIVITY

2008-03-11

Gibberellins (GA) are phytohormones that regulate growth and development of plants. GA homeostasis is maintained by feedback regulation of GA metabolism genes. To understand this regulation, we manipulated the GA pathway in tobacco and studied its effects on the morphological phenotype, GA levels and also on the expression of endogenous GA metabolism genes. The over-expression of a GA 3-oxidase (biosynthesis gene) in tobacco (3ox-OE) induced slight variations in phenotype and active GA₁levels, but we also found an increase in GA₈ levels (GA₁ inactivation product) and a conspicuous induction of GA 2-oxidases (catabolism genes; NtGA2ox3, and -5) suggesting an important role for these particular genes in the control of GA homeostasis. The effect of simultaneous over-expression of two biosynthesis genes, a GA 3-oxidase and a GA 20-oxidase (20ox/3ox-OE), on phenotype and GA content, suggests that GA 3-oxidases are non-limiting enzymes in tobacco, even in a GA20ox-OE background. Moreover, the expression analysis of GA metabolism genes in transgenic plants (3ox-OE, 20ox-OE and hybrid 3ox/20ox-OE), and in response to application of different GA₁concentrations, showed genes with different GA sensitivity. GA biosynthesis genes (NtGA20ox1 and NtGA3ox1) are negatively feedback regulated mainly by high GA levels. In contrast, GA catabolism genes which have positive feedback regulation are sensitive to high (NtGA2ox1) or to low (NtGA2ox3, and -5) GA concentrations. These two last GA2ox genes seem to play a predominant role in GA homeostasis under mild GA variations but not under large GA changes, where biosynthesis genes GA20ox and GA3ox may be more important.