Plant and Cell Physiology - Advance Access

Functional Analysis of FT and TFL1 Orthologues from Orchid (Oncidium Gower Ramsey) that Regulate the Vegetative to Reproductive Transition

Hou, C.-J., Yang, C.-H. — 2009-07-01

The FLOWERING LUCUS T (FT) and TERMINAL FLOWER 1 (TFL1) genes play crucial roles in regulating the vegetative to reproductive phase transition. Orthologues of FT/TFL1 (OnFT and OnTFL1) were isolated and characterized from Oncidium Gower Ramsey. OnFT mRNA was detected in axillary buds, leaves, pseudobulb and flowers. In flowers, OnFT was expressed higher in young flower buds than in mature flowers and was predominantly expressed in sepals and petals. The expression of OnFT was regulated by photoperiod, with the highest expression from the 8th to 12th hours of the light period and the lowest expression at dawn. In contrast, the expression of OnTFL1 was only detected in axillary bud and pseudobulb, and was not influenced by light. Ectopic expression of OnFT in transgenic Arabidopsis plants showed novel phenotypes by flowering early and losing inflorescence indeterminacy. In addition, ectopic expression of OnFT was able to partially complement the late flowering defect in transgenic Arabidopsis ft-1 mutants. In transgenic tfl1-11 mutant plants, 35S::OnTFL1 delayed flowering and rescued the phenotype of terminal flowers. Furthermore, substitution of the key single amino acid His85 to Tyr was able to convert the OnTFL1 function to OnFT by promoting flowering in 35S::OnTFL1-H85Y transgenic Arabidopsis plants. Further analysis indicated that the expression of APETALA1 (AP1) was significantly up-regulated in 35S::OnFT and 35S::OnTFL1-H85Y plants, and was down-regulated in 35S::OnTFL1 transgenic Arabidopsis plants. Our data indicated that OnFT and OnTFL1 are putative PEBP genes in orchids that regulate flower transition similar to their orthologues in Arabidopsis.

Overexpression of BiP has inhibitory effects on the accumulation of seed storage proteins in endosperm cells of rice

Yasuda, H., Hirose, S., Kawakatsu, T., Wakasa, Y., Takaiwa, F. — 2009-06-29

Seed storage proteins are specifically and highly synthesized during seed maturation and are deposited into protein bodies via the ER lumen. The accumulation process is mediated by ER chaperones such as BiP and PDI. To examine the role of ER chaperones and the relationship between ER chaperones and accumulation levels of seed storage proteins, we generated transgenic rice plants in which the rice BiP and PDI genes were overexpressed in an endosperm-specific manner under the control of the rice seed storage protein glutelin promoter. The seed phenotype of the PDI-overexpressing transformant was almost identical with that of the wild type, whereas overexpression of BiP resulted in transgenic rice seed that displayed an opaque phenotype with floury and shrunken features. In the BiP-overexpressing line, the accumulation levels of seed storage proteins and starch contents were significantly lower compared with wild type. Interestingly, overproduction of BiP in the endosperm of the transformant not only altered the morphological structure of ER-derived PB-I, but also generated unusual new protein body (PB)-like structures composed of a high-electron density matrix containing glutelin and BiP and a low-electron density matrix containing prolamins. Notably, polysomes were attached around the aberrant PB-like structures, indicating that this aberrant structure is an ER-derived PB-I derivative. These results suggested that the PB-like structure may be formed in the ER lumen, resulting in inhibition of translation, folding and transport of seed proteins.

Differential Gene Expression Profiles of the Mitochondrial Respiratory Components in Illuminated Arabidopsis Leaves.

Yoshida, K., Noguchi, K. — 2009-06-29

Plant mitochondria have multiple energy-dissipating components in the respiratory chain. It is known that these components are induced under several stress conditions. Here we examined whether the gene expression pattern and its regulatory mechanism under high-light (HL) conditions are different among the respiratory components in Arabidopsis leaves. The alternative oxidase (AOX) gene expression (AOX1a and AOX1c) and protein amount were elevated after exposure to HL. In addition to AOX, the expression of other respiratory genes, including NDA1, NDB2, NDC1, UCP1, UCP5, COX6b, and CI76, was also HL-induced. NDB2 was co-expressed with AOX1a, but other HL-induced genes showed a distinct expression pattern. Manipulation of photosynthesis or respiration using several chemicals revealed that, while the expression of AOX1a and NDB2 was mainly induced by the inhibition of the respiratory chain, NDA1 expression was affected by photosynthesis-related reactive oxygen species. The expression of AOX1c, NDC1, COX6b, and CI76 was not induced by these manipulations. When plants were exposed to HL under high CO₂ environment, the expression of several respiratory genes was more strongly induced, suggesting that modulations of cellular carbon status by elevated photosynthesis are involved in respiratory gene expression. Based on these results, we propose a mechanistic model of respiratory gene expression in illuminated leaves.

A novel carotenoid derivative, lutein 3-acetate, accumulates in senescent leaves of rice

Kusaba, M., Maoka, T., Morita, R., Takaichi, S. — 2009-06-27

The biosynthetic pathway of lutein, the most abundant carotenoid in leaves, has been extensively studied, while its degradation pathway during senescence is poorly understood. We found that a novel carotenoid derivative, lutein 3-acetate, accumulates in senescent leaves of rice (Oryza sativa L.). The change in contents of lutein and lutein 3-acetate suggests that lutein is converted to lutein 3-acetate during senescence. The analyses of mutants involved in LHCII accumulation and degradation suggests that the converting activity is induced during senescence and that only free lutein, not lutein bound to LHCII, is converted into lutein 3-acetate.

Identification and Expression Analysis of Light-Dependent and Light-Independent Protochlorophyllide Oxidoreductases in the Chromatically Adapting Cyanobacterium Fremyella diplosiphon UTEX 481.

2009-06-27

The cyanobacteria Fremyella diplosiphon can alternate its light harvesting pigments, a process called complimentary chromatic adaptation (CCA), allowing it to photosynthesize in green light (GL) and in fluctuating light conditions. Nevertheless, F. diplosiphon requires chlorophylls for photosynthesis under all light conditions. Two alternate enzymes catalyze the penultimate step of chlorophyll synthesis, Light-dependent protochlorophyllide oxidoreductase (LPOR) and Dark-operative protochlorophyllide Oxidoreductase (DPOR). DPOR enzymatic activity is light independent, while LPOR requires light. Therefore, we hypothesize that F. diplosiphon up regulates DPOR gene expression in GL, so that DPOR is more abundant when LPOR is less functional. We cloned the genes encoding the three subunits of DPOR, chlL, chlN, and chlB, and the LPOR gene, por, to determined abundance of the transcripts under RL, GL, and dark conditions. We found that F. diplosiphon chlL and chlN genes are transcribed as parts of a single operon, a gene structure that is conserved within cyanobacteria. Transcripts levels of all DPOR genes are up regulated approximately 2-fold in GL relative to levels in RL, whereas LPOR transcript levels are reduced in GL. Moreover, mutations in CCA regulators, RcaE and CpeR, modify DPOR and LPOR transcript levels under specific light conditions. Finally, both DPOR and LPOR transcripts are down regulated 2 to 5 fold in the dark. These results provide the first evidence that light quality and CCA affect the genetic regulation of chlorophyll biosynthesis in freshwater cyanobacteria, ecologically important photosynthetic organisms.

Dynamic bridges - a calponin-domain kinesin from rice links actin filaments and microtubules in both cycling and non-cycling cells

Frey, N., Klotz, J., Nick, P. — 2009-06-27

Interaction and cross-talk between microtubules and actin microfilaments is important for cell axis and polarity during plant cell growth and development, but little is known about the molecular components of this interaction. Plant kinesins with calponin-homology domain (KCH) were recently identified and associated with a putative role in microtubule-microfilament cross-linking. KCHs belong to a distinct branch of the minus end-directed kinesin subfamily and so far have only been identified in land plants including the mosses. Here we report the identification of a new KCH from rice (Oryza sativa), OsKCH1, and show that OsKCH1 is associated with cortical microtubules and actin microfilaments in vivo. Furthermore, OsKCH1 is shown to bind to microtubules and actin microfilaments in vitro in a domain dependent way. Additionally, this unique type of kinesin is shown to oligomerize both in vivo and in vitro. These findings are discussed with respect to a general role of KCHs as linkers between actin filaments and microtubules in both cell elongation and division.

Functional and structural characterization of a flavonoid glucoside 1,6-glucosyltransferase from Catharanthus Roseus

Masada, S., Terasaka, K., Oguchi, Y., Okazaki, S., Mizushima, T., Mizukami, H. — 2009-06-27

Sugar-sugar glycosyltransferases play an important role in structural diversity of small molecule glycosides in higher plants. We isolated a cDNA clone encoding a sugar-sugar glucosyltransferase (CaUGT3) catalyzing 1,6-glucosylation of flavonol- and flavone glucosides for the first time from Catharanthus roseus. CaUGT3 exhibited a unique glucosyl chain elongation activity forming not only gentiobioside but also gentiotrioside and gentiotetroside in a sequential manner. We investigated the functional properties of CaUGT3 using homology modeling and site-directed mutagenesis, and identified amino acids positioned in the acceptor binding pocket as crucial for providing enough space to accommodate flavonoid glucosides instead of flavonoid aglycones. These results provide basic information for understanding and engineering the catalytic functions of sugar-sugar glycosyltransferases involved in biosynthesis of plant glycosides.

Survey for rice proteins interacting with OsFCA and OsFY proteins which are homologous to the Arabidopsis flowering time proteins, FCA and FY

2009-06-26

The FCA protein is involved in controlling flowering time and plays more general roles in RNA-mediated chromatin silencing in Arabidopsis. It contains two RNA binding domains and a WW domain. The FCA protein interacts with FY, a polyadenylation factor, via its WW domain. We previously characterized a rice gene, OsFCA, which was homologous to FCA. Here, we found that the OsFCA protein could interact through its WW domain with following proteins: OsFY, a protein containing a CID domain present in RNA-processing factors such as Pcf11 and Nrd1, a protein similar to splicing factor SF1, a protein similar to FUSE splicing factor, and OsMADS8. The FY protein is associated with the 3' end processing machinery in Arabidopsis. Thus, we examined interactions between the OsFY and the rice homologs (OsCstF-50, -64, -77) of the AtCstF-50, -64, and -77 proteins. We found that OsFY could bind OsCstF50, whereas the OsCstF77 protein could bridge the interaction between OsCstF50 and OsCstF64. Taken together, our data suggest that OsFCA could interact with several proteins other than OsFY through its WW domain and may play several roles in rice.

Physiological and Molecular Approaches to Improve Drought Resistance in Soybean

Manavalan, L. P., Guttikonda, S. K., Tran, L.-S. P., Nguyen, H. T. — 2009-06-22

Drought stress is a major constraint to the production and yield stability of soybean (Glycine max (L.) Merr.). For developing high yielding varieties under drought conditions, the most widely employed criterion has traditionally been direct selection for yield stability over multiple locations. However, this approach is time consuming and labor intensive, because yield is a highly quantitative trait with low heritability, and influenced by differences arising from soil heterogeneity and environmental factors. The alternative strategy of indirect selection using secondary traits has succeeded only in a few crops, due to problems with repeatability and lack of phenotyping strategies, especially for root related traits. Considerable efforts have been directed towards identifying traits associated with drought resistance in soybean. With the availability of the whole genome sequence, physical maps, genetics and functional genomics tools, integrated approaches using molecular breeding and genetic engineering offer new opportunities for improving drought resistance in soybean. Genetic engineering for drought resistance with candidate genes has been reported in the major food crops, and efforts for developing drought resistant soybean lines are in progress. The objective of this article is to consolidate the current knowledge of physiology, molecular breeding and functional genomics which may be influential in integrating breeding and genetic engineering approaches for drought resistance in soybean.

Regulation of HSD1 in seeds of Arabidopsis thaliana

2009-06-20

The hydroxysteroid dehydrogenase HSD1, identified in the proteome of oil bodies from mature Arabidopsis seeds, is encoded by At5g50600 and At5g50700, two gene copies anchored on a duplicated region of chromosome 5. Using a real-time quantitative reverse transcriptase (RT)-PCR approach, the accumulation of HSD1 mRNA was shown to be specifically and highly induced in oil-accumulating tissues of maturing seeds. HSD1 mRNA disappeared during germination. The activity of HSD1 promoter and the localization of HSD1 transcripts by in situ hybridization were consistent with this pattern. A complementary set of molecular and genetic analyses showed that HSD1 is a target of LEAFY COTYLEDON2, a transcriptional regulator able to bind the promoter of HSD1. Immunoblot analyses and immunolocalization experiments using anti-AtHSD1 antibodies established that the pattern of HSD1 deposition was faithfully reflecting mRNA accumulation. At the subcellular level, the study of HSD1:GFP fusion proteins showed the targeting of HSD1 to the surface of oil bodies. Transgenic lines over expressing HSD1 were then obtained to test the importance of a proper transcriptional regulation of HSD1 in seeds. Whereas no impact on oil accumulation could be detected, transgenic seeds exhibited lower cold and light requirements to break dormancy, germinate, and mobilize storage lipids. Interestingly, over expressors of HSD1 over accumulated HSD1 protein in seeds but not in vegetative organs, suggesting that post-transcriptional regulations exist that prevent HSD1 accumulation in tissues deprived of oil bodies.

d14, a Strigolactone Insensitive Mutant of Rice, Shows an Accelerated Outgrowth of Tillers

2009-06-19

Recent studies using highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones, a group of terpenoid lactones, act as a new hormone class, or its biosynthetic precursors, in inhibiting shoot branching. Here, we provide evidence that DWARF14 (D14) inhibits rice tillering and may act as a new component of the strigolactone-dependent branching inhibition pathway. The d14 mutant exhibits increased shoot branching with reduced plant height as previously characterized strigolactone-deficient and -insensitive mutants, d10 and d3, respectively. The d10-1 d14-1 double mutant is phenotypically indistinguishable from the d10-1 and d14-1 single mutants, consistent with the idea that D10 and D14 function in the same pathway. However unlike with d10, the d14 branching phenotype could not be rescued by exogenous strigolactones. In addition, the d14 mutant contained a higher level of 2'-epi-5-deoxystrigol than the wild type. Positional cloning revealed that D14 encodes a protein of the /β-fold hydrolase superfamily, some members of which play a role in metabolism or signaling of plant hormones. We propose that D14 functions downstream of strigolactone synthesis, as a component of hormone signaling or as an enzyme that participates in the conversion of strigolactones to the bioactive form.

A Synechocystis Homolog of SipA Protein, Ssl3451, Enhances the Activity of the Histidine Kinase Hik33

Sakayori, T., Shiraiwa, Y., Suzuki, I. — 2009-06-19

In the cyanobacterium Synechocystis sp. PCC 6803, the histidine kinase Hik33 regulates the expression of several stress-inducible genes. Recently, a yeast two-hybrid screen revealed a specific interaction between Hik33 and a small protein, Ssl3451 [S. Sato et al., DNA Res. (2007) 14, 207-214]. To investigate the function of Ssl3451, we developed an assay to monitor the autophosphorylation of Hik33 in vitro. Addition of Ssl3451 to the reaction mixture dramatically enhanced the autophosphorylation activity of Hik33. Pulse-chase experiments revealed that Ssl3451 stimulated the autophosphorylation of Hik33 but did not affect its dephosphorylation. These findings indicated that Ssl3451 might be an activator of Hik33. When the amount of Hik33 was kept constant and the amount of Ssl3451 was increased in the reaction mixture, the extent of autophosphorylation of Hik33 reached a plateau when equimolar concentrations were present, suggesting that Ssl3451 enhances the activity of Hik33 by associating with it with a 1:1 stoichiometry. Disruption of the gene for Ssl3451 resulted in increased expression of the hliB gene, which is induced by Hik33 under standard growth conditions, but it did not affect the levels of the hliB mRNA at low temperature. Together, these results suggest that Ssl3451 might enhance the activity of Hik33 both in vitro and in vivo.

Four Orchid (Ondicium Gower Ramsey) AP1/AGL9-like MADS Box Genes Show Novel Expression Patterns and Cause Different Effects on Floral Transition and Formation in Arabidopsis thaliana

Chang, Y.-Y., Chiu, Y.-F., Wu, J.-W., Yang, C.-H. — 2009-06-18

Four AP1/AGL9 functional MADS box genes were characterized from the orchid (Ondicium Gower Ramsey). OMADS6 is a SEP3 orthologue, OMADS11 is a SEP1/2 orthologue, OMADS7 is an AGL6-like gene and OMADS10 is a putative paleoAP1 orthologue. The identity of these four genes was further supported by the presence of conserved motifs in the C-terminal regions of the proteins. OMADS6 showed an expression pattern different from SEP3 orthologues, with expression in the sepal, petal, lip and carpel, and was barely detected in the stamen. The expression pattern for OMADS11 was similar to OMADS6 and different from SEP1/2 orthologues since its expression was undetectable in the stamen. The expression pattern for OMADS7 was nearly identical to OMADS6. The similarities in the expression patterns of the SEP/AGL6-like genes OMADS6, 11 and 7 indicated that their transcriptional regulation is highly evolutionary conserved in the orchid. Unlike OMADS6/11/7, OMADS10 was only expressed in vegetative leaves and in the lip and carpel of mature flowers, which distinguishes it from most genes in the SQUA subfamily. Ectopic expression of OMADS6, 11 or 7 caused extremely early flowering, whereas 35S::OMADS10 only caused moderately early flowering in transgenic Arabidopsis plants. In addition, flower organ conversions, such as carpelloid sepals and staminoid petals, were observed in 35S::OMADS6 and carpelloid sepals were produced in 35S::OMADS7, while flower organ conversions were not observed in 35S::OMADS11 or 35S::OMADS10 transgenic flowers. This result reveals possible functional diversification of the orchid AP1/AGL9 genes OMADS6, 11, 7 and 10 in regulating flower transition and formation.

Three Arabidopsis SnRK2 Protein Kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3 Involved in ABA-Signaling are Essential for the Control of Seed Development and Dormancy

2009-06-18

Abscisic acid (ABA) is an important phytohormone regulating various plant processes, including stress tolerance, seed development and germination. SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3 are redundant ABA-activated SNF1-related protein kinases 2 (SnRK2s) in Arabidopsis thaliana. We examined the role of these protein kinases in seed development and germination. These SnRK2 proteins were mainly expressed in the nucleus during seed development and germination. The triple mutant (srk2d srk2e srk2i) was sensitive to desiccation and showed severe growth defects during seed development. The triple mutant exhibited a loss of dormancy and elevated seed ABA content relative to wild type plants. The severity of these phenotypes was far stronger than that of any single or double SRK2D, SRK2E and SRK2I mutants, including the srk2d srk2i mutant. The triple mutant had greatly reduced phosphorylation activity of in-gel kinase experiments using bZIP transcription factors including ABI5. Microarray experiments revealed that 48% and 30% of the down-regulated genes in abi5 and abi3 seeds were suppressed in the triple mutant seeds, respectively. Moreover, disruption of the three protein kinases induced global changes in the up-regulation of ABA-repressive gene expression, as well as the down-regulation of ABA-inducible gene expression. These alterations of gene expression result in a loss of dormancy and severe growth defects during seed development. Collectively, these results indicate that SRK2D, SRK2E and SRK2I protein kinases involved in ABA-signaling are essential for the control of seed development and dormancy through the extensive control of gene expression.

PosMed-plus: an Intelligent Search Engine that Inferentially Integrates Cross-species Information Resources for Molecular Breeding of Plants

2009-06-16

Molecular breeding of crops is an efficient way to upgrade plant functions useful to mankind. A key step is forward genetics or positional cloning to identify the genes that confer useful functions. In order to accelerate the whole research process, we have developed an integrated database system powered by an intelligent data-retrieval engine termed PosMed-plus (Positional Medline for plant upgrading science), allowing us to prioritize highly promising candidate genes in a given chromosomal interval(s) of Arabidopsis thaliana and rice, Oryza sativa. By inferentially integrating cross-species information resources including genomes, transcriptomes, proteomes, localizomes, phenomes and literature, the system compares a user's query, such as phenotypic or functional keywords, with the literature associated with the relevant genes located within the interval. By utilizing orthologous and paralogous correspondences, PosMed-plus efficiently integrates cross-species information to facilitate the ranking of rice candidate genes based on evidence from other model species such as Arabidopsis. PosMed-plus is a plant science version of the PosMed system widely used by mammalian researchers, and provides both a powerful integrative search function and a rich integrative display of the integrated databases. PosMed-plus is the first cross-species integrated database that inferentially prioritizes candidate genes for forward genetics approaches in plant science, and will be expanded for wider use in plant-upgrading in many species.

Mitochondrial damage in soybean seed axis during imbibition at chilling temperatures

Yin, G., Sun, H., Xin, X., Qin, G., Liang, Z., Jing, X. — 2009-06-11

The development of mitochondria during seed germination is essential for plant growth. However, the developmental process is still poorly understood. The temperature plays a key role for soybean germination and in this study we characterized the mitochondrial ultrastructure and proteome after imbibition at 22°C, 10°C and 4°C for 24 h. The mitochondria from the soybean seed axis can be divided into light and heavy mitochondria by Percoll density gradient centrifugation. The 4°C-imbibed axes mainly contained light mitochondria, which had lower levels of specific mitochondrial enzymes and oxidative phosphorylation activity. In contrast, the 22°C-imbibed axes mainly contained heavy mitochondria, which exhibited higher metabolism. Electron microscopy revealed that mitochondria in the 4°C-imbibed axes had a poorly developed internal membrane system with few cristae, while the mitochondria in the 22°C-imbibed axis developed more normally. Furthermore, we compared the axis mitochondrial proteomes during imbibition at different temperature. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Proteins involved in mitochondrial metabolites including malate dehydrogenase (tricarboxylic acid cycle enzyme), putative ATP synthase subunit (oxidative phosphorylation complex subunits), mitochondrial chaperonin-60 (heat shock protein), arginase (urea cycle enzyme), and mitochondrial elongation factor Tu (mitochondrial genome transcript enzyme) were identified. The reduced expression of these proteins might not support the normal mitochondrial metabolism. We conclude that chilling during imbibition causes mitochondrial damage at ultrastructural and metabolic levels.

Transcript profiling during fibre development identifies pathways in secondary metabolism and cell wall structure that may contribute to cotton fibre quality.

Al-Ghazi, Y., Bourot, S., Arioli, T., Dennis, E. S., Llewellyn, D. J. — 2009-06-10

A global gene expression profiling study at different stages of fibre development was undertaken on two cotton species cultivated for fibre, Gossypium hirsutum (L.) and G. barbadense (L.). A large proportion of the genome was expressed during both fibre elongation and subsequent secondary cell wall thickening.

There was a major shift in abundance of transcripts for gene regulation, cell organisation and metabolism between fibre elongation and fibre thickening that was fundamentally similar in both species. Each stage had its own distinctive features represented by specific metabolic and regulatory genes, a number of which have been noted previously. Many of the genes expressed in the fibres were of a similar type and developmental expression to those seen in other fibre producing plants indicating a conservation of mechanisms of cell elongation and wall thickening across diverse plant genera.

Secondary metabolism and pectin synthesis and modification genes were amongst the most statistically significant differentially expressed categories between the two species during fibre elongation. The gene profiles of the fibre thickening stage, however, were almost identical between the two species suggesting that their different final fibre quality properties may be established at earlier stages of fibre development. Expression levels of representative phenylpropanoid and pectin modification genes showed high correlations with specific fibre properties in an inter-specific cotton RIL population, supporting a role in determining fibre quality.

Activation of sucrose transport in defoliated Lolium perenne L.: an example of apoplastic phloem loading plasticity

2009-06-10

The pathway of carbon phloem loading was examined in leaf tissues of the forage grass Lolium perenne. The effect of defoliation (leaf blade removal) on sucrose transport capacity was assessed in leaf sheaths as the major carbon source for regrowth. The pathway of carbon transport was assessed via a combination of electron microscopy, plasmolysis experiments, and plasma membrane vesicles (PMV) purified by aqueous two-phase partitioning from the microsomal fraction. Results support an apoplastic phloem loading mechanism. Imposition of an artificial proton-motive-force to PMV from leaf sheaths energized an active, transient and saturable uptake of Sucrose (Suc). The affinity of Suc carriers for Suc was 580 µM in leaf sheaths of undefoliated plants. Defoliation induced a decrease of K_M followed by an increase of V_max. A transporter was isolated from stubble (including leaf sheaths) cDNA libraries and functionally expressed in yeast. The level of Lolium perenne SUcrose Transporter 1 (LpSUT1) expression increased in leaf sheaths in response to defoliation. All together, results indicate that Suc transport capacity increased in leaf sheaths of L. perenne in response to leaf blade removal. This increase might imply de novo synthesis of Suc transporters, including LpSUT1, and may represent one of the mechanisms contributing to rapid refoliation.

Omics-based approaches to methionine side-chain elongation in Arabidopsis: characterization of the genes encoding methylthioalkylmalate isomerase and methylthioalkylmalate dehydrogenase

2009-06-05

Glucosinolates (GSLs) are secondary metabolites in Brassicaceae plants synthesized from amino acids. Met-derived GSLs (Met-GSLs) with diverse side chains of various lengths are the major GSLs in Arabidopsis. Met chain elongation enzymes are responsible for variations in chain length in Met-GSL biosynthesis. The genes encoding Met chain elongation enzymes are considered to have been recruited from the Leu biosynthetic pathway in the course of evolution. Among them, the genes encoding methylthioalkylmalate synthases and aminotransferases have been identified; however, the remaining genes that encode methylthioalkylmalate isomerase (MAM-I) and methylthioalkylmalate dehydrogenase (MAM-D) remain to be identified. In our previous study based on transcriptome coexpression analysis, we identified candidate genes for a large subunit of MAM-I and MAM-D. In this study, we confirmed their predicted functions by targeted GSL analysis of the knockout mutants, and named the respective genes MAM-IL1/AtleuC1 and MAM-D1/AtIMD1. Metabolic profiling of the knockout mutants of Met chain elongation enzymes, conducted by means of widely targeted metabolomics, implied the roles of these enzymes in controlling metabolism from Met to primary and Met-related secondary metabolites. As shown here, an omics-based approach is an efficient strategy for the functional elucidation of genes involved in metabolism.

Phenome analysis in plant species using loss-of-function and gain-of-function mutants

Kuromori, T., Takahashi, S., Kondou, Y., Shinozaki, K., Matsui, M. — 2009-06-05

Analysis of genetic mutations is one of the most effective ways to investigate gene function. We now have methods that allow for mass production of mutant lines and cells in a variety of model species. Recently, large numbers of mutant lines have been generated both by ‘loss-of-function’ and by ‘gain-of-function’ techniques. In parallel, phenotypic information covering various mutant resources has been acquired and released in web-based databases. As a result, significant progress in comprehensive phenotype analysis is being made through the use of these tools. Arabidopsis and rice are two major model plant species in which genome sequencing projects have been completed. Arabidopsis is the most widely used experimental plant, with a large number of mutant resources and several examples of systematic phenotype analysis. Rice is a major crop species and is used as a model plant, with an increasing number of mutant resources. Other plant species are also being employed in functional genetics research. In this review, the present status of mutant resources for large-scale studies of gene function in plant research and the current perspective on using loss-of-function and gain-of-function mutants in phenome research will be discussed.

Proteome analysis of detergent resistant membranes (DRMs) associated with OsRac1 mediated innate immunity in rice

Fujiwara, M., Hamada, S., Hiratsuka, M., Fukao, Y., Kawasaki, T., Shimamoto, K. — 2009-06-05

OsRac1, a member of the Rac/Rop GTPase family, plays important roles as a molecular switch in rice innate immunity, and the active form of OsRac1 functions in the plasma membrane (PM). To study the precise localization of OsRac1 in the PM and its possible association with other signaling components, we performed proteomic analysis of DRMs (detergent-resistant membranes) isolated from rice suspension cells transformed with myc-tagged constitutively active (CA) OsRac1. DRMs are regions of the PM that are insoluble after Triton X-100 treatment under cold conditions and are thought to be involved in various signaling processes in animal, yeast, and plant cells. We identified 192 proteins in DRMs that included receptor-like kinases (RLK) such as Xa21, NB-LRR type disease resistance proteins, a glycosylphosphatidylinositol (GPI)-anchored protein, syntaxin, NADPH oxidase, a WD-40 repeat family protein, and various GTP-binding proteins. Many of these proteins have been previously identified in the DRMs isolated from other plant species, and animal and yeast cells, validating the methods used in our study. To examine the possible association of DRMs and OsRac1-mediated innate immunity, we used rice suspension cells transformed with myc-tagged wild type (WT)-OsRac1 and found that OsRac1 and RACK1A, an effector of OsRac1, shifted to the DRMs after chitin elicitor treatment. These results suggest that OsRac1-mediated innate immunity is associated with DRMs in the PM.

Common Sets of Promoter Elements Determine the Expression Characteristics of Three Arabidopsis Genes Encoding Isoforms of Mitochondrial Cytochrome c Oxidase Subunit 6b

Mufarrege, E. F., Curi, G. C., Gonzalez, D. H. — 2009-06-03

The promoters of the three Arabidopsis nuclear genes encoding mitochondrial cytochrome c oxidase subunit 6b (AtCOX6b) originate similar expression patterns, with preferential expression in anthers and meristems, and are induced by sucrose and etiolation. Additionally, induction of AtCOX6b-1 by GA3 and AtCOX6b-3 by 6-benzylaminopurine was observed. Site II elements (TGGGCC/T) present in the three promoters bind common nuclear proteins and are important for basal and induced expression. Induction by sucrose requires, in addition, the integrity of elements with the sequence TACTAA. The results imply the participation of common regulatory factors in the expression of the three Arabidopsis COX6b genes.

Vacuolar SNAREs Function in the Formation of the Leaf Vascular Network by Regulating Auxin Distribution

Shirakawa, M., Ueda, H., Shimada, T., Nishiyama, C., Hara-Nishimura, I. — 2009-06-03

In normal leaf development, a two-dimensional pattern of leaf veins is known to form by differentiation of vascular cells from ground meristem cells in a manner that is regulated by the polar flow of auxin. However, the mechanisms regulating the distribution of auxin in the leaf primordium are largely unknown. Here we show that vacuolar SNAREs, VAM3 and VTI11, are required for the formation of the leaf vascular network in a dosage-dependent manner. This is the first report to show that the prevacuolar compartment (PVC)-vacuole traffic pathway is required for the formation of the leaf vascular network. vam3-4, a VAM3-defective mutant, was found to have an immature vascular network. An analysis of the DR5 reporter in indicated that VAM3 is involved in the proper pattern formation of auxin maxima in the leaf primordium. This suggests that the immature vascular network in vam3-4 was mainly determined at the stage of procambium formation in the leaf primordium. The abnormal distribution of auxin maxima was caused by the non-polarized localization of the auxin efflux carrier PIN1 in leaf primordium cells. VAM3 is the first key protein which is required for the proper localization of PIN1 in leaf cells. Finally, we found that PIN1 proteins were constitutively transported to vacuoles in leaf and roots cells. Our findings demonstrate that the PVC-vacuole pathway is required for the formation of auxin maxima, which regulates the polar localization of PIN1, which, in turn, is required for the formation of the leaf vascular network.

Functional analysis of transcription factors in Arabidopsis

Mitsuda, N., Ohme-Takagi, M. — 2009-05-28

Transcription factors (TFs) regulate the expression of genes at the transcriptional level. Modification of TF activity dynamically alters the transcriptome, which leads to metabolic and phenotypic changes. Thus, functional analysis of TFs using ‘omics-based’ methodologies is one of the most important areas of the post-genome era. In this mini-review, we present an overview of Arabidopsis TFs and introduce strategies for the functional analysis of plant TFs, which include both traditional and recently developed technologies. These strategies can be assigned to five categories: bioinformatic analysis; analysis of molecular function; expression analysis; phenotype analysis and network analysis for the description of entire transcriptional regulatory networks.

Membrane-associated, Boron Interacting Proteins Isolated By Boronate Affinity Chromatography

Wimmer, M. A., Lochnit, G., Bassil, E., Muhling, K. H., Goldbach, H. E. — 2009-05-28

Boron deficiency symptoms point to a role of boron in plant membranes, but the molecular partners interacting with boron have not yet been identified. The objective of the present study was to isolate and identify membrane-associated proteins with an ability to interact with boron.

Boron-interacting proteins were isolated from root microsomal preparations of arabidopsis (Arabidopsis thaliana) and maize (Zea mays) using phenylboronate affinity chromatography, subsequently separated by two-dimensional gel electrophoresis and identified using MALDI-TOF peptide mass fingerprinting.

Sixteen boron-binding membrane-associated proteins were identified in A. thaliana, and nine in Z. mays roots. Additional un-identified proteins were also present. Common to both species were the beta subunit of mitochondrial ATP synthase, several beta-glucosidases, a luminal binding protein and fructose bisphosphate aldolase. In A. thaliana, binding of these proteins to boron was significantly reduced after 4 days of boron deprivation.

The relatively high number of diverse proteins identified as boron interacting, many of which are usually enriched in membrane microdomains, supports the hypothesis that boron plays a function in plant membranes by crosslinking glycoproteins, and may be involved in their recruiting to membrane microdomains.

Differential effects of a transgene to confer low phytic acid in caryopses located at different positions in rice panicles

Kuwano, M., Takaiwa, F., Yoshida, K. T. — 2009-05-22

In previous studies, we attempted to reduce phytic acid in rice seeds by silencing the 1D-myo-inositol 3-phosphate synthase gene, RINO1, using an antisense sequence under control of the rice glutelin GluB-1 promoter. The stable transgenic line showed a weak low-phytic-acid phenotype. In this study, we show that position of the caryopsis in the panicle might affect the level of gene silencing through a difference in temporal and spatial expression patterns between RINO1 and GluB-1 promoters, resulting in a large variation in Pi levels and a small increase in Pi in the transgenic seeds.

Characterization of Arabidopsis 6-Phosphogluconolactonase T-DNA Insertion Mutants Reveals an Essential Role of the Oxidative Section of the Plastidic Pentose Phosphate Pathway in Plant Growth and Development

Xiong, Y., DeFraia, C., Williams, D., Zhang, X., Mou, Z. — 2009-05-20

The Arabidopsis PGL1, PGL2, PGL4 and PGL5 are predicted to encode cytosolic isoforms of 6-phosphogluconolactonase (6PGL), whereas PGL3 is predicted to encode a 6PGL that has been shown to localize in both plastids and peroxisomes. Therefore, 6PGL may exist in the cytosol, plastids, and peroxisomes. However, the function of 6PGL in the three subcellular locations has not been well defined. Here we show that PGL3 is essential, whereas PGL1, PGL2, and PGL5 are individually dispensable for plant growth and development. Knockdown of PGL3 in the pgl3 mutant leads to a dramatic decrease in plant size, a significant increase in total glucose-6-phosphate dehydrogenase activity, and a marked decrease in cellular redox potential. Interestingly, the pgl3 plants exhibit constitutive pathogenesis-related gene expression and enhanced resistance to Pseudomonas syringae pv. maculicola ES4326 and Hyaloperonospora arabidopsidis Noco2. We found that, though pgl3 does not spontaneously accumulate elevated levels of free salicylic acid (SA), the constitutive defense responses in pgl3 plants are almost completely suppressed by the npr1 and sid2/eds16/ics1 mutations, suggesting that the pgl3 mutation activates NPR1- and SID2/EDS16/ICS1-dependent defense responses. We demonstrate that plastidic (not peroxisomal) localization and 6PGL activity of the PGL3 protein are essential for complementing all pgl3 phenotypes, indicating that the oxidative section of the plastidic pentose phosphate pathway (PPP) is required for plant normal growth and development. Thus, pgl3 provides a useful tool not only for defining the role of the PPP in different subcellular compartments, but also for dissecting the SA/NPR1-mediated signaling pathway.

Arabidopsis Mutants carrying Chimeric Sigma Factor Genes reveal Regulatory Determinants for Plastid Gene Expression

Schweer, J., Geimer, S., Meurer, J., Link, G. — 2009-05-13

Like bacteria, plastids contain sigma factors for promoter binding and transcription initiation. Accumulating evidence suggests that members of the plant sigma factor family can have specialized non-redundant roles in terms of promoter preference in various developmental and environmental situations. To specify regulatory determinants, we have chosen pairwise exchange of portions of Arabidopsis sigma coding regions, followed by transformation of the chimeric constructs into a sigma 6 knockout line. The resulting phenotypes and plastid RNA patterns point to an important though not exclusive role of the highly variable amino-terminal portion of plant sigma proteins.

Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: an application for hormone profiling in Oryza sativa

2009-04-20

We have developed a highly sensitive and high-throughput method for the simultaneous analysis of 43 molecular species of cytokinins, auxins, ABA, and gibberellins (GAs). This method consists of an automatic liquid handling system for solid phase extraction and ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI, UPLC-ESI-qMS/MS). In order to improve the detection limit of negatively charged compounds, such as GAs, we chemically derivatized fractions containing auxin, ABA, and GAs with bromocholine that has a quaternary ammonium functional group. This modification, that we call "MS-probe", makes these hormone-derivatives have a positive ion charge and permits all compounds to be measured in the positive ion mode with UPLC-ESI-qMS/MS in a single run. Consequently, quantification limits of GAs increased up to 50-fold. Our current method needs less than 100 mg (fresh weight) of plant tissues to determine phytohormone profiles and enables us to analyze simultaneously more than 180 plant samples. Application of this method to plant hormone profiling enabled us to draw organ-distribution maps of hormone species in rice and also to identify interactions among the 4 major hormones in the rice GA-signaling mutants, gid1-3, gid2-1, and slr1. Combining the results of hormone profiling data with transcriptome data in the GA signaling mutants allows us to analyze relationships between changes in gene expression and hormone metabolism.