The Plant Journal

Miao, Ying; Zentgraf, Ulrike

doi: 10.1111/j.1365-313X.2010.04233.xpmid: 20409006

WRKY transcription factors play a central role in controlling leaf senescence in Arabidopsis. One important member, WRKY53, is tightly regulated by various mechanisms, and is a convergence node between senescence and pathogen responses. Using WRKY53 in a yeast two‐hybrid screen, we isolated the HECT domain E3 ubiquitin ligase UPL5. In contrast to mammals, Arabidopsis contains only seven HECT E3 ubiquitin ligases, whose targets and functions are largely unknown. In yeast cells, UPL5 interacts with WRKY53 via its leucine zipper domain, and this interaction was confirmed in the cytoplasm of plant cells by a bimolecular fluorescence complementation assay. UPL5 was able to use the WRKY53 protein as a substrate for polyubiquitination in an in vitro system, and induction of UPL5 expression by an ethanol‐inducible system in upl5 plants led to degradation of the WRKY53 protein. Expression of both genes is regulated antagonistically in response to hydrogen peroxide, jasmonic acid and plant development. Two T‐DNA insertion lines (upl5‐1 and upl5‐2) showed the same senescence phenotype as WRKY53 over‐expressers. Over‐expression of WRKY53 in the upl5 background enhanced the accelerated senescence phenotype of WRKY53 over‐expressers. Therefore, we conclude that UPL5 regulates leaf senescence in Arabidopsis through degradation of WRKY53 and ensures that senescence is executed in the correct time frame.

Xia, Chuan; Wang, Yu‐Jiao; Li, Wen‐Qing; Chen, Yi‐Ran; Deng, Yi; Zhang, Xue‐Qin; Chen, Li‐Qun; Ye, De

doi: 10.1111/j.1365-313X.2010.04237.xpmid: 20444226

Previous studies have shown that subunits E (eIF3e), F (eIF3f) and H (elF3h) of eukaryotic translation initiation factor 3 play important roles in cell development in humans and yeast. eIF3e and eIF3h have also been reported to be important for normal cell growth in Arabidopsis. However, the functions of subunit eIF3f remain largely unknown in plant species. Here we report characterization of mutants for the Arabidopsis eIF3f (AteIF3f) gene. AteIF3f encodes a protein that is highly expressed in pollen grains, developing embryos and root tips, and interacts with Arabidopsis eIF3e and eIF3h proteins. A Ds insertional mutation in AteIF3f disrupted pollen germination and embryo development. Expression of some of the genes that are essential for pollen tube growth and embryogenesis is down‐regulated in ateif3f‐1 homozygous seedlings obtained by pollen rescue. These results suggested that AteIF3f might play important roles in Arabidopsis cell growth and differentiation in combination with eIF3e and eIF3h.

Peng, Lianwei; Cai, Wenhe; Shikanai, Toshiharu

doi: 10.1111/j.1365-313X.2010.04240.xpmid: 20444231

In higher plants, the chloroplast NAD(P)H dehydrogenase (NDH) complex mediates chlororespiration and photosystem I (PSI) cyclic electron transport in thylakoid membranes. Because of its low abundance and fragility, our knowledge on the assembly of chloroplast NDH is very limited, and some nuclear‐encoded factors may be involved in this process. We show here that two Arabidopsis proteins, CHLORORESPIRATORY REDUCTION 6 (CRR6) and CRR7, which were previously identified in mutants specifically defective in NDH accumulation, are present in the stroma, and their stability is independent of the NDH complex, suggesting that they are unlikely to be NDH subunits. Blue native PAGE analysis showed that the accumulation of NDH subcomplex A, which is a core part of NDH that is conserved in divergent species, was specifically impaired in the crr6 and crr7‐1 mutants. However, the expression of plastid‐encoded genes encoding the subcomplex A subunits was not affected, suggesting that CRR6 and CRR7 are involved in post‐translational steps during the biogenesis of subcomplex A. We also discovered that a substantial quantity of NdhH is present in several protein complexes in the chloroplast stroma, possibly as early assembly intermediates of subcomplex A. Although the accumulation of these stromal complexes was not affected in crr6 or crr7‐1, CRR6 was co‐purified with NdhH, implying that CRR6 functions in the later step of subcomplex‐A biogenesis. Accumulation of CRR7 was independent of that of CRR6; we propose that CRR7 functions in a different step in subcomplex‐A biogenesis from CRR6.

Rodriguez, Maria C. Suarez; Edsgärd, Daniel; Hussain, Syed S.; Alquezar, David; Rasmussen, Morten; Gilbert, Thomas; Nielsen, Bjørn H.; Bartels, Dorothea; Mundy, John

doi: 10.1111/j.1365-313X.2010.04243.xpmid: 20444235

Studies of the resurrection plant Craterostigma plantagineum have revealed some of the mechanisms which these desiccation‐tolerant plants use to survive environments with extreme dehydration and restricted seasonal water. Most resurrection plants are polyploid with large genomes, which has hindered efforts to obtain whole genome sequences and perform mutational analysis. However, the application of deep sequencing technologies to transcriptomics now permits large‐scale analyses of gene expression patterns despite the lack of a reference genome. Here we use pyro‐sequencing to characterize the transcriptomes of C. plantagineum leaves at four stages of dehydration and rehydration. This reveals that genes involved in several pathways, such as those required for vitamin K and thiamin biosynthesis, are tightly regulated at the level of gene expression. Our analysis also provides a comprehensive picture of the array of cellular responses controlled by gene expression that allow resurrection plants to survive desiccation.

Bhattarai, Kishor K.; Atamian, Hagop S.; Kaloshian, Isgouhi; Eulgem, Thomas

doi: 10.1111/j.1365-313X.2010.04232.xpmid: 20409007

WRKY transcription factors play a central role in transcriptional reprogramming associated with plant immune responses. However, due to functional redundancy, typically the contribution of individual members of this family to immunity is only subtle. Using microarray analysis, we found that the paralogous tomato WRKY genes SlWRKY72a and b are transcriptionally up‐regulated during disease resistance mediated by the R gene Mi‐1. Virus‐induced gene silencing of these two genes in tomato resulted in a clear reduction of Mi‐1‐mediated resistance as well as basal defense against root‐knot nematodes (RKN) and potato aphids. Using Arabidopsis T‐DNA insertion mutants, we found that their Arabidopsis ortholog, AtWRKY72, is also required for full basal defense against RKN as well as to the oomycete Hyaloperonospora arabidopsidis. Despite their similar roles in basal defense against RKN in both tested plant species, WRKY72‐type transcription factors in tomato, but not in Arabidopsis, clearly contributed to basal defense against the bacterial pathogen Pseudomonas syringae. Of the five R genes that we tested in tomato and Arabidopsis, only Mi‐1 appeared to be dependent on WRKY72‐type transcription factors. Interestingly, AtWRKY72 target genes, identified by microarray analysis of H. arabidopsidis‐triggered transcriptional changes, appear to be largely non‐responsive to analogs of the defense hormone salicylic acid (SA). Thus, similarly to Mi‐1, which in part acts independently of SA, AtWRKY72 appears to utilize SA‐independent defense mechanisms. We propose that WRKY72‐type transcription factors play a partially conserved role in basal defense in tomato and Arabidopsis, a function that has been recruited to serve Mi‐1‐dependent immunity.

Yoo, Seong Jeon; Chung, Kyung Sook; Jung, Seung Hye; Yoo, So Yeon; Lee, Jong Seob; Ahn, Ji Hoon

doi: 10.1111/j.1365-313X.2010.04234.xpmid: 20409005

The FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family is a small gene family that encodes important regulators that control flower development in Arabidopsis. Here, we investigated the biological role of the product of BROTHER OF FT AND TFL1 (BFT), a member of this family, whose function remains unknown. Comparison of the critical residues that play a role in distinguishing FT‐ or TFL1‐like activity revealed that BFT is more similar to FT. Similar to FT expression, BFT expression showed a diurnal oscillation pattern, peaking in the evening. In situ hybridization revealed BFT expression in the shoot apical meristem, young leaf and axillary inflorescence meristem. Transgenic plants over‐expressing BFT exhibited delayed flowering and severe floral defects (floral indeterminacy and compact inflorescences surrounded by serrate leaves), similar to 35S::TFL1 plants. LEAFY (LFY) and APETALA1 (AP1) expression was significantly reduced in 35S::BFT plants. BFT over‐expression failed to rescue the terminal flower phenotype of tfl1 mutants; however, it delayed both terminal flower formation in the primary inflorescence and axillary inflorescence development in the tfl1 mutant background. Consistent with this, the loss‐of‐function BFT alleles, bft‐2 and an BFT RNAi line, accelerated termination of the primary inflorescence and formation of axillary inflorescences in the tfl1 mutant background. Taken together, our results suggest that, despite similarities in the critical residues of BFT and FT, BFT possesses a TFL1‐like activity and functions redundantly with TFL1 in inflorescence meristem development, and possibly contributes to the regulation of plant architecture.

Griebel, Thomas; Zeier, Jürgen

doi: 10.1111/j.1365-313X.2010.04235.xpmid: 20444228

Upon inoculation with pathogenic microbes, plants induce an array of metabolic changes that potentially contribute to induced resistance or even enhance susceptibility. When analysing leaf lipid composition during the Arabidopsis thaliana–Pseudomonas syringae interaction, we found that accumulation of the phytosterol stigmasterol is a significant plant metabolic process that occurs upon bacterial leaf infection. Stigmasterol is synthesized from β‐sitosterol by the cytochrome P450 CYP710A1 via C22 desaturation. Arabidopsis cyp710A1 mutant lines impaired in pathogen‐inducible expression of the C22 desaturase and concomitant stigmasterol accumulation are more resistant to both avirulent and virulent P. syringae strains than wild‐type plants, and exogenous application of stigmasterol attenuates this resistance phenotype. These data indicate that induced sterol desaturation in wild‐type plants favours pathogen multiplication and plant susceptibility. Stigmasterol formation is triggered through perception of pathogen‐associated molecular patterns such as flagellin and lipopolysaccharides, and through production of reactive oxygen species, but does not depend on the salicylic acid, jasmonic acid or ethylene defence pathways. Isolated microsomal and plasma membrane preparations exhibited a similar increase in the stigmasterol/β‐sitosterol ratio as whole‐leaf extracts after leaf inoculation with P. syringae, indicating that the stigmasterol produced is incorporated into plant membranes. The increased contents of stigmasterol in leaves after pathogen attack do not influence salicylic acid‐mediated defence signalling but attenuate pathogen‐induced expression of the defence regulator flavin‐dependent monooxygenase 1. P. syringae thus promotes plant disease susceptibility through stimulation of sterol C22 desaturation in leaves, which increases the stigmasterol to β‐sitosterol ratio in plant membranes.

Konishi, Mineko; Yanagisawa, Shuichi

doi: 10.1111/j.1365-313X.2010.04239.xpmid: 20444232

Nitrate is a major nitrogen source for land plants and also acts as a signaling molecule that induces changes in growth and gene expression. To identify the cis‐acting DNA element involved in nitrate‐responsive gene expression, we analyzed the promoter of the Arabidopsis gene encoding nitrite reductase (NIR1). A region from positions −188 to −1, relative to the translation start site, was found to contain at least one cis‐element necessary for the nitrate‐dependent activation of the promoter, in which the activity of nitrate transporter NRT2.1 and/or NRT2.2 plays a critical role. To define this nitrate‐responsive cis‐element (NRE), we compared the sequences of several nitrite reductase gene promoters from various higher plants and identified a conserved sequence motif as the putative NRE. A synthetic promoter in which the four copies of a 43‐bp sequence containing the motif were fused to the 35S minimal promoter was found to direct nitrate‐responsive transcription. Furthermore, mutations within this conserved motif in the native NIR1 promoter markedly reduced the nitrate‐responsive activity of the promoter, indicating that the 43‐bp sequence is an NRE that is both necessary and sufficient for nitrate‐responsive transcription. We also show that both the native NIR1 promoter and the synthetic promoter display a similar level of sensitivity to nitrate, but respond differentially to exogenously supplied glutamine, indicating independent modulation of NIR1 expression by NRE‐mediated nitrate induction and feedback repression mediated by other cis‐element(s). These findings thus define the presence of multiple cis‐elements involved in the nitrogen response in Arabidopsis.

Yang, Haibian; Shi, Yiting; Liu, Jingyan; Guo, Lin; Zhang, Xiaoyan; Yang, Shuhua

doi: 10.1111/j.1365-313X.2010.04241.xpmid: 20444230

Low temperature is one of environmental factors that restrict plant growth homeostasis and plant–pathogen interactions. Recent studies suggest a link between temperature responses and defense responses; however, the underlying molecular mechanisms remain unclear. In this study, the chilling sensitive 3 (chs3‐1) mutant in Arabidopsis was characterized. chs3‐1 plants showed arrested growth and chlorosis when grown at 16°C or when shifted from 22 to 4°C. chs3‐1 plants also exhibited constitutively activated defense responses at 16°C, which were alleviated at a higher temperature (22°C). Map‐based cloning of CHS3 revealed that it encodes an unconventional disease resistance (R) protein belonging to the TIR‐NB‐LRR class with a zinc‐binding LIM domain (Lin‐11, Isl‐1 and Mec‐3 domains) at the carboxyl terminus. The chs3‐1 mutation in the conserved LIM‐containing domain led to the constitutive activation of the TIR‐NB‐LRR domain. Consistently, the growth and defense phenotypes of chs3‐1 plants were completely suppressed by eds1, sgt1b and rar1, partially by pad4 and nahG, but not by npr1 and ndr1. Intriguingly, chs3‐1 plants grown at 16°C showed enhanced tolerance to freezing temperatures. This tolerance was correlated with growth defect and cell death phenotypes caused by activated defense responses. Other mutants with activated defense responses, including cpr1, cpr5 and slh1 also displayed enhanced freezing tolerance. These findings revealed a role of an unconventional mutant R gene in plant growth, defense response and cold stress, suggesting a mutual interaction between cold signaling and defense responses.

Aronsson, Henrik; Combe, Jonathan; Patel, Ramesh; Agne, Birgit; Martin, Meryll; Kessler, Felix; Jarvis, Paul

doi: 10.1111/j.1365-313X.2010.04242.xpmid: 20444229

The atToc33 protein is one of several pre‐protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis‐related pre‐proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild‐type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159‐deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.