Abscisic Acid Regulation Research Articles

Developmental and stress regulation of gene expression for a 9-cis-epoxycarotenoid dioxygenase, CstNCED, isolated from Crocus sativus stigmas

Oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. ABA has been associated with dormancy and flower senescence, while also regulating plant adaptive responses to various environmental stresses. An NCED gene, CstNCED, was cloned from Crocus sativus stigmas. The deduced amino acid sequence of the CstNCED protein shared high identity with other monocot NCEDs, and was closely related to the liliopsida enzymes. At the N-terminus of CstNCED a chloroplast transit peptide sequence is located. However, its expression in chloroplast-free tissues suggested localization in other plastid types. The relationship between expression of CstNCED and the endogenous ABA level was investigated in the stigma and corms, where it was developmentally regulated. The senescence of the unpollinated stigma is preceded by an increase in ABA levels and CstNCED expression. In corms, a correlation was observed between CstNCED expression and dormancy. Furthermore, CstNCED expression was correlated with the presence of zeaxanthin in the dormant corms. When detached C. sativus leaves and stigmas were water and salt stressed, increases in CstNCED mRNA were observed. The results provided evidence of the involvement of CstNCED in the regulation of ABA-associated processes such as flower senescence and corm dormancy in monocotyledonous saffron.

A Nucleotide Metabolite Controls Stress-Responsive Gene Expression and Plant Development

Abiotic stress, such as drought and high salinity, activates a network of signaling cascades that lead to the expression of many stress-responsive genes in plants. The Arabidopsis FIERY1 (FRY1) protein is a negative regulator of stress and abscisic acid (ABA) signaling and exhibits both an inositol polyphosphatase and a 3′,5′-bisphosphate nucleotidase activity in vitro. The FRY1 nucleotidase degrades the sulfation byproduct 3′-phosphoadenosine-5′-phosphate (PAP), yet its in vivo functions and particularly its roles in stress gene regulation remain unclear. Here we developed a LC-MS/MS method to quantitatively measure PAP levels in plants and investigated the roles of this nucleotidase activity in stress response and plant development. It was found that PAP level was tightly controlled in plants and did not accumulate to any significant level either under normal conditions or under NaCl, LiCl, cold, or ABA treatments. In contrast, high levels of PAP were detected in multiple mutant alleles of FRY1 but not in mutants of other FRY1 family members, indicating that FRY1 is the major enzyme that hydrolyzes PAP in vivo. By genetically reducing PAP levels in fry1 mutants either through overexpression of a yeast PAP nucleotidase or by generating a triple mutant of fry1 apk1 apk2 that is defective in the biosynthesis of the PAP precursor 3′-phosphoadenosine-5′-phosphosulfate (PAPS), we demonstrated that the developmental defects and superinduction of stress-responsive genes in fry1 mutants correlate with PAP accumulation in planta. We also found that the hypersensitive stress gene regulation in fry1 requires ABH1 but not ABI1, two other negative regulators in ABA signaling pathways. Unlike in yeast, however, FRY1 overexpression in Arabidopsis could not enhance salt tolerance. Taken together, our results demonstrate that PAP is critical for stress gene regulation and plant development, yet the FRY1 nucleotidase that catabolizes PAP may not be an in vivo salt toxicity target in Arabidopsis.

Regulation of carotenoid accumulation and the expression of carotenoid metabolic genes in citrus juice sacs in vitro

In the present study, to investigate the mechanisms regulating carotenoid accumulation in citrus, a culture system was set up in vitro with juice sacs of three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). The juice sacs of all the three varieties enlarged gradually with carotenoid accumulation. The changing patterns of carotenoid content and the expression of carotenoid metabolic genes in juice sacs in vitro were similar to those ripening on trees in the three varieties. Using this system, the changes in the carotenoid content and the expression of carotenoid metabolic genes in response to environmental stimuli were investigated. The results showed that carotenoid accumulation was induced by blue light treatment, but was not affected by red light treatment in the three varieties. Different regulation of CitPSY expression, which was up-regulated by blue light while unaffected by red light, led to different changes in carotenoid content in response to these two treatments in Satsuma mandarin and Valencia orange. In all three varieties, increases in carotenoid content were observed with sucrose and mannitol treatments. However, the accumulation of carotenoid in the two treatments was regulated by distinct mechanisms at the transcriptional level. With abscisic acid (ABA) treatment, the expression of the genes investigated in this study was up-regulated in Satsuma mandarin and Lisbon lemon, indicating that ABA induced its own biosynthesis at the transcriptional level. This feedback regulation of ABA led to decreases in carotenoid content. With gibberellin (GA) treatment, carotenoid content was significantly decreased in the three varieties. Changes in the expression of genes related to carotenoid metabolism varied among the three varieties in response to GA treatment. These results provided insights into improving carotenoid content and composition in citrus during fruit maturation.

Importance of ABA homeostasis under terminal drought stress in regulating grain filling events

Recent studies suggest that abscisic acid (ABA) at its basal level plays an important role during seed set and grain filling events. Under drought stress ABA levels were found to be significantly enhanced in the developing seed. Until now we lack an understanding of (A) ABA homeostasis in developing seeds under terminal drought and (B) the interactive role of ABA in regulating the starch biosynthesis pathway in developing grains under terminal drought. We have recently reported the possible regulation of ABA homeostasis in source (flag leaf) and sink (developing grains) tissues under post-anthesis drought stress in barley and concluded that significantly enhanced ABA levels in developing grains are due to strong activation of the ABA deconjugation pathway and fine regulation of the ABA biosynthesis-degradation pathway.1 Additionally, we provided evidence for the role of ABA in differential regulation of starch biosynthesis genes and a significant upregulation of starch degradation beta amylase genes under drought, i.e. ABA not only influences the rate of starch accumulation but also starch quality.

Effect of Exogenous Abscisic Acid on psbA Expression at Grain Filling Stage in Two Wheat Cultivars Under Drought Stress

Abscisic acid (ABA) is an important plant hormone and may be involved in drought resistance in crops. The object of this study was to understand the effects of exogenous ABA on wheat (Triticum aestivum L.) plant under drought stress. After drought stress simulated with polyethylene glycol (PEG) for 72 h at grain-filling stage, flag leaves of Yumai 949 and Shaanmai 5 showed significant increase in malondialdehyde (MDA) content and decreases in relative water content (RWC), chlorophyll content, and yield. Meanwhile, the transcription of psbA gene in photosystem II system was also depressed according to the analysis of reverse transcriptional PCR. Application of exogenous ABA had a significant effect on alleviation of these reactions from PEG stress. Compared to Yumai 949, Shaanmai 5 received smaller injury in plasma membranes and less reduces in RWC, chlorophyll content, yield, and psbA transcriptional level after PEG stress. Moreover, exogenous ABA treatment resulted in restoration of all parameters tested to the levels before stress in Shaanmai 5. This result indicated that the expression of psbA gene was closely related to drought stress resistance subject to wheat cultivar. The primary evidence of ABA regulation on psbA expression may enrich the mechanism of drought resistance and suggest the potential of exogenous ABA to enhance resistance to drought stress in wheat during grain filling period.

Common and unique elements of the ABA-regulated transcriptome of Arabidopsis guard cells

BackgroundIn the presence of drought and other desiccating stresses, plants synthesize and redistribute the phytohormone abscisic acid (ABA). ABA promotes plant water conservation by acting on specialized cells in the leaf epidermis, guard cells, which border and regulate the apertures of stomatal pores through which transpirational water loss occurs. Following ABA exposure, solute uptake into guard cells is rapidly inhibited and solute loss is promoted, resulting in inhibition of stomatal opening and promotion of stomatal closure, with consequent plant water conservation. There is a wealth of information on the guard cell signaling mechanisms underlying these rapid ABA responses. To investigate ABA regulation of gene expression in guard cells in a systematic genome-wide manner, we analyzed data from global transcriptomes of guard cells generated with Affymetrix ATH1 microarrays, and compared these results to ABA regulation of gene expression in leaves and other tissues.ResultsThe 1173 ABA-regulated genes of guard cells identified by our study share significant overlap with ABA-regulated genes of other tissues, and are associated with well-defined ABA-related promoter motifs such as ABREs and DREs. However, we also computationally identified a unique cis-acting motif, GTCGG, associated with ABA-induction of gene expression specifically in guard cells. In addition, approximately 300 genes showing ABA-regulation unique to this cell type were newly uncovered by our study. Within the ABA-regulated gene set of guard cells, we found that many of the genes known to encode ion transporters associated with stomatal opening are down-regulated by ABA, providing one mechanism for long-term maintenance of stomatal closure during drought. We also found examples of both negative and positive feedback in the transcriptional regulation by ABA of known ABA-signaling genes, particularly with regard to the PYR/PYL/RCAR class of soluble ABA receptors and their downstream targets, the type 2C protein phosphatases. Our data also provide evidence for cross-talk at the transcriptional level between ABA and another hormonal inhibitor of stomatal opening, methyl jasmonate.ConclusionsOur results engender new insights into the basic cell biology of guard cells, reveal common and unique elements of ABA-regulation of gene expression in guard cells, and set the stage for targeted biotechnological manipulations to improve plant water use efficiency.

Quantitative transcriptomic analysis of abscisic acid‐induced and reactive oxygen species‐dependent expression changes and proteomic profiling in Arabidopsis suspension cells

SummaryEarly rapid changes in response to the phytohormone abscisic acid (ABA) have been observed at the transcript level, but little is known how these transcript changes translate to changes in protein abundance under the same conditions. Here we have performed a global quantitative analysis of transcript and protein changes in Arabidopsis suspension cells in response to ABA using microarrays and quantitative proteomics. In summary, 3494 transcripts and 50 proteins were significantly regulated by ABA over a treatment period of 20–24 h. Abscisic acid also caused a rapid and strong increase in production of extracellular reactive oxygen species (ROS) with an average half‐rise time of 33 sec. A subset of ABA‐regulated transcripts were differentially regulated in the presence of the ROS scavenger dimethylthiourea (DMTU) as compared with ABA alone, suggesting a role for ROS in the regulation of these ABA‐induced genes. Transcript changes showed an overall poor correlation to protein changes (r = 0.66). Only a subset of genes was regulated at the transcript and protein level, including known ABA marker genes. We furthermore identified ABA regulation of proteins that function in a branch of glucosinolate catabolism previously not associated with ABA signaling. The discovery of genes that were differentially regulated at the transcript and at the protein level emphasizes the strength of our combined approach. In summary, our dataset not only expands previous studies on gene and protein regulation in response to ABA, but rather uncovers unique aspects of the ABA regulon and gives rise to additional mechanisms regulated by ABA.

Proteomic analysis of heterosis during maize seed germination

Heterosis is observed for most phenotypic traits and developmental stages in many plants. In this study, the embryos, from germinating seeds after 24 h of soaking, for five elite maize hybrids and their parents were selected to unravel the genetic basis of heterosis using 2-D proteomic method. In total, 257 (80.06%), 363 (58.74%), 351 (79.95%), 242 (54.50%), and 244 (46.30%) nonadditively expressed proteins were identified in hybrids Zhengdan 958, Nongda 108, Yuyu 22, Xundan 20, and Xundan 18, respectively. The nonadditive proteins were divided into above high-parent (++; 811, 55.66%), high-parent (+; 121, 8.30%), partial dominance (+-; 249, 17.09%), low-parent (-; 30, 2.06%), below low-parent (- -; 62, 4.26%), and D (different; 184, 12.63%) expression patterns. The observed patterns indicate the important roles of dominance, partial dominance, and overdominance in regulating seed germination in maize. Additionally, 54 different proteins were identified by mass spectrometry and classified into nine functional groups: metabolism (9), cell detoxification (8), unknown functional proteins (8), chaperones (7), signal transduction (6), development process (5), other (5), transporter (3), and stress response (3). Of these, the most interesting are those involved with germination-related hormone signal transduction and the abscisic acid and gibberellin regulation networks.

Exogenous Applications of Abscisic Acid Increase Curing of Pierce's Disease-Affected Grapevines Growing in Pots.

Foliar and drench applications of the plant growth regulator abscisic acid (ABA) were applied to 1-year-old potted Vitis vinifera 'Pinot Noir' and 'Cabernet Sauvignon' vines infected with Xylella fastidiosa, the bacterial pathogen that causes Pierce's disease (PD). A naturally occurring ABA and a synthetic ABA were applied, and both materials showed some effectiveness at increasing curing rates of PD-affected grapevines. Pinot Noir grapevines treated with the drench ABA treatments had significantly greater disease curing effects than the unsprayed control plants. It has been shown that plant phenolics have antimicrobial properties, and we found a positive correlation between effective ABA treatments and the total phenolic compound content of xylem sap extracted from Pinot Noir vines.

Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells

Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are important signalling agents in both animals and plants. In plants, G proteins modulate numerous responses, including abscisic acid (ABA) and pathogen-associated molecular pattern (PAMP) regulation of guard cell ion channels and stomatal apertures. Previous analyses of mutants deficient in the sole canonical Arabidopsis Gα subunit, GPA1, have shown that Gα-deficient guard cells are impaired in ABA inhibition of K(+) influx channels, and in pH-independent activation of anion efflux channels. ABA-induced Ca(2+) uptake through ROS-activated Ca(2+)-permeable channels in the plasma membrane is another key component of ABA signal transduction in guard cells, but the question of whether these channels are also dependent on Gα for their ABA response has not been evaluated previously. We used two independent Arabidopsis T-DNA null mutant lines, gpa1-3 and gpa1-4, to investigate this issue. We observed that gpa1 mutants are disrupted both in ABA-induced Ca(2+)-channel activation, and in production of reactive oxygen species (ROS) in response to ABA. However, in response to exogenous H(2)O(2) application, I(Ca) channels are activated normally in gpa1 guard cells. In addition, H(2)O(2) inhibition of stomatal opening and promotion of stomatal closure are not disrupted in gpa1 mutant guard cells. These data indicate that absence of GPA1 interrupts ABA signalling between ABA reception and ROS production, with a consequent impairment in Ca(2+)-channel activation.

Transcriptional regulatory programs underlying barley germination and regulatory functions of Gibberellin and abscisic acid

BackgroundSeed germination is a complex multi-stage developmental process, and mainly accomplished through concerted activities of many gene products and biological pathways that are often subjected to strict developmental regulation. Gibberellins (GA) and abscisic acid (ABA) are two key phytohormones regulating seed germination and seedling growth. However, transcriptional regulatory networks underlying seed germination and its associated biological pathways are largely unknown.ResultsThe studies examined transcriptomes of barley representing six distinct and well characterized germination stages and revealed that the transcriptional regulatory program underlying barley germination was composed of early, late, and post-germination phases. Each phase was accompanied with transcriptional up-regulation of distinct biological pathways. Cell wall synthesis and regulatory components including transcription factors, signaling and post-translational modification components were specifically and transiently up-regulated in early germination phase while histone families and many metabolic pathways were up-regulated in late germination phase. Photosynthesis and seed reserve mobilization pathways were up-regulated in post-germination phase. However, stress related pathways and seed storage proteins were suppressed through the entire course of germination. A set of genes were transiently up-regulated within three hours of imbibition, and might play roles in initiating biological pathways involved in seed germination. However, highly abundant transcripts in dry barley and Arabidopsis seeds were significantly conserved. Comparison with transcriptomes of barley aleurone in response to GA and ABA identified three sets of germination responsive genes that were regulated coordinately by GA, antagonistically by ABA, and coordinately by GA but antagonistically by ABA. Major CHO metabolism, cell wall degradation and protein degradation pathways were up-regulated by both GA and seed germination. Those genes and metabolic pathways are likely to be important parts of transcriptional regulatory networks underlying GA and ABA regulation of seed germination and seedling growth.ConclusionsThe studies developed a model depicting transcriptional regulatory programs underlying barley germination and GA and ABA regulation of germination at gene, pathway and systems levels, and established a standard transcriptome reference for further integration with various -omics and biological data to illustrate biological networks underlying seed germination. The studies also generated a great amount of systems biological evidence for previously proposed hypotheses, and developed a number of new hypotheses on transcriptional regulation of seed germination for further experimental validation.

Involvement of Arabidopsis RACK1 in protein translation and its regulation by abscisic acid.

Earlier studies have shown that RACK1 functions as a negative regulator of abscisic acid (ABA) responses in Arabidopsis (Arabidopsis thaliana), but the molecular mechanism of the action of RACK1 in these processes remains elusive. Global gene expression profiling revealed that approximately 40% of the genes affected by ABA treatment were affected in a similar manner by the rack1 mutation, supporting the view that RACK1 is an important regulator of ABA responses. On the other hand, coexpression analysis revealed that more than 80% of the genes coexpressed with RACK1 encode ribosome proteins, implying a close relationship between RACK1's function and the ribosome complex. These results implied that the regulatory role for RACK1 in ABA responses may be partially due to its putative function in protein translation, which is one of the major cellular processes that mammalian and Saccharomyces cerevisiae RACK1 is involved in. Consistently, all three Arabidopsis RACK1 homologous genes, namely RACK1A, RACK1B, and RACK1C, complemented the growth defects of the S. cerevisiae cross pathway control2/rack1 mutant. In addition, RACK1 physically interacts with Arabidopsis Eukaryotic Initiation Factor6 (eIF6), whose mammalian homolog is a key regulator of 80S ribosome assembly. Moreover, rack1 mutants displayed hypersensitivity to anisomycin, an inhibitor of protein translation, and displayed characteristics of impaired 80S functional ribosome assembly and 60S ribosomal subunit biogenesis in a ribosome profiling assay. Gene expression analysis revealed that ABA inhibits the expression of both RACK1 and eIF6. Taken together, these results suggest that RACK1 may be required for normal production of 60S and 80S ribosomes and that its action in these processes may be regulated by ABA.

Molecular cloning and characterization of a group 3 LEA gene from Agropyron mongolicum Keng

Late embryogenesis-abundant (LEA) protein is one of the components involved in desiccation tolerance (DT) by maintaining cellular structures in the dry state. In this study, a member of the group 3 LEA, MwLEA1, was cloned from Mongolian wheatgrass ( Agropyron mongolium Keng) based on a homologous sequence from wheat ( Triticum aestivum L .). Its full-length cDNA sequence was 705 bp, encoding a protein of 187 amino acids. The amino acid sequence comparison revealed its high homology with LEA proteins from other plant species. The deduced MwLEA1 protein had five repeat 11- amino-acid motifs, with a molecular weight of 19.4 kDa and a theoretical isoelectric point of 8.8. Subcellular localization indicated that the MwLEA1 was localized in the nucleus of the onion epithelial cell. Under water stress conditions, MwLEA1 exhibited different expression levels, which was higher in root and shoot but lowest in leaf. The expression profiling under different stresses indicated that MwLEA1 played roles in responses to water, salt stresses as well as abscisic acid (ABA) regulation. The gene of MwLEA1 was transformed into tobaccos by Agrobacterium tumefaciens -mediated method. Eleven regenerated plants were analyzed by polymerase chain reaction (PCR) and southern blotting, and 6 of them were proved to be transgenic plants. Key words: Agropyron mongolium Keng, cloning, late embryogenesis abundant, subcellular localization, expression, transformation.

Improvement of Tolerance to Osmotic Stress in Arabidopsis Plants Ectopically Expressing Peanut AhNCED1 Gene

Gene AhNCED1 plays a vital role in regulation of abscisic acid (ABA) biosynthesis in peanut ( Arachis hypogaea L.) plants in response to drought stress. Two binary vectors harboring AhNCED1 gene, p35S::ORF and pAtNCED3p::ORF, were established, which were driven by the CaMV 35S promoter from pCAMBIA1301 and the AtNCED3 gene promoter from wild type Arabidopsis, respectively. Wild type and 129B08/ nced3 mutant Arabidopsis plants were separately transformed with Agrobacterium harboring p35S::ORF or pAtNCED3p::ORF vector, resulting in 35S::ORF-WT and A3p::ORF-B08 transgenic plants, accordingly. The stable expression of AhNCED1 gene in Arabidopsis plants was confirmed with duplex reverse transcription PCR (RT-PCR). The wild type, 129B08/ nced3 mutant, and the transgenic Arabidopsis plants were subsequently tested for sensitivity to exogenous ABA and tolerance to osmotic stress. The results showed that the ABA sensitivity was declined in the 129B08/ nced3 mutant and increased in the Arabidopsis plants ectopically expressing AhNCED1 gene. Under sorbitol stress, the relative germination rate of 129B08/ nced3 mutant seeds was far lower than that of the wild type seeds. However, the relative germination rate of A3p::ORF-B08 transgenic seeds was close to that of the wild type seeds, which was significantly higher than that of 129B08/ nced3 mutant seeds. The relative germination rate of 35S::ORF-WT transgenic seeds was higher than that of the wild type seeds in the treatment of 300 mmol L −1 sorbitol. When treated with 300 mmol L −1 sorbitol, the leaves of 129B08/ nced3 mutant plants were highly chlorotic, and root formation and seedling growth were severely inhibited. In contrast, at this concentration of sorbitol, the leaves of A3p::ORF-B08 transgenic seedlings were only slightly chlorotic, just similar to those of the wild type plants. There was almost no influence on the growth of 35S::ORF-WT transgenic seedlings under the sorbitol treatment. Based on the results of germination assay and phenotypic evaluation, the 129B08/ nced3 mutant was hypersensitive to the nonionic osmotic stress induced by sorbitol, and ectopic expression of peanut AhNCED1 gene reverted the hypersensitivity of 129B08/ nced3 mutant Arabidopsis to sorbitol and conferred enhanced osmotic stress tolerance in transgenic Arabidopsis plants.

ABA overly-sensitive 5 (ABO5), encoding a pentatricopeptide repeat protein required for cis-splicing of mitochondrial nad2 intron 3, is involved in the abscisic acid response in Arabidopsis

To study the molecular mechanism of abscisic acid (ABA) regulation of root development, we screened the root growth of Arabidopsis mutants for sensitivity to ABA. ABA overly-sensitive 5 (ABO5/At1g51965) was identified, and was determined to encode a pentatricopeptide repeat protein required for cis-splicing of mitochondrial nad2 intron 3 (nad2 is one subunit in complex I). Under constant light conditions (24-h light/0-h dark photoperiod), abo5 mutants exhibited various phenotypes and expressed lower transcripts of stress-inducible genes, such as RD29A, COR47 and ABF2, and photosynthesis-related genes proton gradient regulation 5 (PGR5) and PGR5-likephotosynthetic phenotype (PGRL1), but higher levels of nuclear-encoded genes alternative oxidase 1a (AOX1a) and oxidative signal-inducible 1 (OXI1). Prolonged ABA treatment increased the expression of the cox2 gene in complex IV and nad genes in complex I to a higher level than no ABA treatment in the wild type, but only to a moderate level in abo5, probably because abo5 already expressed high levels of mitochondrial-encoded cox2 and nad genes under no ABA treatment. More H(2) O(2) accumulated in the root tips of abo5 than in the wild type, and H(2) O(2) accumulation was further enhanced by ABA treatment. However, these growth phenotypes and gene-expression defects were attenuated by growing abo5 plants under short-day conditions (12-h light/12-h dark photoperiod). Our results indicate that ABO5 is important in the plant response to ABA.

Abscisic Acid Increases Arabidopsis ABI5 Transcription Factor Levels by Promoting KEG E3 Ligase Self-Ubiquitination and Proteasomal Degradation

The Arabidopsis thaliana RING-type E3 ligase KEEP ON GOING (KEG) is a negative regulator of abscisic acid (ABA) signaling. Seedlings homozygous for T-DNA insertions in KEG accumulate high levels of the ABA-responsive transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5). Here, we demonstrate that KEG E3 ligase activity is required for the regulation of ABI5 abundance. KEG ubiquitinates ABI5 in vitro, and a functional KEG RING domain is required to restore the levels of ABI5 in keg-1 to that of the wild type. Overexpression of KEG leads to ABA insensitivity, which correlates with KEG protein levels. In the presence of ABA, ABI5 levels increase drastically via a decrease in ubiquitin-meditated proteasomal degradation. Our results indicate that ABA promotes ABI5 accumulation by inducing the ubiquitination and proteasomal degradation of KEG. A functional RING domain is required for the ABA-induced degradation of KEG, suggesting that the loss is due to self-ubiquitination. Mutations within KEG's kinase domain or treatments with kinase inhibitors prohibit the ABA-induced ubiquitination and degradation of KEG, indicating that phosphorylation, possibly self-phosphorylation, is involved in the ABA regulation of KEG protein levels. We discuss a model for how ABA may negatively regulate KEG protein abundance, leading to accumulation of ABI5 and ABA-dependent cellular responses.

The Arabidopsis thaliana NAC transcription factor family: structure–function relationships and determinants of ANAC019 stress signalling

TFs (transcription factors) are modular proteins minimally containing a DBD (DNA-binding domain) and a TRD (transcription regulatory domain). NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] proteins comprise one of the largest plant TF families. They are key regulators of stress perception and developmental programmes, and most share an N-terminal NAC domain. On the basis of analyses of gene expression data and the phylogeny of Arabidopsis thaliana NAC TFs we systematically decipher structural and functional specificities of the conserved NAC domains and the divergent C-termini. Nine of the ten NAC domains analysed bind a previously identified conserved DNA target sequence with a CGT[GA] core, although with different affinities. Likewise, all but one of the NAC proteins analysed is dependent on the C-terminal region for transactivational activity. In silico analyses show that the NAC TRDs contain group-specific sequence motifs and are characterized by a high degree of intrinsic disorder. Furthermore, ANAC019 was identified as a new positive regulator of ABA (abscisic acid) signalling, conferring ABA hypersensitivity when ectopically expressed in plants. Interestingly, ectopic expression of the ANAC019 DBD or TRD alone also resulted in ABA hypersensitivity. Expression of stress-responsive marker genes [COR47 (cold-responsive 47), RD29b (responsive-to-desiccation 29b) and ERD11 (early-responsive-to-dehydration 11)] were also induced by full-length and truncated ANAC019. Domain-swapping experiments were used to analyse the specificity of this function. Chimaeric proteins, where the NAC domain of ANAC019 was replaced with the analogous regions from other NAC TFs, also have the ability to positively regulate ABA signalling. In contrast, replacing the ANAC019 TRD with other TRDs abolished ANAC019-mediated ABA hypersensitivity. In conclusion, our results demonstrate that the biochemical and functional specificity of NAC TFs is associated with both the DBDs and the TRDs.

MYB96‐mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis

*The Arabidopsis MYB96 transcription factor plays a role in abscisic acid (ABA)-mediated drought response. Notably, anthocyanins accumulate in the activation-tagging myb96-1d line, suggesting a role of MYB96 in biotic and abiotic stress responses in plants. Here, we investigate the role of MYB96 in salicylic acid (SA) biosynthesis and plant defense and explore the mechanisms underlying the ABA-SA interaction. *myb96-1d and myb96-1 were subject to pathogen infection assays, and expression of SA biosynthetic and defense genes was examined. myb96-1d was crossed with the NahG transgenic plants to investigate the role of MYB96 in ABA regulation of SA biosynthesis. *Whereas myb96-1d exhibited an enhanced disease resistance, myb96-1 was susceptible to pathogen infection. A subset of pathogenesis-related (PR) genes was up-regulated in myb96-1d. However, PR transcript abundances were reduced in myb96-1d X NahG. Interestingly, a SA biosynthetic gene SALICYLIC ACID INDUCTION DEFICIENT2 (SID2) was up-regulated, and concentrations of SA and SA-beta-glucoside (SAG) were elevated in myb96-1d. In addition, the inductive effects of abiotic stresses on SID2 were reduced in aba3-1. *Our observations indicate that MYB96-mediated ABA signals enhance plant disease resistance by inducing SA biosynthesis. It is envisioned that MYB96 is a molecular link that mediates ABA-SA crosstalks.

Overproduction of the Membrane-bound Receptor-like Protein Kinase 1, RPK1, Enhances Abiotic Stress Tolerance in Arabidopsis

RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H(2)O(2)-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.

The Nuclear Interactor PYL8/RCAR3 ofFagus sylvaticaFsPP2C1 Is a Positive Regulator of Abscisic Acid Signaling in Seeds and Stress

The functional protein phosphatase type 2C from beechnut (Fagus sylvatica; FsPP2C1) was a negative regulator of abscisic acid (ABA) signaling in seeds. In this report, to get deeper insight on FsPP2C1 function, we aim to identify PP2C-interacting partners. Two closely related members (PYL8/RCAR3 and PYL7/RCAR2) of the Arabidopsis (Arabidopsis thaliana) BetV I family were shown to bind FsPP2C1 in a yeast two-hybrid screening and in an ABA-independent manner. By transient expression of FsPP2C1 and PYL8/RCAR3 in epidermal onion (Allium cepa) cells and agroinfiltration in tobacco (Nicotiana benthamiana) as green fluorescent protein fusion proteins, we obtained evidence supporting the subcellular localization of both proteins mainly in the nucleus and in both the cytosol and the nucleus, respectively. The in planta interaction of both proteins in tobacco cells by bimolecular fluorescence complementation assays resulted in a specific nuclear colocalization of this interaction. Constitutive overexpression of PYL8/RCAR3 confers ABA hypersensitivity in Arabidopsis seeds and, consequently, an enhanced degree of seed dormancy. Additionally, transgenic 35S:PYL8/RCAR3 plants are unable to germinate under low concentrations of mannitol, NaCl, or paclobutrazol, which are not inhibiting conditions to the wild type. In vegetative tissues, Arabidopsis PYL8/RCAR3 transgenic plants show ABA-resistant drought response and a strong inhibition of early root growth. These phenotypes are strengthened at the molecular level with the enhanced induction of several ABA response genes. Both seed and vegetative phenotypes of Arabidopsis 35S:PYL8/RCAR3 plants are opposite those of 35S:FsPP2C1 plants. Finally, double transgenic plants confirm the role of PYL8/RCAR3 by antagonizing FsPP2C1 function and demonstrating that PYL8/RCAR3 positively regulates ABA signaling during germination and abiotic stress responses.