Overexpression of a pearl millet WRKY transcription factor gene, PgWRKY74, in Arabidopsis retards shoot growth under dehydration and salinity-stressed conditions.

Pearl millet (Cenchrus americanus) is a cereal crop that can tolerate high temperatures, drought, and low-fertility conditions where other crops lose productivity. However, genes regulating this ability are largely unknown. Transcription factors (TFs) regulate transcription of their target genes, regulate downstream biological processes, and thus are candidates for regulators of such tolerance of pearl millet. PgWRKY74 encodes a group IIc WRKY TF in pearl millet and is downregulated by drought. PgWRKY74 may have a role in drought tolerance. The objective of this study was to gain insights into the physiological and biochemical functions of PgWRKY74. Yeast one-hybrid and gel shift assays were performed to examine transcriptional activation potential and deoxyribonucleic acid (DNA)-binding ability, respectively. Transgenic Arabidopsis thaliana plants overexpressing PgWRKY74-green fluorescent protein (GFP) fusion gene were generated and tested for growth and stress-responsive gene expression under mannitol and NaCl-stressed conditions. A construct with PgWRKY74 enabled yeast reporter cells to survive on test media in the yeast one-hybrid assays. The electrophoretic mobility of DNA with putative WRKY TF-binding motifs was lower in the presence of a recombinant PgWRKY74 protein than its absence. The PgWRKY74-GFP-overexpressing Arabidopsis plants exhibited smaller rosette areas than did wild-type plants under mannitol-stressed and NaCl-stressed conditions, and exhibited weaker expression of RD29B, which is induced by the stress-related phytohormone abscisic acid (ABA), under the mannitol-stressed condition. PgWRKY74 have transcriptional activation potential and DNA-binding ability, and can negatively regulate plant responses to mannitol and NaCl stresses, possibly by decreasing ABA levels or ABA sensitivity.

Identification of OsPK5 involved in rice glycolytic metabolism and GA/ABA balance for improving seed germination via genome-wide association study.

Seed germination plays a pivotal role in the plant life cycle, and its precise regulatory mechanisms are not clear. In this study, 19 quantitative trait loci (QTLs) associated with rice seed germination were identified through genome-wide association studies (GWAS) of the following traits in 2016 and 2017: germination rate (GR) at 3, 5, and 7 days after imbibition (DAI) and germination index (GI). Two major stable QTLs, qSG4 and qSG11.1, were found to be associated with GR and GI over 2 continuous years. Furthermore, OsPK5, encoding a pyruvate kinase, was shown to be a crucial regulator of seed germination in rice, and might be a causal gene of the key QTL qSG11.1, on chromosome 11. Natural variation in OsPK5 function altered the activity of pyruvate kinase. The disruption of OsPK5 function resulted in slow germination and seedling growth during seed germination, blocked glycolytic metabolism, caused glucose accumulation, decreased energy levels, and affected the GA/ABA balance. Taken together, our results provide novel insights into the roles of OsPK5 in seed germination, and facilitate its application in rice breeding to improve seed vigour.

Physiological and transcriptional responses of the ectomycorrhizal fungus Cenococcum geophilum to salt stress.

Ectomycorrhizal (ECM) fungi improve the host plant's tolerance to abiotic and biotic stresses. Cenococcum geophilum (Cg) is among the most common ECM fungi worldwide and often grows in saline environments. However, the physiological and molecular mechanisms of salt tolerance in this fungus are largely unknown. In the present study, 12 isolates collected from different ecogeographic regions were used to investigate the mechanism of salt tolerance of Cg. The isolates were classified into four groups (salt-sensitive, moderately salt-tolerant, salt-tolerant, and halophilic) based on their in vitro mycelial growth under 0, 50, 125, 250, and 500 mM NaCl concentrations. Hence, the Na, Ca, P, and K concentrations of mycelia and the pH of the culture solution were determined. Compared with salt-tolerant isolates, treatment with 250 mM NaCl significantly increased the sodium concentration and decreased the potassium concentration of salt-sensitive isolates. RNA-sequencing and qRT-PCR analysis were conducted to identify differentially expressed genes (DEGs) involved in transmembrane transport and oxidoreductase activity pathways. The hydrogen peroxide concentration and activities of peroxidase and superoxide dismutase in mycelia were determined, and the accumulation and scavenging of reactive oxygen species in the salt-sensitive isolates were more active than those in the salt-tolerant isolates. The results supply functional validations to RNA-seq and qRT-PCR analysis. This study provides novel insights into the salt-stress response of Cg isolates and provides a foundation for elucidation of the salt-tolerance mechanism of ECM fungi.

RNA-Binding Protein MAC5A Is Required for Gibberellin-Regulated Stamen Development.

The development of floral organs is coordinated by an elaborate network of homeotic genes, and gibberellin (GA) signaling is involved in floral organ development; however, the underlying molecular mechanisms remain elusive. In the present study, we found that MOS4-ASSOCIATED COMPLEX 5A (MAC5A), which is a protein containing an RNA-binding motif, was involved in the development of sepals, petals, and stamens; either the loss or gain of MAC5A function resulted in stamen malformation and a reduced seed set. The exogenous application of GA considerably exacerbated the defects in mac5a null mutants, including fewer stamens and male sterility. MAC5A was predominantly expressed in pollen grains and stamens, and overexpression of MAC5A affected the expression of homeotic genes such as APETALA1 (AP1), AP2, and AGAMOUS (AG). MAC5A may interact with RABBIT EARS (RBE), a repressor of AG expression in Arabidopsis flowers. The petal defect in rbe null mutants was at least partly rescued in mac5a rbe double mutants. These findings suggest that MAC5A is a novel factor that is required for the normal development of stamens and depends on the GA signaling pathway.

Comprehensive analysis of NAC transcription factor family uncovers drought and salinity stress response in pearl millet (Pennisetum glaucum).

BACKGROUND: Pearl millet (Pennisetum glaucum) is a cereal crop that possesses the ability to withstand drought, salinity and high temperature stresses. The NAC [NAM (No Apical Meristem), ATAF1 (Arabidopsis thaliana Activation Factor 1), and CUC2 (Cup-shaped Cotyledon)] transcription factor family is one of the largest transcription factor families in plants. NAC family members are known to regulate plant growth and abiotic stress response. Currently, no reports are available on the functions of the NAC family in pearl millet. RESULTS: Our genome-wide analysis found 151 NAC transcription factor genes (PgNACs) in the pearl millet genome. Thirty-eight and 76 PgNACs were found to be segmental and dispersed duplicated respectively. Phylogenetic analysis divided these NAC transcription factors into 11 groups (A-K). Three PgNACs (- 073, - 29, and - 151) were found to be membrane-associated transcription factors. Seventeen other conserved motifs were found in PgNACs. Based on the similarity of PgNACs to NAC proteins in other species, the functions of PgNACs were predicted. In total, 88 microRNA target sites were predicted in 59 PgNACs. A previously performed transcriptome analysis suggests that the expression of 30 and 42 PgNACs are affected by salinity stress and drought stress, respectively. The expression of 36 randomly selected PgNACs were examined by quantitative reverse transcription-PCR. Many of these genes showed diverse salt- and drought-responsive expression patterns in roots and leaves. These results confirm that PgNACs are potentially involved in regulating abiotic stress tolerance in pearl millet. CONCLUSION: The pearl millet genome contains 151 NAC transcription factor genes that can be classified into 11 groups. Many of these genes are either upregulated or downregulated by either salinity or drought stress and may therefore contribute to establishing stress tolerance in pearl millet.

The B″-family subunits of protein phosphatase 2A are necessary for in-vitro dephosphorylation of the Arabidopsis mechanosensory transcription factor VIP1.

Protein phosphatase 2A (PP2A) B″-family subunits have Ca2+-binding EF-hand motifs and can bind PP2A substrates. Arabidopsis thaliana PP2A B″-family subunits are encoded by six genes, and bind a transcription factor, VIP1. VIP1 is dephosphorylated and nuclear-localized by hypo-osmotic stress. However, whether PP2A B″-family subunits mediate the VIP1 dephosphorylation is unclear. Here, we show by yeast two-hybrid and in vitro pull down assays that Arabidopsis PP2A B″-family subunits bind Arabidopsis PP2A A (scaffold) subunits. We also show that VIP1 dephosphorylation in vitro can be induced by a PP2A B″-family subunit.

A GDSL-type esterase/lipase gene, GELP77, is necessary for pollen dissociation and fertility in Arabidopsis.

Pollen wall characteristics are dramatically changed during pollen maturation. Many genes have been identified as regulators of such changes in pollen wall characteristics, but mechanisms of such changes have not been completely understood. Here, a GDSL-type esterase/lipase gene, GELP77, is shown to regulate such changes in Arabidopsis thaliana. GELP77-deficient (gelp77) plants exhibited male sterility, and this phenotype was suppressed by introduction of a GELP77 genomic fragment. Mature pollen grains of wild-type Arabidopsis plants have an organized reticulate surface structure and are dissociated from each other. In contrast, pollen grains of gelp77 lacked such a structure and were shrunken and stuck to each other. Nuclei were not detectable in gelp77 microspores at a putative uninucleate stage, suggesting that GELP77 is required as early as this stage. In plants that have the GELP77 promoter-GELP77-GFP transgene, the GELP77-GFP fusion protein was detected in microspores, tapetal cells and middle layer cells in anthers at post-meiotic stages, whereas not anthers at pre-meiotic stages. Analysis of amino acid sequences suggests that GELP77 is phylogenetically distant from the other 104 GDSL-type esterase/lipase genes in Arabidopsis and that GELP77 orthologs are present in various plant species. Together, these results indicate that GELP77 regulates pollen wall characteristics in Arabidopsis.

A putative AGAMOUS ortholog is a candidate for the gene determining ease of dehulling in Tartary buckwheat (Fagopyrum tataricum).

MAIN CONCLUSION: Tartary buckwheat rice-type cultivars, which allow easy dehulling, lacked periclinal cell divisions that proceed underneath the epidermis in the proximity of ovary midribs in non-rice-type cultivars. The easy dehulling in these cultivars was associated with a G→A substitution in an AGAMOUS ortholog. Ease of dehulling in Tartary buckwheat (Fagopyrum tataricum) can affect the quality of its products. Tartary buckwheat cultivars that allow easy dehulling are called rice-type cultivars. The rice and non-rice hull types are determined by a single gene, but this gene is unclear. Here, we show that cells underneath the epidermis in the proximity of ovary midribs undergo periclinal cell divisions in non-rice-type cultivars but do not in a rice-type cultivar. The cells that arose from the periclinal cell divisions later underwent lignification, which should increase mechanical strength of hulls. In RNA sequencing, a partial mRNA of an AGAMOUS ortholog in Tartary buckwheat (FtAG) was found to be absent in the rice-type cultivar. Cloning of this gene revealed that this is a 42-bp deletion due to a G→A substitution at a splice acceptor site in the FtAG genomic region. In F2 progeny derived from a cross between non-rice-type and rice-type cultivars, all the rice-type plants exhibited the homozygous A/A allele at this site, whereas all the Tartary-type plants exhibited either the homozygous G/G allele or the heterozygous A/G allele. These results suggest that FtAG is a candidate for the gene that determines ease of dehulling in Tartary buckwheat. The DNA marker that we developed to distinguish the FtAG alleles can be useful in breeding Tartary buckwheat cultivars.

Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress.

VIP1 is a bZIP transcription factor in Arabidopsis thaliana. When cells are exposed to mechanical stress, VIP1 transiently accumulates in the nucleus, where it regulates the expression of its target genes and suppresses mechanical stress-induced root waving. The nuclear-cytoplasmic shuttling of VIP1 is regulated by phosphorylation and calcium-dependent signaling, but specific regulators of these processes remain to be identified. Here, inhibitors of protein phosphatase 2A (PP2A) are shown to inhibit both the mechanical stress-induced dephosphorylation and nuclear accumulation of VIP1. The PP2A B subunit, which recruits substrates of PP2A holoenzyme, is classified into B, B', B'', and B''' families. Using bimolecular fluorescence complementation, in vitro pull-down, and yeast two-hybrid assays, we show that VIP1 interacts with at least two of the six members of the Arabidopsis PP2A B''-family subunit, which have calcium-binding EF-hand motifs. VIP1AAA, a constitutively nuclear-localized VIP1 variant with substitutions in putative phosphorylation sites of VIP1, suppressed the root waving induced by VIP1-SRDX (a repression domain-fused variant of VIP1). These results support the idea that VIP1 is dephosphorylated by PP2A and that the dephosphorylation suppresses the root waving. The phosphorylation sites of VIP1 and its homologs were narrowed down by in vitro phosphorylation, yeast two-hybrid, and protein subcellular localization assays.

B-family subunits of protein phosphatase 2A are necessary for pollen development but not for female gametophyte development in Arabidopsis.

Protein phosphatase 2A (PP2A) is a heterotrimeric protein complex conserved among eukaryotes. The B subunit of PP2A determines the substrate specificity of the PP2A holoenzyme, and is classified into the B, B', B″ and B‴ families. Arabidopsis thaliana has two isoforms of the B-family subunit (ATBA and ATBB). A double knockout of their genes is lethal, but which developmental process is primarily impaired by the double knockout is unclear. Identifying such a process helps understand PP2A-mediated signaling more deeply. Here, genetic characterization of new knockout mutants for these genes shows that they are necessary for pollen development but not for female gametophyte development. Compared to wild-type pollen grains, the mutant pollen grains exhibited lower enzyme activities, germinated less frequently on stigmas, and exhibited the aberrant numbers of sperm cell nuclei, suggesting that ATBA and ATBB play pleiotropic roles in pollen development. The amino acids stabilizing the interaction between the human PP2A A and B-family subunits are conserved in an Arabidopsis A subunit (AtPP2AA2), ATBA and ATBB. His-tagged AtPP2AA2 co-immunoprecipitated with either Myc-tagged ATBA or Myc-tagged ATBB in vitro, confirming their interactions. Proteins that regulate pollen development and that undergo dephosphorylation are likely primary targets of ATBA and ATBB.

Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana.

Background and Aims: VIP1 is a bZIP transcription factor in Arabidopsis thaliana. VIP1 and its close homologues transiently accumulate in the nucleus when cells are exposed to hypo-osmotic and/or mechanical stress. Touch-induced root bending is enhanced in transgenic plants overexpressing a repression domain-fused form of VIP1 (VIP1-SRDXox), suggesting that VIP1, possibly with its close homologues, suppresses touch-induced root bending. The aim of this study was to identify regulators of these functions of VIP1 in mechanical stress responses. Methods: Co-immunoprecipitation analysis using VIP1-GFP fusion protein expressed in Arabidopsis plants identified calmodulins as VIP1-GFP interactors. In vitro crosslink analysis was performed using a hexahistidine-tagged calmodulin and glutathione S-transferase-fused forms of VIP1 and its close homologues. Plants expressing GFP-fused forms of VIP1 and its close homologues (bZIP59 and bZIP29) were submerged in hypotonic solutions containing divalent cation chelators, EDTA and EGTA, and a potential calmodulin inhibitor, chlorpromazine, to examine their effects on the nuclear-cytoplasmic shuttling of those proteins. VIP1-SRDXox plants were grown on medium containing 40 mm CaCl2, 40 mm MgCl2 or 80 mm NaCl. MCA1 and MCA2 are mechanosensitive calcium channels, and the hypo-osmotic stress-dependent nuclear-cytoplasmic shuttling of VIP1-GFP in the mca1 mca2 double knockout mutant background was examined. Key Results: In vitro crosslink products were detected in the presence of CaCl2, but not in its absence. EDTA, EGTA and chlorpromazine all inhibited both the nuclear import and the nuclear export of VIP1-GFP, bZIP59-GFP and bZIP29-GFP. Either 40 mm CaCl2or 80 mm NaCl enhanced the VIP-SRDX-dependent root bending. The nuclear-cytoplasmic shuttling of VIP1 was observed even in the mca1 mca2 mutant. Conclusions: VIP1 and its close homologues can interact with calmodulins. Their nuclear-cytoplasmic shuttling requires neither MCA1 nor MCA2, but does require calcium signalling. Salt stress affects the VIP1-dependent regulation of root bending.

The bZIP Protein VIP1 Is Involved in Touch Responses in Arabidopsis Roots.

VIP1 is a bZIP transcription factor in Arabidopsis (Arabidopsis thaliana). VIP1 transiently accumulates in the nucleus when cells are exposed to hypoosmotic conditions, but its physiological relevance is unclear. This is possibly because Arabidopsis has approximately 10 close homologs of VIP1 and they function redundantly. To examine their physiological roles, transgenic plants overexpressing a repression domain-fused form of VIP1 (VIP1-SRDXox plants), in which the gene activation mediated by VIP1 is expected to be repressed, were generated. Because hypoosmotic stress can mimic mechanical stimuli (e.g. touch), the touch-induced root-waving phenotypes and gene expression patterns in those transgenic plants were examined. VIP1-SRDXox plants exhibited more severe root waving and lower expression of putative VIP1 target genes. The expression of the VIP1-green fluorescent protein (GFP) fusion protein partially suppressed the VIP1-SRDX-induced increase in root waving when expressed in the VIP1-SRDXox plants. These results suggest that VIP1 can suppress the touch-induced root waving. The VIP1-SRDX-induced increase in root waving was also suppressed when the synthetic auxin 2,4-dichlorophenoxy acetic acid or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, which is known to activate auxin biosynthesis, was present in the growth medium. Root cap cells with the auxin marker DR5rev::GFP were more abundant in the VIP1-SRDXox background than in the wild-type background. Auxin is transported via the root cap, and the conditions of outermost root cap layers were abnormal in VIP1-SRDXox plants. These results raise the possibility that VIP1 influences structures of the root cap and thereby regulates the local auxin responses in roots.

Arabidopsis G-protein ß subunit AGB1 interacts with NPH3 and is involved in phototropism.

Heterotrimeric G proteins (Gα, Gß and Gγ) have pleiotropic roles in plants, but molecular mechanisms underlying them remain to be elucidated. Here we show that Arabidopsis Gß (AGB1) interacts with NPH3, a regulator of phototropism. Yeast two-hybrid assays, in vitro pull-down assays and bimolecular fluorescence complementation assays showed that AGB1 and NPH3 physically interact. NPH3-null mutation (nph3) is known to completely abolish hypocotyl phototropism. Loss-of-function mutants of AGB1 (agb1-1 and agb1-2) showed decreased hypocotyl phototropism, and agb1/nph3 double mutants showed no hypocotyl phototropism. These results suggest that AGB1 is involved in the NPH3-mediated regulation of phototropism.

Data of RNA sequencing of pearl millet panicles treated with a high temperature.

Pearl millet (Pennisetum glaucum) is a cereal crop that can grow and set seeds even under drought, high temperatures and nutrient-poor conditions. Panicles of two pearl millet cultivars that differ in seed-setting rates were exposed to two different high-temperature treatments at three different developmental stages with three replicates, and RNA was prepared from these panicles. The resulting RNA samples were subjected to sequencing with the Illumina NovaSeq 6000 sequencer. The obtained data were 150-base-paired-end reads and were approximately 5 Gb/sample in total. These read data were deposited as those for a project in the NCBI (National Center for Biotechnology Information) BioProject database.

Arabidopsis heterotrimeric G protein ß subunit interacts with a plasma membrane 2C-type protein phosphatase, PP2C52.

Heterotrimeric G proteins (Gα, Gß, Gγ) play important roles in signal transduction among various eukaryotic species. G proteins transmit signals by regulating the activities of effector proteins, but only a few Gß-interacting effectors have been identified in plants. Here we show by a yeast two-hybrid screen that a putative myristoylated 2C-type protein phosphatase, PP2C52, is an Arabidopsis Gß (AGB1)-interacting partner. The interaction between AGB1 and PP2C52 was confirmed by an in vitro pull-down assay and a bimolecular fluorescence complementation assay. PP2C52 transcripts were detected in many tissues. PP2C52 was localized to the plasma membrane and a mutation in the putative myristoylation site of PP2C52 disrupted its plasma membrane localization. Our results suggest that PP2C52 interacts with AGB1 on the plasma membrane and transmits signals via dephosphorylation of other proteins.

A bZIP protein, VIP1, is a regulator of osmosensory signaling in Arabidopsis.

Abscisic acid is a stress-related phytohormone that has roles in dehydration and rehydration. In Arabidopsis (Arabidopsis thaliana), two genes that inactivate abscisic acid, CYP707A1 and CYP707A3, are rapidly up-regulated upon rehydration. The factors that regulate CYP707A1/3 are not well characterized. We expressed a bZIP protein, VIP1, as a green fluorescent protein fusion protein in Arabidopsis and found that the nuclear localization of VIP1 was enhanced within 10 min after rehydration. A yeast one-hybrid assay revealed that the amino-terminal region of VIP1 has transcriptional activation potential. In a transient reporter assay using Arabidopsis protoplasts, VIP1 enhanced the promoter activities of CYP707A1/3. In gel shift and chromatin immunoprecipitation analyses, VIP1 directly bound to DNA fragments of the CYP707A1/3 promoters. Transgenic plants expressing VIP1-green fluorescent protein were found to overexpress CYP707A1/3 mRNAs. The time course of nuclear-cytoplasmic shuttling of VIP1 was consistent with the time courses of the expression of CYP707A1/3. These results suggest that VIP1 functions as a regulator of osmosensory signaling in Arabidopsis.

Arabidopsis heterotrimeric G protein ß subunit, AGB1, regulates brassinosteroid signalling independently of BZR1.

The Arabidopsis thaliana heterotrimeric G protein ß subunit, AGB1, is involved in both abscisic acid (ABA) signalling and brassinosteroid (BR) signalling, but it is unclear how AGB1 regulates these signalling pathways. A key transcription factor downstream of BR, BZR1, and its gain-of-function mutant, bzr1-1, were overexpressed in an AGB1-null mutant, agb1-1, to examine their effects on the BR hyposensitivity and the ABA hypersensitivity of agb1-1, and to examine whether AGB1 regulates the functions of BZR1. Because the amino acid sequence of AGB1 contains 17 putative modification motifs of glycogen synthase kinase 3/SHAGGY-like protein kinases (GSKs), which are known components of BR signalling, the interaction between AGB1 and one of the Arabidopsis GSKs, BIN2, was examined. Expression of bzr1-1 alleviated the effects of a BR biosynthesis inhibitor, brassinazole, in both the wild type and agb1-1, and overexpression of BZR1 alleviated the effects of ABA in both the wild type and agb1-1. AGB1 did not affect the phosphorylation state of BZR1 in vivo. AGB1 interacted with BIN2 in vitro, but did not affect the phosphorylation state of BIN2. The results suggest that AGB1 interacts with BIN2, but regulates the BR signalling in a BZR1-independent manner.

The Arabidopsis adaptor protein AP-3µ interacts with the G-protein ß subunit AGB1 and is involved in abscisic acid regulation of germination and post-germination development.

Heterotrimeric G-proteins (G-proteins) have been implicated in ubiquitous signalling mechanisms in eukaryotes. In plants, G-proteins modulate hormonal and stress responses and regulate diverse developmental processes. However, the molecular mechanisms of their functions are largely unknown. A yeast two-hybrid screen was performed to identify interacting partners of the Arabidopsis G-protein ß subunit AGB1. One of the identified AGB1-interacting proteins is the Arabidopsis adaptor protein AP-3µ. The interaction between AGB1 and AP-3µ was confirmed by an in vitro pull-down assay and bimolecular fluorescence complementation assay. Two ap-3µ T-DNA insertional mutants were found to be hyposensitive to abscisic acid (ABA) during germination and post-germination growth, whereas agb1 mutants were hypersensitive to ABA. During seed germination, agb1/ap-3µ double mutants were more sensitive to ABA than the wild type but less sensitive than agb1 mutants. However, in post-germination growth, the double mutants were as sensitive to ABA as agb1 mutants. These data suggest that AP-3µ positively regulates the ABA responses independently of AGB1 in seed germination, while AP-3µ does require AGB1 to regulate ABA responses during post-germination growth.

Phylogenetic trees, conserved motifs and predicted subcellular localization for transcription factor families in pearl millet.

OBJECTIVES: Pearl millet (Pennisetum glaucum) is a cereal crop that is tolerant to a high temperature, a drought and a nutrient-poor condition. Characterizing pearl millet proteins can help to improve productivity of pearl millet and other crops. Transcription factors in general are proteins that regulate transcription of their target genes and thereby regulate diverse processes. Some transcription factor families in pearl millet were characterized in previous studies, but most of them are not. The objective of the data presented was to characterize amino acid sequences for most transcription factors in pearl millet. DATA DESCRIPTION: Sequences of 2395 pearl millet proteins that have transcription factor-associated domains were extracted. Subcellular and suborganellar localization of these proteins was predicted by MULocDeep. Conserved domains in these sequences were confirmed by CD-Search. These proteins were classified into 85 families on the basis of those conserved domains. A phylogenetic tree including pearl millet proteins and their counterparts in Arabidopsis thaliana and rice was constructed for each of these families. Sequence motifs were identified by MEME for each of these families.

Drought-induced activation and rehydration-induced inactivation of MPK6 in Arabidopsis.

Mitogen-activated protein kinases (MPKs) have roles in regulating developmental processes and responses to various stimuli in plants. Activations of some MPKs are necessary for proper responses to hyperosmolarity and to a stress-related phytohormone, abscisic acid (ABA). However, there is no direct evidence that MPK activations are regulated by drought and rehydration. Here we show that the activation state of one of the Arabidopsis MPKs, MPK6, is directly regulated by drought and rehydration. An immunoblot analysis using an anti-active MPK antibody detected drought-induced activation and rehydration-induced inactivation of MPK6. MPK6 was activated by drought even in an ABA-deficient mutant, aba2-4. In addition, exogenously added ABA failed to suppress the rehydration-dependent inactivation of MPK6. Under drought conditions, elevated levels of reactive oxygen species (ROS), which are known elicitors of MPK6 activation, were detected in both wild type and an MPK6-deficient mutant, mpk6-4. These results suggest that ROS, but not ABA, induces MPK6 activation as an upstream signal under drought conditions.