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.

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.

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.

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.

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.

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.

NDR/LATS-family protein kinase genes are indispensable for embryogenesis in Arabidopsis.

NDR/LATS-family protein kinases are conserved among eukaryotes. These protein kinases in yeast and animals phosphorylate specific targets and regulate the cell cycle. Arabidopsis thaliana has eight NDR/LATS-family protein kinase genes (NDR1-8), of which NDR2, NDR4, and NDR5 are involved in regulating pollen development. However, the functions of the other NDR/LATS-family protein kinase genes in plants are unclear. Here, we show that three putative phosphorylation sites of an Arabidopsis basic leucine zipper transcription factor, VIP1, correspond to NDR/LATS-family protein kinase phosphorylation motifs and that two of these three sites are phosphorylated by NDR2, NDR3, or NDR8 in vitro. Expression of NDR1-8 was detected in various tissues. An NDR4 NDR6 NDR7 NDR8 quadruple mutation caused embryonic lethality These results suggest that different NDR/LATS-family protein kinases in plants have distinct physiological roles.

TGIF-DB: terse genomics interface for developing botany.

OBJECTIVES: Pearl millet (Pennisetum glaucum) is a staple cereal crop for semi-arid regions. Its whole genome sequence and deduced putative gene sequences are available. However, the functions of many pearl millet genes are unknown. Situations are similar for other crop species such as garden asparagus (Asparagus officinalis), chickpea (Cicer arietinum) and Tartary buckwheat (Fagopyrum tataricum). The objective of the data presented here was to improve functional annotations of genes of pearl millet, garden asparagus, chickpea and Tartary buckwheat with gene annotations of model plants, to systematically provide such annotations as well as their sequences on a website, and thereby to promote genomics for those crops. DATA DESCRIPTION: Sequences of genomes and transcripts of pearl millet, garden asparagus, chickpea and Tartary buckwheat were downloaded from a public database. These transcripts were associated with functional annotations of their Arabidopsis thaliana and rice (Oryza sativa) counterparts identified by BLASTX. Conserved domains in protein sequences of those species were identified by the HMMER scan with the Pfam database. The resulting data was deposited in the figshare repository and can be browsed on the Terse Genomics Interface for Developing Botany (TGIF-DB) website ( http://webpark2116.sakura.ne.jp/rlgpr/ ).

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.

Biotin plays an important role in Arabidopsis thaliana seedlings under carbonate stress.

Globally, many saline-alkali soils are rich in NaHCO3 and Na2CO3, which are characterized by a high pH Carbonate stress caused by this kind of soil severely damages plant cells and inhibits plant growth. Biotin and HCO3- participate in the first and rate-limiting reaction of the fatty acid biosynthesis pathway, but whether biotin contributes to plant responses to carbonate stress is unclear. In this study, we revealed that high carbonate and biotin concentrations inhibited Arabidopsis (Arabidopsis thaliana) seedling growth. However, specific concentrations of carbonate and biotin decreased the inhibitory effects of the other compound at the germination and seedling stages. Additionally, a carbonate treatment increased the endogenous biotin content and expression of AtBIO2, which encodes a biotin synthase. Moreover, phenotypic analyses indicated that the overexpression of AtBIO2 in Arabidopsis enhanced the tolerance to carbonate stress, whereas mutations to AtBIO2 had the opposite effect. Furthermore, the carbonate stress-induced accumulation of reactive oxygen species was lower in plants overexpressing AtBIO2 than in the wild-type and bio2 mutants. Accordingly, biotin, which is an essential vitamin for plants, can enhance the resistance to carbonate stress.

Developing a tool to shoot genes by a man-made air pressure.

BACKGROUND: Biolistic systems are used to shoot exogenous DNA, RNA, protein, and other macromolecules to transfer them into cells for genetic transformation, genome editing, and drug delivery. Such systems are especially useful for plants and other organisms that are incompatible with other macromolecule delivery methods. Commercially available, conventional biolistic systems consist of a shooting device (or "gun") and a cylinder bottle for high-pressure helium gas. These cost a lot for installation and have low portability. RESULTS: We assembled an inexpensive air duster gun and a hand pump into a portable tool to shoot genes by a man-made air pressure (TSGAMAP). TSGAMAP allows to shoot DNA-coated gold particles with the 3-MPa maximum air pressure. When DNA with a fluorescent protein gene, GFP, was shot by TSGAMAP into leaf epidermal cells of onion, leaf lettuce, and Chinese cabbage, for all of these species, some cells in all became to exhibit GFP signals. When GFP was shot with another fluorescent protein gene, mCherry, into Chinese cabbage cells, both GFP and mCherry signals were detected in some cells. When a transcription factor gene AoAMS was fused with GFP and shot into Chinese cabbage cells, nuclear-localized GFP signals were detected in some cells. These results suggest that TSGAMAP can be used for protein coexpression and protein subcellular localization analyses. CONCLUSIONS: TSGAMAP is a cost-saving and portable tool to shoot DNA and other microparticles into cells. This can expand the use of biolistics in research and education.

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.

VIP1, a bZIP protein, interacts with the catalytic subunit of protein phosphatase 2A in Arabidopsis thaliana.

VirE2-INTERACTING PROTEIN1 (VIP1) is a basic leucine zipper protein in Arabidopsis thaliana. VIP1 changes its subcellular localization from the cytoplasm to the nucleus when cells are exposed to mechanical or hypo-osmotic stress. The nuclear localization of VIP1 is inhibited either by inhibitors of calcium signaling or by inhibitors of protein phosphatases 1, 2A and 4 (PP1, PP2A and PP4, respectively). VIP1 binds to the PP2A B"-family subunits, which have calcium-binding EF-hand motifs and which act as the regulatory, substrate-recruiting B subunit of PP2A. The VIP1 de-phosphorylation can therefore be mediated by PP2A. However, details of the PP2A-mediated de-phosphorylation of VIP1 are unclear. Here, with yeast two-hybrid assays and in-vitro pull-down assays, we show that VIP1 does not interact with the scaffolding A subunit of PP2A, but that VIP1 does interact with the catalytic C subunits. Our data raise the possibility that not only the B"-family B subunit of PP2A but also its C subunit contributes to the PP2A-mediated de-phosphorylation of VIP1.

Data of whole genome sequencing of five garden asparagus (Asparagus officinalis) individuals with the MinION nanopore sequencer.

Garden asparagus (Asparagus officinalis) is a perennial, dioecious crop. Genomic DNA samples were prepared from five A. officinalis individuals that differ in sex and phenotypes, and sequenced with the MinION nanopore sequencer. The obtained data were 1.5-5 Gb/sample, and the average read length was larger than 1.4 kb for all the samples. The resulting reads were mapped to the existing A. officinalis genome sequence. The existing A. officinalis transcript sequences were mapped to the MinION-derived reads. On the basis of these mapping results, flanking sequences of five partial gene fragments that previously had not been mapped to any region of the existing genome were determined by genomic PCR followed by Sanger sequencing. These sequences enabled to estimate the genomic positions of those five partial gene fragments. The MinION-derived data and the flanking sequences of the five gene fragments were deposited in the NCBI (National Center for Biotechnology Information) SRA (Sequence Read Archive) database and the NCBI Nucleotide database, respectively.

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.

Death of female flower microsporocytes progresses independently of meiosis-like process and can be accelerated by specific transcripts in Asparagus officinalis.

Asparagus officinalis (garden asparagus) is a dioecious perennial crop, and the dioecy (i.e., sex) of A. officinalis can affect its productivity. In A. officinalis, flower anthers in female plants fail to accumulate callose around microsporocytes, fail to complete meiosis, and degenerate due to cell death. Although 13 genes have been implicated in the anther development of male and female flowers, it is unclear how these genes regulate the cell death in female flower anthers. The aim of this study was to narrow down factors involved in this process. TUNEL staining and Feulgen staining of female flower microsporocytes suggest that female microsporocytes enter a previously undetected meiosis-like process, and that the cell death occurs independently of this meiosis-like process, excluding the possibility that the cell death is caused by the cessation of meiosis. RNA sequencing with individual floral organs (tepals, pistils and stamens) revealed that several genes possibly regulating the cell death, such as metacaspase genes and a Bax inhibitor-1 gene, are differentially regulated between female and male flower anthers, and that genes involved in callose accumulation are up-regulated only in male flower anthers. These genes are likely involved in regulating the cell death in female flower anthers in A. officinalis.

Pearl millet stress-responsive NAC transcription factor PgNAC21 enhances salinity stress tolerance in Arabidopsis.

Pearl millet (Pennisetum glaucum) is the sixth-leading cereal crop and a staple food crop. It is known for its high tolerance to abiotic stress and good nutrient profile. NAC (NAM, ATAF1/2 and CUC) transcription factors (TFs) play an important role in abiotic stress tolerance. In our study, the pearl millet stress-responsive NAC TF gene PgNAC21 was characterized. Gene expression analysis revealed that PgNAC21 expression is induced by salinity stress and abscisic acid (ABA) treatment. In silico promoter analysis showed the presence of ABA response elements (ABREs) and MYB TF binding sites. A yeast one-hybrid assay indicated that a putative MYB TF in pearl millet, PgMYB1, binds to the promoter of PgNAC21. A transactivation assay in yeast cells revealed that PgNAC21 functions as a transcription activator and that its activation domain is located in its C-terminus. Relative to control plants, Arabidopsis plants overexpressing PgNAC21 exhibited better seed germination, heavier fresh weight and greater root length under salinity stress. Overexpression of PgNAC21 in Arabidopsis plants also enhanced the expression of stress-responsive genes such as GSTF6 (GLUTATHIONE S-TRANSFERASE 6), COR47 (COLD-REGULATED 47) and RD20 (RESPONSIVE TO DEHYDRATION 20). Our data demonstrate that PgNAC21 functions as a stress-responsive NAC TF and can be utilized in transgenic approaches for developing salinity stress tolerance in crop plants.

Possible inhibition of Arabidopsis VIP1-mediated mechanosensory signaling by streptomycin.

VIP1 (VIRE2-INTERACTING PROTEIN 1) and its close homologues are Arabidopsis thaliana bZIP proteins regulating stress responses and root tropisms. They are present in the cytoplasm under steady conditions, but transiently accumulate in the nucleus when cells are exposed to mechanical stress such as hypo-osmotic stress and touch. This pattern of changes in subcellular localization is unique to VIP1 and its close homologues, and can be useful to further characterize mechanical stress signaling in plants. A recent study showed that calcium signaling regulates this pattern of subcellular localization. Here, we show that a possible calcium channel inhibitor, streptomycin, also inhibits the nuclear accumulation of VIP1. Candidates for the specific regulators of the mechanosensitive calcium signaling are further discussed.