Blue light receptor CRY1 regulates HSFA1d nuclear localization to promote plant thermotolerance.

Temperature increases as light intensity rises, but whether light signals can be directly linked to high temperature response in plants is unclear. Here, we find that light pre-treatment enables plants to survive better under high temperature, designated as light-induced thermotolerance (LIT). With short-term light treatment, plants induce light-signaling pathway genes and heat shock genes. Blue light photoreceptor cryptochrome 1 (CRY1) is required for LIT. We also find that CRY1 physically interacts with the heat shock transcription factor A1d (HsfA1d) and that HsfA1d is involved in thermotolerance under light treatment. Furthermore, CRY1 promotes HsfA1d nuclear localization through importin alpha 1 (IMPα1). Consistent with this, CRY1 shares more than half of the chromatin binding sites with HsfA1d. Mutation of CRY1 (cry1-304) diminishes a large number of HsfA1d binding sites that are shared with CRY1. We present a model where, by coupling light sensing to high-temperature stress, CRY1 confers thermotolerance in plants via HsfA1d.

Abscisic acid alleviates mercury toxicity in wheat (Triticum aestivum L.) by promoting cell wall formation.

Mercury (Hg) is a heavy metal (HM) that affects crop growth and productivity. In a previous study, we found that application of exogenous abscisic acid (ABA) alleviated growth inhibition in Hg-stressed wheat seedlings. However, the physiological and molecular mechanisms underlying ABA-mediated Hg detoxification remained unclear. In this study, Hg exposure reduced the plant fresh and dry weights and root numbers. Exogenous ABA treatment significantly resumed the plant growth, increased the plant height and weight, and enriched the roots numbers and biomass. The application of ABA enhanced Hg absorption and raised the Hg levels in the roots. In addition, exogenous ABA decreased Hg-induced oxidative damage and significantly brought down the activities of antioxidant enzymes, such as SOD, POD and CAT. Global gene expression patterns in the roots and leaves exposed to HgCl2 and ABA treatments were examined via RNA-Seq. The data showed that genes related to ABA-mediated Hg detoxification were enriched in functions related to cell wall formation. Weighted gene co-expression network analysis (WGCNA) further indicated that the genes implicated in Hg detoxification were related to cell wall synthesis. Under Hg stress, ABA significantly induced expression of the genes encoding cell wall synthesis enzymes, regulated the activity of hydrolase, and increased the concentrations of cellulose and hemicellulose, hence promoting cell wall synthesis. Taken together, these results suggest that exogenous ABA could alleviate Hg toxicity in wheat by promoting cell wall formation and suppressing translocation of Hg from roots to shoots.

HY5-HDA9 orchestrates the transcription of HsfA2 to modulate salt stress response in Arabidopsis.

Integration of light signaling and diverse abiotic stress responses contribute to plant survival in a changing environment. Some reports have indicated that light signals contribute a plant's ability to deal with heat, cold, and stress. However, the molecular link between light signaling and the salt-response pathways remains unclear. We demonstrate here that increasing light intensity elevates the salt stress tolerance of plants. Depletion of HY5, a key component of light signaling, causes Arabidopsis thaliana to become salinity sensitive. Interestingly, the small heat shock protein (sHsp) family genes are upregulated in hy5-215 mutant plants, and HsfA2 is commonly involved in the regulation of these sHsps. We found that HY5 directly binds to the G-box motifs in the HsfA2 promoter, with the cooperation of HISTONE DEACETYLASE 9 (HDA9), to repress its expression. Furthermore, the accumulation of HDA9 and the interaction between HY5 and HDA9 are significantly enhanced by salt stress. On the contrary, high temperature triggers HY5 and HDA9 degradation, which leads to dissociation of HY5-HDA9 from the HsfA2 promoter, thereby reducing salt tolerance. Under salt and heat stress conditions, fine tuning of protein accumulation and an interaction between HY5 and HDA9 regulate HsfA2 expression. This implies that HY5, HDA9, and HsfA2 play important roles in the integration of light signaling with salt stress and heat shock response.

PIF4 Promotes Expression of HSFA2 to Enhance Basal Thermotolerance in Arabidopsis.

Heat stress (HS) seriously restricts the growth and development of plants. When plants are exposed to extreme high temperature, the heat stress response (HSR) is activated to enable plants to survive. Sessile plants have evolved multiple strategies to sense and cope with HS. Previous studies have established that PHYTOCHROME INTERACTING FACTOR 4 (PIF4) acts as a key component in thermomorphogenesis; however, whether PIF4 regulates plant thermotolerance and the molecular mechanism linking this light transcriptional factor and HSR remain unclear. Here, we show that the overexpression of PIF4 indeed provides plants with a stronger basal thermotolerance and greatly improves the survival ability of Arabidopsis under severe HS. Via phylogenetic analysis, we identified two sets (six) of PIF4 homologs in wheat, and the expression patterns of the PIF4 homologs were conservatively induced by heat treatment in both wheat and Arabidopsis. Furthermore, the PIF4 protein was accumulated under heat stress and had an identical expression level. Additionally, we found that the core regulator of HSR, HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), was highly responsive to light and heat. Followed by promoter analysis and ChIP-qPCR, we further found that PIF4 can bind directly to the G-box motifs of the HSFA2 promoter. Via effector-reporter assays, we found that PIF4 binding could activate HSFA2 gene expression, thereby resulting in the activation of other HS-inducible genes, such as heat shock proteins. Finally, the overexpression of PIF4 led to a stronger basal thermotolerance under non-heat-treatment conditions, thereby resulting in an enhanced tolerance to severe heat stress. Taken together, our findings propose that PIF4 is linked to heat stress signaling by directly binding to the HSFA2 promoter and triggering the HSR at normal temperature conditions to promote the basal thermotolerance. These functions of PIF4 provide a candidate direction for breeding heat-resistant crop cultivars.

ZmPP2C26 Alternative Splicing Variants Negatively Regulate Drought Tolerance in Maize.

Serine/threonine protein phosphatase 2C (PP2C) dephosphorylates proteins and plays crucial roles in plant growth, development, and stress response. In this study, we characterized a clade B member of maize PP2C family, i.e., ZmPP2C26, that negatively regulated drought tolerance by dephosphorylating ZmMAPK3 and ZmMAPK7 in maize. The ZmPP2C26 gene generated ZmPP2C26L and ZmPP2C26S isoforms through untypical alternative splicing. ZmPP2C26S lost 71 amino acids including an MAPK interaction motif and showed higher phosphatase activity than ZmPP2C26L. ZmPP2C26L directly interacted with, dephosphorylated ZmMAPK3 and ZmMAPK7, and localized in chloroplast and nucleus, but ZmPP2C26S only dephosphorylated ZmMAPK3 and localized in cytosol and nucleus. The expression of ZmPP2C26L and ZmPP2C26 was significantly inhibited by drought stress. Meanwhile, the maize zmpp2c26 mutant exhibited enhancement of drought tolerance with higher root length, root weight, chlorophyll content, and photosynthetic rate compared with wild type. However, overexpression of ZmPP2C26L and ZmPP2C26S significantly decreased drought tolerance in Arabidopsis and rice with lower root length, chlorophyll content, and photosynthetic rate. Phosphoproteomic analysis revealed that the ZmPP2C26 protein also altered phosphorylation level of proteins involved in photosynthesis. This study provides insights into understanding the mechanism of PP2C in response to abiotic stress.

Catalase (CAT) Gene Family in Wheat (Triticum aestivum L.): Evolution, Expression Pattern and Function Analysis.

Catalases (CATs) are present in almost all living organisms and play important roles in plant development and response to various stresses. However, there is relatively little information on CAT genes in wheat and related Triticeae species. A few studies on CAT family genes in wheat have been reported. In this study, ten CAT proteins (TaCATs) were identified in wheat and classified into three groups based on their phylogenetic features and sequence analysis. The analysis of the structure and motif composition of the TaCAT proteins suggested that a segmental duplication event occurred in the TaCAT gene family. Collinearity relationship analysis among different species showed that there were three orthologous CAT genes in rice and in maize. By analyzing the cis-elements in the promoter regions, we speculated that TaCAT genes expression might be regulated by light, oxygen deficit, methyl jasmonate and abscisic acid, and by transcription factors such as MYB. A Gene Ontology (GO)-based analysis showed that TaCAT proteins may be related to the response to various stresses, are cytoplasm localized, and may function as antioxidant enzymes. RT-qPCR and transcriptome data analyses exhibited distinct expression patterns of TaCAT genes in different tissues and in response to various treatments. In this study, a comprehensive analysis of wheat CAT genes was performed, enriching our knowledge of CAT genes and providing a foundation for further functional analyses of this gene family in wheat.

Phylogenetic and expression analyses of HSF gene families in wheat (Triticum aestivum L.) and characterization of TaHSFB4-2B under abiotic stress.

The heat shock transcription factors (HSFs) family is widely present in eukaryotes including plants. Recent studies have indicated that HSF is a multifunctional group of genes involved in plant growth and development, as well as response to abiotic stresses. Here we combined the bioinformatic, molecular biology way to dissect the function of Hsf, specifically HsfB4 in wheat under abiotic stresses. In this study, we identified 78 TaHSF genes in wheat (Triticum aestivum) and analyzed their phylogenetic relationship and expression regulation motifs. Next, the expression profiles of TaHSFs and AtHSFs were analyzed in different tissues as well as in response to abiotic stress. Furthermore, to explore the role of HSFB4 in abiotic stress response, we cloned TaHSFB4-2B from the wheat variety, Chinese Spring. Subcellular localization analysis showed that TaHSFB4-2B was localized in the nucleus. In addition, We observed TaHSFB4-2B was highly expressed in the root and stem, its transcription was induced under long-term heat shock, cold, and salinity stress. Additionally, overexpression of TaHSFB4-2B suppressed seed germination and growth in Arabidopsis with salinity and mannitol treatment. It also modulated the expression of stress-responsive genes, including AtHSP17.8, AtHSP17.6A, AtHSP17.6C, CAT2, and SOS1, under both normal and stress conditions. From these finding, we propose that TaHSFB4-2B act as a negative regulator of abiotic stress response in the plant.

Maize brachytic2 (br2) suppresses the elongation of lower internodes for excessive auxin accumulation in the intercalary meristem region.

BACKGROUND: Short internodes contribute to plant dwarfism, which is exceedingly beneficial for crop production. However, the underlying mechanisms of internode elongation are complicated and have been not fully understood. RESULTS: Here, we report a maize dwarf mutant, dwarf2014 (d2014), which displays shortened lower internodes. Map-based cloning revealed that the d2014 gene is a novel br2 allele with a splicing variation, resulting in a higher expression of BR2-T02 instead of normal BR2-T01. Then, we found that the internode elongation in d2014/br2 exhibited a pattern of inhibition-normality-inhibition (transient for the ear-internode), correspondingly, at the 6-leaf, 12-leaf and 14-leaf stages. Indeed, BR2 encodes a P-glycoprotein1 (PGP1) protein that functions in auxin efflux, and our in situ hybridization assay showed that BR2 was mainly expressed in vascular bundles of the node and internode. Furthermore, significantly higher auxin concentration was detected in the stem apex of d2014 at the 6-leaf stage and strictly in the node region for the ear-internode at the 14-leaf stage. In such context, we propose that BR2/PGP1 transports auxin from node to internode through the vascular bundles, and excessive auxin accumulation in the node (immediately next to the intercalary meristem) region suppresses internode elongation of d2014. CONCLUSIONS: These findings suggest that low auxin levels mediated by BR2/PGP1 in the intercalary meristem region are crucial for internode elongation.

miR167 limits anther growth to potentiate anther dehiscence.

In flowering plants, anther dehiscence and pollen release are essential for sexual reproduction. Anthers dehisce after cell wall degradation weakens stomium cell junctions in each anther locule, and desiccation creates mechanical forces that open the locules. Either effect or both together may break stomium cell junctions. The microRNA miR167 negatively regulates ARF6 and ARF8, which encode auxin response transcription factors. Arabidopsis mARF6 or mARF8 plants with mutated miR167 target sites have defective anther dehiscence and ovule development. Null mir167a mutations recapitulated mARF6 and mARF8 anther and ovule phenotypes, indicating that MIR167a is the main miR167 precursor gene that delimits ARF6 and ARF8 expression in these organs. Anthers of mir167a or mARF6/8 plants overexpressed genes encoding cell wall loosening functions associated with cell expansion, and grew larger than wild-type anthers did starting at flower stage 11. Experimental desiccation enabled dehiscence of miR167-deficient anthers, indicating competence to dehisce. Conversely, high humidity conditions delayed anther dehiscence in wild-type flowers. These results support a model in which miR167-mediated anther growth arrest permits anther dehiscence. Without miR167 regulation, excess anther growth delays dehiscence by prolonging desiccation.

The miR164-dependent regulatory pathway in developing maize seed.

MicroRNA164 (miR164) plays a key role in leaf and flower development, lateral root initiation, and stress responses. However, little is known about the regulatory roles of miR164 during seed development, particularly in maize. The aim of this study was to discover the developmental function of miR164 in maize seed. Small RNA sequencing (sRNA-seq) was performed at two key stages. The results indicated that miR164 was down-regulated during maize seed development. In addition, degradome library sequencing and transient expression assays identified the target genes for miR164. Two microRNA (miRNA) pairs, miR164-NAM, ATAF, and CUC32 (NAC32) and miR164-NAC40, were isolated. The developmental function of miR164 was determined by analyzing the differentially expressed genes (DEGs) between the wild-type and miR164 transgenic lines using RNA sequencing (RNA-seq) and by screening the DEGs related to NAC32 and NAC40 via co-expression and transient expression analysis. These results identified two beta-expansin genes, EXPB14 and EXPB15, which were located downstream of the NAC32 and NAC40 genes. This study revealed, for the first time, a miR164-dependent regulatory pathway, miR164-NAC32/NAC40-EXPB14/EXPB15, which participates in maize seed expansion. These findings highlight the significance of miR164 in maize seed development, and can be used to explore the role of miRNA in seed development.

Identification and characterization of microRNAs in maize endosperm response to exogenous sucrose using small RNA sequencing.

Sucrose acts as a signaling molecule for genes critical to starch biosynthesis in maize endosperm. Previously, we showed that sucrose could regulate starch biosynthesis in maize via transcription factors. To better understand the complex regulation of starch biosynthesis, the 10days after pollination endosperm from Zea mays L. B73 inbred line was collected and treated with sucrose for small RNA sequencing. The sequencing results revealed that 24 known miRNAs and 190 novel miRNAs were significantly differentially expressed in response to sucrose. In addition, most of target mRNAs were characterized as transcription factors, mainly including, MYB, ARF, NAC, AP2/ERF, WRKY, and GRAS, which play important roles in starch biosynthesis and seed development in maize endosperm. The expression profiles of miR398a/b and miR159b/j/k followed opposite expression trends to their target genes when analyzed by qPCR. In conclusion, these results show that sucrose regulates the expression of starch synthetic genes through miRNAs.

Sucrose and ABA regulate starch biosynthesis in maize through a novel transcription factor, ZmEREB156.

Sucrose is not only the carbon source for starch synthesis, but also a signal molecule. Alone or in coordination with ABA, it can regulate the expression of genes involved in starch synthesis. To investigate the molecular mechanisms underlying this effect, maize endosperms were collected from Zea mays L. B73 inbred line 10 d after pollination and treated with sucrose, ABA, or sucrose plus ABA at 28 °C in the dark for 24 h. RNA-sequence analysis of the maize endosperm transcriptome revealed 47 candidate transcription factors among the differentially expressed genes. We therefore speculate that starch synthetic gene expression is regulated by transcription factors induced by the combination of sucrose and ABA. ZmEREB156, a candidate transcription factor, is induced by sucrose plus ABA and is involved in starch biosynthesis. The ZmEREB156-GFP-fused protein was localized in the nuclei of onion epidermal cells, and ZmEREB156 protein possessed strong transcriptional activation activity. Promoter activity of the starch-related genes Zmsh2 and ZmSSIIIa increased after overexpression of ZmEREB156 in maize endosperm. ZmEREB156 could bind to the ZmSSIIIa promoter but not the Zmsh2 promoter in a yeast one-hybrid system. Thus, ZmEREB156 positively modulates starch biosynthetic gene ZmSSIIIa via the synergistic effect of sucrose and ABA.

ZmbZIP91 regulates expression of starch synthesis-related genes by binding to ACTCAT elements in their promoters.

Starch synthesis is a key process that influences crop yield and quality, though little is known about the regulation of this complex metabolic pathway. Here, we present the identification of ZmbZIP91 as a candidate regulator of starch synthesis via co-expression analysis in maize (Zea mays L.). ZmbZIP91 was strongly associated with the expression of starch synthesis genes. Reverse tanscription-PCR (RT-PCR) and RNA in situ hybridization indicated that ZmbZIP91 is highly expressed in maize endosperm, with less expression in leaves. Particle bombardment-mediated transient expression in maize endosperm and leaf protoplasts demonstrated that ZmbZIP91 could positively regulate the expression of starch synthesis genes in both leaves and endosperm. Additionally, the Arabidopsis mutant vip1 carried a mutation in a gene (VIP1) that is homologous to ZmbZIP91, displayed altered growth with less starch in leaves, and ZmbZIP91 was able to complement this phenotype, resulting in normal starch synthesis. A yeast one-hybrid experiment and EMSAs showed that ZmbZIP91 could directly bind to ACTCAT elements in the promoters of starch synthesis genes (pAGPS1, pSSI, pSSIIIa, and pISA1). These results demonstrate that ZmbZIP91 acts as a core regulatory factor in starch synthesis by binding to ACTCAT elements in the promoters of starch synthesis genes.