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.

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.

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.

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 miR167deficient 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.

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.

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.

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BackgroundShort 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.ResultsHere, 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.ConclusionsThese findings suggest that low auxin levels mediated by BR2/PGP1 in the intercalary meristem region are crucial for internode elongation.