No abstract

The BES1/BZR1 transcription factors regulate the expression of brassinosteroid responsive genes and play pivotal roles in plant development. However, the function of BES1/BZR1 regulating kernel development remains unclear. In this study, maize ZmBES1/BZR1-5 is found to positively regulate kernel size. Candidate-gene association analysis showed that four and three SNPs related to ZmBES1/BZR1-5 were significantly associated with kernel width and 100-kernel weight in 513 diverse maize inbred lines, respectively. Overexpression of ZmBES1/BZR1-5 gene in Arabidopsis and rice both significantly increased seed size and weight, as well as smaller kernel produced in maize Mu transposon insertion and EMS mutants. The ZmBES1/BZR1-5 protein contains bHLH and BAM domains, shows no transcriptional activity as monomer but forms homodimer through BAM domain, and locates in nucleus. Chromatin immunoprecipitation sequencing (ChIP-seq), yeast one-hybrid (Y1H) and dual-luciferase assay demonstrate that ZmBES1/BZR1-5 protein binds to the promoter of AP2/EREBP genes (Zm00001d010676 and Zm00001d032077) and inhibits their transcription. cDNA library screening shows that ZmBES1/BZR1-5 interacts with casein kinase II subunit β4 (ZmCKIIβ4) and ferredoxin 2 (ZmFdx2) in vitro and vivo, respectively. Taken together, the study suggests that ZmBES1/BZR1-5 positively regulates kernel size and provides new insights into understanding the mechanism of kernel development 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.

The clade A members of serine/threonine protein phosphatase 2Cs (PP2Cs) play crucial roles in plant growth, development, and stress response via the ABA signaling pathway. But little is known about other PP2C clades in plants. Our previous study showed that maize the ZmPP2C26, a clade B member of ZmPP2Cs, negatively regulated drought tolerance in transgenic Arabidopsis. However, the upstream regulatory mechanism of ZmPP2C26 remains unclear. In the present study, the expression of ZmPP2C26 gene in maize was analyzed by quantitative real time PCR (qRT-PCR). The results showed that the expression of ZmPP2C26 in shoot and root was both significantly inhibited by drought stress. Subsequently, a 2175 bp promoter of ZmPP2C26 was isolated from maize genome (P 2175 ). To validate whether the promoter possess some key cis-element and negatively drive ZmPP2C26 expression in drought stress, three 5-deletion fragments of 1505, 1084 and 215 bp was amplified from P 2175 and were fused to b-glucuronidase (GUS) and luciferase gene (LUC) to produce promoter::GUS and promoter::LUC constructs, and transformed into tobacco, respectively. Transient expression assays indicated that all promoters could drive GUS and LUC expression. The GUS and LUC activity were both significantly inhibited by PEG-6000 treatment. Notably, the -1084 to -215 bp promoter possess one MBS element and inhibits the expression of GUS and LUC under drought stress. Meanwhile, we found that the 215 bp length is enough to drive ZmPP2C26 expression. These findings will provide insights into understanding the transcription-regulatory mechanism of ZmPP2C26 negatively regulating drought tolerance.

To avoid the unregulated overexpression of the exogenous genes, specific or inducible expression is necessary for some exogenous genes in transgenic plants. But little is known about organ-or tissue-specific promoters in maize. In the present study, the expression of a maize pentatricopeptide repeat (PPR) protein encoding gene, GRMZM2G129783, was analyzed by RNA-sequencing data and confirmed by quantitative real time PCR. The results showed that the PPR GRMZM2G129783 gene specifically expressed in vegetative organs. Consequently, a 1830 bp sequence upstream of the start codon of the promoter for GRMZM2G129783 gene was isolated from maize genome (P 1830 ). To validate whether the promoter possesses the vegetative organ-specificity, the full-length and three 5 0 -end deletion fragments of P 1830 of different length (1387, 437, and 146 bp) were fused with glucuronidase (GUS) gene to generate promoter::GUS constructs and transformed into tobacco. The transient expression and fluorometric GUS assay in transgenic tobacco showed that all promoter could drive the expression of the GUS gene, the -437 to -146 bp region possessed some crucial elements for root-specific expression, and the shortest and optimal sequence to maintain transcription activity was 146 and 437 bp in length, respectively. These results indicate that the promoter of the PPR GRMZM2G129783 gene is a vegetative organ-specific promoter and will be useful in transgenic modification of commercial crops for moderate specific expression after further evaluation in monocotyledons.