No abstract

Previous studies have identified miR169/NF-YA modules are important regulators of plant development and stress responses. Currently, reported genome sequence data offers an opportunity for global characterization of miR169 and NF-YA genes, which may provide insights into the molecular mechanisms of the miR169/NF-YA modules in maize. In our study, fourteen NF-YA transcription factors with conserved domains were identified based on maize genome loci. The miR169 gene family has 18 members that generate 10 mature products, and 8 of these mature miR169 members could target 7 of 14 ZmNF-YA genes in maize. The seven ZmNF-YA proteins were localized to the nucleus while lacked transcriptional activity. We investigated the expression patterns of the zma-miR169 members and their targeted ZmNF-YA genes in maize roots treated by drought stress (polyethylene glycol, PEG), hormone stress (abscisic acid, ABA), and salt stress (NaCl). The zma-miR169 family members were downregulated in short term (0∼48 h) and generally upregulated over the long term (15 days) in response to the three abiotic stress conditions. Most of the targeted ZmNF-YA genes exhibited a reverse correlation with zma-miR169 gene expression over both the short term and long term. Maize root elongation was promoted by PEG and ABA but repressed by NaCl over the long term. Apparently, ZmNF-YA14 expression perfectly matched the zma-miR169 expression and corresponded to root growth reversely.

SummaryLong hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification-mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down-regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double-stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross-silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA-derived siRNAs were characteristic of Argonaute-binding small RNAs. Our results indicate that RNAi mutant library is a high-efficient approach for genomewide gene identification in plants.

Jasmonate (JA) is an important signaling molecule involved in the regulation of many physiological and stress-related processes in plants. Jasmonate ZIM-domain (JAZ) proteins have been implicated in regulating JA signaling pathways and the cross talk between various phytohormones. Maize is not only an important cereal crop, but also a model plant for monocotyledon studies. Although many JAZ proteins have been characterized in Arabidopsis and rice, few reports have examined the function of JAZ proteins in maize. In this report, we examined the phylogenetic relationship and expression pattern of JAZ family genes in maize. In addition, a tassel and endosperm-specific JAZ gene, ZmJAZ14, was identified using microarray data analysis and real-time RT-PCR, and its expression was induced by polyethylene glycol (PEG), jasmonate (JA), abscisic acid (ABA), and gibberellins (GAs). ZmJAZ14 was shown to be localized in the nucleus and possessed no transcriptional activating activity, suggesting that it functions as a transcriptional regulator. We found that overexpression of ZmJAZ14 in Arabidopsis enhanced plant tolerance to JA and ABA treatment, as well as PEG stress, while it promoted growth under GA stimulus. Moreover, ZmJAZ14 interacted with a subset of transcription factors in Arabidopsis, and the accumulation of several marker genes involved in JA, ABA, and GA signaling pathways were altered in the overexpression lines. These results suggest that ZmJAZ14 may serve as a hub for the cross talk among the JA, ABA, and GA signaling pathways. Our results can be used to further characterize the function of JAZ family proteins in maize, and the gene cloned in this study may serve as a candidate for drought tolerance and growth promotion regulation in maize.

Maize (Zea mays) originated in southern Mexico and has spread over a wide latitudinal range. Maize expansion from tropical to temperate regions has necessitated a reduction of its photoperiod sensitivity. In this study, we cloned a quantitative trait locus (QTL) regulating flowering time in maize and show that the maize ortholog of Arabidopsis thaliana EARLY FLOWERING3, ZmELF3.1, is the causal locus. We demonstrate that ZmELF3.1 and ZmELF3.2 proteins can physically interact with ZmELF4.1/4.2 and ZmLUX1/2, to form evening complex(es) (EC) in the maize circadian clock. Loss-of-function mutants for ZmELF3.1/3.2 and ZmLUX1/2 exhibited delayed flowering under long-day and short-day conditions. We show that EC directly represses the expression of several flowering suppressor genes, such as the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) genes ZmCCT9 and ZmCCT10, ZmCONSTANS-LIKE 3, and the PSEUDORESPONSE REGULATOR (PRR) genes ZmPRR37a and ZmPRR73, thus alleviating their inhibition, allowing florigen gene expression and promoting flowering. Further, we identify two closely linked retrotransposons located in the ZmELF3.1 promoter that regulate the expression levels of ZmELF3.1 and may have been positively selected during post-domestication spread of maize from tropical to temperate regions during the pre-Columbian era. These findings provide insights into circadian clock-mediated regulation of photoperiodic flowering in maize and new targets of genetic improvement for breeding.

Salt stress significantly reduces the productivity of crop plants including maize (Zea mays). miRNAs are major regulators of plant growth and stress responses, but few studies have examined the potential impacts of miRNAs on salt stress responses in maize. Here, we show that ZmmiR169q is responsive to stress-induced ROS signals. After detecting that salt stress and exogenous H2O2 treatment reduced the accumulation of ZmmiR169q, stress assays with transgenic materials showed that depleting ZmmiR169q increased seedling salt tolerance whereas overexpressing ZmmiR169q decreased salt tolerance. Helping explain these observations, we found that ZmmiR169q repressed the transcript abundance of its target NUCLEAR FACTOR YA8 (ZmNF-YA8), and overexpression of ZmNF-YA8 in maize improved salt tolerance, specifically by transcriptionally activating the expression of the efficient antioxidant enzyme PEROXIDASE1. Our study reveals a direct functional link between salt stress and a miR169q-NF-YA8 regulatory module that plants use to manage ROS stress and strongly suggests that ZmNF-YA8 can be harnessed as a resource for developing salt-tolerant crop varieties.

Long non-coding RNAs (lncRNAs) have been reported to be involved in the development of maize plant. However, few focused on seed development of maize. Here, we identified 753 lncRNA candidates in maize genome from six seed samples. Similar to the mRNAs, lncRNAs showed tissue developmental stage specific and differential expression, indicating their putative role in seed development. Increasing evidence shows that crosstalk among RNAs mediated by shared microRNAs (miRNAs) represents a novel layer of gene regulation, which plays important roles in plant development. Functional roles and regulatory mechanisms of lncRNAs as competing endogenous RNAs (ceRNA) in plants, particularly in maize seed development, are unclear. We combined analyses of consistently altered 17 lncRNAs, 840 mRNAs and known miRNA to genome-wide investigate potential lncRNA-mediated ceRNA based on “ceRNA hypothesis”. The results uncovered seven novel lncRNAs as potential functional ceRNAs. Functional analyses based on their competitive coding-gene partners by Gene Ontology (GO) and KEGG biological pathway demonstrated that combined effects of multiple ceRNAs can have major impacts on general developmental and metabolic processes in maize seed. These findings provided a useful platform for uncovering novel mechanisms of maize seed development and may provide opportunities for the functional characterization of individual lncRNA in future studies.

In plants, leaf is crucial for photosynthesis and respiration. Leaf area and quantity are important for leaf vegetables to increase biomass. The process of leaf development involves coordinated regulation among small RNAs, transcription factors and hormones. Here, we found leaf size were regulated by transcription factors NF-YA2 and NF-YA10 in Arabidopsis. NF-YA2 and NF-YA10 overexpression increased biomass accumulation through promoting leaf growth and cell expansion. NF-YA2 and NF-YA10 were expressed in SAM and leaf vasculature. Endogenous IAA content reduced by 20% and 24% in transgenic Arabidopsis plants overexpressing NF-YA2 and NF-YA10 compared to wild-type plants. Chromatin immunoprecipitation assays revealed that NF-YA2 and NF-YA10 bound directly to the cis-element CCAAT in the promoter of the YUC2, and decreased the expression of YUC2, a YUCCA family gene. The auxin transporter gene PIN1 and auxin response factor1 and 2 (ARF1 and ARF2) genes, transcriptional repressors, were downregulated. These findings showed leaf development was regulated by NF-YA2 and NF-YA10 through the auxin-signaling pathway and may provide a new insight into the genetic engineering of vegetables biomass and crop productivity.