Summary: Over the past two decades, Zea mays (maize) has been established as a model system for the study of indirect plant defense against herbivores. When attacked by lepidopteran larvae, maize leaves emit a complex blend of volatiles, mainly composed of sesquiterpenes, to attract the natural enemies of the herbivores. This is associated with a swift transcriptional induction of terpene synthases such as TPS10; however, the molecular components controlling the complex transcriptional reprogramming in this process are still obscure. Here, by exploiting the finding that the maize TPS10 promoter retained its full responsiveness to herbivory in Arabidopsis, we identified the region from −300 to −200 of the TPS10 promoter as both necessary and sufficient for its herbivore inducibility through 5′ deletion mapping. A high‐throughput screening of an Arabidopsis transcription factor library using this promoter region as the bait identified seven AP2/ERF family transcription factors. Among their close homologs in maize, EREB58 was the only gene responsive to herbivory, with a spatiotemporal expression pattern highly similar to that of TPS10. Meanwhile, EREB58 was also responsive to Jasmonate. In vivo and in vitro assays indicated that EREB58 promotes TPS10 expression by directly binding to the GCC‐box within the region from −300 to −200 of the TPS10 promoter. Transgenic maize plants overexpressing EREB58 constitutively over‐accumulate TPS10 transcript, and also (E)‐β‐farnesene and (E)‐α‐bergamotene, two major sesquiterpenes produced by TPS10. In contrast, jasmonate induction of TPS10 and its volatiles was abolished in EREB58‐RNAi transgenic lines. In sum, these results demonstrate that EREB58 is a positive regulator of sesquiterpene production by directly promoting TPS10 expression.

It has long been recognized that stomatal movement modulates CO₂ availability and as a consequence the photosynthetic rate of plants, and that this process is feedback-regulated by photoassimilates. However, the genetic components and mechanisms underlying this regulatory loop remain poorly understood, especially in monocot crop species. Here, we report the cloning and functional characterization of a maize (Zea mays) mutant named closed stomata1 (cst1). Map-based cloning of cst1 followed by confirmation with the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 system identified the causal mutation in a Clade I Sugars Will Eventually be Exported Transporters (SWEET) family gene, which leads to the E81K mutation in the CST1 protein. CST1 encodes a functional glucose transporter expressed in subsidiary cells, and the E81K mutation strongly impairs the oligomerization and glucose transporter activity of CST1. Mutation of CST1 results in reduced stomatal opening, carbon starvation, and early senescence in leaves, suggesting that CST1 functions as a positive regulator of stomatal opening. Moreover, CST1 expression is induced by carbon starvation and suppressed by photoassimilate accumulation. Our study thus defines CST1 as a missing link in the feedback-regulation of stomatal movement and photosynthesis by photoassimilates in maize.

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.

Optimization of shade avoidance response (SAR) is crucial for enhancing crop yield in high-density planting conditions in modern agriculture, but a comprehensive study of the regulatory network of SAR is still lacking in monocot crops. In this study, the genome-wide early responses in maize seedlings to the simulated shade (low red/far-red ratio) and also to far-red light treatment were transcriptionally profiled. The two processes were predominantly mediated by phytochrome B and phytochrome A, respectively. Clustering of differentially transcribed genes (DTGs) along with functional enrichment analysis identified important biological processes regulated in response to both treatments. Co-expression network analysis identified two transcription factor modules as potentially pivotal regulators of SAR and de-etiolation, respectively. A comprehensive cross-species comparison of orthologous DTG pairs between maize and Arabidopsis in SAR was also conducted, with emphasis on regulatory circuits controlling accelerated flowering and elongated growth, two physiological hallmarks of SAR. Moreover, it was found that the genome-wide distribution of DTGs in SAR and de-etiolation both biased toward the maize1 subgenome, and this was associated with differential retention of various cis-elements between the two subgenomes. The results provide the first transcriptional picture for the early dynamics of maize phytochrome signaling. Candidate genes with regulatory functions involved in maize shade avoidance response have been identified, offering a starting point for further functional genomics investigation of maize adaptation to heavily shaded field conditions.

Chewing insects cause severe yield losses in crop production worldwide. Crop plants counteract chewing insects by transcriptionally promoting a repertoire of defense gene products that are either toxic to, or attractive to the natural enemies of, pest insects. However, the complexity of the transcriptional reprogramming in plant defense response against chewing insects is still not well understood. In this study, the genome-wide early responses in maize seedlings to Asian corn borer (ACB, Ostrinia furnacalis) and also to jasmonic acid(JA), the pivotal phytohormone controlling plant defense response against herbivory, were transcriptionally profiled by RNA-Seq. Clustering of differentially expressed genes (DEGs) along with functional enrichment analysis revealed important biological processes regulated in response to ACB infestation and/or jasmonic acid. Moreover, DEGs with distinct expression patterns were differentially enriched with diverse families of cis-elements on their promoters. Multiple inventories of differentially expressed transcription factors (DETFs) in each DEG group were also analyzed. A transient expression assay using transfected maize protoplastswas established to examine the potential roles of DETFs in maize defense response and JA signaling, and this was used to show that ZmNAC60, an ACB- and JA-inducible DETF, represented a novel positive regulator of JA and defense pathway genes. This study provided a comprehensive transcriptional picture for the early dynamics of maize defense responses and JA signaling, and the identification of DETFs offered potential targets for further functional genomics investigation of master regulators in maize defense responses against herbivory.

Herbicide tolerance has been the dominant trait introduced during the global commercialization of genetically modified (GM) crops. Herbicide-tolerant crops, especially glyphosate-resistant crops, offer great advantages for weed management; however, despite these benefits, glyphosate-resistant maize (Zea mays L.) has not yet been commercially deployed in China. To develop a new bio-breeding resource for glyphosate-resistant maize, we introduced a codon-optimized glyphosate N-acetyltransferase gene, gat, and the enolpyruvyl-shikimate-3-phosphate synthase gene, gr79-epsps, into the maize variety B104. We selected a genetically stable high glyphosate resistance (GR) transgenic event, designated GG2, from the transgenic maize population through screening with high doses of glyphosate. A molecular analysis demonstrated that single copy of gat and gr79-epsps were integrated into the maize genome, and these two genes were stably transcribed and translated. Field trials showed that the transgenic event GG2 could tolerate 9000 g acid equivalent (a.e.) glyphosate per ha with no effect on phenotype or yield. A gas chromatography-mass spectrometry (GC–MS) analysis revealed that, shortly after glyphosate application, the glyphosate (PMG) and aminomethylphosphonic acid (AMPA) residues in GG2 leaves decreased by more than 90% compared to their levels in HGK60 transgenic plants, which only harbored the epsps gene. Additionally, PMG and its metabolic residues (AMPA and N-acetyl-PMG) were not detected in the silage or seeds of GG2, even when far more than the recommended agricultural dose of glyphosate was applied. The co-expression of gat and gr79-epsps, therefore, confers GG2 with high GR and a low risk of herbicide residue accumulation, making this germplasm a valuable GR event in herbicide-tolerant maize breeding.