The APETALA2/Ethylene-Responsive Transcriptional Factors containing conservative AP2/ERF domains constituted a plant-specific transcription factor (TF) superfamily, called AP2/ERF. The configuration of the AP2/ERF superfamily in maize has remained unresolved. In this study, we identified the 229 AP2/ERF genes in the latest (B73 RefGen_v5) maize reference genome. Phylogenetic classification of the ZmAP2/ERF family members categorized it into five clades, including 27 AP2 (APETALA2), 5 RAV (Related to ABI3/VP), 89 DREB (dehydration responsive element binding), 105 ERF (ethylene responsive factors), and a soloist. The duplication events of the paralogous genes occurred from 1.724–25.855 MYA, a key route to maize evolution. Structural analysis reveals that they have more introns and few exons. The results showed that 32 ZmAP2/ERFs regulate biotic stresses, and 24 ZmAP2/ERFs are involved in responses towards abiotic stresses. Additionally, the expression analysis showed that DREB family members are involved in plant sex determination. The real-time quantitative expression profiling of ZmAP2/ERFs in the leaves of the maize inbred line B73 under ABA, JA, salt, drought, heat, and wounding stress revealed their specific expression patterns. Conclusively, this study unveiled the evolutionary pathway of ZmAP2/ERFs and its essential role in stress and developmental processes. The generated information will be useful for stress resilience maize breeding programs.

Jasmonates (JAs) together with jasmonic acid and its offshoots are lipid-derived endogenous hormones that play key roles in both developmental processes and different defense responses in plants. JAs have been studied intensively in the past decades for their substantial roles in plant defense comebacks against diverse environmental stresses among model plants. However, the role of this phytohormone has been poorly investigated in the monocotyledonous species against abiotic stresses. In this study, a JA biosynthesis mutant opr7opr8 was used for the investigation of JA roles in the salt stress responses of maize seedlings, whose roots were exposed to 0 to 300 mM NaCl. Foliar stomatal observation showed that opr7opr8 had a larger stomatal aperture than wild type (WT) (B73) under salinity stress, indicating that JA positively regulates guard cell movement under salt stress. The results regarding chlorophyll content and leaf senescence showed that opr7opr8 exhibited delayed leaf senescence under salt stress as compared to WT, indicating that JA plays a role in salt-inducing cell death and subsequent leaf senescence. Moreover, the morphological parameters, including the length of the shoots and roots, and the fresh and dry weights of the shoots and roots, showed that after 7 days of salt treatment, opr7opr8 had heavier and longer shoots than WT but slighter and shorter roots than WT. In addition, ion analysis showed that opr7opr8 accumulated less sodium but more potassium in the leaves than WT but more sodium and less potassium in the roots than WT, suggesting that JA deficiency causes higher salt stress to the roots but less stress to the leaves of the seedlings. Reactive oxygen species (ROS) analysis showed that opr7opr8 produced less H₂O₂ than WT in the leaves but more H₂O₂ in the roots under salt treatment, and correspondingly, ROS-scavenging enzymes superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) showed a similar variation, i.e., opr7opr8 has lower enzymatic activities in the shoots but higher activities in the roots than WT under salt treatment. For osmotic adjustment, opr7opr8 produced less proline in the shoots at 100 and 300 mM NaCl treatments but more in the roots than the WT roots under all salt treatments. In addition, the gene expression for abscisic acid (ABA) biosynthesis under salt stress was investigated. Results showed that the expression levels of four key enzymes of ABA biosynthesis, ZEP1, NCED5, AO1, and VP10, were significantly downregulated in the shoots as compared to WT under salt treatment. Putting all the data together, we concluded that JA-deficiency in maize seedlings reduced the salt-stress responses in the shoots but exaggerated the responses in the roots. In addition, endogenous JA acted as a positive regulator for the transportation of sodium ions from the roots to the shoots because the mutant opr7opr8 had a higher level of sodium in the roots but a significantly lower level in the shoots than WT. Furthermore, JA may act as a positive regulator for ABA biosynthesis in the leaves under salt stress.

Jasmonates (JAs) play an important role in many developmental processes, such as root growth, leaf senescence, male fertility, and defense responses against insects and pathogens. The F-box protein COI1, which plays a central role in JA signal transduction, perceives the JA signal and is required for all the JA-mediated defense responses against biotic and abiotic stresses. JA signaling elements including COI1 have been extensively investigated in Arabidopsise. However, the elements of the JA signaling pathway in maize are largely unknown. In this study, we identified four F-box protein genes from the maize genome, which share high homology with AtCOI1, designated as ZmCOI1a, ZmCOI1b, ZmCOI1c, and ZmCOI2, collectively ZmCOIs. To test whether or not the homologous genes of maize are functionally conservative in JA signaling, we over-expressed ZmCOIs in the Arabidopsis coi1-1 mutant. The results showed that over-expression of ZmCOI1a, ZmCOI1b or ZmCOI1c in the coi1-1 mutant resulted in the restoration of male fertility, indicating successful complementation of coi1-1 sterility by ZmCOI1a, ZmCOI1b, and ZmCOI1c. However, ZmCOI2 was not able to restore male fertility of the mutant, indicating that ZmCOI2 has a function diverged from JA signaling. Furthermore, over-expression of the ZmCOI1a, ZmCOI1b, and ZmCOI1c genes, except ZmCOI2, which, in the coi1-1 mutant, caused restoration of resistance to the leaf pathogen Botrytis cinerea and the soil-borne pathogen Pythium aristosporum. In addition, a set of JA-dependent genes are highly induced by wounding in the transformants of ZmCOIa, ZmCOI1b, and ZmCOI1c, but not inducible in transformants of ZmCOI2 or in the coi1-1 mutant, indicating that ZmCOIa, ZmCOI1b, and ZmCOI1c, except ZmCOI2, which can compensate coi1-1 mutation of Arabidopsis for the stress defense response. Putting all the data together, our results suggested that ZmCOIa, ZmCOI1b, and ZmCOI1c, but not ZmCOI2, act as AtCOI1 orthologues in maize for JA signal transduction.

The TIFY gene family is a plant-specific gene family encoding a group of proteins characterized by its namesake, the conservative TIFY domain and members can be organized into four subfamilies: ZML, TIFY, PPD and JAZ (Jasmonate ZIM-domain protein) by presence of additional conserved domains. The TIFY gene family is intensively explored in several model and agriculturally important crop species and here, yet the composition of the TIFY family of maize has remained unresolved. This study increases the number of maize TIFY family members known by 40%, bringing the total to 47 including 38 JAZ, 5 TIFY, and 4 ZML genes. The majority of the newly identified genes were belonging to the JAZ subfamily, six of which had aberrant TIFY domains, suggesting loss JAZ-JAZ or JAZ-NINJA interactions. Six JAZ genes were found to have truncated Jas domain or an altered degron motif, suggesting resistance to classical JAZ degradation. In addition, seven membranes were found to have an LxLxL-type EAR motif which allows them to recruit TPL/TPP co-repressors directly without association to NINJA. Expression analysis revealed that ZmJAZ14 was specifically expressed in the seeds and ZmJAZ19 and 22 in the anthers, while the majority of other ZmJAZs were generally highly expressed across diverse tissue types. Additionally, ZmJAZ genes were highly responsive to wounding and JA treatment. This study provides a comprehensive update of the maize TIFY/JAZ gene family paving the way for functional, physiological, and ecological analysis.

Recently, crop breeders have widely adopted a new biotechnology-based process, termed Seed Production Technology (SPT), to produce hybrid varieties. The SPT does not produce nuclear male-sterile lines, and instead utilizes transgenic SPT maintainer lines to pollinate male-sterile plants for propagation of nuclear-recessive male-sterile lines. A late-stage pollen-specific promoter is an essential component of the pollen-inactivating cassette used by the SPT maintainers. While a number of plant pollen-specific promoters have been reported so far, their usefulness in SPT has remained limited. To increase the repertoire of pollen-specific promoters for the maize community, we conducted a comprehensive comparative analysis of transcriptome profiles of mature pollen and mature anthers against other tissue types. We found that maize pollen has much less expressed genes (>1 FPKM) than other tissue types, but the pollen grain has a large set of distinct genes, called pollen-specific genes, which are exclusively or much higher (100 folds) expressed in pollen than other tissue types. Utilizing transcript abundance and correlation coefficient analysis, 1215 mature pollen-specific (MPS) genes and 1009 mature anther-specific (MAS) genes were identified in B73 transcriptome. These two gene sets had similar GO term and KEGG pathway enrichment patterns, indicating that their members share similar functions in the maize reproductive process. Of the genes, 623 were shared between the two sets, called mature anther- and pollen-specific (MAPS) genes, which represent the late-stage pollen-specific genes of the maize genome. Functional annotation analysis of MAPS showed that 447 MAPS genes (71.7% of MAPS) belonged to genes encoding pollen allergen protein. Their 2-kb promoters were analyzed for cis-element enrichment and six well-known pollen-specific cis-elements (AGAAA, TCCACCA, TGTGGTT, [TA]AAAG, AAATGA, and TTTCT) were found highly enriched in the promoters of MAPS. Interestingly, JA-responsive cis-element GCC box (GCCGCC) and ABA-responsive cis-element-coupling element1 (ABRE-CE1, CCACC) were also found enriched in the MAPS promoters, indicating that JA and ABA signaling likely regulate pollen-specific MAPS expression. This study describes a robust and straightforward pipeline to discover pollen-specific promotes from publicly available data while providing maize breeders and the maize industry a number of late-stage (mature) pollen-specific promoters for use in SPT for hybrid breeding and seed production.

The doubled haploid (DH) technology is employed worldwide in various crop-breeding programs, especially maize. Still, restoring tassel fertility is measured as one of the major restrictive factors in producing DH lines. Colchicine, nitrous oxide, oryzalin, and amiprophosmethyl are common chromosome-doubling agents that aid in developing viable diploids (2n) from sterile haploids (n). Although colchicine is the most widely used polyploidy-inducing agent, it is highly toxic to mammals and plants. Therefore, there is a dire need to explore natural, non-toxic, or low-toxic cheaper and accessible substitutes with a higher survival and fertility rate. To the best of our knowledge, the advanced usage of human anticancer drugs “Paclitaxel (PTX)” and “Caffeine–Taurine (CAF–T)” for in vivo maize haploids doubling is being disclosed for the first time. These two antimitotic and antimicrotubular agents (PTX and CAF–T) were assessed under various treatment conditions compared to colchicine. As a result, the maximum actual doubling rates (ADR) for PTX versus colchicine in maize haploid seedlings were 42.1% (400 M, 16 h treatment) versus 31.9% (0.5 mM, 24 h treatment), respectively. In addition, the ADR in maize haploid seeds were CAF–T 20.0% (caffeine 2 g/L + taurine 12 g/L, 16 h), PTX 19.9% (100 μM, 24 h treatment), and colchicine 26.0% (2.0 mM, 8 h treatment). Moreover, the morphological and physiological by-effects in haploid plants by PTX were significantly lower than colchicine. Hence, PTX and CAF–T are better alternatives than the widely used traditional colchicine to improve chromosome-doubling in maize crop.