Paclitaxel and Caffeine-Taurine, New Colchicine Alternatives for Chromosomes Doubling in Maize Haploid Breeding.

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.

Wide-Range Portrayal of AP2/ERF Transcription Factor Family in Maize (Zea mays L.) Development and Stress Responses.

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.

A Rapid Pipeline for Pollen- and Anther-Specific Gene Discovery Based on Transcriptome Profiling Analysis of Maize Tissues.

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.

An updated census of the maize TIFY family.

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.

Green leaf volatiles and jasmonic acid enhance susceptibility to anthracnose diseases caused by Colletotrichum graminicola in maize.

Colletotrichum graminicola is a hemibiotrophic fungus that causes anthracnose leaf blight (ALB) and anthracnose stalk rot (ASR) in maize. Despite substantial economic losses caused by these diseases, the defence mechanisms against this pathogen remain poorly understood. Several hormones are suggested to aid in defence against C. graminicola, such as jasmonic acid (JA) and salicylic acid (SA), but supporting genetic evidence was not reported. Green leaf volatiles (GLVs) are a group of well-characterized volatiles that induce JA biosynthesis in maize and are known to function in defence against necrotrophic pathogens. Information regarding the role of GLVs and JA in interactions with (hemi)biotrophic pathogens remains limited. To functionally elucidate GLVs and JA in defence against a hemibiotrophic pathogen, we tested GLV- and JA-deficient mutants, lox10 and opr7 opr8, respectively, for resistance to ASR and ALB and profiled jasmonates and SA in their stalks and leaves throughout infection. Both mutants were resistant and generally displayed elevated levels of SA and low amounts of jasmonates, especially at early stages of infection. Pretreatment with GLVs restored susceptibility of lox10 mutants, but not opr7 opr8 mutants, which coincided with complete rescue of JA levels. Exogenous methyl jasmonate restored susceptibility in both mutants when applied before inoculation, whereas methyl salicylate did not induce further resistance in either of the mutants, but did induce mutant-like resistance in the wild type. Collectively, this study reveals that GLVs and JA contribute to maize susceptibility to C. graminicola due to suppression of SA-related defences.

Interruption of Jasmonic Acid Biosynthesis Causes Differential Responses in the Roots and Shoots of Maize Seedlings against Salt Stress.

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.

Comparative analysis of the male inflorescence transcriptome profiles of an ms22 mutant of maize.

In modern agricultural production, maize is the most successful crop utilizing heterosis. 712C-ms22 is an important male sterile material in maize. In this study, we performed transcriptome sequencing analysis of the V10 stage of male inflorescence. Through this analysis, 27.63 million raw reads were obtained, and trimming of the raw data revealed 26.63 million clean reads, with an average match rate of 94.64%. Using Tophat software, we matched these clean reads to the maize reference genome. The abundance of 39,622 genes was measured, and 35,399 genes remained after filtering out the non-expressed genes across all the samples. These genes were classified into 19 categories by clusters of orthologous groups of protein annotation. Transcriptome sequencing analysis of the male sterile and fertile 712C-ms22 maize revealed some key DEGs that may be related to metabolic pathways. qRT-PCR analysis validated the gene expression patterns identified by RNA-seq. This analysis revealed some of the essential genes responsible for pollen development and for pollen tube elongation. Our findings provide useful markers of male sterility and new insights into the global mechanisms mediating male sterility in maize.

Methane protects against polyethylene glycol-induced osmotic stress in maize by improving sugar and ascorbic acid metabolism.

Although aerobic methane (CH4) release from plants leads to an intense scientific and public controversy in the recent years, the potential functions of endogenous CH4 production in plants are still largely unknown. Here, we reported that polyethylene glycol (PEG)-induced osmotic stress significantly increased CH4 production and soluble sugar contents in maize (Zea mays L.) root tissues. These enhancements were more pronounced in the drought stress-tolerant cultivar Zhengdan 958 (ZD958) than in the drought stress-sensitive cultivar Zhongjiangyu No.1 (ZJY1). Exogenously applied 0.65 mM CH4 not only increased endogenous CH4 production, but also decreased the contents of thiobarbituric acid reactive substances. PEG-induced water deficit symptoms, such as decreased biomass and relative water contents in both root and shoot tissues, were also alleviated. These beneficial responses paralleled the increases in the contents of soluble sugar and the reduced ascorbic acid (AsA), and the ratio of AsA/dehydroascorbate (DHA). Further comparison of transcript profiles of some key enzymes in sugar and AsA metabolism suggested that CH4 might participate in sugar signaling, which in turn increased AsA production and recycling. Together, these results suggested that CH4 might function as a gaseous molecule that enhances osmotic stress tolerance in maize by modulating sugar and AsA metabolism.

New perspectives into jasmonate roles in maize.

It is well-known from the model dicotyledonous plants, Arabidopsis and tomato, that jasmonates (JAs) act as defense hormones in planta due to their potent ability to mediate defensive responses against insect/pathogen attacks or harsh environmental conditions. JA is also required for various developmental processes such as male fertility, seed maturation, root extension, and leaf senescence. In our recently published Plant Cell paper, the multiple roles of JA in the monocotyledonous agro-economically important model plant, maize, were investigated by comprehensive analysis of JA-deficient double mutant disrupted in the two oxophytodienoate reductase genes, OPR7 and OPR8. These two genes are the closest orthologs of the Arabidopsis JA-producing OPR3 and are the only maize OPRs required for JA biosynthesis. With this mutant, we previously showed that JA is essential for both male and female reproductive development, and required for the regulation of brace root pigmentation, leaf senescence, and defense against oomycete Pythium aristosporum, and beet armyworm (Spodoptera exigua). In this addendum, we expanded the investigation into the function of JA in elongation of sheaths, leaves, and roots, and its involvement in photomorphogenesis of seedlings.

Characterization of genetic diversity and linkage disequilibrium of ZmLOX4 and ZmLOX5 loci in maize.

Maize (Zea mays L.) lipoxygenases (ZmLOXs) are well recognized as important players in plant defense against pathogens, especially in cross kingdom lipid communication with pathogenic fungi. This study is among the first to investigate genetic diversity at important gene paralogs ZmLOX4 and ZmLOX5. Sequencing of these genes in 400 diverse maize lines showed little genetic diversity and low linkage disequilibrium in the two genes. Importantly, we identified one inbred line in which ZmLOX5 has a disrupted open reading frame, a line missing ZmLOX5, and five lines with a duplication of ZmLOX5. Tajima's D test suggests that both ZmLOX4 and ZmLOX5 have been under neutral selection. Further investigation of haplotype data revealed that within the ZmLOX family members only ZmLOX12, a monocot specific ZmLOX, showed strong linkage disequilibrium that extends further than expected in maize. Linkage disequilibrium patterns at these loci of interest are crucial for future candidate gene association mapping studies. ZmLOX4 and ZmLOX5 mutations and copy number variants are under further investigation for crop improvement.

Disruption of OPR7 and OPR8 reveals the versatile functions of jasmonic acid in maize development and defense.

Here, multiple functions of jasmonic acid (JA) in maize (Zea mays) are revealed by comprehensive analyses of JA-deficient mutants of the two oxo-phytodienoate reductase genes, OPR7 and OPR8. Single mutants produce wild-type levels of JA in most tissues, but the double mutant opr7 opr8 has dramatically reduced JA in all organs tested. opr7 opr8 displayed strong developmental defects, including formation of a feminized tassel, initiation of female reproductive buds at each node, and extreme elongation of ear shanks; these defects were rescued by exogenous JA. These data provide evidence that JA is required for male sex determination and suppression of female reproductive organ biogenesis. Moreover, opr7 opr8 exhibited delayed leaf senescence accompanied by reduced ethylene and abscisic acid levels and lack of anthocyanin pigmentation of brace roots. Remarkably, opr7 opr8 is nonviable in nonsterile soil and under field conditions due to extreme susceptibility to a root-rotting oomycete (Pythium spp), demonstrating that these genes are necessary for maize survival in nature. Supporting the importance of JA in insect defense, opr7 opr8 is susceptible to beet armyworm. Overall, this study provides strong genetic evidence for the global roles of JA in maize development and immunity to pathogens and insects.

A downstream mediator in the growth repression limb of the jasmonate pathway.

Wounding plant tissues initiates large-scale changes in transcription coupled to growth arrest, allowing resource diversion for defense. These processes are mediated in large part by the potent lipid regulator jasmonic acid (JA). Genes selected from a list of wound-inducible transcripts regulated by the jasmonate pathway were overexpressed in Arabidopsis thaliana, and the transgenic plants were then assayed for sensitivity to methyl jasmonate (MeJA). When grown in the presence of MeJA, the roots of plants overexpressing a gene of unknown function were longer than those of wild-type plants. When transcript levels for this gene, which we named JASMONATE-ASSOCIATED1 (JAS1), were reduced by RNA interference, the plants showed increased sensitivity to MeJA and growth was inhibited. These gain- and loss-of-function assays suggest that this gene acts as a repressor of JA-inhibited growth. An alternative transcript from the gene encoding a second protein isoform with a longer C terminus failed to repress jasmonate sensitivity. This identified a conserved C-terminal sequence in JAS1 and related genes, all of which also contain Zim motifs and many of which are jasmonate-regulated. Both forms of JAS1 were found to localize to the nucleus in transient expression assays. Physiological tests of growth responses after wounding were consistent with the fact that JAS1 is a repressor of JA-regulated growth retardation.

Cloning and characterization of a bamboo LEAFY HULL STERILE1 homologous gene.

A cDNA named DlMADS8 was isolated from the young spikelets of the sweet bamboo, Dendrocalamus latiflorus by rapid amplification of cDNA end (RACE). DNA sequence analysis showed that DlMADS8 was composed of full ORF and 3'UTR, but without 5'UTR. The cDNA contained 1059 nucleotides and encoded a putative protein of 244 amino acid residues. The gene displayed the structure of a typical plant MADS-box gene, which consisted of a MADS domain, K domain, a short I region, and the C-terminal region. Phylogenetic analysis of plant MADS-box genes based on amino acid sequences revealed that DlMADS8 was grouped into the AGAMOUS-LIKE 2 (AGL2)-like subfamily. It was homologous to the LEAFY HULL STERILE1 (LHS1) genes of grasses. To study the functions of it, DlMADS8 cDNA clone driven by the CaMV 35S promoter was transformed into Arabidopsis thaliana. Transgenic plants of DlMADS8 exhibited the phenotypes of curled leaves and early flowering. After bolting, three novel phenotypes related to inflorescence development were observed in different transgenic plants. No obvious homeotic conversions of floral organs were observed in all of the 35S::DllMADS8 transgenic Arabidopsis plants. These results indicated that DlMADS8 probably plays a role in floral meristem determinacy and is involved in controlling the flowering time of D. latiflorus.