Molecular mechanism analysis of ZmRL6 positively regulating drought stress tolerance in maize.

MYB-related genes, a subclass of MYB transcription factor family, have been documented to play important roles in biological processes such as secondary metabolism and stress responses that affect plant growth and development. However, the regulatory roles of MYB-related genes in drought stress response remain unclear in maize. In this study, we discovered that a 1R-MYB gene, ZmRL6, encodes a 96-amino acid protein and is highly drought-inducible. We also found that it is conserved in both barley (Hordeum vulgare L.) and Aegilops tauschii. Furthermore, we observed that overexpression of ZmRL6 can enhance drought tolerance while knock-out of ZmRL6 by CRISPR-Cas9 results in drought hypersensitivity. DAP-seq analyses additionally revealed the ZmRL6 target genes mainly contain ACCGTT, TTACCAAAC and AGCCCGAG motifs in their promoters. By combining RNA-seq and DAP-seq results together, we subsequently identified eight novel target genes of ZmRL6 that are involved in maize's hormone signal transduction, sugar metabolism, lignin synthesis, and redox signaling/oxidative stress. Collectively, our data provided insights into the roles of ZmRL6 in maize's drought response.

Wheat gibberellin oxidase genes and their functions in regulating tillering.

Multiple genetic factors control tillering, a key agronomy trait for wheat (Triticum aestivum L.) yield. Previously, we reported a dwarf-monoculm mutant (dmc) derived from wheat cultivar Guomai 301, and found that the contents of gibberellic acid 3 (GA3) in the tiller primordia of dmc were significantly higher. Transcriptome analysis indicated that some wheat gibberellin oxidase (TaGAox) genes TaGA20ox-A2, TaGA20ox-B2, TaGA3ox-A2, TaGA20ox-A4, TaGA2ox-A10 and TaGA2ox-B10 were differentially expressed in dmc. Therefore, this study systematically analyzed the roles of gibberellin oxidase genes during wheat tillering. A total of 63 TaGAox genes were identified by whole genome analysis. The TaGAoxs were clustered to four subfamilies, GA20oxs, GA2oxs, GA3oxs and GA7oxs, including seven subgroups based on their protein structures. The promoter regions of TaGAox genes contain a large number of cis-acting elements closely related to hormone, plant growth and development, light, and abiotic stress responses. Segmental duplication events played a major role in TaGAoxs expansion. Compared to Arabidopsis, the gene collinearity degrees of the GAoxs were significantly higher among wheat, rice and maize. TaGAox genes showed tissue-specific expression patterns. The expressions of TaGAox genes (TaGA20ox-B2, TaGA7ox-A1, TaGA2ox10 and TaGA3ox-A2) were significantly affected by exogenous GA3 applications, which also significantly promoted tillering of Guomai 301, but didn't promote dmc. TaGA7ox-A1 overexpression transgenic wheat lines were obtained by Agrobacterium mediated transformation. Genomic PCR and first-generation sequencing demonstrated that the gene was integrated into the wheat genome. Association analysis of TaGA7ox-A1 expression level and tiller number per plant demonstrated that the tillering capacities of some TaGA7ox-A1 transgenic lines were increased. These data demonstrated that some TaGAoxs as well as GA signaling were involved in regulating wheat tillering, but the GA signaling pathway was disturbed in dmc. This study provided valuable clues for functional characterization of GAox genes in wheat.

A natural promoter variation of SlBBX31 confers enhanced cold tolerance during tomato domestication.

Cold stress affects crop growth and productivity worldwide. Understanding the genetic basis of cold tolerance in germplasms is critical for crop improvement. Plants can coordinate environmental stimuli of light and temperature to regulate cold tolerance. However, it remains unknown which gene in germplasms could have such function. Here, we utilized genome-wide association study (GWAS) to investigate the cold tolerance of wild and cultivated tomato accessions and discovered that increased cold tolerance is accompanied with tomato domestication. We further identified a 27-bp InDel in the promoter of the CONSTANS-like transcription factor (TF) SlBBX31 is significantly linked with cold tolerance. Coincidentally, a key regulator of light signalling, SlHY5, can directly bind to the SlBBX31 promoter to activate SlBBX31 transcription while the 27-bp InDel can prevent S1HY5 from transactivating SlBBX31. Parallel to these findings, we observed that the loss of function of SlBBX31 results in impaired tomato cold tolerance. SlBBX31 can also modulate the cold-induced expression of several ERF TFs including CBF2 and DREBs. Therefore, our study has uncovered that SlBBX31 is possibly selected during tomato domestication for cold tolerance regulation, providing valuable insights for the development of hardy tomato varieties.

The wheat leaf delayed virescence of mutant dv4 is associated with the abnormal photosynthetic and antioxidant systems.

Chlorophyll (Chl) is a key pigment for wheat (Triticum aestivum L.) photosynthesis, consequently impacts grain yield. A wheat mutant named as delayed virescence 4 (dv4) was obtained from cultivar Guomai 301 (wild type, WT) treated with ethyl methane sulfonate (EMS). The seedling leaves of dv4 were shallow yellow, apparently were chlorophyll deficient. They started to turn green at the jointing stage and returned to almost ordinary green at the heading stage. Leaf transcriptome comparison of Guomai 301 and dv4 at the jointing stage showed that most differentially expressed genes (DEGs) of transcription and translation were highly expressed in dv4, one key gene nicotianamine aminotransferase A (NAAT-A) involved in the synthesis and metabolism pathways of tyrosine, methionine and phenylalanine was significantly lowly expressed. The expression levels of the most photosynthesis related genes, such as photosystem I (PS I), ATPase and light-harvesting chlorophyll protein complex-related homeotypic genes, and protochlorophyllide reductase A (PORA) were lower; but macromolecule degradation and hypersensitivity response (HR) related gene heat shock protein 82 (HSP82) was highly expressed. Compared to WT, the contents of macromolecules such as proteins and sugars were reduced; the contents of Chl a, Chl b, total Chl, and carotenoids in leaves of dv4 were significantly less at the jointing stage, while the ratio of Chl a / Chl b was the same as that of WT. The net photosynthetic rate, stomatal conductance and transpiration rate of dv4 were significantly lower. The H2O2 content were higher, while the contents of total phenol and malondialdehyde (MDA), antioxidant enzyme activities were lower in leaves of dv4. In conclusion, the reduced contents of macromolecules and photosynthetic pigments, the abnormal photosynthetic and antioxidant systems were closely related to the phenotype of dv4.

Key auxin response factor (ARF) genes constraining wheat tillering of mutant dmc.

Tillering ability is a key agronomy trait for wheat (Triticum aestivum L.) production. Studies on a dwarf monoculm wheat mutant (dmc) showed that ARF11 played an important role in tillering of wheat. In this study, a total of 67 ARF family members were identified and clustered to two main classes with four subgroups based on their protein structures. The promoter regions of T. aestivum ARF (TaARF) genes contain a large number of cis-acting elements closely related to plant growth and development, and hormone response. The segmental duplication events occurred commonly and played a major role in the expansion of TaARFs. The gene collinearity degrees of the ARFs between wheat and other grasses, rice and maize, were significantly high. The evolution distances among TaARFs determine their expression profiles, such as homoeologous genes have similar expression profiles, like TaARF4-3A-1, TaARF4-3A-2 and their homoeologous genes. The expression profiles of TaARFs in various tissues or organs indicated TaARF3, TaARF4, TaARF9 and TaARF22 and their homoeologous genes played basic roles during wheat development. TaARF4, TaARF9, TaARF12, TaARF15, TaARF17, TaARF21, TaARF25 and their homoeologous genes probably played basic roles in tiller development. qRT-PCR analyses of 20 representative TaARF genes revealed that the abnormal expressions of TaARF11 and TaARF14 were major causes constraining the tillering of dmc. Indole-3-acetic acid (IAA) contents in dmc were significantly less than that in Guomai 301 at key tillering stages. Exogenous IAA application significantly promoted wheat tillering, and affected the transcriptions of TaARFs. These data suggested that TaARFs as well as IAA signaling were involved in controlling wheat tillering. This study provided valuable clues for functional characterization of ARFs in wheat.

Enhanced SA and Ca2+ signaling results in PCD-mediated spontaneous leaf necrosis in wheat mutant wsl.

Leaf is the major photosynthesis organ and the key source of wheat (Triticum aestivum L.) grain. Spotted leaf (spl) mutant is a kind of leaf lesion mimic mutants (LMMs) in plants, which is an ideal material for studying the mechanisms of leaf development. In this study, we report the leaf abnormal development molecular mechanism of a spl mutant named white stripe leaf (wsl) derived from wheat cultivar Guomai 301 (WT). Histochemical observation indicated that the leaf mesophyll cells of the wsl were destroyed in the necrosis regions. To explore the molecular regulatory network of the leaf development in mutant wsl, we employed transcriptome analysis, histochemistry, quantitative real-time PCR (qRT-PCR), and observations of the key metabolites and photosynthesis parameters. Compared to WT, the expressions of the chlorophyll synthesis and photosynthesis-related homeotic genes were repressed; many genes in the WRKY transcription factor (TF) families were highly expressed; the salicylic acid (SA) and Ca2+ signal transductions were enhanced in wsl. Both the chlorophyll contents and the photosynthesis rate were lower in wsl. The contents of SA and reactive oxygen species (ROS) were significantly higher, and the leaf rust resistance was enhanced in wsl. Based on the experimental data, a primary molecular regulatory model for leaf development in wsl was established. The results indicated that the SA accumulation and enhanced Ca2+ signaling led to programmed cell death (PCD), and ultimately resulted in spontaneous leaf necrosis of wsl. These results laid a solid foundation for further research on the molecular mechanism of leaf development in wheat.

Key wheat GRF genes constraining wheat tillering of mutant dmc.

Tillering is a key agronomy trait for wheat (Triticum aestivum L.) production. Previously, we have reported a dwarf-monoculm wheat mutant (dmc) obtained from cultivar Guomai 301 (wild type, WT), and found growth regulating factors (GRFs) playing important roles in regulating wheat tillering. This study is to systematically investigate the roles of all the wheat GRFs (T. aestivum GRFs, TaGRFs) in regulating tillering, and screen out the key regulators. A total of 30 TaGRFs were identified and their physicochemical properties, gene structures, conserved domains, phylogenetic relationships and tissue expression profiles were analyzed. The expression levels of all the TaGRFs were significantly lower in dmc than those in WT at early tillering stage, and the abnormal expressions of TaGRF2-7(A, B, D), TaGRF5-7D, TaGRF10-6(A, B, D) and TaGRF11-2A were major causes constraining the tillering of dmc. The transcriptions of TaGRFs were significantly affected by exogenous indole acetic acid (IAA) and gibberellin acid (GA3) applications, which suggested that TaGRFs as well as IAA, GA signaling were involved in controlling wheat tillering. This study provided valuable clues for functional characterization of GRF genes in wheat.

Cytological and molecular characterizations of a novel 2A nullisomic line derived from a widely-grown wheat cultivar Zhoumai 18 conferring male sterility.

A dwarf, multi-pistil and male sterile dms mutant was previously reported by us. However, the genetic changes in this dms are unclear. To examine the genetic changes, single nucleotide polymorphism (SNP) association, chromosome counting, and high-resolution chromosome fluorescence in situ hybridization (FISH) techniques were employed. By comparing tall plants (T) with dwarf plants (D) in the offspring of dms mutant plants, SNP association analysis indicated that most SNPs were on chromosome 2A. There were three types in offspring of dms plants, with 42, 41 and 40 chromosomes respectively. High-resolution chromosome painting analysis demonstrated that T plants had all 42 wheat chromosomes; the medium plants (M) had 41 chromosomes, lacking one chromosome 2A; while D plants had 40 wheat chromosomes, and lacked both 2A chromosomes. These data demonstrated that dms resulted from a loss of chromosome 2A. We identified 23 genes on chromosome 2A which might be involved in the development of stamens or pollen grains. These results lay a solid foundation for further analysis of the molecular mechanisms of wheat male sterility. Because D plants can be used as a female parent to cross with other wheat genotypes, dms is a unique germplasm for any functional study of chromosome 2A and wheat breeding specifically targeting genes on 2A.

The Major Factors Causing the Microspore Abortion of Genic Male Sterile Mutant NWMS1 in Wheat (Triticum aestivum L.).

Male sterility is a valuable trait for genetic research and production application of wheat (Triticum aestivum L.). NWMS1, a novel typical genic male sterility mutant, was obtained from Shengnong 1, mutagenized with ethyl methane sulfonate (EMS). Microstructure and ultrastructure observations of the anthers and microspores indicated that the pollen abortion of NWMS1 started at the early uninucleate microspore stage. Pollen grain collapse, plasmolysis, and absent starch grains were the three typical characteristics of the abnormal microspores. The anther transcriptomes of NWMS1 and its wild type Shengnong 1 were compared at the early anther development stage, pollen mother cell meiotic stage, and binucleate microspore stage. Several biological pathways clearly involved in abnormal anther development were identified, including protein processing in endoplasmic reticulum, starch and sucrose metabolism, lipid metabolism, and plant hormone signal transduction. There were 20 key genes involved in the abnormal anther development, screened out by weighted gene co-expression network analysis (WGCNA), including SKP1B, BIP5, KCS11, ADH3, BGLU6, and TIFY10B. The results indicated that the defect in starch and sucrose metabolism was the most important factor causing male sterility in NWMS1. Based on the experimental data, a primary molecular regulation model of abnormal anther and pollen developments in mutant NWMS1 was established. These results laid a solid foundation for further research on the molecular mechanism of wheat male sterility.

Enhanced Senescence Process is the Major Factor Stopping Spike Differentiation of Wheat Mutant ptsd1.

Complete differentiation of the spikes guarantees the final wheat (Triticum aestivum L.) grain yield. A unique wheat mutant that prematurely terminated spike differentiation (ptsd1) was obtained from cultivar Guomai 301 treated with ethyl methane sulfonate (EMS). The molecular mechanism study on ptsd1 showed that the senescence-associated genes (SAGs) were highly expressed, and spike differentiation related homeotic genes were depressed. Cytokinin signal transduction was weakened and ethylene signal transduction was enhanced. The enhanced expression of Ca2+ signal transduction related genes and the accumulation of reactive oxygen species (ROS) caused the upper spikelet cell death. Many genes in the WRKY, NAC and ethylene response factor (ERF) transcription factor (TF) families were highly expressed. Senescence related metabolisms, including macromolecule degradation, nutrient recycling, as well as anthocyanin and lignin biosynthesis, were activated. A conserved tae-miR164 and a novel-miR49 and their target genes were extensively involved in the senescence related biological processes in ptsd1. Overall, the abnormal phytohormone homeostasis, enhanced Ca2+ signaling and activated senescence related metabolisms led to the spikelet primordia absent their typical meristem characteristics, and ultimately resulted in the phenotype of ptsd1.

The miRNA-mRNA Networks Involving Abnormal Energy and Hormone Metabolisms Restrict Tillering in a Wheat Mutant dmc.

Tillers not only determine plant architecture but also influence crop yield. To explore the miRNA regulatory network restraining tiller development in a dwarf-monoculm wheat mutant (dmc) derived from Guomai 301 (wild type, WT), we employed miRNome and transcriptome integrative analysis, real-time qRT-PCR, histochemistry, and determinations of the key metabolites and photosynthesis parameters. A total of 91 differentially expressed miRNAs (DEMs) were identified between dmc and WT. Among them, 40 key DEMs targeted 45 differentially expressed genes (DEGs) including the key DEGs encode growth-regulating factors (GRF), auxin response factors (ARF), and other proteins involved in the metabolisms of hormones and carbohydrates, etc. Compared with WT, both the chlorophyll contents and the photosynthesis rate were lower in dmc. The contents of glucose, sucrose, fructose, and maltose were lower in dmc. The contents of auxin (IAA) and zeatin (ZA) were significantly lower, but gibberellin (GA) was significantly higher in the tiller tissues of dmc. This research demonstrated that the DEMs regulating hormone and carbohydrate metabolisms were important causes for dmc to not tiller. A primary miRNA-mRNA regulatory model for dmc tillering was established. The lower photosynthesis rate, insufficient energy, and abnormal hormone metabolisms restrict tillering in dmc.

Gene Expression Profiles and microRNA Regulation Networks in Tiller Primordia, Stem Tips, and Young Spikes of Wheat Guomai 301.

Tillering and spike differentiation are two key events for wheat (Triticum aestivum L.). A study on the transcriptomes and microRNA group profiles of wheat at the two key developmental stages will bring insight into the molecular regulation mechanisms. Guomai 301 is a representative excellent new high yield wheat cultivar in the Henan province in China. The transcriptomes and microRNA (miRNA) groups of tiller primordia (TPs), stem tips (STs), and young spikes (YSs) in Guomai 301 were compared to each other. A total of 1741 tillering specifically expressed and 281 early spikes differentiating specifically expressed differentially expressed genes (DEGs) were identified. Six major expression profile clusters of tissue-specific DEGs for the three tissues were classified by gene co-expression analysis using K-means cluster. The ribosome (ko03010), photosynthesis-antenna proteins (ko00196), and plant hormone signal transduction (ko04075) were the main metabolic pathways in TPs, STs, and YSs, respectively. Similarly, 67 TP specifically expressed and 19 YS specifically expressed differentially expressed miRNAs were identified, 65 of them were novel. The roles of 3 well known miRNAs, tae-miR156, tae-miR164, and tae-miR167a, in post-transcriptional regulation were similar to that of other researches. There were 651 significant negative miRNA-mRNA interaction pairs in TPs and YSs, involving 63 differentially expressed miRNAs (fold change > 4) and 416 differentially expressed mRNAs. Among them 12 key known miRNAs and 16 novel miRNAs were further analyzed, and miRNA-mRNA regulatory networks during tillering and early spike differentiating were established.

Quantitative Changes in the Transcription of Phytohormone-Related Genes: Some Transcription Factors Are Major Causes of the Wheat Mutant dmc Not Tillering.

Tiller number is an important agronomic trait for grain yield of wheat (Triticum aestivum L.). A dwarf-monoculm wheat mutant (dmc) was obtained from cultivar Guomai 301 (wild type, WT). Here, we explored the molecular basis for the restrained tiller development of the mutant dmc. Two bulked samples of the mutant dmc (T1, T2 and T3) and WT (T4, T5 and T6) with three biological replicates were comparatively analyzed at the transcriptional level by bulked RNA sequencing (RNA-Seq). In total, 68.8 Gb data and 463 million reads were generated, 80% of which were mapped to the wheat reference genome of Chinese Spring. A total of 4904 differentially expressed genes (DEGs) were identified between the mutant dmc and WT. DEGs and their related major biological functions were characterized based on GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) categories. These results were confirmed by quantitatively analyzing the expression profiles of twelve selected DEGs via real-time qRT-PCR. The down-regulated gene expressions related to phytohormone syntheses of auxin, zeatin, cytokinin and some transcription factor (TF) families of TALE, and WOX might be the major causes of the mutant dmc, not tillering. Our work provides a foundation for subsequent tiller development research in the future.