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.

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.

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.

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.

Transcriptome analysis for the restrained stem development of the wheat mutant dms / Desenvolvimento e análise de transcriptoma de uma haste de trigo mutante dms

ABSTRACT: Wheat (Triticum aestivum L.) stem development significantly affects grain yield. The dwarf plants (D) of wheat mutant dms was less than 30cm. Here, we were to explore the molecular basis for the restrained stem development of the dwarf plants. The results were reached by compare the young spikes and stems transcriptomes of the tall (T) and D plants of mutant dms. We identified 663 genes highly expressed in stem tips. We identified 997 differentially expressed genes (DEGs) in stem tips between T and D, 403 DEGs were significantly related with stem development. Most biological processes in stem tips on dwarf plants were significantly suppressed, such as phytohormone signaling etc. The sequencing analysis results were confirmed by quantitatively analysis the expression profiles of fourteen key DEGs via real-time QRT-PCR. We identified a group genes related to wheat stem development, identified a group DEGs related to the restrained stem development of D plants of dms. The suppressed phytohormone signaling, carbohydrate transport and metabolism were the major causal factors leading to dwarf plants of D. Our dataset provides a useful resource for investigating wheat stem development.

RESUMO: O desenvolvimento da haste do trigo (Triticum aestivum L.) afeta significativamente o rendimento dos grãos. A partir disso, empregou-se a base molecular para o desenvolvimento de uma haste de variedade de trigo. Os resultados foram alcançados ao conferir as hastes de um mutante de trigo DMS. Identificou-se 663 genes altamente expressos na haste; 997 genes (DEGs) diferentemente expressos em hastes entre T e D e; 403 DEGs foram significativamente diferentes. A maioria dos processos biológicos de caule tipo não plantas foram significativamente suprimidas, com o fito hormônio de sinalização. Os resultados da análise de sequencia foram confirmados pela expressão quantitativa de perfis de catorze chave DEGs através de qRT-PCR em tempo real. Nota-se um grupo de genes relacionados com a haste de trigo de desenvolvimento. Identificou-se um grupo DEGs relacionados com o desenvolvimento de um tronco D plantas de DMS. O fito hormônio de sinalização, transporte e metabolismo de hidratos de carbono foram os principais fatores suscetíveis das plantas anão de D. Nosso conjunto de dados fornece um recurso útil para investigar o desenvolvimento do caule de trigo.

Transcriptome Analysis for Abnormal Spike Development of the Wheat Mutant dms.

BACKGROUND: Wheat (Triticum aestivum L.) spike development is the foundation for grain yield. We obtained a novel wheat mutant, dms, characterized as dwarf, multi-pistil and sterility. Although the genetic changes are not clear, the heredity of traits suggests that a recessive gene locus controls the two traits of multi-pistil and sterility in self-pollinating populations of the medium plants (M), such that the dwarf genotype (D) and tall genotype (T) in the progeny of the mutant are ideal lines for studies regarding wheat spike development. The objective of this study was to explore the molecular basis for spike abnormalities of dwarf genotype. RESULTS: Four unigene libraries were assembled by sequencing the mRNAs of the super-bulked differentiating spikes and stem tips of the D and T plants. Using integrative analysis, we identified 419 genes highly expressed in spikes, including nine typical homeotic genes of the MADS-box family and the genes TaAP2, TaFL and TaDL. We also identified 143 genes that were significantly different between young spikes of T and D, and 26 genes that were putatively involved in spike differentiation. The result showed that the expression levels of TaAP1-2, TaAP2, and other genes involved in the majority of biological processes such as transcription, translation, cell division, photosynthesis, carbohydrate transport and metabolism, and energy production and conversion were significantly lower in D than in T. CONCLUSIONS: We identified a set of genes related to wheat floral organ differentiation, including typical homeotic genes. Our results showed that the major causal factors resulting in the spike abnormalities of dms were the lower expression homeotic genes, hormonal imbalance, repressed biological processes, and deficiency of construction materials and energy. We performed a series of studies on the homeotic genes, however the other three causal factors for spike abnormal phenotype of dms need further study.