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SummaryA better understanding of wheat functional genomics could facilitate the targeted breeding for agronomic traits improvement and environmental adaptation. With the release of reference genomes and extensive re-sequencing data of wheat and relatives, wheat functional genomics enters a new era. However, limited transformation efficiency in wheat hampers in-depth gene functional study and genetic manipulation for breeding. Here, we generated an EMS mutagenesis library of KN9204, a widely grown elite wheat variety in northern China, with available reference genome, transcriptome, and epigenome of various tissues. The library harbors enormous developmental diversity covering important tissues and transition stages. Exome capture sequencing of 2,090 mutant lines, with probes designed by KN9204 genome, revealed that 98.79% of coding genes have mutations and 1,383 EMS-type SNPs per line averagely. Novel allelic variations for important agronomic trait-related genes, such asRht-D1, Q, TaTB1, andWFZP, were identified. We tested 100 lines with severe mutations in 80 NAC TFs under drought and salinity stresses, and found 13 lines with altered sensitivity. Three lines were further analyzed for the regulation insight of NAC TFs in stress response by combing transcriptome and available chromatin accessibility data. Hundreds of direct targets of NAC with altered transcriptional pattern in mutant lines under salt or drought stress induction were identified, includingSNAC1, DREB2B, CML16andZFP182, knowing factors in abiotic stresses response. Thus, we have generated and indexed KN9204 EMS mutant library which would facilitate functional genomics study and provide resources for genetic manipulation in wheat.

Background Heat shock transcription factors (Hsfs) are present in majority of plants and play central roles in thermotolerance, transgenerational thermomemory, and many other stress responses. Our previous paper identified at least 82 Hsf members in a genome-wide study on wheat (Triticum aestivum L.). In this study, we analyzed the Hsf expression profiles in the advanced development stages of wheat, isolated the markedly heat-responsive gene TaHsfA2–10 (GenBank accession number MK922287), and characterized this gene and its role in thermotolerance regulation in seedlings of Arabidopsis thaliana (L. Heynh.). Results In the advanced development stages, wheat Hsf family transcription profiles exhibit different expression patterns and varying heat-responses in leaves and roots, and Hsfs are constitutively expressed to different degrees under the normal growth conditions. Overall, the majority of group A and B Hsfs are expressed in leaves while group C Hsfs are expressed at higher levels in roots. The expression of a few Hsf genes could not be detected. Heat shock (HS) caused upregulation about a quarter of genes in leaves and roots, while a number of genes were downregulated in response to HS. The highly heat-responsive gene TaHsfA2–10 was isolated through homeologous cloning. qRT-PCR revealed that TaHsfA2–10 is expressed in a wide range of tissues and organs of different development stages of wheat under the normal growth conditions. Compared to non-stress treatment, TaHsfA2–10 was highly upregulated in response to HS, H2O2, and salicylic acid (SA), and was downregulated by abscisic acid (ABA) treatment in two-leaf-old seedlings. Transient transfection of tobacco epidermal cells revealed subcellular localization of TaHsfA2–10 in the nucleus under the normal growth conditions. Phenotypic observation indicated that TaHsfA2–10 could improve both basal thermotolerance and acquired thermotolerance of transgenic Arabidopsis thaliana seedlings and rescue the thermotolerance defect of the T-DNA insertion mutant athsfa2 during HS. Compared to wild type (WT) seedlings, the TaHsfA2–10-overexpressing lines displayed both higher chlorophyll contents and higher survival rates. Yeast one-hybrid assay results revealed that TaHsfA2–10 had transactivation activity. The expression levels of thermotolerance-related AtHsps in the TaHsfA2–10 transgeinc Arabidopsis thaliana were higher than those in WT after HS. Conclusions Wheat Hsf family members exhibit diversification and specificity of transcription expression patterns in advanced development stages under the normal conditions and after HS. As a markedly responsive transcriptional factor to HS, SA and H2O2, TaHsfA2–10 involves in thermotolerance regulation of plants through binding to the HS responsive element in promoter domain of relative Hsps and upregulating the expression of Hsp genes.

Plant heat shock transcription factors (Hsfs) are important regulators of heat shock signal transduction pathway. There are 30 members of the Hsf family in maize, only two of which, ZmHsf06 and ZmHsf12, belong to subclass A1. Our previous work demonstrated that ZmHsf06-overexpressing Arabidopsis lines showed improved tolerance to heat and drought stresses. In this study, we isolated ZmHsf12 from young leaves of maize (Zea mays L.) using homologous cloning methods. The CDS (coding sequence) of ZmHsf12 is 1,494 bp and encodes a putative protein consisting of 497 amino acids which possesses domains such as DBD (DNA-binding domain), OD (oligomerization domain), NLS (nuclear localization signal), NES (nuclear export signal), and an AHA (activator) motif. The ZmHsf12-GFP fusion protein is localized to the cell nucleus. ZmHsf12 was expressed in many maize organs, and its expression was up-regulated by heat shock. Furthermore, we characterized the function of ZmHsf12 in yeast and Arabidopsis. Yeast cells overexpressing ZmHsf12 showed enhanced heat tolerance. ZmHsf12-overexpressing Arabidopsis seedlings displayed significant increases in both basal and acquired thermotolerance. Compared to WT seedlings, the ZmHsf12-overexpressing lines displayed both increased chlorophyll contents and higher survival rates. Also, the expression of AtHsps was increased higher in the ZmHsf12-overexpressing Arabidopsis lines after heat stress. The results of our study strongly suggested that ZmHsf12 may take part in plant response to heat stress.

Heat shock transcription factor (Hsf) plays a transcriptional regulatory role in plants during heat stress and other abiotic stresses. 31 non-redundant ZmHsf genes from maize were identified and clustered in the reference genome sequenced by Single Molecule Real Time (SMRT). The amino acid length, chromosome location, and presence of functional domains and motifs of all ZmHsfs sequences were analyzed and determined. Phylogenetics and collinearity analyses reveal gene duplication events in Hsf family and collinearity blocks shared by maize, rice and sorghum. The results of RNA-Seq analysis of anthesis and post-anthesis periods in maize show different expression patterns of ZmHsf family members. Specially, ZmHsf26 of A2 subclass and ZmHsf23 of A6 subclass were distinctly up-regulated after heat shock (HS) at post-anthesis stage. Nanopore transcriptome sequencing of maize seedlings showed that alternative splicing (AS) events occur in ZmHsf04 and ZmHsf17 which belong to subclass A2 after heat shock. Through sequence alignment, semi-quantitative and quantitative RT-PCR, we found that intron retention events occur in response to heat shock, and newly splice isoforms, ZmHsf04-II and ZmHsf17-II, were transcribed. Both new isoforms contain several premature termination codons in their introns which may lead to early termination of translation. The ZmHsf04 expression was highly increased than that of ZmHsf17, and the up-regulation of ZmHsf04-I transcription level were significantly higher than that of ZmHsf04-II after HS.

Background. The yield of maize crop is influenced seriously by heat waves. Plant heat shock transcription factors (Hsfs) play a key regulatory role in heat shock signal transduction pathway. Method. In this study, a new heat shock transcription factor gene, ZmHsf01 (accession number: MK888854) , was cloned from maize young leaves using homologous cloning method. The transcriptional level of ZmHsf01 were detected by qRT-PCR in different tissues or under heat shock, abscisic acid (ABA) and hydrogen peroxide (H2O2) treatment. The transgenic yeast and Arabidopsis were used to study the gene function of ZmHsf01. Result. The coding sequence (CDS) of ZmHsf01 was 1176 bp and encoded a protein that consisted of 391 amino acids. The homologous analysis result showed that ZmHsf01 and SbHsfA2d had the highest protein sequence identity. Subcellular localization experiments demonstrated that ZmHsf01 is localized to the nucleus. ZmHsf01 was expressed in many maize tissues and was up-regulated by heat stress. ZmHsf01 was up-regulated in roots and down-regulated in leaves by ABA and H2O2treatments. In yeast, ZmHsf01-overexpressing cells showed increased thermotolerance. In Arabidopsis seedlings, ZmHsf01 complemented the thermotolerance defects of athsfa2 mutant and ZmHsf01-overexpressing lines presented enhanced basal and acquired thermotolerance. Compared to wild type (WT) seedlings, ZmHsf01-overexpressing lines showed increased chlorophyll content after heat stress. The expression level of heat shock protein genes was up-regulated higher in ZmHsf01-overexpressing Arabidopsis seedlings than that in WT. These results suggested that ZmHsf01 plays a vital role in plant response to heat stress.

Background Heat waves can critically influence maize crop yields. Plant heat shock transcription factors (HSFs) play a key regulating role in the heat shock (HS) signal transduction pathway. Method In this study, a homologous cloning method was used to clone HSF gene ZmHsf01 (accession number: MK888854) from young maize leaves. The transcript levels of ZmHsf01 were detected using qRT-PCR in different tissues and treated by HS, abscisic acid (ABA), hydrogen peroxide (H2O2), respectively, and the functions of gene ZmHsf01 were studied in transgenic yeast and Arabidopsis. Result ZmHsf01 had a coding sequence (CDS) of 1176 bp and encoded a protein consisting of 391 amino acids. The homologous analysis results showed that ZmHsf01 and SbHsfA2d had the highest protein sequence identities. Subcellular localization experiments confirmed that ZmHsf01 was localized in the nucleus. ZmHsf01 was expressed in many maize tissues. It was up-regulated by HS, and up-regulated in roots and down-regulated in leaves under ABA and H2O2treatments. ZmHsf01-overexpressing yeast cells showed increased thermotolerance. In Arabidopsis seedlings, ZmHsf01 compensated for the thermotolerance defects of mutant athsfa2, and ZmHsf01-overexpressing lines showed enhanced basal and acquired thermotolerance. When compared to wild type (WT) seedlings, ZmHsf01-overexpressing lines showed higher chlorophyll content and survival rates after HS. Heat shock protein (HSP) gene expression levels were more up-regulated in ZmHsf01-overexpressing Arabidopsis seedlings than WT seedlings. These results suggest that ZmHsf01 plays a vital role in response to HS in plant.

Background Enhancement of crop productivity under various abiotic stresses is a major objective of agronomic research. Wheat (Triticum aestivum L.) as one of the world’s staple crops is highly sensitive to heat stress, which can adversely affect both yield and quality. Plant heat shock factors (Hsfs) play a crucial role in abiotic and biotic stress response and conferring stress tolerance. Thus, multifunctional Hsfs may be potentially targets in generating novel strains that have the ability to survive environments that feature a combination of stresses. Result In this study, using the released genome sequence of wheat and the novel Hsf protein HMM (Hidden Markov Model) model constructed with the Hsf protein sequence of model monocot (Oryza sativa) and dicot (Arabidopsis thaliana) plants, genome-wide TaHsfs identification was performed. Eighty-two non-redundant and full-length TaHsfs were randomly located on 21 chromosomes. The structural characteristics and phylogenetic analysis with Arabidopsis thaliana, Oryza sativa and Zea mays were used to classify these genes into three major classes and further into 13 subclasses. A novel subclass, TaHsfC3 was found which had not been documented in wheat or other plants, and did not show any orthologous genes in A. thaliana, O. sativa, or Z. mays Hsf families. The observation of a high proportion of homeologous TaHsf gene groups suggests that the allopolyploid process, which occurred after the fusion of genomes, contributed to the expansion of the TaHsf family. Furthermore, TaHsfs expression profiling by RNA-seq revealed that the TaHsfs could be responsive not only to abiotic stresses but also to phytohormones. Additionally, the TaHsf family genes exhibited class-, subclass- and organ-specific expression patterns in response to various treatments. Conclusions A comprehensive analysis of Hsf genes was performed in wheat, which is useful for better understanding one of the most complex Hsf gene families. Variations in the expression patterns under different abiotic stress and phytohormone treatments provide clues for further analysis of the TaHsfs functions and corresponding signal transduction pathways in wheat. Electronic supplementary material The online version of this article (10.1186/s12864-019-5617-1) contains supplementary material, which is available to authorized users.

Table S5. Motif sequences identified by MEME tools. (DOC 838 kb)

High temperature directly affects the yield and quality of crops. Plant Hsfs play vital roles in plant response to heat shock. In the present study, ZmHsf05 was isolated from maize (Zea mays L.) using homologous cloning methods. The sequencing analysis demonstrated that CDS of ZmHsf05 was 1080 bp length and encoded a protein containing 359 amino acids. The putative amino acid sequence of ZmHsf05 contained typical Hsf domains, such as DBD, OD, NLS and AHA motif. Subcellular localization assays displayed that the ZmHsf05 is localized to the nucleus. ZmHsf05 was expressed in many maize tissues and its expression level was increased by heat stress treatment. ZmHsf05 rescued the reduced thermotolerance of the athsfa2 mutant in Arabidopsis seedlings. Arabidopsis seedlings of ZmHsf05-overexpressing increased both the basal and acquired thermotolerances. After heat stress, the ZmHsf05-overexpressing lines showed enhanced survival rate and chlorophyll content compared with WT seedlings. The expression of Hsps was up-regulated in the ZmHsf05-overexpressing Arabidopsis lines after heat stress treatment. These results suggested that ZmHsf05 plays an important role in both basal and acquired thermotolerance in plants.

Background Heat waves can critically influence maize crop yields. Plant heat shock transcription factors (HSFs) play a key regulating role in the heat shock (HS) signal transduction pathway. Method In this study, a homologous cloning method was used to clone HSF gene ZmHsf01 (accession number: MK888854) from young maize leaves. The transcript levels of ZmHsf01 were detected using qRT-PCR in different tissues and treated by HS, abscisic acid (ABA), hydrogen peroxide (H2O2), respectively, and the functions of gene ZmHsf01 were studied in transgenic yeast and Arabidopsis. Result ZmHsf01 had a coding sequence (CDS) of 1176 bp and encoded a protein consisting of 391 amino acids. The homologous analysis results showed that ZmHsf01 and SbHsfA2d had the highest protein sequence identities. Subcellular localization experiments confirmed that ZmHsf01 was localized in the nucleus. ZmHsf01 was expressed in many maize tissues. It was up-regulated by HS, and up-regulated in roots and down-regulated in leaves under ABA and H2O2treatments. ZmHsf01-overexpressing yeast cells showed increased thermotolerance. In Arabidopsis seedlings, ZmHsf01 compensated for the thermotolerance defects of mutant athsfa2, and ZmHsf01-overexpressing lines showed enhanced basal and acquired thermotolerance. When compared to wild type (WT) seedlings, ZmHsf01-overexpressing lines showed higher chlorophyll content and survival rates after HS. Heat shock protein (HSP) gene expression levels were more up-regulated in ZmHsf01-overexpressing Arabidopsis seedlings than WT seedlings. These results suggest that ZmHsf01 plays a vital role in response to HS in plant.

Heat shock transcription factors (Hsfs) play an important role in regulating heat stress response in plants. Our previous study found that there were 82 non-redundant Hsfs in wheat, 18 of which belonged to subclass A2. In this study, we cloned an A2 member, TaHsfA2-1, which encoded a protein of 346 amino acid residues in wheat. The fusion protein TaHsfA2-1-GFP was localized in the nucleus under normal growth conditions. TaHsfA2-1 was expressed in nearly all the measured tissues, most highly in mature leaves. The expression level of TaHsfA2-1 can be enhanced by heat stress, PEG stress, and signal molecules such as H2O2 and SA. Yeast cells transformed with TaHsfA2-1 improved thermotolerance compared to those with the empty vector. TaHsfA2-1-overexpressing Arabidopsis displayed a better growth state with more green leaves than wild-type seedlings after heat stress. Accordingly, the chlorophyll content and survival rate in the transgenic lines were higher than in the wild type, and relative conductivity in the transgenic lines was lower than in the wild type. Further research found that TaHsfA2-1-overexpressing Arabidopsis up-regulated the expression of some heat shock protein genes (Hsps) compared to wild type after heat stress. These results suggested that TaHsfA2-1 is a new gene that improves thermotolerance in plants by mediating the expression of Hsps. A functional gene was provided for molecular breeding in the subsequent research.

Using homology cloning method, a heat shock transcription factor (Hsf) like gene, ZmHsf-like , was cloned from maize ( Zea mays ) leaves. Sequence analyses showed that the open reading frame (ORF) of the gene ZmHsf-like is 1 404 bp long, encoding 467 amino acids. The sequence of amino acids encoded by ZmHsf-like contains the most conserved and typical DNA-binding domain of Hsf family. By bombardment into onion epidermis, we firstly found that the ZmHsf-like was subcellular-located in nucleus. NucPred analysis revealed there is a classic NLS of KKRR peptide in protein. Real-time PCR showed that ZmHsf-like gene expressed in leaves, stems and roots of maize seedlings under normal growth conditions, and the highest expression level was in roots, lower in leaves and the lowest in stems. The ZmHsf-like gene expression could be up-regulated by heat shock, PEG, ABA, and H 2 O 2 in different degrees, among which the heat shock and ABA worked more efficiently. Obvious differences of the peak value and its corresponding time point of ZmHsf-like gene expression were observed among treatments. Experiments with inhibitor further suggested that the up-regulation ZmHsf-like gene expression of heat shock was H 2 O 2 -dependent while the induction of ZmHsf-like with PEG did not depend on the existence of H 2 O 2 . These results pointed out that ZmHsf-like gene probably regulates responsive reactions to abiotic stresses especially heat shock and drought through different signal transduction pathways.

为了从分子水平上鉴别不同的转基因株系，以转 BADH 基因苜蓿的T 0 代基因组DNA为模版，采用热不对称交错PCR(TAIL-PCR)方法分离其外源基因插入位点的侧翼序列，获得了B127株系的左翼序列和右翼序列，以及B125、B138、B295和B196株系的左翼序列。侧翼序列特征分析表明，有的T-DNA边界序列被删除，有的边界序列被保留，并填充了一段未知来源的核苷酸序列。根据侧翼序列中插入载体序列和紧邻插入序列的基因组序列特征，分别设计PCR扩增的上、下游引物，并对获得的42个转 BADH 株系分别进行左、右翼序列的扩增，结果表明，转基因植株B106、B125、B138、B157、B158、B289、B295、B305和B127具有相同的扩增条带，B203、B220、B223和B196具有相同的扩增条带，说明这些株系可能仅来源于2个转化事件。本研究建立的事件特异性检测方法可以准确地将不同的转化株系区别开来。

The gene encoding betaine aldehyde dehydrogenase (BADH) has been transformed into alfalfa (Medicago sativa L.) and resulted in 42 transgenic plants with improved salt tolerance. However, these transgenic lines were derived from the same transformant vector, which were unable to distinguish them from each other using common methods. For differentiating these transformants at molecular level, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to separate the T-DNA flanking sequences for identifying transgenic plants in event-specific detection. A total of 6 sequences flanking either the left or the right borders of the T-DNA were obtained. The left border sequence of T-DNA was completely deleted from the vector and not integrated into the genome of alfalfa in the transgenic plant B196. Although the left border flanking sequence in the transgenic plant B127 was reserved, it was filled with a DNA sequence of unknown origin. The forward and backward primers for PCR were designed based on the characteristics of the flanking sequences originating from the vector sequence and the alfalfa genomic sequence adjacent to the integrated vector sequence, respectively. According to the result of PCR amplification in the 42 BADH-transgenic lines, plants B106, B125, B127, B138, B157, B158, B289, B295, and B305 presented the same amplification banding pattern. Plants B196, B203, B220, and B223 exhibited the same banding pattern, which was different from that amplified from other plants. These results indicated that the plants with identical amplification banding patterns may come from the same transformation event.

WXP1 is an ethylene‐responsive element‐binding transcription factor that has been shown to lead to improved drought tolerance and increased wax accumulation when overexpressed in transgenic alfalfa under the control of the CaMV35S promoter. In this study, alfalfa was transformed with the WXP1 gene, driven by the epidermis‐specific promoter CER6. The transgenic CER6::WXP1 alfalfa plants and a previously tested CaMV35S::WXP1 line were subjected to water stress for 3 d and then rewatered. Gas exchange, chlorophyll fluorescence, relative water content, and water potential were measured. Compared with the controls, the transgenic lines showed higher net photosynthetic rate, transpiration rate, and stomatal conductance and higher efficiency of photosystem II, quantum yield of photosystem II, coefficient of photochemical quenching, and apparent electron transport rate under water stress and after rewatering. The transgenic lines also showed higher relative water content and leaf water potential under water defic...

White clover (Trifolium repens L.) is an important pasture legume in many regions of the world. A commercial cultivar of white clover (cv. Patriot) was transformed with a Medicago truncatula L. transcription factor gene, WXP1, and a reporter gene, β-glucuronidase (GUS). The WXP1 gene and the GUS gene were placed under control of the Arabidopsis CER6 promoter. GUS staining and cross-section analysis revealed the CER6 promoter directed constitutive expression in leaves and epidermis preferential expression in petioles of white clover. Independent transgenic WXP1 lines, empty vector and wild-type controls were subjected to drought stress treatment. The plants were characterised by measuring several physiological parameters including gas exchange, chlorophyll fluorescence, relative water content and leaf water potential. The WXP1 transgenic lines had higher net photosynthetic rates, higher efficiency of PSII, higher relative water content and leaf water potential under drought-stressed conditions. Consistent with the results from physiological analyses, the transgenic white clover plants carrying WXP1 showed improved tolerance to drought stress.