TaSINA2B, interacting with TaSINA1D, positively regulates drought tolerance and root growth in wheat (Triticum aestivum L.).

Wheat (Triticum aestivum L.) is an important food crop mainly grown in arid and semiarid regions worldwide, whose productivity is severely limited by drought stress. Although various E3 ubiquitin (Ub) ligases regulate drought stress, only a few SINA-type E3 Ub ligases are known to participate in such responses. Herein, we identified and cloned 15 TaSINAs from wheat. The transcription level of TaSINA2B was highly induced by drought, osmotic and abscisic acid treatments. Two-type promoters of TaSINA2B were found in 192 wheat accessions; furthermore wheat accessions with promoter TaSINA2BII showed a considerably higher level of drought tolerance and gene expression levels than those characterizing accessions with promoter TaSINA2BI that was mainly caused by a 64 bp insertion in its promoter. Enhanced drought tolerance of TaSINA2B-overexpressing (OE) transgenic wheat lines was found to be associated with root growth promotion. Further, an interaction between TaSINA2B and TaSINA1D was detected through yeast two-hybrid and bimolecular fluorescence complementation assays. And TaSINA1D-OE transgenic wheat lines showed similar drought tolerance and root growth phenotype to those observed when TaSINA2B was overexpressed. Therefore, the variation of TaSINA2B promoter contributed to the drought stress regulation, while TaSINA2B, interacting with TaSINA1D, positively regulated drought tolerance by promoting root growth.

TaTIP41 and TaTAP46 positively regulate drought tolerance in wheat by inhibiting PP2A activity.

Drought is a major environmental stress limiting global wheat (Triticum aestivum) production. Exploring drought tolerance genes is important for improving drought adaptation in this crop. Here, we cloned and characterized TaTIP41, a novel drought tolerance gene in wheat. TaTIP41 is a putative conserved component of target of rapamycin (TOR) signaling, and the TaTIP41 homoeologs were expressed in response to drought stress and abscisic acid (ABA). The overexpression of TaTIP41 enhanced drought tolerance and the ABA response, including ABA-induced stomatal closure, while its downregulation using RNA interference (RNAi) had the opposite effect. Furthermore, TaTIP41 physically interacted with TaTAP46, another conserved component of TOR signaling. Like TaTIP41, TaTAP46 positively regulated drought tolerance. Furthermore, TaTIP41 and TaTAP46 interacted with type-2A protein phosphatase (PP2A) catalytic subunits, such as TaPP2A-2, and inhibited their enzymatic activities. Silencing TaPP2A-2 improved drought tolerance in wheat. Together, our findings provide new insights into the roles of TaTIP41 and TaTAP46 in the drought tolerance and ABA response in wheat, and their potential application in improving wheat environmental adaptability.

Genome-wide association study reveals the genetic variation and candidate gene for grain calcium content in bread wheat.

KEY MESSAGE: This study provides important information on the genetic basis of GCaC in wheat, thus contributing to breeding efforts to improve the nutrient quality of wheat. Calcium (Ca) plays important roles in the human body. Wheat grain provides the main diet for billions of people worldwide but is low in Ca content. Here, grain Ca content (GCaC) of 471 wheat accessions was determined in four field environments. A genome-wide association study (GWAS) was performed to reveal the genetic basis of GCaC using the phenotypic data form four environments and a wheat 660 K single nucleotide polymorphism (SNP) array. Twelve quantitative trait locus (QTLs) for GCaC were identified on chromosomes 1A, 1D, 2A, 3B, 6A, 6D, 7A, and 7D, which was significant in at least two environments. Haplotype analysis revealed that the phenotypic difference between the haplotypes of TraesCS6D01G399100 was significant (P ≤ 0.05) across four environments, suggesting it as an important candidate gene for GCaC. This research enhances our understanding of the genetic architecture of GCaC for further improving the nutrient quality of wheat.

Genome-wide association study for grain zinc concentration in bread wheat (Triticum aestivum L.).

Introduction: Zinc (Zn) deficiency causes serious diseases in people who rely on cereals as their main food source. However, the grain zinc concentration (GZnC) in wheat is low. Biofortification is a sustainable strategy for reducing human Zn deficiency. Methods: In this study, we constructed a population of 382 wheat accessions and determined their GZnC in three field environments. Phenotype data was used for a genome-wide association study (GWAS) using a 660K single nucleotide polymorphism (SNP) array, and haplotype analysis identified an important candidate gene for GZnC. Results: We found that GZnC of the wheat accessions showed an increasing trend with their released years, indicating that the dominant allele of GZnC was not lost during the breeding process. Nine stable quantitative trait loci (QTLs) for GZnC were identified on chromosomes 3A, 4A, 5B, 6D, and 7A. And an important candidate gene for GZnC, namely, TraesCS6D01G234600, and GZnC between the haplotypes of this gene showed, significant difference (P ≤ 0.05) in three environments. Discussion: A novel QTL was first identified on chromosome 6D, this finding enriches our understanding of the genetic basis of GZnC in wheat. This study provides new insights into valuable markers and candidate genes for wheat biofortification to improve GZnC.

Trade-offs between grain yields and ecological efficiencies in a wheat-maize cropping system using optimized tillage and fertilization management on the North China Plain.

Optimized fertilizer and tillage management can be an effective strategy for high ecological efficiencies as well as crop yields. The objective of this study was to assess the impact of diverse management practices on carbon footprint, and ecosystem services in a wheat-maize cropping system. An in situ field experiment field was conducted from 2018 to 2020 on the North China Plain, and six treatments were established: deep tillage (DT), shallow tillage (ST), no tillage (NT), deep tillage + adding organic fertilizer (DTF), shallow tillage + adding organic fertilizer (STF), and no tillage + adding organic fertilizer (NTF). The results showed that adding organic fertilizer and the deeper tillage depth caused higher direct CO2 and N2O emission fluxes. DTF treatment significantly increased carbon footprint either per-unit area (CFa) or per-unit net income (CFe). Compared with DT treatment, STF treatment had higher CFa but lower CFe by increasing net income through boosted crop yields. Besides, the highest ecosystem service values (ESV) were present in STF treatment during both 2 years (42,017.13 CNY ha-1 and 43,352.03 CNY ha-1). In conclusion, STF treatment was an optimal management practice to trade-off grain yields and ecological efficiencies in a wheat-maize cropping system. Furthermore, this study highlights that adding organic fertilizer could be an efficient option toward sustainable farmland utilization with high soil carbon sequestration capacity and high ESV.

A genome-wide association study revealed the genetic variation and candidate genes for grain copper content in bread wheat (Triticum aestivum L.).

As an essential microelement, copper plays a crucial role in the human body. However, the grains of bread wheat, a major crop food, contain a low copper content. Here, a diversity panel of 443 wheat accessions cultivated in four environments was used to analyse grain copper content by ICAP-7000, and the genetic variation in grain copper content was examined using a 660 K single nucleotide polymorphism chip. Phenotypic analysis indicated that the grain copper content varied between 2.58 mg kg-1 and 13.65 mg kg-1. A genome-wide association study identified 12 QTLs associated with grain copper content that showed significance in at least two environments on chromosomes 1A, 1D, 3D, 4A, 5A, 5D, 6B, 6D, 7A and 7D. Through haplotype analysis, the phenotypic difference between the haplotypes of three genes, TraesCS5D01G282300, TraesCS6B01G052900 and TraesCS7D01G146600, showed significance (P ⩽ 0.05) in four environments. They were considered to be important candidate genes for grain copper content in wheat. In addition, we detected that the grain copper content gradually decreased with release years among wheat accessions in China, and the percentage of favourable alleles showed a similar trend. Analysing the changes in grain copper content with yield factors, we found that the dilute effect was mainly caused by thousand kernel weight. This study provides useful information on the genetic basis for grain copper content, and thus helps in improving the wheat grain quality.

Genome-Wide Association Study on Root System Architecture and Identification of Candidate Genes in Wheat (Triticum aestivum L.).

The root tissues play important roles in water and nutrient acquisition, environmental adaptation, and plant development. In this study, a diversity panel of 388 wheat accessions was collected to investigate nine root system architecture (RSA) traits at the three-leaf stage under two growing environments: outdoor pot culture (OPC) and indoor pot culture (IPC). Phenotypic analysis revealed that root development was faster under OPC than that under IPC and a significant correlation was observed between the nine RSA traits. The 660K single-nucleotide polymorphism (SNP) chip was used for a genome-wide association study (GWAS). Significant SNPs with a threshold of -log10 (p-value) ≥ 4 were considered. Thus, 36 quantitative trait loci (QTLs), including 13 QTL clusters that were associated with more than one trait, were detected, and 31 QTLs were first identified. The QTL clusters on chromosomes 3D and 5B were associated with four and five RSA traits, respectively. Two candidate genes, TraesCS2A01G516200 and TraesCS7B01G036900, were found to be associated with more than one RSA trait using haplotype analysis, and preferentially expressed in the root tissues. These favourable alleles for RSA traits identified in this study may be useful to optimise the root system in wheat.

Temporal expression study of miRNAs in the crown tissues of winter wheat grown under natural growth conditions.

BACKGROUND: Winter wheat requires prolonged exposure to low temperature to initiate flowering (vernalization). Shoot apical meristem of the crown is the site of cold perception, which produces leaf primordia during vegetative growth before developing into floral primordia at the initiation of the reproductive phase. Although many essential genes for winter wheat cold acclimation and floral initiation have been revealed, the importance of microRNA (miRNA) meditated post-transcriptional regulation in crowns is not well understood. To understand the potential roles of miRNAs in crown tissues, we performed a temporal expression study of miRNAs in crown tissues at the three-leaf stage, winter dormancy stage, spring green-up stage, and jointing stage of winter wheat grown under natural growth conditions. RESULTS: In total, 348 miRNAs belonging to 298 miRNA families, were identified in wheat crown tissues. Among them, 92 differentially expressed miRNAs (DEMs) were found to be significantly regulated from the three-leaf stage to the jointing stage. Most of these DEMs were highly expressed at the three-leaf stage and winter dormancy stage, and then declined in later stages. Six DEMs, including miR156a-5p were markedly induced during the winter dormancy stage. Eleven DEMs, including miR159a.1, miR390a-5p, miR393-5p, miR160a-5p, and miR1436, were highly expressed at the green-up stage. Twelve DEMs, such as miR172a-5p, miR394a, miR319b-3p, and miR9676-5p were highly induced at the jointing stage. Moreover, 14 novel target genes of nine wheat or Pooideae-specific miRNAs were verified using RLM-5' RACE assay. Notably, six mTERFs and two Rf1 genes, which are associated with mitochondrial gene expression, were confirmed as targets of three wheat-specific miRNAs. CONCLUSIONS: The present study not only confirmed the known miRNAs associated with phase transition and floral development, but also identified a number of wheat or Pooideae-specific miRNAs critical for winter wheat cold acclimation and floral development. Most importantly, this study provided experimental evidence that miRNA could regulate mitochondrial gene expression by targeting mTERF and Rf1 genes. Our study provides valuable information for further exploration of the mechanism of miRNA mediated post-transcriptional regulation during winter wheat vernalization and inflorescent initiation.

[Effects of tillage and fertility on soil nitrogen balance and greenhouse gas emissions of wheat-maize rotation system in Central Henan Province, China.] / 耕作和培肥对豫中区小麦-玉米轮作系统土壤氮平衡和温室气体排放的影响.

Energy saving, emission reduction, and efficiency improvement are important directions for agricultural development in Central Henan Province, the main grain production area in the Huang-huai-hai Plain. Based on the tillage and fertilization positioning experiment in 2010, we investigated the effects of three tillage methods (deep tillage, shallow tillage, and no-tillage) and two fertilization methods (nitrogen fertilizer and nitrogen fertilizer+organic fertilizer) on soil nitrogen balance and greenhouse gas emissions from 2018 to 2019. The results showed that soil nitrogen accumulation increased with organic fertilizer addition. During wheat and maize maturation, soil total nitrogen accumulation in the 0-60 cm layer was the highest under the treatment of shallow tillage+organic fertilizer, being 8058.53 and 8299 kg N·hm-2, respectively, being 3.2%-27.4% and 4.3%-7.2% higher than other treatments. The treatment with organic fertilizer addition resulted in nitrogen surplus. The shallow tillage+organic fertilizer treatment led to the highest nitrogen surplus (13.57 kg N·hm-2), which was 9.52 and 0.18 kg N·hm-2 higher than deep tillage+organic fertilizer and no tillage+organic fertilizer treatments. Nitrate leaching was the main way of nitrogen losses, accounting for 73.4%-76.9% of the total losses. The amount of nitrate leaching was the highest in deep tillage+organic fertilizer treatment (48.37 kg N·hm-2), being 18.9%-35.1% higher than other treatments. Results of greenhouse gases emission during 2018-2019 showed that global warming potential was the highest under the treatment of deep tillage+organic fertilizer, which was 33070 kg N·hm-2, being 6.6%-26.8% higher than other treatments. The treatment of organic fertilizer addition increased the emission of N2O and CO2 and reduced the absorption of CH4. The annual grain yield was highest under the treatment of deep tillage+organic fertilizer, which was 5.0%-17.1% higher than other treatments. The crop harvest index was the highest under the treatment of shallow tillage+organic fertilizer. The recommended cropping mode in Central Henan Pro-vince is shallow tillage+organic fertilizer, which could ensure crop yields, maintain soil nitrogen balance, and reduce greenhouse gas emissions.

Evolutionary Divergence and Biased Expression of NAC Transcription Factors in Hexaploid Bread Wheat (Triticum aestivum L.).

The NAC genes, a large plant-specific family of transcription factors, regulate a wide range of pathways involved in development and response to biotic and abiotic stress. In this study, the NAC transcription factors were identified in 27 green plants, and the results showed that NAC transcription factors in plants undergo an appearance stage from water to land and a number expansion stage from gymnosperm to angiosperm. Investigating the evolutionary process of the NAC transcription factors from diploid species to hexaploid wheat revealed that tandem replications during the polyploidization process is an important event for increasing the number of NAC transcription factors in wheat. Then, the molecular characteristics, phylogenetic relationships, and expression patterns of 462 NAC transcription factors of hexaploid wheat (TaNACs) were analyzed. The protein structure results showed that TaNAC was relatively conservative at the N-terminal that contains five subdomains. All these TaNACs were divided into Group I and Group II by phylogenetic analysis, and the TaNACs in Group I should undergo strong artificial selection based on single nucleotide polymorphism (SNP) analysis. Through genome synteny and phylogenetic analysis, these TaNACs were classified into 88 groups and 9 clusters. The biased expression results of these TaNACs showed that there are 24 groups and 67 groups of neofunctionalization genes under biotic and abiotic stress, respectively, and 16 groups and 59 groups of subfunctionalization genes. This shows that neofunctionalization plays an important role in coping with different stresses. Our study provides new insights into the evolution of NAC transcription factors in hexaploid wheat.

Identification and Temporal Expression Analysis of Conserved and Novel MicroRNAs in the Leaves of Winter Wheat Grown in the Field.

Cold acclimation and vegetative/reproductive transition are two important evolutionary adaptive mechanisms for winter wheat surviving the freezing temperature in winter and successful seeds setting in the next year. MicroRNA (miRNA) is a class of regulatory small RNAs (sRNAs), which plays critical roles in the growth and development of plants. However, the regulation mechanism of miRNAs during cold acclimation and vegetative/reproductive transition of winter wheat is not much understood. In this study, four sRNA libraries from leaves of winter wheat grown in the field at the three-leaf stage, winter dormancy stage, spring green-up stage, and jointing stage were analyzed to identify known and novel miRNAs and to understand their potential roles in the growth and development of winter wheat. We examined miRNA expression using a high-throughput sequencing technique. A total of 373 known, 55 novel, and 27 putative novel miRNAs were identified. Ninety-one miRNAs were found to be differentially expressed at the four stages. Among them, the expression of six known and eight novel miRNAs was significantly suppressed at the winter dormancy stage, whereas the expression levels of seven known and eight novel miRNAs were induced at this stage; three known miRNAs and three novel miRNAs were significantly induced at the spring green-up stage; six known miRNAs were induced at the spring green-up stage and reached the highest expression level at the jointing stage; and 20 known miRNAs and 10 novel miRNAs were significantly induced at the jointing stage. Expression of a number of representative differentially expressed miRNAs was verified using quantitative real-time polymerase chain reaction (qRT-PCR). Potential target genes for known and novel miRNAs were predicted. Moreover, six novel target genes for four Pooideae species-specific miRNAs and two novel miRNAs were verified using the RNA ligase-mediated 5'-rapid amplification of cDNA ends (RLM-5'RACE) technique. These results indicate that miRNAs are key non-coding regulatory factors modulating the growth and development of wheat. Our study provides valuable information for in-depth understanding of the regulatory mechanism of miRNAs in cold acclimation and vegetative/reproductive transition of winter wheat grown in the field.

Transcriptome analysis of osmotic-responsive genes in ABA-dependent and -independent pathways in wheat (Triticum aestivum L.) roots.

Bread wheat is one of the most important crops in the world. However, osmotic stress significantly inhibits wheat growth and development, and reduces crop yield and quality. Plants respond to osmotic stress mainly through abscisic acid (ABA)-dependent and -independent pathways. In this study, root transcriptome profiles of wheat seedlings exposed to osmotic stress and exogenous ABA were analysed to identify osmotic-responsive genes belonging to the ABA-dependent or -independent pathways. We found that osmotic stress promoted proline biosynthesis in the ABA-dependent pathway, and trehalose biosynthesis is likely promoted among soluble sugars to maintain protein bioactivity under osmotic stress. In wheat roots subjected to osmotic stress, calcium ions, and glutathione exert their functions mainly through calcium-binding protein (CaM/CML) and glutathione-S-transferase, respectively, depending on both pathways. In addition, a complex relationship among phytohormones signal transduction was observed in response to osmotic stress. The findings of this study deepen our understanding of the molecular mechanisms of osmotic-stress resistance, and provide several candidate osmotic-responsive genes for further study.

Overexpression of TaWRKY146 Increases Drought Tolerance through Inducing Stomatal Closure in Arabidopsis thaliana.

As a superfamily of transcription factors, the tryptophan-arginine-lysine-tyrosine (WRKY) transcription factors have been found to be essential for abiotic and biotic stress responses in plants. Currently, only 76 WRKY transcription factors in wheat could be identified in the NCBI database, among which only a few have been functionally analyzed. Herein, a total of 188 WRKY transcription factors were identified from the wheat genome database, which included 123 full-length coding sequences, and all of them were used for detailed evolution studies. By bioinformatics analysis, a WRKY transcription factor, named TaWRKY146, was found to be the homologous gene of AtWRKY46, overexpression of which leads to hypersensitivity to drought and salt stress in Arabidopsis. Consequently, the full length of TaWRKY146 was cloned, and the expression levels of TaWRKY146 were found significantly up-regulated in the leaves and roots of wheat seedlings, which were subjected to osmotic stress. Overexpression of TaWRKY146 in Arabidopsis was shown to enhance drought tolerance by the induction of stomatal closure that reduced the transpiration rate. All these results provide a firm foundation for further identification of WRKY transcription factors with important functions in wheat.

Proteomic profiling analysis reveals that glutathione system plays important roles responding to osmotic stress in wheat (Triticum aestivum L.) roots.

Wheat is one of the most important crops in the world, and osmotic stress has become one of the main factors affecting wheat production. Understanding the mechanism of the response of wheat to osmotic stress would be greatly significant. In the present study, isobaric tag for relative and absolute quantification (iTRAQ) was used to analyze the changes of protein expression in the wheat roots exposed to different osmotic stresses. A total of 2,228 expressed proteins, including 81 differentially expressed proteins, between osmotic stress and control, were found. The comprehensive analysis of these differentially expressed proteins revealed that osmotic stress increased the variety of expressed proteins and suppressed the quantity of expressed proteins in wheat roots. Furthermore, the proteins for detoxifying and reactive oxygen species scavenging, especially the glutathione system, played important roles in maintaining organism balance in response to osmotic stress in wheat roots. Thus, the present study comprehensively describes the protein expression changes in wheat roots in response to osmotic stress, providing firmer foundation to further study the mechanism of osmotic resistance in wheat.

[Identification and analysis of the NAC transcription factor family in Triticum urartu].

NAC transcription factors are one of plant-specific gene families with diverse functions, and they regulate plant development, organ formation and stress responses. Currently, the researches about NAC transcription factors mainly focus on model plants, Arabidopsis and rice, whereas such studies are hardly reported in wheat and other plants. In this study, the full-length coding sequences (CDS) of NAC transcription factors from Triticum urartu (TuNAC) were identified through bioinformatic analysis. Their biological function, evolutionary relationship, gene duplication and chromosomal locations were further predicted and analyzed. The quantitative real-time PCR (qRT-PCR) assay was used to verify the expression pattern of abiotic-related TuNAC transcription factors. A total of 87 TuNAC transcription factors with full-length CDS were identified, which were divided into seven subgroups through phylogenetic analysis. Thirty-nine TuNAC transcription factors were located on seven chromosomes, and five pairs of TuNAC transcription factors were duplicated. The expression of four TuNAC transcription factors was consistently increased under diverse abiotic stress by qRT-PCR assay. Our study thus provides basis for further functional investigations of TuNAC transcription factors.

[Effects of silicon on the ultrastructures of wheat radical cells under copper stress].

To explore the alleviation effect of silicon on wheat growth under copper stress, cultivar Aikang 58 was chosen as the experimental material. The growth, root activities and root tip ultrastructures of wheat seedlings, which were cultured in Hoagland nutrient solution with five different treatments (control, 15 mg x L(-1) Cu2+, 30 mg x L(-1) Cu2+, 15 mg x L(-1) Cu2+ and 50 mg x L(-1) silicon, 30 mg x L(-1) Cu2+ and 50 mg x L(-1) silicon), were fully analyzed. The results showed that root length, plant height and root activities of wheat seedlings were significantly restrained under the copper treatments compared with the control (P < 0.01), while these restraining effects were alleviated after adding silicon to copper-stress Hoagland nutrient solution. Under copper stress, the cell wall and cell membrane of wheat seedling root tips suffered to varying degrees of destruction, which caused the increase of intercellular space and the disappearance of some organelles. After adding silicon, the cell structure was maintained intact, although some cells and organelles were still slightly deformed compared with the control. In conclusion, exogenous silicon could alleviate the copper stress damages on wheat seedlings and cellular components to some extent.

Genome-wide analysis of the WRKY transcription factors in aegilops tauschii.

The WRKY transcription factors (TFs) play important roles in responding to abiotic and biotic stress in plants. However, due to its unfinished genome sequencing, relatively few WRKY TFs with full-length coding sequences (CDSs) have been identified in wheat. Instead, the Aegilops tauschii genome, which is the D-genome progenitor of the hexaploid wheat genome, provides important resources for the discovery of new genes. In this study, we performed a bioinformatics analysis to identify WRKY TFs with full-length CDSs from the A. tauschii genome. A detailed evolutionary analysis for all these TFs was conducted, and quantitative real-time PCR was carried out to investigate the expression patterns of the abiotic stress-related WRKY TFs under different abiotic stress conditions in A. tauschii seedlings. A total of 93 WRKY TFs were identified from A. tauschii, and 79 of them were found to be newly discovered genes compared with wheat. Gene phylogeny, gene structure and chromosome location of the 93 WRKY TFs were fully analyzed. These studies provide a global view of the WRKY TFs from A. tauschii and a firm foundation for further investigations in both A. tauschii and wheat.

[Application of ICP-MS/ICP-AES to detecting nutrition and heavy metal contents in grain of detached wheat spikes in vitro culture].

The heavy metals in the environment led to the defect of nutrient contents in grains and metabolic disturbance seriously. In order to research the effect of heavy metal copper and cadmium on nutrient contents and heavy metal contents of wheat grain, the present paper studied the nutrition element (P, K, Ca, Mg, Na, Fe, Mn, Zn and B) and contents of heavy metal (Pb, Cr, Hg, Cu and Cd) in organs of detached wheat spikes in vitro culture by ICP-MS/ICP-AES. The results showed that the weight of grain reduced significantly with copper and cadmium treatment. Under different copper treatment, the contents of P, K, Mg, Ca, Na, Mn and Zn increased, but Fe and B contents reduced. Exogenous cadmium promoted the absorption of P, K, Ca, Mg and Mn while interrupted the absorption of Na, Fe, Zn and B. Exogenous copper and cadmium treatment reduced the Hg content in wheat grain, but the Cu and Cd contents were 55-folds and 62 folds of the national food sanitation standard. All the data showed that the effects of copper and cadmium are disadvantageous to the accumulation of nutrient and heavy metal contents.