The miR166d/TaCPK7-D Signaling Module Is a Critical Mediator of Wheat (Triticum aestivum L.) Tolerance to K+ Deficiency.

It is well established that potassium (K+) is an essential nutrient for wheat (Triticum aestivum L.) growth and development. Several microRNAs (miRNAs), including miR166, are reportedly vital roles related to plant growth and stress responses. In this study, a K+ starvation-responsive miRNA (miR166d) was identified, which showed increased expression in the roots of wheat seedlings exposed to low-K+ stress. The overexpression of miR166d considerably increased the tolerance of transgenic Arabidopsis plants to K+ deprivation treatment. Furthermore, disrupting miR166d expression via virus-induced gene silencing (VIGS) adversely affected wheat adaptation to low-K+ stress. Additionally, miR166d directly targeted the calcium-dependent protein kinase 7-D gene (TaCPK7-D) in wheat. The TaCPK7-D gene expression was decreased in wheat seedling roots following K+ starvation treatment. Silencing TaCPK7-D in wheat increased K+ uptake under K+ starvation. Moreover, we observed that the miR166d/TaCPK7-D module could affect wheat tolerance to K+ starvation stress by regulating TaAKT1 and TaHAK1 expression. Taken together, our results indicate that miR166d is vital for K+ uptake and K+ starvation tolerance of wheat via regulation of TaCPK7-D.

Proteomic Analysis of Roots Response to Potassium Deficiency and the Effect of TaHAK1-4A on K+ Uptake in Wheat.

Potassium (K+) is essential for plant growth and stress responses. A deficiency in soil K+ contents can result in decreased wheat quality and productivity. Thus, clarifying the molecular mechanism underlying wheat responses to low-K+ (LK) stress is critical. In this study, a tandem mass tag (TMT)-based quantitative proteomic analysis was performed to investigate the differentially abundant proteins (DAPs) in roots of the LK-tolerant wheat cultivar "KN9204" at the seedling stage after exposure to LK stress. A total of 104 DAPs were identified in the LK-treated roots. The DAPs related to carbohydrate and energy metabolism, transport, stress responses and defense, and post-translational modifications under LK conditions were highlighted. We identified a high-affinity potassium transporter (TaHAK1-4A) that was significantly up-regulated after the LK treatment. Additionally, TaHAK1-4A was mainly expressed in roots, and the encoded protein was localized in the plasma membrane. The complementation assay in yeast suggested that TaHAK1-4A mediates K+ uptake under extreme LK conditions. The overexpression of TaHAK1-4A increased the fresh weight and root length of Arabidopsis under LK conditions and improved the growth of Arabidopsis athak5 mutant seedlings, which grow poorly under LK conditions. Moreover, silencing of TaHAK1-4A in wheat roots treated with LK stress decreased the root length, dry weight, K+ concentration, and K+ influx. Accordingly, TaHAK1-4A is important for the uptake of K+ by roots exposed to LK stress. Our results reveal the protein metabolic changes in wheat induced by LK stress. Furthermore, we identified a candidate gene potentially relevant for developing wheat lines with increased K+ use efficiency.

Evaluation of Salt Tolerance Mechanism and Study on Salt Tolerance Relationship of Different Salt-Tolerant Wheat Varieties.

The physiological and biochemical indexes of different salt-tolerant wheat cultivars under salt stress are affected to different degrees. The changes in physiological and biochemical indexes in salt-tolerant wheat varieties are moderate, while salt-sensitive wheat varieties have bigger changes. In this article, through comprehensive utilization of germination and seedling indoor test morphological indexes, physiological and biochemical index, and output index, combined with the evaluation mechanism of wheat's salt resistance, different salt tolerance types in the middle and lower reaches of the Yangtze River in Shandong province were studied; a collection of 100 wheat varieties and 11 wheat varieties planted over a large area were classified into three groups: strong salt resistance, medium salt resistance, and weak salt resistance. Comparative analysis of different wheat varieties' salt resistance evaluation mechanism was performed, as well as the analysis of the germination rate of wheat varieties, coleoptile growth situation, emergence rate, protect wheat seeding rate, tillering rate, seedling height, root length, seedling dry weight, wet weight, number of leaves, plant growth situation, agronomic characters of the Na+/K+ ratio, and other physiological and biochemical indexes such as salt resistance index. The evaluation mechanism of salt tolerance and the relationship of salt tolerance of different wheat varieties were obtained. The results showed that there were significant differences in salt tolerance among the 100 wheat varieties at the germination stage. Among them, three varieties, YM (Yangmai) 25, YM (Yangmai) 24, and EM (Emai) 25, had the strongest salt tolerance at the germination stage, reaching the level of the salt tolerance variety DK 961, and the salt tolerance was in the first level, while NM 17, NM 23, and other 21 varieties reached the level of the salt tolerance. These 24 varieties with strong salt tolerance at the germination stage can be used to screen the salt tolerance of wheat planted in the saline soil of Jiangsu coastal beach.

Proteomic analysis reveals the molecular mechanism underlying the cold acclimation and freezing tolerance of wheat (Triticum aestivum L.).

Cold acclimation (CA) is an important evolutionary adaptive mechanism for wheat freezing resistence. To clarify the molecular basis of wheat CA and freezing tolerance, the effects of CA (4 °C) and non-CA (20 °C) treatments and freezing stress (-5 °C) on the proteins in the wheat crown were characterized via an iTRAQ-based proteomic analysis. A total of 669 differentially accumulated proteins (DAPs) were identified after the CA, of which seven were also DAPs in the CA plants exposed to freezing stress. Additionally, the 15 DAPs in the CA group and the 23 DAPs in the non-CA group after the freezing treatment differed substantially. Functional analyses indicated that CA enhanced freezing tolerance by regulating proteins involved in signal transduction, carbohydrate metabolism, stress and defense responses, and phenylpropanoid biosynthesis. An integrated transcriptomic, proteomic, and metabolomic analysis revealed significant changes in various components of the glutathione metabolic pathway. The overexpression and silencing of Wdhn13 in Arabidopsis and wheat resulted in increased tolerance and sensitivity to freezing stress, respectively, suggesting Wdhn13 promotes freezing tolerance. Overall, our study offers insights into the regulatory network underlying the CA and freezing tolerance of wheat, which may be useful for elucidating wheat freezing resistance.

Genome-Wide Identification and Low Temperature Responsive Pattern of Actin Depolymerizing Factor (ADF) Gene Family in Wheat (Triticum aestivum L.).

The actin depolymerizing factor (ADF) gene family, which is conserved in eukaryotes, is important for plant development, growth, and stress responses. Cold stress restricts wheat growth, development, and distribution. However, genome-wide identification and functional analysis of the ADF family in wheat is limited. Further, because of the promising role of ADF genes in cold response, there is need for an understanding of the function of this family on wheat under cold stress. In this study, 25 ADF genes (TaADFs) were identified in the wheat genome and they are distributed on 15 chromosomes. The TaADF gene structures, duplication events, encoded conversed motifs, and cis-acting elements were investigated. Expression profiles derived from RNA-seq data and real-time quantitative PCR analysis revealed the tissue- and temporal-specific TaADF expression patterns. In addition, the expression levels of TaADF13/16/17/18/20/21/22 were significantly affected by cold acclimation or freezing conditions. Overexpression of TaADF16 increased the freezing tolerance of transgenic Arabidopsis, possibly because of enhanced ROS scavenging and changes to the osmotic regulation in cells. The expression levels of seven cold-responsive genes were up-regulated in the transgenic Arabidopsis plants, regardless of whether the plants were exposed to low temperature. These findings provide fundamental information about the wheat ADF genes and may help to elucidate the regulatory effects of the encoded proteins on plant development and responses to low-temperature stress.

Genome-wide association study reveals the genetic basis of yield- and quality-related traits in wheat.

BACKGROUND: Identifying the loci and dissecting the genetic architecture underlying wheat yield- and quality-related traits are essential for wheat breeding. A genome-wide association study was conducted using a high-density 90 K SNP array to analyze the yield- and quality-related traits of 543 bread wheat varieties. RESULTS: A total of 11,140 polymorphic SNPs were distributed on 21 chromosomes, including 270 significant SNPs associated with 25 yield- and quality-related traits. Additionally, 638 putative candidate genes were detected near the significant SNPs based on BLUP data, including three (TraesCS7A01G482000, TraesCS4B01G343700, and TraesCS6B01G295400) related to spikelet number per spike, diameter of the first internode, and grain volume. The three candidate genes were further analyzed using stage- and tissue- specific gene expression data derived from an RNA-seq analysis. These genes are promising candidates for enhancing yield- and quality-related traits in wheat. CONCLUSIONS: The results of this study provide a new insight to understand the genetic basis of wheat yield and quality. Furthermore, the markers detected in this study may be applicable for marker-assisted selection in wheat breeding programs.

Multi-Omics Analyses Reveal the Molecular Mechanisms Underlying the Adaptation of Wheat (Triticum aestivum L.) to Potassium Deprivation.

Potassium (K) is essential for regulating plant growth and mediating abiotic stress responses. Elucidating the biological mechanism underlying plant responses to K-deficiency is crucial for breeding new cultivars with improved K uptake and K utilization efficiency. In this study, we evaluated the extent of the genetic variation among 543 wheat accessions differing in K-deficiency tolerance at the seedling and adult plant stages. Two accessions, KN9204 and BN207, were identified as extremely tolerant and sensitive to K-deficiency, respectively. The accessions were exposed to normal and K-deficient conditions, after which their roots underwent ionomic, transcriptomic, and metabolomic analyses. Under K-deficient conditions, KN9204 exhibited stronger root growth and maintained higher K concentrations than BN207. Moreover, 19,440 transcripts and 162 metabolites were differentially abundant in the roots of both accessions according to transcriptomic and metabolomic analyses. An integrated analysis of gene expression and metabolite profiles revealed that substantially more genes, including those related to ion homeostasis, cellular reactive oxygen species homeostasis, and the glutamate metabolic pathway, were up-regulated in KN9204 than in BN207 in response to low-K stress. Accordingly, these candidate genes have unique regulatory roles affecting plant K-starvation tolerance. These findings may be useful for further clarifying the molecular changes underlying wheat root adaptations to K deprivation.

Global identification and characterization of miRNA family members responsive to potassium deprivation in wheat (Triticum aestivum L.).

Potassium (K) is essential for plant growth and stress responses. MicroRNAs (miRNAs) are involved in adaptation to nutrient deprivation through modulating gene expression. Here, we identified the miRNAs responsive to K deficiency in Triticum aestivum based on high-throughput small RNA sequencing analyses. Eighty-nine miRNAs, including 68 previously reported ones and 21 novel ones, displayed differential expression under K deficiency. In Gene Ontology and Kyoto Encyclopedia and Genome analyses, the putative target genes of the differentially expressed miRNAs were categorized into functional groups associated with ADP-binding activity, secondary metabolic pathways, and biosynthesis and metabolism. Functional characterization of tae-miR408, an miRNA significantly down-regulated under K deficiency, revealed its important role in mediating low-K tolerance. Compared with wild type, transgenic tobacco lines overexpressing tae-miR408 showed significantly improved K uptake, biomass, photosynthesis, and reactive oxygen species scavenging under K deficiency. These results show that distinct miRNAs function in the plant response to K deficiency through regulating target genes involved in energy metabolism and various secondary metabolic pathways. Our findings shed light on the plant response to K deficiency mediated by miRNAs in T. aestivum. Distinct miRNAs, such as tae-miR408, are valuable targets for generating crop varieties with improved K-use efficiency.

[A simple error-prone PCR method through dATP reduction].

UNLABELLED: The error-prone PCR is one of the main methods for in vitro gene mutagenesis, usually through adding Mn2+ increasing Mg2+ and dCTP/dTTP concentration. OBJECTIVE AND METHODS: In this study, both the antifungal protein gene Ace-AMP1 from Allium cepa and the Bt toxin gene cry1A(c) from Bacillus thuringiensis were subjected to PCR mutagenesis through reducing the dATP concentration, but without adding Mn2+ or adjusting other PCR components. RESULTS: The result showed that the rates of base mutation and sequence variation were increased along with the decrease of dATP concentrations. When dTTP/dCTP/dGTP: dATP equaled 20:1-40:1, the rate of base mutation was between 1.4% and 1.8%, and the rate of sequence variation was between 77.8% and 100%. DISCUSSION AND CONCLUSION: This method is simple and practical, and enables the process optimization of several mutagenic factors in conventional error-prone PCR. Moreover, as the resulting base mutations were mainly A x T-->G x C transition, the present method provides a new way to improve the GC content of gene by in vitro mutagenesis. The mutagenesis method of simply reducing single dNTP concentration could improve AT or GC content of the target gene, it is an expansion of error-prone PCR.

Fine mapping a domestication-related QTL for spike-related traits in a synthetic wheat.

QTL analysis using a BC5F2:3 mapping population derived from a cross between Am3, a synthetic hexaploid wheat as a donor parent, and Laizhou953, a Chinese winter wheat cultivar as a recurrent parent, showed that variation at the microsatellite locus Xgwm113 on chromosome 4B was associated with variation in grain number per spike (GN), spike length (SL), and spikelet number per spike (SPI). The Qgn.caas-4B, Qsl.caas-4B, and Qspi.caas-4B were responsible for 16.6%-35.6%, 18.0%-32.3%, and 23.7%-25.9% of the phenotypic variation present in two environments, respectively. Segregation for GN fit a Mendelian monogenic ratio. A subpopulation consisting of 497 plants was used to map the QTL to a 1.2 cM interval between Xgwm113 and Xgwm857. The three spike traits, GN, SL, and SPI, were correlated and were thus probably under the pleiotropic control of the QTL. The Am3 allele had a reduction effect on all three spike traits. Evidence for positive selective history on SSR locus Xgwm113 was supported using Ewens-Watterson's statistic test on a germplasm panel of wild and landrace entries, suggesting that this genomic region may contain genes under selection during wheat domestication.