Transcriptome based identification and validation of heat stress transcription factors in wheat progenitor species Aegilops speltoides.

Wheat, one of the major cereal crops worldwide, get adversely affected by rising global temperature. We have identified the diploid B genome progenitor of wheat, Aegilops speltoides (SS), as a potential donor for heat stress tolerance. Therefore, the present work was planned to study the total transcriptome profile of heat stress-tolerant Ae. speltoides accession pau3809 (AS3809) and compare with that of tetraploid and hexaploid wheat cultivars PDW274 and PBW725, respectively. The comparative transcriptome was utilized to identify and validate heat stress transcription factors (HSFs), the key genes involved in imparting heat stress tolerance. Transcriptome analysis led to the identification of a total of 74 K, 68 K, and 76 K genes in AS3809, PDW274, and PBW725, respectively. There was a high uniformity of GO profiles under the biological, molecular, and cellular functions across the three wheat transcriptomes, suggesting the conservation of gene function. Twelve HSFs having the highest FPKM value were identified in the AS3809 transcriptome data, while six of these HSFs namely HSFA3, HSFA5, HSFA9, HSFB2a, HSFB2b, and HSFC1b, were validated with qRT PCR. These six HSFs were identified as an important component of thermotolerance in AS3809 as evident from their comparative higher expression under heat stress.

Selection and Validation of Reference Genes for RT-qPCR Analysis in Aegilops tauschii (Coss.) under Different Abiotic Stresses.

Aegilops tauschii (Coss.) is an aggressive and serious annual grass weed in China. Its DD genome is a rich source of genetic material and performs better under different abiotic stress conditions (salinity, drought, temperature, etc.). Reverse-transcribed quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for reference gene selection and validation. This work aimed to evaluate the stability of reference gene expression in Ae. tauschii under different abiotic stresses (salinity, drought, hot, and cold) and developmental stages (seedling and development). The results show that the ubiquitin-conjugating enzyme E2 36-like (UBC36) and protein microrchidia 2-like (HSP) are the most stable genes under control and salinity conditions, respectively. Under drought stress conditions, UBC36 is more stable as compared with others. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is the most stable reference gene during heat stress conditions and thioredoxin-like protein (YLS) under cold stress condition. Phosphate2A serine/threonine-protein phosphatase 2A (PP2A) and eukaryotic translation initiation factor 3 (ETIF3) are the most stable genes at seedling and developmental stages. Intracellular transport protein (CAC) is recommended as the most stable gene under different abiotic stresses and at developmental stages. Furthermore, the relative expression levels of NHX1 and DREB under different levels of salinity and drought stress conditions varied with the most (HSP and UBC36) and least (YLS and ACT) stable genes. This study provides reliable reference genes for understanding the tolerance mechanisms in Ae. tauschii under different abiotic stress conditions.

Identification and validation of genetic locus Rfk1 for wheat fertility restoration in the presence of Aegilops kotschyi cytoplasm.

KEY MESSAGE: Major fertility restorer locus for Aegilops kotschyi cytoplasm in wheat, Rfk1, was mapped to chromosome arm 1BS. Most likely candidate gene is TraesCS1B02G197400LC, which is predicted to encode a pectinesterase/pectinesterase inhibitor. Cytoplasmic male sterility (CMS) is widely used for heterosis and hybrid seed production in wheat. Genes related to male fertility restoration in the presence of Aegilops kotschyi cytoplasm have been reported, but the fertility restoration-associated gene loci have not been investigated systematically. In this study, a BC1F1 population derived from a backcross between KTP116A, its maintainer line TP116B, and its restorer line LK783 was employed to map fertility restoration by bulked segregant RNA-Seq (BSR-Seq). A major fertility allele restorer locus for Ae. kotschyi cytoplasm in wheat, Rfk1, was mapped to chromosome arm 1BS, and it was contributed by LK783. Morphological and cytological studies showed that male fertility restoration occurred mainly after the late uninucleate stage. Based on simple sequence repeat and single-nucleotide polymorphism genotyping, the gene locus was located between Xnwafu_6 and Xbarc137 on chromosome arm 1BS. To further isolate the specific region, six Kompetitive allele-specific polymerase chain reaction markers derived from BSR-Seq were developed to delimit Rfk1 within physical intervals of 26.0 Mb. After searching for differentially expressed genes within the candidate interval in the anthers and sequencing analysis, TraesCS1B02G197400LC was identified as a candidate gene for Rfk1 and it was predicted to encode a pectinesterase/pectinesterase inhibitor. Expression analysis also confirmed that it was specifically expressed in the anthers, and its expression level was higher in fertile lines compared with sterile lines. Thus, TraesCS1B02G197400LC was identified as the most likely candidate gene for Rfk1, thereby providing insights into the fertility restoration mechanism for K-type CMS in wheat.

Effects of plant density on tillering in the weed grass Aegilops tauschii Coss. and its phytohormonal regulation.

Aegilops tauschii Coss, a notorious wheat field weed, poses a serious threat to wheat in China. Tillers are an important agronomic tool for yield. In this study, a total of 12 Ae. tauschii populations were collected from China to investigate the effect of plant density on tiller occurrence and its phytohormonal regulation. We assayed the growth parameters of Ae. tauschii and the levels of endogenous hormones at different plant densities. The results showed that plant density had a significant effect on the quantity and quality of Ae. tauschii seeds produced per plant. In particular, the tiller and spike numbers per plant were negatively affected by plant density (P < 0.0001). The contents of 13 endogenous hormones in the tiller nodes changed in response to plant density. Among them, indole-3-acetic acid (IAA) and gibberellin (GA) positively responded to plant density. However, the reverse result was found for cytokinin (CTK). Interestingly, phylogenetic tree analysis of auxin (AeYUCCA), CK (AeIPT) and GA (AeCPS) biosynthesis related genes found that phylogenies in the Gramineae for the three different genes were various, hinting at horizontal gene transfer. Moreover, the dynamics of the expression of AeYUCCA, AeIPT and AeCPS were roughly consistent with their phytohormone contents during tillering stage. When externally sprayed on plants of Ae. tauschii, 2,4-D isooctyl ester and GA3 markedly reduced its tillering while 6-BA had no significant effect.

New gene functions are involved in the thermotolerance of the wild wheat relative Aegilops umbellulata.

Wheat is one of the most important food crops in the world for human consumption, like all plants it is exposed to environmental stresses including high temperatures. The deleterious effect of high temperatures negatively affects plant growth and development, leading to reduced viability and yield. These effects can be reduced by improvement of thermotolerance through innovative breeding strategies, based on the expansion of the genetic pool available, by exploring important genetic functions from wheat wild progenitors. Improving the genetic thermotolerance characteristics of wheat requires greater understanding of genetic bases of thermotolerance, through identification of high temperature stress related genes. A good source of new useful alleles is given by Aegilops species characterized by thermotolerant habits. In this study we have classified as thermotolerant or thermosensitive, on the basis of physiologic tests, some accessions of wheat wild relative species belonging to Aegilops and Triticum genera. A thermotolerant accession of Aegilops umbellulata (AUM5) was selected, subjected to different thermal treatments and analyzed at transcriptional level. By differential display reverse transcriptase polymerase chain reaction (DDRT-PCR), we investigated modulation of gene expression elicited by heat treatments. This approach allowed the identification of various transcript-derived fragments (TDFs) produced by AUM5 in response to different thermal treatments. The functions of the inducible unique genes in the molecular determination of thermotolerance process are discussed.

Comparative Transcriptome Analysis of Two Aegilops tauschii with Contrasting Drought Tolerance by RNA-Seq.

As the diploid progenitor of common wheat, Aegilops tauschii is considered to be a valuable resistance source to various biotic and abiotic stresses. However, little has been reported concerning the molecular mechanism of drought tolerance in Ae. tauschii. In this work, the drought tolerance of 155 Ae. tauschii accessions was firstly screened on the basis of their coleoptile lengths under simulated drought stress. Subsequently, two accessions (XJ002 and XJ098) with contrasting coleoptile lengths were selected and intensively analyzed on rate of water loss (RWL) as well as physiological characters, confirming the difference in drought tolerance at the seedling stage. Further, RNA-seq was utilized for global transcriptome profiling of the two accessions seedling leaves under drought stress conditions. A total of 6969 differentially expressed genes (DEGs) associated with drought tolerance were identified, and their functional annotations demonstrated that the stress response was mediated by pathways involving alpha-linolenic acid metabolism, starch and sucrose metabolism, peroxisome, mitogen-activated protein kinase (MAPK) signaling, carbon fixation in photosynthetic organisms, and glycerophospholipid metabolism. In addition, DEGs with obvious differences between the two accessions were intensively analyzed, indicating that the expression level of DEGs was basically in alignment with the physiological changes of Ae. tauschii under drought stress. The results not only shed fundamental light on the regulatory process of drought tolerance in Ae. tauschii, but also provide a new gene resource for improving the drought tolerance of common wheat.

Cellular and physiological responses to drought stress in Aegilops tauschii genotypes.

Drought stress is one of the most important limiting factors in crop yield through impact on the cellular and physiological functions of the plant. Therefore, the study of physiological responses of plants can help to better understanding the drought tolerance mechanisms. In this experiment, 125 wild diploid wheat genotypes of Aegilops tauschii were evaluated for the physiological responses under rainfed and supplemental irrigation conditions. The physiological characteristics such as leaf relative water content (RWC), excised leaf water retention (ELWR), relative water loss (RWL), chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, ion leakage, membrane stability index (MSI) and proline content were measured. The results showed that the higher proline content, lower chlorophyll degradation rate and low amount of the membrane stability index (MSI) may inhibit the grain yield reduction under rainfed conditions. It was also found that the lower ion leakage due to the low cell membrane damage may led to the higher yield under rain-fed conditions. The results of regression analysis in both rainfed and supplemental irrigation conditions showed that proline content and total chlorophyll were introduced into the model, and explained the most variation in the grain yield. So, considering the above traits, the genotypes 16, 22, 43, 66 and 106 seems to be more drought tolerant and could be exploited in wheat breeding programs after further assessments.

Early Sibling Conflict May Ultimately Benefit the Family.

Relatives often interact differently with each other than with nonrelatives, and whether kin cooperate or compete has important consequences for the evolution of mating systems, seed size, dispersal, and competition. Previous research found that the larger of the size dimorphic seeds produced by the annual plant Aegilops triuncialis suppressed germination of their smaller sibs by 25%-60%. Here, we found evidence for kin recognition and sibling rivalry later in life among Aegilops seedlings that places seed-seed interactions in a broader context. In experiments with size dimorphic seeds, seedlings reduced the growth of sibling seedlings by ∼40% but that of nonsibling seedlings by ∼25%. These sequential antagonistic interactions between seeds and then seedlings provide insight into conflict and cooperation among kin. Kin-based conflict among seeds may maintain dormancy for some seeds until the coast is clear of more competitive siblings. If so, biotically induced seed dormancy may be a unique form of cooperation, which increases the inclusive fitness of maternal plants and offspring by minimizing competition among kin.

Genome-wide and evolutionary analysis of the class III peroxidase gene family in wheat and Aegilops tauschii reveals that some members are involved in stress responses.

BACKGROUND: The class III peroxidase (PRX) gene family is a plant-specific member of the PRX superfamily that is closely related to various physiological processes, such as cell wall loosening, lignification, and abiotic and biotic stress responses. However, its classification, evolutionary history and gene expression patterns are unclear in wheat and Aegilops tauschii. RESULTS: Here, we identified 374, 159 and 169 PRXs in Triticum aestivum, Triticum urartu and Ae. tauschii, respectively. Together with PRXs detected from eight other plants, they were classified into 18 subfamilies. Among subfamilies V to XVIII, a conserved exon-intron structure within the "001" exon phases was detected in the PRX domain. Based on the analysis, we proposed a phylogenetic model to infer the evolutionary history of the exon-intron structures of PRX subfamilies. A comparative genomics analysis showed that subfamily VII could be the ancient subfamily that originated from green algae (Chlamydomonas reinhardtii). Further integrated analysis of chromosome locations and collinearity events of PRX genes suggested that both whole genome duplication (WGD) and tandem duplication (TD) events contributed to the expansion of T. aestivum PRXs (TaePRXs) during wheat evolution. To validate functions of these genes in the regulation of various physiological processes, the expression patterns of PRXs in different tissues and under various stresses were studied using public microarray datasets. The results suggested that there were distinct expression patterns among different tissues and PRXs could be involved in biotic and abiotic responses in wheat. qRT-PCR was performed on samples exposed to drought, phytohormone treatments and Fusarium graminearum infection to validate the microarray predictions. The predicted subcellular localizations of some TaePRXs were consistent with the confocal microscopy results. We predicted that some TaePRXs had hormone-responsive cis-elements in their promoter regions and validated these predicted cis-acting elements by sequencing promoters. CONCLUSION: In this study, identification, classification, evolution, and expression patterns of PRXs in wheat and relative plants were performed. Our results will provide information for further studies on the evolution and molecular mechanisms of wheat PRXs.