Genome-wide analysis, expansion and expression of the NAC family under drought and heat stresses in bread wheat (T. aestivum L.).

The NAC family is one of the largest plant-specific transcription factor families, and some of its members are known to play major roles in plant development and response to biotic and abiotic stresses. Here, we inventoried 488 NAC members in bread wheat (Triticum aestivum). Using the recent release of the wheat genome (IWGS RefSeq v1.0), we studied duplication events focusing on genomic regions from 4B-4D-5A chromosomes as an example of the family expansion and neofunctionalization of TaNAC members. Differentially expressed TaNAC genes in organs and in response to abiotic stresses were identified using publicly available RNAseq data. Expression profiling of 23 selected candidate TaNAC genes was studied in leaf and grain from two bread wheat genotypes at two developmental stages in field drought conditions and revealed insights into their specific and/or overlapping expression patterns. This study showed that, of the 23 TaNAC genes, seven have a leaf-specific expression and five have a grain-specific expression. In addition, the grain-specific genes profiles in response to drought depend on the genotype. These genes may be considered as potential candidates for further functional validation and could present an interest for crop improvement programs in response to climate change. Globally, the present study provides new insights into evolution, divergence and functional analysis of NAC gene family in bread wheat.

Coexpression network and phenotypic analysis identify metabolic pathways associated with the effect of warming on grain yield components in wheat.

Wheat grains are an important source of human food but current production amounts cannot meet world needs. Environmental conditions such as high temperature (above 30°C) could affect wheat production negatively. Plants from two wheat genotypes have been subjected to two growth temperature regimes. One set has been grown at an optimum daily mean temperature of 19°C while the second set of plants has been subjected to warming at 27°C from two to 13 days after anthesis (daa). While warming did not affect mean grain number per spike, it significantly reduced other yield-related indicators such as grain width, length, volume and maximal cell numbers in the endosperm. Whole genome expression analysis identified 6,258 and 5,220 genes, respectively, whose expression was affected by temperature in the two genotypes. Co-expression analysis using WGCNA (Weighted Gene Coexpression Network Analysis) uncovered modules (groups of co-expressed genes) associated with agronomic traits. In particular, modules enriched in genes related to nutrient reservoir and endopeptidase inhibitor activities were found to be positively associated with cell numbers in the endosperm. A hypothetical model pertaining to the effects of warming on gene expression and growth in wheat grain is proposed. Under moderately high temperature conditions, network analyses suggest a negative effect of the expression of genes related to seed storage proteins and starch biosynthesis on the grain size in wheat.

Positional cloning of a candidate gene for resistance to the sunflower downy mildew, Plasmopara halstedii race 300.

The resistance of sunflower to Plasmopara halstedii is conferred by major resistance genes denoted Pl. Previous genetic studies indicated that the majority of these genes are clustered on linkage groups 8 and 13. The Pl6 locus is one of the main clusters to have been identified, and confers resistance to several P. halstedii races. In this study, a map-based cloning strategy was implemented using a large segregating F2 population to establish a fine physical map of this cluster. A marker derived from a bacterial artificial chromosome (BAC) clone was found to be very tightly linked to the gene conferring resistance to race 300, and the corresponding BAC clone was sequenced and annotated. It contains several putative genes including three toll-interleukin receptor-nucleotide binding site-leucine rich repeats (TIR-NBS-LRR) genes. However, only one TIR-NBS-LRR appeared to be expressed, and thus constitutes a candidate gene for resistance to P. halstedii race 300.

Structural, expression and interaction analysis of rice SKP1-like genes.

The degradation of proteins by the 26S proteasome is initiated by protein polyubiquitination mediated by a three-step cascade. The specific ubiquitination of different target proteins is mediated by different classes of E3 ubiquitin ligases, among which the best known are Skp1-Cullin-F-box complexes. Whereas protists, fungi and some vertebrates have a single SKP1 gene, many animal and plant species possess multiple SKP1 homologues. In this paper, we report on the structure, phylogeny and expression of the complete set of rice SKP1 genes (OSKs, Oryza sativa SKP1-like genes). Our analyses indicated that OSK1 and OSK20 belong to a class of SKP1 genes that contain one intron at a conserved position and are highly expressed. In addition, our yeast two-hybrid results revealed that OSK proteins display a differing ability to interact with F-box proteins. However, OSK1 and OSK20 seemed to interact with most of the nine F-box proteins tested. We suggest that rice OSK1 and OSK20 are likely to have functions similar to the Arabidopsis ASK1 and ASK2 genes.

Down-regulation of the TaGW2 gene by RNA interference results in decreased grain size and weight in wheat.

For important food crops such as wheat and rice, grain yield depends on grain number and size. In rice (Oryza sativa), GW2 was isolated from a major quantitative trait locus for yield and encodes an E3 RING ligase that negatively regulates grain size. Wheat (Triticum aestivum) has TaGW2 homologues in the A, B, and D genomes, and polymorphisms in TaGW2-A were associated with grain width. Here, to investigate TaGW2 function, RNA interference (RNAi) was used to down-regulate TaGW2 transcript levels. Transgenic wheat lines showed significantly decreased grain size-related dimensions compared with controls. Furthermore, TaGW2 knockdown also caused a significant reduction in endosperm cell number. These results indicate that TaGW2 regulates grain size in wheat, possibly by controlling endosperm cell number. Wheat and rice GW2 genes thus seem to have divergent functions, with rice GW2 negatively regulating grain size and TaGW2 positively regulating grain size. Analysis of transcription of TaGW2 homoeologues in developing grains suggested that TaGW2-A and -D act in both the division and late grain-filling phases. Furthermore, biochemical and molecular analyses revealed that TaGW2-A is a functional E3 RING ubiquitin ligase with nucleocytoplasmic subcellular partitioning. A functional nuclear export sequence responsible for TaGW2-A export from the nucleus to the cytosol and retention in the nucleolus was identified. Therefore, these results show that TaGW2 acts in the regulation of grain size and may provide an important tool for enhancement of grain yield.

Transcriptional profile analysis of E3 ligase and hormone-related genes expressed during wheat grain development.

BACKGROUND: Wheat grains are an important source of food, stock feed and raw materials for industry, but current production levels cannot meet world needs. Elucidation of the molecular mechanisms underlying wheat grain development will contribute valuable information to improving wheat cultivation. One of the most important mechanisms implicated in plant developmental processes is the ubiquitin-proteasome system (UPS). Among the different roles of the UPS, it is clear that it is essential to hormone signaling. In particular, E3 ubiquitin ligases of the UPS have been shown to play critical roles in hormone perception and signal transduction. RESULTS: A NimbleGen microarray containing 39,179 UniGenes was used to study the kinetics of gene expression during wheat grain development from the early stages of cell division to the mid-grain filling stage. By comparing 11 consecutive time-points, 9284 differentially expressed genes were identified and annotated during this study. A comparison of the temporal profiles of these genes revealed dynamic transcript accumulation profiles with major reprogramming events that occurred during the time intervals of 80-120 and 220-240°Cdays. The list of the genes expressed differentially during these transitions were identified and annotated. Emphasis was placed on E3 ligase and hormone-related genes. In total, 173 E3 ligase coding genes and 126 hormone-related genes were differentially expressed during the cell division and grain filling stages, with each family displaying a different expression profile. CONCLUSIONS: The differential expression of genes involved in the UPS and plant hormone pathways suggests that phytohormones and UPS crosstalk might play a critical role in the wheat grain developmental process. Some E3 ligase and hormone-related genes seem to be up- or down-regulated during the early and late stages of the grain development.

Molecular characterization of two types of resistance in sunflower to Plasmopara halstedii, the causal agent of downy mildew.

Depending on host-pathotype combination, two types of sunflower-Plasmopara halstedii incompatibility reactions have previously been identified. Type I resistance can restrict the growth of the pathogen in the basal region of the hypocotyls, whereas type II cannot, thus allowing the pathogen to reach the cotyledons. In type II resistance, a large portion of the hypocotyls is invaded by the pathogen and, subsequently, a hypersensitive reaction (HR) is activated over a long portion of the hypocotyls. Thus, the HR in type II resistance coincides with a higher induction of hsr203j sunflower homologue in comparison with type I resistance, where the HR is activated only in the basal part of hypocotyls. Although the pathogen was not detected in cotyledons of type I resistant plants, semiquantitative polymerase chain reaction confirmed the early abundant growth of the pathogen in cotyledons of susceptible plants by 6 days postinfection (dpi). This was in contrast to scarce growth of the pathogen in cotyledons of type II-resistant plants at a later time point (12 dpi). This suggests that pathogen growth differs according to the host-pathogen combination. To get more information about sunflower downy mildew resistance genes, the full-length cDNAs of RGC151 and RGC203, which segregated with the PlARG gene (resistance type I) and Pl14 gene (resistance type II), were cloned and sequenced. Sequence analyses revealed that RGC151 belongs to the Toll/interleukin-1 receptor (TIR) nucleotide-binding site leucine-rich repeat (NBS-LRR) class whereas RGC203 belongs to class coiled-coil (CC)-NBS-LRR. This study suggests that type II resistance may be controlled by CC-NBS-LRR gene transcripts which are enhanced upon infection by P. halstedii, rather than by the TIR-NBS-LRR genes that might control type I resistance.

Proteomic and morphological analysis of early stages of wheat grain development.

The identification of 249 proteins in the first 2 wks of wheat grain development enabled the chronological description of the early processes of grain formation. Cell division involved expression of the enzymes and proteins of the cytoskeleton and structure, DNA repair and replication enzymes and cellular metabolism enzymes (synthesis of amino acids, cell wall initiation, carbon fixation and energy production, cofactors and vitamins) with a peak expression at 125 degrees C day (degrees day after anthesis). After the first synthesis of amino acids, protein transport mechanisms, translation signals, sugar metabolism (polymerization of protein) and stress/defence proteins were activated with stable expression between 150 and 280 degrees C day. Proteins responsible for folding and degradation, including different subunits of proteasome, were highly expressed at 195 degrees C day. Proteins associated with starch granules (GBSS type 1) were present at the beginning of grain formation and increased regularly up to 280 degrees C day. Heat shock proteins (HSP70, 80, 90) were expressed throughout the early grain development stages.

Expressed Sequence Tags from the oomycete Plasmopara halstedii, an obligate parasite of the sunflower.

BACKGROUND: Sunflower downy mildew is a major disease caused by the obligatory biotrophic oomycete Plasmopara halstedii. Little is known about the molecular mechanisms underlying its pathogenicity. In this study we used a genomics approach to gain a first insight into the transcriptome of P. halstedii. RESULTS: To identify genes from the obligatory biotrophic oomycete Plasmopara halstedii that are expressed during infection in sunflower (Helianthus annuus L.) we employed the suppression subtraction hybridization (SSH) method from sunflower seedlings infected by P. halstedii. Using this method and random sequencing of clones, a total of 602 expressed sequence tags (ESTs) corresponding to 230 unique sequence sets were identified. To determine the origin of the unisequences, PCR primers were designed to amplify these gene fragments from genomic DNA isolated either from P. halstedii sporangia or from Helianthus annuus. Only 145 nonredundant ESTs which correspond to a total of 373 ESTs (67.7%) proved to be derived from P. halstedii genes and that are expressed during infection in sunflower. A set of 87 nonredundant sequences were identified as showing matches to sequences deposited in public databases. Nevertheless, about 7% of the ESTs seem to be unique to P. halstedii without any homolog in any public database. CONCLUSION: A summary of the assignment of nonredundant ESTs to functional categories as well as their relative abundance is listed and discussed. Annotation of the ESTs revealed a number of genes that could function in virulence. We provide a first glimpse into the gene content of P. halstedii. These resources should accelerate research on this important pathogen.

A sunflower BAC library suitable for PCR screening and physical mapping of targeted genomic regions.

A sunflower BAC library consisting of 147,456 clones with an average size of 118 kb has been constructed and characterized. It represents approximately 5x sunflower haploid genome equivalents. The BAC library has been arranged in pools and superpools of DNA allowing screening with various PCR-based markers. Each of the 32 superpools contains 4,608 clones and corresponds to a 36 matrix pools. Thus, the screening of the entire library could be accomplished in less than 80 PCR reactions including positive and negative controls. As a demonstration of the feasibility of the concept, a set of 24 SSR markers covering about 36 cM in the sunflower SSR map (Tang et al. in Theor Appl Genet 105:1124-1136, 2002) have been used to screen the BAC library. About 125 BAC clones have been identified and then organized in 23 contigs by HindIII digestion. The contigs are anchored on the SSR map and thus constitutes a first-generation physical map of this region. The utility of this BAC library as a genomic resource for physical mapping and map-based cloning in sunflower is discussed.