Loci harboring genes with important role in drought and related abiotic stress responses in flax revealed by multiple GWAS models.

KEY MESSAGE: QTNs associated with drought tolerance traits and indices were identified in a flax mini-core collection through multiple GWAS models and phenotyping at multiple locations under irrigated and non-irrigated field conditions. Drought is a critical phenomenon challenging today's agricultural sector. Crop varieties adapted to moisture deficit are becoming vital. Flax can be greatly affected by limiting moisture conditions, especially during the early development and reproductive stages. Here, a mini-core collection comprising genotypes from more than 20 major growing countries was evaluated for 11 drought-related traits in irrigated and non-irrigated fields for 3 years. Heritability of the traits ranged from 44.7 to 86%. Six of the 11 traits showed significant phenotypic difference between irrigated and non-irrigated conditions. A genome-wide association study (GWAS) was performed for these six traits and their corresponding stress indices based on 106 genotypes and 12,316 single nucleotide polymorphisms (SNPs) using six multi-locus and one single-locus models. The SNPs were then assigned to 8050 linkage disequilibrium (LD) blocks to which a restricted two-stage multi-locus multi-allele GWAS was applied. A total of 144 quantitative trait nucleotides (QTNs) and 13 LD blocks were associated with at least one trait or stress index. Of these, 16 explained more than 15% of the genetic variance. Most large-effect QTN loci harbored gene(s) previously predicted to play role(s) in the associated traits. Genes mediating responses to abiotic stresses resided at loci associated with stress indices. Flax genes Lus10009480 and Lus10030150 that are predicted to encode WAX INDUCER1 and STRESS-ASSOCIATED PROTEIN (SAP), respectively, are among the important candidates detected. Accessions with multiple favorable alleles outperformed others for grain yield, thousand seed weight and fiber/biomass in non-irrigated conditions, suggesting their potential usefulness in breeding and genomic selection.

Genome-wide identification of ATP binding cassette (ABC) transporter and heavy metal associated (HMA) gene families in flax (Linum usitatissimum L.).

BACKGROUND: The recent release of the reference genome sequence assembly of flax, a self-pollinated crop with 15 chromosome pairs, into chromosome-scale pseudomolecules enables the characterization of gene families. The ABC transporter and HMA gene families are important in the control of cadmium (Cd) accumulation in crops. To date, the genome-wide analysis of these two gene families has been successfully conducted in some plant species, but no systematic evolutionary analysis is available for the flax genome. RESULTS: Here we describe the ABC transporter and HMA gene families in flax to provide a comprehensive overview of its evolution and some support towards the functional annotation of its members. The 198 ABC transporter and 12 HMA genes identified in the flax genome were classified into eight ABC transporter and four HMA subfamilies based on their phylogenetic analysis and domains' composition. Nine of these genes, i.e., LuABCC9, LuABCC10, LuABCG58, LuABCG59, LuABCG71, LuABCG72, LuABCG73, LuHMA3, and LuHMA4, were orthologous with the Cd associated genes in Arabidopsis, rice and maize. Ten motifs were identified from all ABC transporter and HMA genes. Also, several motifs were conserved among genes of similar length, but each subfamily each had their own motif structures. Both the ABC transporter and HMA gene families were highly conserved among subfamilies of flax and with those of Arabidopsis. While four types of gene duplication were observed at different frequencies, whole-genome or segmental duplications were the most frequent with 162 genes, followed by 29 dispersed, 14 tandem and 4 proximal duplications, suggesting that segmental duplications contributed the most to the expansion of both gene families in flax. The rates of non-synonymous to synonymous (Ka/Ks) mutations of paired duplicated genes were for the most part lower than one, indicative of a predominant purifying selection. Only five pairs of genes clearly exhibited positive selection with a Ka/Ks ratio greater than one. Gene ontology analyses suggested that most flax ABC transporter and HMA genes had a role in ATP binding, transport, catalytic activity, ATPase activity, and metal ion binding. The RNA-Seq analysis of eight different organs demonstrated diversified expression profiling patterns of the genes and revealed their functional or sub-functional conservation and neo-functionalization. CONCLUSION: Characterization of the ABC transporter and HMA gene families will help in the functional analysis of candidate genes in flax and other crop species.

Evolution of Carthamus species revealed through sequence analyses of the fad2 gene family.

The diversity of 11 fatty acid desaturase (fad2) genes has not been investigated between cultivated and wild species in the Carthamus genus. In this study, 17 C. tinctorius accessions and 28 accessions from other Carthamus species were subjected to sequence analyses of this fad2 gene family. Results showed that among these genes, fad2-1 had a major role in the conversion of oleic acid to linoleic acid. Grouping of all studied wild polyploid species and the wild diploid C. leucocaulos suggested that C. lanatus transferred its fad2-1 gene to C. turkestanicus and C. lanatus. A phylogenetic tree based on fad2-1 gene sequences also showed that C. palaestinus and C. oxyacanthus grouped with C. tinctorius individuals, suggesting that C. tinctorius is closely related to both wild species. A one base pair deletion at position 604 in the fad2-1 gene coding region correlated with high levels of oleic acid content in five mutant phenotypes of the evaluated C. tinctorius accessions. Grouping of fad2-1 and fad2-8 (Ctfad2-10) indicated that both of these genes are involved in oleate desaturases activity. The fad2-3 (Ctfad2-3) and Ctfad2-4 had the highest sequence similarity among the other fad2 genes, indicating the conservative nature of these two genes among all the studied species. Our results suggest that C. lanatus is the likely progenitor of C. turkestanicus and C. creticus (Synonym C. baeticus). Also, C. palaestinus is genetically closer to C. tinctorius but the involvement of C. oxyacanthus cannot be excluded and, this requires further investigation.

The Complex Genetic Architecture of Early Root and Shoot Traits in Flax Revealed by Genome-Wide Association Analyses.

Roots are fundamental organs for water and nutrient uptake as well as for signal transduction in response to biotic and abiotic stresses. Flax has a shallow tap root system that relies mostly on top soil nutrient and moisture resources. The crop can easily be outcompeted by weeds or other crops in intercropping systems, especially in moisture deficit conditions. However, there is a wide range of variation among genotypes in terms of performance under scarce resources such as moisture limitation. Here we phenotyped 15 root, two shoot traits and shoot to root dry weight ratio on 115 flax accessions grown in a hydroponic pouch system and performed a genome-wide association study (GWAS) based on seven different models to identify quantitative trait loci underlying these traits. Significant variation among genotypes was observed for the two shoot and 12 of the 14 root traits. Shoot dry weight was correlated with root network volume, length, surface area, and root dry weight (r > 0.5, P < 0.001) but not significantly correlated with root depth (r = 0.033, P > 0.05). The seven GWAS models detected a total of 228 quantitative trait nucleotides (QTNs) for 16 traits. Most loci, defined by an interval of 100 kb up and downstream of the QTNs, harbored genes known to play role(s) in root and shoot development, suggesting them as candidates. Examples of candidate genes linked to root network QTNs included genes encoding GRAS transcription factors, mitogen-activated protein kinases, and auxin related lateral organ boundary proteins while QTN loci for shoot dry weight harbored genes involved in photomorphogenesis and plant immunity. These results provide insights into the genetic bases of early shoot and root development traits in flax that could be capitalized upon to improve its root architecture, particularly in view of better withstanding water limiting conditions during the cropping season.

MicroRNA-guided regulation of heat stress response in wheat.

BACKGROUND: With rising global temperature, understanding plants' adaptation to heat stress has implications in plant breeding. MicroRNAs (miRNAs) are small, non-coding, regulatory RNAs guiding gene expression at the post-transcriptional level. In this study, small RNAs and the degradome (parallel analysis of RNA ends) of leaf tissues collected from control and heat-stressed wheat plants immediately at the end of the stress period and 1 and 4 days later were analysed. RESULTS: Sequencing of 24 small RNA libraries produced 55.2 M reads while 404 M reads were obtained from the corresponding 24 PARE libraries. From these, 202 miRNAs were ascertained, of which mature miRNA evidence was obtained for 104 and 36 were found to be differentially expressed after heat stress. The PARE analysis identified 589 transcripts targeted by 84 of the ascertained miRNAs. PARE sequencing validated the targets of the conserved members of miRNA156, miR166 and miR393 families as squamosa promoter-binding-like, homeobox leucine-zipper and transport inhibitor responsive proteins, respectively. Heat stress responsive miRNA targeted superoxide dismutases and an array of homeobox leucine-zipper proteins, F-box proteins and protein kinases. Query of miRNA targets to interactome databases revealed a predominant association of stress responses such as signalling, antioxidant activity and ubiquitination to superoxide dismutases, F-box proteins, pentatricopeptide repeat-containing proteins and mitochondrial transcription termination factor-like proteins. CONCLUSION: The interlaced data set generated in this study identified and validated heat stress regulated miRNAs and their target genes associated with thermotolerance. Such accurate identification and validation of miRNAs and their target genes are essential to develop novel regulatory gene-based breeding strategies.

The genetic structure of flax illustrates environmental and anthropogenic selections that gave rise to its eco-geographical adaptation.

Flax, one of the eight founder crops of agriculture, has been cultivated for its oil and/or fiber for millennia. Understanding genetic divergence and geographic origins of germplasm in line with their cultivation history and ecological adaptation are essential for conservation and breeding. Here we performed a genome-wide assessment based on more than 51,000 single nucleotide polymorphic sites defining 383 flax accessions from a core collection representing 37 flax growing countries. Population structure analysis resulted in a total of 12 populations that were pooled into four major groups: Temperate, South Asian, Abyssinian and Mediterranean. The vast majority (n = 335) belonged to the Temperate group that comprised eight populations including one dominated by fiber flax. Genetic variation between fiber and oil morphotypes was less pronounced than variation within morphotypes. The genetic variation among groups and populations was attributed in part to eco-geographic and anthropogenic factors. Genetic signatures indicated loci under strong selection by environmental factors such as day length. A high concentration of private haplotypes were observed in the South Asian, Mediterranean and Abyssinian populations despite their low genotype representation, hinting at the long history of the crop in these regions. The addition of genotypes from these three regions would enrich the core collection by capturing a wider genetic breadth for breeding and conservation.

The RING-Type E3 Ligase XBAT35.2 Is Involved in Cell Death Induction and Pathogen Response.

XBAT35 belongs to a subfamily of Arabidopsis (Arabidopsis thaliana) RING-type E3s that are similar in domain architecture to the rice (Oryza sativa) XA21 Binding Protein3, a defense protein. The XBAT35 transcript undergoes alternative splicing to produce two protein isoforms, XBAT35.1 and XBAT35.2. Here, we demonstrate that XBAT35.2 localizes predominantly to the Golgi and is involved in cell death induction and pathogen response. XBAT35.2, but not XBAT35.1, was found to trigger cell death when overexpressed in tobacco (Nicotiana benthamiana) leaves and does so in a manner that requires its RING domain. Loss of XBAT35 gene function disrupts the plant's ability to defend against pathogen attack, whereas overexpression of XBAT35.2 enhances resistance to pathogens. XBAT35.2 was found to be unstable and promotes its own degradation, suggesting self-regulation. Inoculation with virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000 results in a drastic reduction in the levels of ubiquitinated XBAT35.2 and an increase in the abundance of the E3. This implies that pathogen infection prohibits XBAT35.2 self-regulation and stabilizes the E3. In agreement with a role in defending against pathogens, XBAT35.2 interacts with defense-related Accelerated Cell Death11 (ACD11) in planta and promotes the proteasome-dependent turnover of ACD11 in cell-free degradation assays. In accordance with regulation by a stabilized XBAT35.2, the levels of ubiquitinated ACD11 increased considerably, and the abundance of ACD11 was reduced following pathogen infection. In addition, treatment of transgenic seedlings with a proteasome inhibitor results in the accumulation of ACD11, confirming proteasome-dependent degradation. Collectively, these results highlight a novel role for XBAT35.2 in cell death induction and defense against pathogens.

Microbial Degradation of Lobster Shells to Extract Chitin Derivatives for Plant Disease Management.

Biodegradation of lobster shells by chitinolytic microorganisms are an environment safe approach to utilize lobster processing wastes for chitin derivation. In this study, we report degradation activities of two microbes, "S223" and "S224" isolated from soil samples that had the highest rate of deproteinization, demineralization and chitinolysis among ten microorganisms screened. Isolates S223 and S224 had 27.3 and 103.8 protease units mg-1 protein and 12.3 and 11.2 µg ml-1 of calcium in their samples, respectively, after 1 week of incubation with raw lobster shells. Further, S223 contained 23.8 µg ml-1 of N-Acetylglucosamine on day 3, while S224 had 27.3 µg ml-1 on day 7 of incubation with chitin. Morphological observations and 16S rDNA sequencing suggested both the isolates were Streptomyces. The culture conditions were optimized for efficient degradation of lobster shells and chitinase (∼30 kDa) was purified from crude extract by affinity chromatography. The digested lobster shell extracts induced disease resistance in Arabidopsis by induction of defense related genes (PR1 > 500-fold, PDF1.2 > 40-fold) upon Pseudomonas syringae and Botrytis cinerea infection. The study suggests that soil microbes aid in sustainable bioconversion of lobster shells and extraction of chitin derivatives that could be applied in plant protection.

Deep sequencing of wheat sRNA transcriptome reveals distinct temporal expression pattern of miRNAs in response to heat, light and UV.

Understanding of plant adaptation to abiotic stresses has implications in plant breeding, especially in the context of climate change. MicroRNAs (miRNAs) and short interfering RNAs play a crucial role in gene regulation. Here, wheat plants were exposed to one of the following stresses: continuous light, heat or ultraviolet radiations over five consecutive days and leaf tissues from three biological replicates were harvested at 0, 1, 2, 3, 7 and 10 days after treatment (DAT). A total of 72 small RNA libraries were sequenced on the Illumina platform generating ~524 million reads corresponding to ~129 million distinct tags from which 232 conserved miRNAs were identified. The expression levels of 1, 2 and 79 miRNAs were affected by ultraviolet radiation, continuous light and heat, respectively. Approximately 55% of the differentially expressed miRNAs were downregulated at 0 and 1 DAT including miR398, miR528 and miR156 that control mRNAs involved in activation of signal transduction pathways and flowering. Other putative targets included histone variants and methyltransferases. These results suggest a temporal miRNA-guided post-transcriptional regulation that enables wheat to respond to abiotic stresses, particularly heat. Designing novel wheat breeding strategies such as regulatory gene-based marker assisted selection depends on accurate identification of stress induced miRNAs.

Optimal level of purple acid phosphatase5 is required for maintaining complete resistance to Pseudomonas syringae.

Plants possess an exceedingly complex innate immune system to defend against most pathogens. However, a relative proportion of the pathogens overcome host's innate immunity and impair plant growth and productivity. We previously showed that mutation in purple acid phosphatase (PAP5) lead to enhanced susceptibility of Arabidopsis to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Here, we report that an optimal level of PAP5 is crucial for mounting complete basal resistance. Overexpression of PAP5 impaired ICS1, PR1 expression and salicylic acid (SA) accumulation similar to pap5 knockout mutant plants. Moreover, plant overexpressing PAP5 was impaired in H2O2 accumulation in response to Pst DC3000. PAP5 is localized in to peroxisomes, a known site of generation of reactive oxygen species for activation of defense responses. Taken together, our results demonstrate that optimal levels of PAP5 is required for mounting resistance against Pst DC3000 as both knockout and overexpression of PAP5 lead to compromised basal resistance.

Purple Acid Phosphatase5 is required for maintaining basal resistance against Pseudomonas syringae in Arabidopsis.

BACKGROUND: Plants have evolved an array of constitutive and inducible defense strategies to restrict pathogen ingress. However, some pathogens still manage to invade plants and impair growth and productivity. Previous studies have revealed several key regulators of defense responses, and efforts have been made to use this information to develop disease resistant crop plants. These efforts are often hampered by the complexity of defense signaling pathways. To further elucidate the complexity of defense responses, we screened a population of T-DNA mutants in Colombia-0 background that displayed altered defense responses to virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). RESULTS: In this study, we demonstrated that the Arabidopsis Purple Acid Phosphatse5 (PAP5) gene, induced under prolonged phosphate (Pi) starvation, is required for maintaining basal resistance to certain pathogens. The expression of PAP5 was distinctly induced only under prolonged Pi starvation and during the early stage of Pst DC3000 infection (6 h.p.i). T-DNA tagged mutant pap5 displayed enhanced susceptibility to the virulent bacterial pathogen Pst DC3000. The pap5 mutation greatly reduced the expression of pathogen inducible gene PR1 compared to wild-type plants. Similarly, other defense related genes including ICS1 and PDF1.2 were impaired in pap5 plants. Moreover, application of BTH (an analog of SA) restored PR1 expression in pap5 plants. CONCLUSION: Taken together, our results demonstrate the requirement of PAP5 for maintaining basal resistance against Pst DC3000. Furthermore, our results provide evidence that PAP5 acts upstream of SA accumulation to regulate the expression of other defense responsive genes. We also provide the first experimental evidence indicating the role PAP5 in plant defense responses.