Genome-wide analysis of wall associated kinase (WAK) gene family in barley.

In plants, wall associated kinases (WAKs) form a unique subfamily of receptor like-kinases (RLKs). In Arabidopsis thaliana, WAK-RLKs are known to regulate biotic stress, cell expansion, and metal tolerance, but their detailed characterization in barley is lacking. In this study, we identified a total of 91 WAK genes in the barley genome and classified them into five groups. Evolutionary analysis of HvWAKs with AtWAKs revealed their species-specific expansion. The maximum number (19 to 20) of WAK genes were located on chromosomes 3, 5 and 6. WAK proteins exhibited similar types of motif distribution in their group. Characterization of a Ds transposon insertion mutant of the wak1 revealed differences in the root length. Further, HvSPL23 transcription factor was identified as a positive co-expressing gene with HvWAK1, suggesting its possible upstream regulator. Taken together, our study provides a base for the functional characterization of WAK family members in the future.

Single cell gene regulatory networks in plants: Opportunities for enhancing climate change stress resilience.

Global warming poses major challenges for plant survival and agricultural productivity. Thus, efforts to enhance stress resilience in plants are key strategies for protecting food security. Gene regulatory networks (GRNs) are a critical mechanism conferring stress resilience. Until recently, predicting GRNs of the individual cells that make up plants and other multicellular organisms was impeded by aggregate population scale measurements of transcriptome and other genome-scale features. With the advancement of high-throughput single cell RNA-seq and other single cell assays, learning GRNs for individual cells is now possible, in principle. In this article, we report on recent advances in experimental and analytical methodologies for single cell sequencing assays especially as they have been applied to the study of plants. We highlight recent advances and ongoing challenges for scGRN prediction, and finally, we highlight the opportunity to use scGRN discovery for studying and ultimately enhancing abiotic stress resilience in plants.

Redox-dependent interaction between thaumatin-like protein and ß-glucan influences malting quality of barley.

Barley is the cornerstone of the malting and brewing industry. It is known that 250 quantitative trait loci (QTLs) of the grain are associated with 19 malting-quality phenotypes. However, only a few of the contributing genetic components have been identified. One of these, on chromosome 4H, contains a major malting QTL, QTL2, located near the telomeric region that accounts, respectively, for 28.9% and 37.6% of the variation in the ß-glucan and extract fractions of malt. In the current study, we dissected the QTL2 region using an expression- and microsynteny-based approach. From a set of 22 expressed sequence tags expressed in seeds at the malting stage, we identified a candidate gene, TLP8 (thaumatin-like protein 8), which was differentially expressed and influenced malting quality. Transcript abundance and protein profiles of TLP8 were studied in different malt and feed varieties using quantitative PCR, immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The experiments demonstrated that TLP8 binds to insoluble (1, 3, 1, 4)-ß-D glucan in grain extracts, thereby facilitating the removal of this undesirable polysaccharide during malting. Further, the binding of TLP8 to ß-glucan was dependent on redox. These findings represent a stride forward in our understanding of the malting process and provide a foundation for future improvements in the final beer-making process.

Genomic organization, phylogenetic comparison, and expression profiles of the SPL family genes and their regulation in soybean.

SQUAMOSA Promoter-Binding Protein-Like (SPL) genes form a major family of plant-specific transcription factors and play an important role in plant growth and development. In this study, we report the identification of 41 SPL genes (GmSPLs) in the soybean genome. Phylogenetic analysis revealed that these genes were divided into five groups (groups 1-5). Further, exon/intron structure and motif composition revealed that the GmSPL genes are conserved within their same group. The N-terminal zinc finger 1 (Zn1) of the SBP domain was a CCCH (Cys3His1) and the C terminus zinc finger 2 (Zn2) was a CCHC (Cys2HisCys) type. The 41 GmSPL genes were distributed unevenly on 17 of the 20 chromosomes, with tandem and segmental duplication events. We found that segmental duplication has made an important contribution to soybean SPL gene family expansion. The Ka/Ks ratios revealed that the duplicated GmSPL genes evolved under the effect of purifying selection. In addition, 17 of the 41 GmSPLs were found as targets of miR156; these might be involved in their posttranscriptional regulation through miR156. Importantly, RLM-RACE analysis confirmed the GmmiR156-mediated cleavage of GmSPL2a transcript in 2-4 mm stage of soybean seed. Alternative splicing events in 9 GmSPLs were detected which produces transcripts and proteins of different lengths that may modulate protein signaling, binding, localization, stability, and other properties. Expression analysis of the soybean SPL genes in various tissues and different developmental stages of seed suggested distinct spatiotemporal patterns. Differences in the expression patterns of miR156-targeted and miR156-non-targeted soybean SPL genes suggest that miR156 plays key functions in soybean development. Our results provide an important foundation for further uncovering the crucial roles of GmSPLs in the development of soybean and other biological processes.

Transcriptome dynamics during fibre development in contrasting genotypes of Gossypium hirsutum L.

Understanding the contribution of genetic background in fibre quality traits is important for the development of future cotton varieties with superior fibre quality. We used Affymetrix microarray (Santa Clara, CA) and Roche 454 GSFLX (Branford, CT) for comparative transcriptome analysis between two superior and three inferior genotypes at six fibre developmental stages. Microarray-based analysis of variance (ANOVA) for 89 microarrays encompassing five contrasting genotypes and six developmental stages suggests that the stages of the fibre development have a more pronounced effect on the differentially expressed genes (DEGs) than the genetic background of genotypes. Superior genotypes showed enriched activity of cell wall enzymes, such as pectin methyl esterase, at early elongation stage, enriched metabolic activities such as lipid, amino acid and ribosomal protein subunits at peak elongation, and prolonged combinatorial regulation of brassinosteroid and auxin at later stages. Our efforts on transcriptome sequencing were focused on changes in gene expression at 25 DPA. Transcriptome sequencing resulted in the generation of 475 658 and 429 408 high-quality reads from superior and inferior genotypes, respectively. A total of 24 609 novel transcripts were identified manually for Gossypium hirsutum with no hits in NCBI 'nr' database. Gene ontology analyses showed that the genes for ribosome biogenesis, protein transport and fatty acid biosynthesis were over-represented in superior genotype, whereas salt stress, abscisic acid stimuli and water deprivation leading to the increased proteolytic activity were more pronounced in inferior genotype.

Differential expression and global analysis of miR156/SQUAMOSA promoter binding-like proteins (SPL) module in oat.

SQUAMOSA promoter binding-like proteins (SPLs) are important transcription factors that influence growth phase transition and reproduction in plants. SPLs are targeted by miR156 but the SPL/miR156 module is completely unknown in oat. We identified 28 oat SPL genes (AsSPLs) distributed across all 21 oat chromosomes except for 4C and 6D. The oat- SPL gene family represented six of eight SPL phylogenetic groups, with no AsSPLs in groups 3 and 7. A novel oat miR156 (AsmiR156) family with 21 precursors divided into 7 groups was characterized. A total of 16 AsSPLs were found to be targeted by AsmiR156. Intriguingly, AsSPL3s showed high transcript abundance during early inflorescence (GS-54), as compared to the lower abundance of AsmiR156, indicating their role in reproductive development. Unravelling the SPL/miR156 regulatory hub and alterations in expression patterns of AsSPLs could provide an essential toolbox for genetic improvement in the cultivated oat.

CAMTA 1 regulates drought responses in Arabidopsis thaliana.

BACKGROUND: Transcription factors (TF) play a crucial role in regulating gene expression and are fit to regulate diverse cellular processes by interacting with other proteins. A TF named calmodulin binding transcription activator (CAMTA) was identified in Arabidopsis thaliana (AtCAMTA1-6). To explore the role of CAMTA1 in drought response, the phenotypic differences and gene expression was studied between camta1 and Col-0 under drought condition. RESULTS: In camta1, root development was abolished showing high-susceptibility to induced osmotic stress resulting in small wrinkled rosette leaves and stunted primary root. In camta1 under drought condition, we identified growth retardation, poor WUE, low photosystem II efficiency, decline in RWC and higher sensitivity to drought with reduced survivability. The microarray analysis of drought treated camta1 revealed that CAMTA1 regulates "drought recovery" as most indicative pathway along with other stress response, osmotic balance, apoptosis, DNA methylation and photosynthesis. Interestingly, majority of positively regulated genes were related to plasma membrane and chloroplast. Further, our analysis indicates that CAMTA1 regulates several stress responsive genes including RD26, ERD7, RAB18, LTPs, COR78, CBF1, HSPs etc. and promoter of these genes were enriched with CAMTA recognition cis-element. CAMTA1 probably regulate drought recovery by regulating expression of AP2-EREBP transcription factors and Abscisic acid response. CONCLUSION: CAMTA1 rapidly changes broad spectrum of responsive genes of membrane integrity and photosynthetic machinery by generating ABA response for challenging drought stress. Our results demonstrate the important role of CAMTA1 in regulating drought response in Arabidopsis, thus could be genetically engineered for improving drought tolerance in crop.

Global analysis of SBP gene family in Brachypodium distachyon reveals its association with spike development.

SQUAMOSA-promoter binding like proteins (SBPs/SPLs) are plant specific transcription factors targeted by miR156 and involved in various biological pathways, playing multi-faceted developmental roles. This gene family is not well characterized in Brachypodium. We identified a total of 18 SBP genes in B. distachyon genome. Phylogenetic analysis revealed that SBP gene family in Brachypodium expanded through large scale duplication. A total of 10 BdSBP genes were identified as targets of miR156. Transcript cleavage analysis of selected BdSBPs by miR156 confirmed their antagonistic connection. Alternative splicing was observed playing an important role in BdSBPs and miR156 interaction. Characterization of T-DNA Bdsbp9 mutant showed reduced plant growth and spike length, reflecting its involvement in the spike development. Expression of a majority of BdSBPs elevated during spikelet initiation. Specifically, BdSBP1 and BdSBP3 differentially expressed in response to vernalization. Differential transcript abundance of BdSBP1, BdSBP3, BdSBP8, BdSBP9, BdSBP14, BdSBP18 and BdSBP23 genes was observed during the spike development under high temperature. Co-expression network, protein-protein interaction and biological pathway analysis indicate that BdSBP genes mainly regulate transcription, hormone, RNA and transport pathways. Our work reveals the multi-layered control of SBP genes and demonstrates their association with spike development and temperature sensitivity in Brachypodium.

Genome-wide analysis of the SPL/miR156 module and its interaction with the AP2/miR172 unit in barley.

The SQUAMOSA-promoter binding like (SPL) gene family encodes transcription factors that have been shown in many species to influence plant growth and development, but information about these genes in barley (Hordeum vulgare L.) is limited. This study identified 17 barley SPL genes, within eight distinct groups, that are orthologs of SPL genes described in Arabidopsis, wheat, and rice. Sixteen barley SPLs undergo alternative splicing. Seven SPLs contain a putative miR156 target site and the transcript levels of the miR156-targeted HvSPLs (HvSPL3, 13 and 23) were lower in vegetative than in reproductive phase but this was true also for some SPL genes such as HvSPL6 that were not regulated by miR156. Because SPL gene products regulate miR172, which is also involved in floral development, the expression of miR172 was studied. An antagonistic expression pattern of miR156 and miR172b during the vegetative and the reproductive phases signifies their apparent function in barley growth phase transition. Characterization of a barley mir172 mutant having an abnormal, indeterminate spikelet phenotype suggests the possible feedback role of AP2/miR172 module on HvSPL genes. This is the first comprehensive analysis of the miR156/SPL/miR172 axis in barley that provides a basis to elucidate their roles in various biological processes.