Pangenomics Comes of Age: From Bacteria to Plant and Animal Applications.

The pangenome refers to a collection of genomic sequence found in the entire species or population rather than in a single individual; the sequence can be core, present in all individuals, or accessory (variable or dispensable), found in a subset of individuals only. While pangenomic studies were first undertaken in bacterial species, developments in genome sequencing and assembly approaches have allowed construction of pangenomes for eukaryotic organisms, fungi, plants, and animals, including two large-scale human pangenome projects. Analysis of the these pangenomes revealed key differences, most likely stemming from divergent evolutionary histories, but also surprising similarities.

Circular RNAs modulate the floral fate acquisition in soybean shoot apical meristem.

BACKGROUND: Soybean (Glycine max), a major oilseed and protein source, requires a short-day photoperiod for floral induction. Though key transcription factors controlling flowering have been identified, the role of the non-coding genome is limited. Circular RNAs (circRNAs) recently emerged as a novel class of RNAs with critical regulatory functions. However, a study on circRNAs during the floral transition of a crop plant is lacking. We investigated the expression and potential function of circRNAs in floral fate acquisition by soybean shoot apical meristem in response to short-day treatment. RESULTS: Using deep sequencing and in-silico analysis, we denoted 384 circRNAs, with 129 exhibiting short-day treatment-specific expression patterns. We also identified 38 circRNAs with predicted binding sites for miRNAs that could affect the expression of diverse downstream genes through the circRNA-miRNA-mRNA network. Notably, four different circRNAs with potential binding sites for an important microRNA module regulating developmental phase transition in plants, miR156 and miR172, were identified. We also identified circRNAs arising from hormonal signaling pathway genes, especially abscisic acid, and auxin, suggesting an intricate network leading to floral transition. CONCLUSIONS: This study highlights the gene regulatory complexity during the vegetative to reproductive transition and paves the way to unlock floral transition in a crop plant.

Genome-wide analysis reveals the crucial role of lncRNAs in regulating the expression of genes controlling pollen development.

KEY MESSAGE: The genomic location and stage-specific expression pattern of many long non-coding RNAs reveal their critical role in regulating protein-coding genes crucial in pollen developmental progression and male germ line specification. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with no apparent protein-coding potential. Multiple investigations have revealed high expression of lncRNAs in plant reproductive organs in a cell and tissue-specific manner. However, their potential role as essential regulators of molecular processes involved in sexual reproduction remains largely unexplored. We have used developing field mustard (Brassica rapa) pollen as a model system for investigating the potential role of lncRNAs in reproductive development. Reference-based transcriptome assembly performed to update the existing genome annotation identified novel expressed protein-coding genes and long non-coding RNAs (lncRNAs), including 4347 long intergenic non-coding RNAs (lincRNAs, 1058 expressed) and 2,045 lncRNAs overlapping protein-coding genes on the opposite strand (lncNATs, 780 expressed). The analysis of expression profiles reveals that lncRNAs are significant and stage-specific contributors to the gene expression profile of developing pollen. Gene co-expression networks accompanied by genome location analysis identified 38 cis-acting lincRNA, 31 cis-acting lncNAT, 7 trans-acting lincRNA and 14 trans-acting lncNAT to be substantially co-expressed with target protein-coding genes involved in biological processes regulating pollen development and male lineage specification. These findings provide a foundation for future research aiming at developing strategies to employ lncRNAs as regulatory tools for gene expression control during reproductive development.

Melatonin and Abiotic Stress Tolerance in Crop Plants.

Increasing food demand by the growing human population and declining crop productivity due to climate change affect global food security. To meet the challenges, developing improved crops that can tolerate abiotic stresses is a priority. Melatonin in plants, also known as phytomelatonin, is an active component of the various cellular mechanisms that alleviates oxidative damage in plants, hence supporting the plant to survive abiotic stress conditions. Exogenous melatonin strengthens this defence mechanism by enhancing the detoxification of reactive by-products, promoting physiological activities, and upregulating stress-responsive genes to alleviate damage during abiotic stress. In addition to its well-known antioxidant activity, melatonin protects against abiotic stress by regulating plant hormones, activating ER stress-responsive genes, and increasing protein homoeostasis, heat shock transcription factors and heat shock proteins. Under abiotic stress, melatonin enhances the unfolded protein response, endoplasmic reticulum-associated protein degradation, and autophagy, which ultimately protect cells from programmed cell death and promotes cell repair resulting in increased plant survival.

Pokkali: A Naturally Evolved Salt-Tolerant Rice Shows a Distinguished Set of lncRNAs Possibly Contributing to the Tolerant Phenotype.

Pokkali is a strong representation of how stress-tolerant genotypes have evolved due to natural selection pressure. Numerous omics-based investigations have indicated different categories of stress-related genes and proteins, possibly contributing to salinity tolerance in this wild rice. However, a comprehensive study towards understanding the role of long-noncoding RNAs (lncRNAs) in the salinity response of Pokkali has not been done to date. We have identified salt-responsive lncRNAs from contrasting rice genotypes IR64 and Pokkali. A total of 63 and 81 salinity-responsive lncRNAs were differentially expressed in IR64 and Pokkali, respectively. Molecular characterization of lncRNAs and lncRNA-miRNA-mRNA interaction networks helps to explore the role of lncRNAs in the stress response. Functional annotation revealed that identified lncRNAs modulate various cellular processes, including transcriptional regulation, ion homeostasis, and secondary metabolite production. Additionally, lncRNAs were predicted to bind stress-responsive transcription factors, namely ERF, DOF, and WRKY. In addition to salinity, expression profiling was also performed under other abiotic stresses and phytohormone treatments. A positive modulation in TCONS_00035411, TCONS_00059828, and TCONS_00096512 under both abiotic stress and phytohormone treatments could be considered as being of potential interest for the further functional characterization of IncRNA. Thus, extensive analysis of lncRNAs under various treatments helps to delineate stress tolerance mechanisms and possible cross-talk.

Circular RNAs Repertoire and Expression Profile during Brassica rapa Pollen Development.

Circular RNAs (circRNAs) are covalently closed RNA molecules generated by the back-splicing of exons from linear precursor mRNAs. Though various linear RNAs have been shown to play important regulatory roles in many biological and developmental processes, little is known about the role of their circular counterparts. In this study, we performed high-throughput RNA sequencing to delineate the expression profile and potential function of circRNAs during the five stages of pollen development in Brassica rapa. A total of 1180 circRNAs were detected in pollen development, of which 367 showed stage-specific expression patterns. Functional enrichment and metabolic pathway analysis showed that the parent genes of circRNAs were mainly involved in pollen-related molecular and biological processes such as mitotic and meiotic cell division, DNA processes, protein synthesis, protein modification, and polysaccharide biosynthesis. Moreover, by predicting the circRNA-miRNA network from our differentially expressed circRNAs, we found 88 circRNAs with potential miRNA binding sites, suggesting their role in post-transcriptional regulation of the genes. Finally, we confirmed the back-splicing sites of nine selected circRNAs using divergent primers and Sanger sequencing. Our study presents the systematic analysis of circular RNAs during pollen development and forms the basis of future studies for unlocking complex gene regulatory networks underpinning reproduction in flowering plants.

Correction to: Analysis of the quinoa genome reveals conservation and divergence of the flowering pathways.

The above article was published online with incorrect Fig. 5 legend. The legend for Fig. 4 was repeated in Fig. 5.

Analysis of the quinoa genome reveals conservation and divergence of the flowering pathways.

Quinoa (Chenopodium quinoa Willd.) is a grain crop grown in the Andes renowned as a highly nutritious plant exhibiting tolerance to abiotic stress such as drought, cold and high salinity. Quinoa grows across a range of latitudes corresponding to differing day lengths, suggesting regional adaptations of flowering regulation. Improved understanding and subsequent modification of the flowering process, including flowering time, ensuring high yields, is one of the key factors behind expansion of cultivation zones and goals of the crop improvement programs worldwide. However, our understanding of the molecular basis of flower initiation and development in quinoa is limited. Here, we use a computational approach to perform genome-wide identification and analysis of 611 orthologues of the Arabidopsis thaliana flowering genes. Conservation of the genes belonging to the photoperiod, gibberellin and autonomous pathways was observed, while orthologues of the key genes found in the vernalisation pathway (FRI, FLC) were absent from the quinoa genome. Our analysis indicated that on average each Arabidopsis flowering gene has two orthologous copies in quinoa. Several genes including orthologues of MIF1, FT and TSF were identified as homologue-rich genes in quinoa. We also identified 459 quinoa-specific genes uniquely expressed in the flower and/or meristem, with no known orthologues in other species. The genes identified provide a resource and framework for further studies of flowering in quinoa and related species. It will serve as valuable resource for plant biologists, crop physiologists and breeders to facilitate further research and establishment of modern breeding programs for quinoa.

High temperature susceptibility of sexual reproduction in crop plants.

Climate change-induced increases in the frequency of extreme weather events, particularly heatwaves, are a serious threat to crop productivity. The productivity of grain crops is dependent on the success of sexual reproduction, which is very sensitive to heat stress. Male gametophyte development has been identified as the most heat-vulnerable stage. This review outlines the susceptibility of the various stages of sexual reproduction in flowering plants from the time of floral transition to double fertilization. We summarize current knowledge concerning the molecular mechanisms underpinning the heat stress-induced aberrations and abnormalities at flowering, male reproductive development, female reproductive development, and fertilization. We highlight the stage-specific bottlenecks in sexual reproduction, which regulate seed set and final yields under high-temperature conditions, together with the outstanding research questions concerning genotypic and species-specific differences in thermotolerance observed in crops. This knowledge is essential for trait selection and genetic modification strategies for the development of heat-tolerant genotypes and high-temperature-resilient crops.

MCRiceRepGP: a framework for the identification of genes associated with sexual reproduction in rice.

Rice is an important cereal crop, being a staple food for over half of the world's population, and sexual reproduction resulting in grain formation underpins global food security. However, despite considerable research efforts, many of the genes, especially long intergenic non-coding RNA (lincRNA) genes, involved in sexual reproduction in rice remain uncharacterized. With an increasing number of public resources becoming available, information from different sources can be combined to perform gene functional annotation. We report the development of MCRiceRepGP, a machine learning framework which integrates heterogeneous evidence and employs multicriteria decision analysis and machine learning to predict coding and lincRNA genes involved in sexual reproduction in rice. The rice genome was reannotated using deep-sequencing transcriptomic data from reproduction-associated tissue/cell types identifying previously unannotated putative protein-coding genes and lincRNAs. MCRiceRepGP was used for genome-wide discovery of sexual reproduction associated coding and lincRNA genes. The protein-coding and lincRNA genes identified have distinct expression profiles, with a large proportion of lincRNAs reaching maximum expression levels in the sperm cells. Some of the genes are potentially linked to male- and female-specific fertility and heat stress tolerance during the reproductive stage. MCRiceRepGP can be used in combination with other genome-wide studies, such as genome-wide association studies, giving greater confidence that the genes identified are associated with the biological process of interest. As more data, especially about mutant plant phenotypes, become available, the power of MCRiceRepGP will grow, providing researchers with a tool to identify candidate genes for future experiments. MCRiceRepGP is available as a web application (http://mcgplannotator.com/MCRiceRepGP/).

Genome-wide analysis of the Hsf gene family in Brassica oleracea and a comparative analysis of the Hsf gene family in B. oleracea, B. rapa and B. napus.

The global climate change-induced abiotic and biotic stresses are predicted to affect crop-growing seasons and crop yield. Heat stress transcription factors (Hsfs) have been suggested to play a significant role in various stress responses. They are an integral part of the signal transduction pathways that operate in response to environmental stresses. Brassica oleracea is one of the agronomical important crop species which consists of cabbage, cauliflower, broccoli, Brussels sprout, kohlrabi and kale. The identification and roles of Hsfs in this important Brassica species are unknown. The availability of whole genome sequence of B. oleracea provides us an opportunity for performing in silico analysis of Hsf genes in B. oleracea. Thirty-five putative genes encoding Hsf proteins were identified and classified into A, B and C classes. Their evolution, physical location, gene structure, domain structure and tissue-specific expression patterns were investigated. Further, a comparative analysis of the Hsf gene family in B. oleracea, B. rapa and B. napus highlighted the role of hybridisation and allopolyploidy in the evolution of the largest known Hsf gene family in B. napus. The presence of orthologous gene clusters, found in Brassica species, but not in A. thaliana, suggested that polyploidisation has resulted in the formation of new Brassica-specific orthologous gene clusters. Gene duplication analysis indicated that the evolution of the Hsf gene family was under strong purifying selection in these Brassica species. High-level synteny was observed within the B. napus genome. Conservation of physical location, the similarity of structure and similar expression profiles between the B. napus Hsf genes and the corresponding genes from B. oleracea and B. rapa suggest a high functional similarity between these genes. This study paves the way for further investigation of Hsf genes in improving stress tolerance in B. oleracea. The genes thus identified may be useful for developing crop varieties resilient to the global climate change.

The Long Intergenic Noncoding RNA (LincRNA) Landscape of the Soybean Genome.

Long intergenic noncoding RNAs (lincRNAs) are emerging as important regulators of diverse biological processes. However, our understanding of lincRNA abundance and function remains very limited especially for agriculturally important plants. Soybean (Glycine max) is a major legume crop plant providing over a half of global oilseed production. Moreover, soybean can form symbiotic relationships with Rhizobium bacteria to fix atmospheric nitrogen. Soybean has a complex paleopolyploid genome and exhibits many vegetative and floral development complexities. Soybean cultivars have photoperiod requirements restricting its use and productivity. Molecular regulators of these legume-specific developmental processes remain enigmatic. Long noncoding RNAs may play important regulatory roles in soybean growth and development. In this study, over one billion RNA-seq read pairs from 37 samples representing nine tissues were used to discover 6,018 lincRNA loci. The lincRNAs were shorter than protein-coding transcripts and had lower expression levels and more sample specific expression. Few of the loci were found to be conserved in two other legume species (chickpea [Cicer arietinum] and Medicago truncatula), but almost 200 homeologous lincRNAs in the soybean genome were detected. Protein-coding gene-lincRNA coexpression analysis suggested an involvement of lincRNAs in stress response, signal transduction, and developmental processes. Positional analysis of lincRNA loci implicated involvement in transcriptional regulation. lincRNA expression from centromeric regions was observed especially in actively dividing tissues, suggesting possible roles in cell division. Integration of publicly available genome-wide association data with the lincRNA map of the soybean genome uncovered 23 lincRNAs potentially associated with agronomic traits.

miRNAs in the crosstalk between phytohormone signalling pathways.

Phytohormones are signal molecules produced within the plant that control its growth and development through the regulation of gene expression. Interaction between different phytohormone pathways is essential in coordinating tissue outgrowth in response to environmental changes, such as the adaptation of root development to water deficit or the initiation of seed germination during imbibition. Recently, microRNAs (miRNAs) have emerged as key regulators of phytohormone response pathways in planta by affecting their metabolism, distribution, and perception. Here we review current knowledge on the miRNA-mediated regulations involved in phytohormone crosstalk. We focus on the miRNAs exhibiting regulatory links with more than one phytohormone pathway and discuss their possible implication in coordinating multiple phytohormone responses during specific developmental processes.

Unique and conserved features of floral evocation in legumes.

Legumes, with their unique ability to fix atmospheric nitrogen, play a vital role in ensuring future food security and mitigating the effects of climate change because they use less fossil energy and produce less greenhouse gases compared with N-fertilized systems. Grain legumes are second only to cereal crops as a source of human and animal food, and they contribute approximately one third of the protein consumed by the human population. The productivity of seed crops, such as grain legumes, is dependent on flowering. Despite the genetic variation and importance of flowering in legume production, studies of the molecular pathways that control flowering in legumes are limited. Recent advances in genomics have revealed that legume flowering pathways are divergent from those of such model species as Arabidopsis thaliana. Here, we discuss the current understanding of flowering time regulation in legumes and highlight the unique and conserved features of floral evocation in legumes.

Identifying long non-coding RNAs involved in heat stress response during wheat pollen development.

Introduction: Wheat is a staple food crop for over one-third of the global population. However, the stability of wheat productivity is threatened by heat waves associated with climate change. Heat stress at the reproductive stage can result in pollen sterility and failure of grain development. Methods: This study used transcriptome data analysis to explore the specific expression of long non-coding RNAs (lncRNAs) in response to heat stress during pollen development in four wheat cultivars. Results and discussion: We identified 11,054 lncRNA-producing loci, of which 5,482 lncRNAs showed differential expression in response to heat stress. Heat-responsive lncRNAs could target protein-coding genes in cis and trans and in lncRNA-miRNA-mRNA regulatory networks. Gene ontology analysis predicted that target protein-coding genes of lncRNAs regulate various biological processes such as hormonal responses, protein modification and folding, response to stress, and biosynthetic and metabolic processes. We also noted some paired lncRNA/protein-coding gene modules and some lncRNA-miRNA-mRNA regulatory modules shared in two or more wheat cultivars. These modules were related to regulating plant responses to heat stress, such as heat-shock proteins and transcription factors, and protein domains, such as MADS-box, Myc-type, and Alpha crystallin/Hsp20 domain. Conclusion: Our results provide the basic knowledge and molecular resources for future functional studies investigating wheat reproductive development under heat stress.

Overexpression of GmPIF4b affects morpho-physiological traits to reduce heat-induced grain loss in soybean.

Heat waves associated with climate change seriously threaten crop productivity. Crop seed yield depends on the success of reproduction. However, reproductive development is most vulnerable to heat stress conditions. Perception of heat and its conversion into cellular signals is a complex process. The basic helix loop helix (bHLH) transcription factor, Phytochrome Interacting Factor 4 (PIF4), plays a significant role in this process. However, studies on PIF4- mediated impacts on crop grain yield at a higher temperature are lacking. We investigated the overexpression of GmPIF4b in soybean to alleviate heat-induced damage and yield using a transgenic approach. Our results showed that under high-temperature conditions (38°C/28°C), overexpressing soybeans plants had higher chlorophyll a and b, and lower proline accumulation compared to WT. Further, overexpression of GmPIF4b improved pollen viability under heat stress and reduced heat-induced structural abnormalities in the male and female reproductive organs. Consequently, the transgenic plants produced higher pods and seeds per plant at high temperatures. Quantitative RT-PCR analysis showed that the overexpressing GmPIF4b soybeans had higher transcripts of heat shock factor, GmHSF-34, and heat-shock protein, GmHSP90A2. Collectively, our results suggest that GmPIF4b regulates multiple morpho-physiological traits for better yield under warmer climatic conditions.

Transcriptome-wide profiling and expression analysis of transcription factor families in a liverwort, Marchantia polymorpha.

BACKGROUND: Transcription factors (TFs) are vital elements that regulate transcription and the spatio-temporal expression of genes, thereby ensuring the accurate development and functioning of an organism. The identification of TF-encoding genes in a liverwort, Marchantia polymorpha, offers insights into TF organization in the members of the most basal lineages of land plants (embryophytes). Therefore, a comparison of Marchantia TF genes with other land plants (monocots, dicots, bryophytes) and algae (chlorophytes, rhodophytes) provides the most comprehensive view of the rates of expansion or contraction of TF genes in plant evolution. RESULTS: In this study, we report the identification of TF-encoding transcripts in M. polymorpha for the first time, as evidenced by deep RNA sequencing data. In total, 3,471 putative TF encoding transcripts, distributed in 80 families, were identified, representing 7.4% of the generated Marchantia gametophytic transcriptome dataset. Overall, TF basic functions and distribution across families appear to be conserved when compared to other plant species. However, it is of interest to observe the genesis of novel sequences in 24 TF families and the apparent termination of 2 TF families with the emergence of Marchantia. Out of 24 TF families, 6 are known to be associated with plant reproductive development processes. We also examined the expression pattern of these TF-encoding transcripts in six male and female developmental stages in vegetative and reproductive gametophytic tissues of Marchantia. CONCLUSIONS: The analysis highlighted the importance of Marchantia, a model plant system, in an evolutionary context. The dataset generated here provides a scientific resource for TF gene discovery and other comparative evolutionary studies of land plants.

Novel members of the AGAMOUS LIKE 6 subfamily of MIKCC-type MADS-box genes in soybean.

BACKGROUND: The classical (C) MIKC-type MADS-box transcription factors comprise one gene family that plays diverse roles in the flowering process ranging from floral initiation to the development of floral organs. Despite their importance in regulating developmental processes that impact crop yield, they remain largely unexplored in the major legume oilseed crop, soybean. RESULTS: We identified 57 MIKC(c)-type transcription factors from soybean and determined the in silico gene expression profiles of the soybean MIKC(c)-type genes across different tissues. Our study implicates three MIKC(c)-type transcription factors as novel members of the AGAMOUS LIKE 6 (AGL6) subfamily of the MIKC(C)-type MADS-box genes, and we named this sister clade PsMADS3. While similar genes were identified in other legume species, poplar and grape, no such gene is represented in Arabidopsis thaliana or rice. RT-PCR analysis on these three soybean PsMADS3 genes during early floral initiation processes revealed their temporal expression similar to that of APETALA1, a gene known to function as a floral meristem identity gene. However, RNA in situ hybridisation showed that their spatial expression patterns are markedly different from those of APETALA1. CONCLUSION: Legume flower development system differs from that in the model plant, Arabidopsis. There is an overlap in the initiation of different floral whorls in soybean, and inflorescent meristems can revert to leaf production depending on the environmental conditions. MIKC(C)-type MADS-box genes have been shown to play key regulatory roles in different stages of flower development. We identified members of the PsMADS3 sub-clade in legumes that show differential spatial expression during floral initiation, indicating their potential novel roles in the floral initiation process. The results from this study will contribute to a better understanding of legume-specific floral developmental processes.

Spatial expression of CLAVATA3 in the shoot apical meristem suggests it is not a stem cell marker in soybean.

CLAVATA3 (CLV3), a stem cell marker in Arabidopsis thaliana, encodes a secreted peptide that maintains the stem cell population within the shoot apical meristem. This work investigated the CLV3 orthologue in a major legume crop, soybean (GmCLV3). Instead of being expressed in the three outermost layers of the meristem as in Arabidopsis, GmCLV3 was expressed deeper in the central zone beneath the fourth layer (L4) of the meristem, overlapping with the expression of soybean WUSCHEL. Subsequent investigation using an alternative stem cell marker (GmLOG1) revealed its expression within layers L2-L4, indicating that GmCLV3 is not a stem cell marker. Overexpression studies of GmCLV3 in Arabidopsis and complementation of clv3-2 mutant suggest similar functional capacity to that of Arabidopsis CLV3. The expression of soybean CLV1, which encodes a receptor for CLV3 in Arabidopsis, was not detectable in the central zone of the meristem via reverse-transcription PCR analysis of amplified RNA from laser-microdissected samples or in situ, implicating a diverged pathway in soybean. This study also reports the novel expression of GmLOG1 in initials of axillary meristem in the boundary region between the SAM and developing leaf primordia, before the expression of GmWUS or GmCLV3, indicating cytokinin as one of the earliest signals in initiating and specifying the stem cell population.

Functional analysis of soybean miR156 and miR172 in tobacco highlights their role in plant morphology and floral transition.

Soybean (Glycine max), a significant oilseed and protein source for humans and livestock feed, needs short day photoperiod for floral induction. Further, soybean has a paleopolyploid genome with multiple copies of flowering genes adding to the complexity of genetic regulation of flowering, and seed set, especially in investigating the role of the noncoding genome. microRNAs, a class of noncoding RNA, play a regulatory role in plant development. miR156 and miR172 are major components of the essential regulatory hub controlling juvenile and vegetative developments and initiation of reproductive phase change leading to flowering. These microRNAs have been originally isolated and studied from model plant, Arabidopsis. However, a study on soybean microRNAs is lacking. We investigated the temporal expression patterns of gma-miR156a and gma-miR172a and found inversely related - gma-miR156a expression was higher in the vegetative stage, and gma-miR172a expression was elevated under inductive flowering conditions. The functions of gma-miR156a and gma-miR172a were evaluated via heterologous expressions in transgenic tobacco plants (Nicotiana tabacum L.). The analysis of overexpression transgenic lines highlighted that gma-miR156a plays a role in juvenile development via repression of the SPL transcription factor family. In contrast, gma-miR172a plays a pivotal role in the reproductive development phase by down-regulating its target genes, AP2. In addition, ectopic expression of gma-miR156a and gma-miR172a affected plant morphology and physiology during plant growth. Collectively, our results suggest that gma-miR156a and gma-miR172a regulate multiple morpho-physiological traits that could be used to enhance crop yield under changing climate conditions.