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.

Rapid Transcriptional Reprogramming Associated With Heat Stress-Induced Unfolded Protein Response in Developing Brassica napus Anthers.

Climate change associated increases in the frequency and intensity of extreme temperature events negatively impact agricultural productivity and global food security. During the reproductive phase of a plant's life cycle, such high temperatures hinder pollen development, preventing fertilization, and seed formation. At the molecular level, heat stress-induced accumulation of misfolded proteins activates a signaling pathway called unfolded protein response (UPR) in the endoplasmic reticulum (ER) and the cytoplasm to enhance the protein folding capacity of the cell. Here, we report transcriptional responses of Brassica napus anthers exposed to high temperature for 5, 15, and 30 min to decipher the rapid transcriptional reprogramming associated with the unfolded protein response. Functional classification of the upregulated transcripts highlighted rapid activation of the ER-UPR signaling pathway mediated by ER membrane-anchored transcription factor within 5 min of heat stress exposure. KEGG pathway enrichment analysis also identified "Protein processing in ER" as the most significantly enriched pathway, indicating that the unfolded protein response (UPR) is an immediate heat stress-responsive pathway during B. napus anther development. Five minutes of heat stress also led to robust induction of the cytosolic HSF-HSP heat response network. Our results present a perspective of the rapid and massive transcriptional reprogramming during heat stress in pollen development and highlight the need for investigating the nature and function of very early stress-responsive networks in plant cells. Research focusing on very early molecular responses of plant cells to external stresses has the potential to reveal new stress-responsive gene networks that can be explored further for developing climate change resilient crops.

Biological Parts for Engineering Abiotic Stress Tolerance in Plants.

It is vital to ramp up crop production dramatically by 2050 due to the increasing global population and demand for food. However, with the climate change projections showing that droughts and heatwaves becoming common in much of the globe, there is a severe threat of a sharp decline in crop yields. Thus, developing crop varieties with inbuilt genetic tolerance to environmental stresses is urgently needed. Selective breeding based on genetic diversity is not keeping up with the growing demand for food and feed. However, the emergence of contemporary plant genetic engineering, genome-editing, and synthetic biology offer precise tools for developing crops that can sustain productivity under stress conditions. Here, we summarize the systems biology-level understanding of regulatory pathways involved in perception, signalling, and protective processes activated in response to unfavourable environmental conditions. The potential role of noncoding RNAs in the regulation of abiotic stress responses has also been highlighted. Further, examples of imparting abiotic stress tolerance by genetic engineering are discussed. Additionally, we provide perspectives on the rational design of abiotic stress tolerance through synthetic biology and list various bioparts that can be used to design synthetic gene circuits whose stress-protective functions can be switched on/off in response to environmental cues.

The Role of Endoplasmic Reticulum Stress Response in Pollen Development and Heat Stress Tolerance.

Endoplasmic reticulum (ER) stress is defined by a protracted disruption in protein folding and accumulation of unfolded or misfolded proteins in the ER. This accumulation of unfolded proteins can result from excessive demands on the protein folding machinery triggered by environmental and cellular stresses such as nutrient deficiencies, oxidative stress, pathogens, and heat. The cell responds to ER stress by activating a protective pathway termed unfolded protein response (UPR), which comprises cellular mechanisms targeted to maintain cellular homeostasis by increasing the ER's protein folding capacity. The UPR is especially significant for plants as being sessile requires them to adapt to multiple environmental stresses. While multiple stresses trigger the UPR at the vegetative stage, it appears to be active constitutively in the anthers of unstressed plants. Transcriptome analysis reveals significant upregulation of ER stress-related transcripts in diploid meiocytes and haploid microspores. Interestingly, several ER stress-related genes are specifically upregulated in the sperm cells. The analysis of gene knockout mutants in Arabidopsis has revealed that defects in ER stress response lead to the failure of normal pollen development and enhanced susceptibility of male gametophyte to heat stress conditions. In this mini-review, we provide an overview of the role of ER stress and UPR in pollen development and its protective roles in maintaining male fertility under heat stress conditions.

A dynamic intron retention program regulates the expression of several hundred genes during pollen meiosis.

KEY MESSAGE: Intron retention is a stage-specific mechanism of functional attenuation of a subset of co-regulated, functionally related genes during early stages of pollen development. To improve our understanding of the gene regulatory mechanisms that drive developmental processes, we performed a genome-wide study of alternative splicing and isoform switching during five key stages of pollen development in field mustard, Brassica rapa. Surprisingly, for several hundred genes (12.3% of the genes analysed), isoform switching results in stage-specific expression of intron-retaining transcripts at the meiotic stage of pollen development. In such cases, we report temporally regulated switching between expression of a canonical, translatable isoform and an intron-retaining transcript that is predicted to produce a truncated and presumably inactive protein. The results suggest a new pervasive mechanism underlying modulation of protein levels in a plant developmental program. The effect is not based on gene expression induction but on the type of transcript produced. We conclude that intron retention is a stage-specific mechanism of functional attenuation of a subset of co-regulated, functionally related genes during meiosis, especially genes related to ribosome biogenesis, mRNA transport and nuclear envelope architecture. We also propose that stage-specific expression of a non-functional isoform of Brassica rapa BrSDG8, a non-redundant member of histone methyltransferase gene family, linked to alternative splicing regulation, may contribute to the intron retention observed.

Genome-Wide In Silico Identification and Comparative Analysis of Dof Gene Family in Brassica napus.

DNA binding with one finger (DOF) proteins are plant-specific transcription factors that play roles in diverse plant functions. However, little is known about the DOF protein repertoire of the allopolyploid crop, Brassica napus. This in silico study identified 117 Brassica napus Dof genes (BnaDofs) and classified them into nine groups (A, B1, B2, C1, C2.1, C2.2, C3, D1, and D2), based on phylogenetic analysis. Most members belonging to a particular group displayed conserved gene structural organisation and protein motif distribution. Evolutionary analysis exemplified that the divergence of the Brassica genus from Arabidopsis, the whole-genome triplication event, and the hybridisation of Brassica oleracea and Brassica rapa to form B. napus, followed by gene loss and rearrangements, led to the expansion and divergence of the Dof transcription factor (TF) gene family in B. napus. So far, this is the largest number of Dof genes reported in a single eudicot species. Functional annotation of BnaDof proteins, cis-element analysis of their promoters, and transcriptomic analysis suggested potential roles in organ development, the transition from the vegetative to the reproductive stage, light responsiveness, phytohormone responsiveness, as well as potential regulatory roles in abiotic stress. Overall, our results provide a comprehensive understanding of the molecular structure, evolution, and possible functional roles of Dof genes in plant development and abiotic stress response.

Engineering Multiple Abiotic Stress Tolerance in Canola, Brassica napus.

Impacts of climate change like global warming, drought, flooding, and other extreme events are posing severe challenges to global crop production. Contribution of Brassica napus towards the oilseed industry makes it an essential component of international trade and agroeconomics. Consequences from increasing occurrences of multiple abiotic stresses on this crop are leading to agroeconomic losses making it vital to endow B. napus crop with an ability to survive and maintain yield when faced with simultaneous exposure to multiple abiotic stresses. For an improved understanding of the stress sensing machinery, there is a need for analyzing regulatory pathways of multiple stress-responsive genes and other regulatory elements such as non-coding RNAs. However, our understanding of these pathways and their interactions in B. napus is far from complete. This review outlines the current knowledge of stress-responsive genes and their role in imparting multiple stress tolerance in B. napus. Analysis of network cross-talk through omics data mining is now making it possible to unravel the underlying complexity required for stress sensing and signaling in plants. Novel biotechnological approaches such as transgene-free genome editing and utilization of nanoparticles as gene delivery tools are also discussed. These can contribute to providing solutions for developing climate change resilient B. napus varieties with reduced regulatory limitations. The potential ability of synthetic biology to engineer and modify networks through fine-tuning of stress regulatory elements for plant responses to stress adaption is also highlighted.

RNA-Seq Highlights Molecular Events Associated With Impaired Pollen-Pistil Interactions Following Short-Term Heat Stress in Brassica napus.

The global climate change is leading to increased frequency of heatwaves with crops getting exposed to extreme temperature events. Such temperature spikes during the reproductive stage of plant development can harm crop fertility and productivity. Here we report the response of short-term heat stress events on the pollen and pistil tissues in a commercially grown cultivar of Brassica napus. Our data reveals that short-term temperature spikes not only affect pollen fitness but also impair the ability of the pistil to support pollen germination and pollen tube growth and that the heat stress sensitivity of pistil can have severe consequences for seed set and yield. Comparative transcriptome profiling of non-stressed and heat-stressed (40°C for 30 min) pollen and pistil (stigma + style) highlighted the underlying cellular mechanisms involved in heat stress response in these reproductive tissues. In pollen, cell wall organization and cellular transport-related genes possibly regulate pollen fitness under heat stress while the heat stress-induced repression of transcription factor encoding transcripts is a feature of the pistil response. Overall, high temperature altered the expression of genes involved in protein processing, regulation of transcription, pollen-pistil interactions, and misregulation of cellular organization, transport, and metabolism. Our results show that short episodes of high-temperature exposure in B. napus modulate key regulatory pathways disrupted reproductive processes, ultimately translating to yield loss. Further investigations on the genes and networks identified in the present study pave a way toward genetic improvement of the thermotolerance and reproductive performance of B. napus varieties.

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.

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.