Expression Atlas to Accelerate Gene Discovery in Difficult-to-breed Plants, for example Cacao
Abstract
Background
Theobroma cacao, the cocoa tree, is a tropical crop grown for its highly valuable cocoa solids and fat which are the basis of a 200-billion-dollar annual chocolate industry. However, the long generation time and difficulties associated with breeding a tropical tree crop have limited the progress of breeders to develop high-yielding disease-resistant varieties. Development of marker-assisted breeding methods for cacao requires discovery of genomic regions and specific alleles of genes encoding important traits of interest. To accelerate gene discovery, we developed a gene atlas composed of a large dataset of replicated transcriptomes with the long-term goal of progressing breeding towards developing high-yielding elite varieties of cacao.
Results
We describe the creation of the Cacao Transcriptome Atlas, its global characterization and define sets of genes co-regulated in highly organ- and temporally-specific manners. RNAs were extracted and transcriptomes sequenced from 123 different tissues and stages of development representing major organs and developmental stages of the cacao lifecycle. In addition, several experimental treatments and time courses were performed to measure gene expression in tissues responding to biotic and abiotic stressors. Samples were collected in replicates (3–5) to enable statistical analysis of gene expression levels for a total of 390 transcriptomes. To promote wide use of these data, all raw sequencing data, expression read mapping matrices, scripts, and other information used to create the resource are freely available online. We verified our atlas by analyzing the expression of genes with known functions and expression patterns in Arabidopsis (ACT7, LEA19, AGL16, TIP13, LHY, MYB2) and found their expression profiles to be generally similar between both species. We also successfully identified tissue-specific genes at two thresholds in many tissue types represented and a set of genes highly conserved across all tissues.
Conclusions
The Cacao Gene Atlas consists of a gene expression browser with graphical user interface and open access to raw sequencing data files as well as the unnormalized and CPM normalized read count data mapped to several cacao genomes. The gene atlas is a publicly available resource to allow rapid mining of cacao gene expression profiles. We hope this resource will be used to help accelerate the discovery of important genes for key cacao traits such as disease resistance and contribute to the breeding of elite varieties to help farmers increase yields.
Rare Genetic Variation with Extreme Gene Expression - Crop Fitness and Survival in Corn
Traits or expression quantitaive trait loci (eQTLs) associated with fitness and survival are among the most interesting to define by RNA-Seq expression profiling as they are directly related to crop productivity. In a study from 2018, the authors generated a multi-tissue gene expression resource capturing the genotypic and phenotypic diversity of modern inbred maize. The study mapped eQTLs using QuantSeq 3′ mRNA-Seq expression profiling and characterized the influence of rare genetic variants. To determine whether rare alleles contribute to extreme expression, rare-allele abundance was investigated and dysregulation could be connected to seed weight.
Abstract
Here we report a multi-tissue gene expression resource that represents the genotypic and phenotypic diversity of modern inbred maize, and includes transcriptomes in an average of 255 lines in seven tissues. We mapped expression quantitative trait loci and characterized the contribution of rare genetic variants to extremes in gene expression. Some of the new mutations that arise in the maize genome can be deleterious; although selection acts to keep deleterious variants rare, their complete removal is impeded by genetic linkage to favourable loci and by finite population size1,2,3,4. Modern maize breeders have systematically reduced the effects of this constant mutational pressure through artificial selection and self-fertilization, which have exposed rare recessive variants in elite inbred lines5. However, the ongoing effect of these rare alleles on modern inbred maize is unknown. By analysing this gene expression resource and exploiting the extreme diversity and rapid linkage disequilibrium decay of maize6, we characterize the effect of rare alleles and evolutionary history on the regulation of expression. Rare alleles are associated with the dysregulation of expression, and we correlate this dysregulation to seed-weight fitness. We find enrichment of ancestral rare variants among expression quantitative trait loci mapped in modern inbred lines, which suggests that historic bottlenecks have shaped regulation. Our results suggest that one path for further genetic improvement in agricultural species lies in purging the rare deleterious variants that have been associated with crop fitness.
Nutritious Content in Ecologically Important Crops
As the global population steadily increases, the pressure rises to produce not only enough food, but also elevate nutritious content. Crops rich in vitamins, minerals, and essential nutrients are not only critical for human health and well-being, they also contribute to the health of livestock impacting the quality of animal produce. Focusing on breeding and developing crops with enhanced nutritional value is essential. This ensures a more sustainable food system that can combat malnutrition and provide a healthier future for a growing population.
Differential Expression in Soybeans
With over 350 million tons produced annually, soybean is among the most valuable crops based on its nutritious seed protein and oil. Soy protein is used as an alternative to animal protein for humans, as animal feed, and for biofuels. Differences in protein-content have been observed in soybean grown in different geographic locations although environmental conditions, such as temperature, humidity, rain fall, and soil composition were quite similar in both habitats. In several studies published in 2022 and 2023, RNA-Seq and differential gene expression analysis were employed to assess the differences in seed protein content between soybeans grown in eastern and western Canada. Gene ontology analyses identified the lipid and carbohydrate metabolic pathways as differentially expressed between high and low protein-producing soybean cultivars
Abstract
Soybean (Glycine max (L.) Merr.) is among the most valuable crops based on its nutritious seed protein and oil. Protein quality, evaluated as the ratio of glycinin (11S) to β-conglycinin (7S), can play a role in food and feed quality. To help uncover the underlying differences between high and low protein soybean varieties, we performed differential expression analysis on high and low total protein soybean varieties and high and low 11S soybean varieties grown in four locations across Eastern and Western Canada over three years (2018–2020). Simultaneously, ten individual differential expression datasets for high vs. low total protein soybeans and ten individual differential expression datasets for high vs. low 11S soybeans were assessed, for a total of 20 datasets. The top 15 most upregulated and the 15 most downregulated genes were extracted from each differential expression dataset and cross-examination was conducted to create shortlists of the most consistently differentially expressed genes. Shortlisted genes were assessed for gene ontology to gain a global appreciation of the commonly differentially expressed genes. Genes with roles in the lipid metabolic pathway and carbohydrate metabolic pathway were differentially expressed in high total protein and high 11S soybeans in comparison to their low total protein and low 11S counterparts. Expression differences were consistent between East and West locations with the exception of one, Glyma.03G054100. These data are important for uncovering the genes and biological pathways responsible for the difference in seed protein between high and low total protein or 11S cultivars.
Abstract
Over the past two decades soybeans grown in western Canada have persistently had lower seed protein than those grown in eastern Canada. To understand the discrepancy in seed protein content between eastern- and western-grown soybeans, RNA-seq and differential expression analysis have been investigated. Ten soybean genotypes, ranging from low to high in seed protein content, were grown in four locations across eastern (Ottawa) and western (Morden, Brandon, and Saskatoon) Canada. Differential expression analysis revealed 34 differentially expressed genes encoding Glycine max Sugars Will Eventually be Exported Transporters (GmSWEETs), including paralogs GmSWEET29 and GmSWEET34 (AtSWEET2 homologs) that were consistently upregulated across all ten genotypes in each of the western locations over three years. GmSWEET29 and GmSWEET34 are likely candidates underlying the lower seed protein content of western soybeans. GmSWEET20 (AtSWEET12 homolog) was downregulated in the western locations and may also play a role in lower seed protein content. These findings are valuable for improving soybean agriculture in western growing regions, establishing more strategic and efficient agricultural practices.
Abstract
Unraveling Spatiotemporal Expression in Rice Development and Seed Tissues
Global rice production provides one of the most important food sources for humans with more than 800 million tons annually. As the population is growing, a stable or increased rice production will get more important in the near future. In this highlighted study from 2022, researchers investigated the small RNA repertoire in root and leaves tissues at different plant life-stages with the aim to identify small RNA regulators of rice yield and quality. The authors provide an in-depth analysis of the sRNA profile throughout rice development and could add several novel sRNA and tsRNA loci to the rice genome annotation. The focus on embryo and endosperm tissues has the potential to aid optimization of rice production through future functional research.
Abstract
Background
Small RNAs (sRNAs) regulate numerous plant processes directly related to yield, such as disease resistance and plant growth. To exploit this yield-regulating potential of sRNAs, the sRNA profile of one of the world’s most important staple crops – rice – was investigated throughout plant development using next-generation sequencing.
Results
Root and leaves were investigated at both the vegetative and generative phase, and early-life sRNA expression was characterized in the embryo and endosperm. This led to the identification of 49,505 novel sRNAs and 5581 tRNA-derived sRNAs (tsRNAs). In all tissues, 24 nt small interfering RNAs (siRNAs) were highly expressed and associated with euchromatic, but not heterochromatic transposable elements. Twenty-one nt siRNAs deriving from genic regions in the endosperm were exceptionally highly expressed, mimicking previously reported expression levels of 24 nt siRNAs in younger endosperm samples. In rice embryos, sRNA content was highly diverse while tsRNAs were underrepresented, possibly due to snoRNA activity. Publicly available mRNA expression and DNA methylation profiles were used to identify putative siRNA targets in embryo and endosperm. These include multiple genes related to the plant hormones gibberellic acid and ethylene, and to seed phytoalexin and iron content.
Conclusions
This work introduces multiple sRNAs as potential regulators of rice yield and quality, identifying them as possible targets for the continuous search to optimize rice production.
Stress Adaptation to Environmental Stressors (Salt, Elevated Temperature, and Drought)
RNA-Seq, expression profiling, and time-course expression studies provide the foundation to understanding the genetic control of stress adaptation and for the genetic improvement of crop resistance to various environmental stressors.
Adaptation to Salt Stress
Adaptation to varying environmental conditions is closely related to the fitness of crops. Identification of cultivars with advantages in stress adaptation allows to further enhance resilience and select for better adapted crops which can still thrive under challenging conditions. Salinity stress for example causes great losses in crop production and affects major crops such as sorghum and cotton. Sorghum, which is among the most important cereals and an important source for food, animal fodder, and biofuels is known for its exceptional adaptability to saline stress. Although cotton is a relatively salt-tolerant species often used as model system for salt stress research, growth and development are still affected by adverse salt conditions impacting on the yield of the more salt-sensitive cultivars. The highlighted publications in this section used gene expression profiling with QuantSeq to identify regulatory pathways involved in saline stress response and adaptation.
Abstract
Abstract
Cotton is an economically important crop throughout the world and is a pioneer crop in salt stress tolerance research. Investigation of the genetic regulation of salinity tolerance will provide information for salt stress-resistant breeding. Here, we employed next-generation RNA-Seq technology to elucidate the salt-tolerant mechanisms in cotton using the diploid cotton species Gossypium davidsonii which has superior stress tolerance. A total of 4744 and 5337 differentially expressed genes (DEGs) were found to be involved in salt stress tolerance in roots and leaves, respectively. Gene function annotation elucidated salt overly sensitive (SOS) and reactive oxygen species (ROS) signaling pathways. Furthermore, we found that photosynthesis pathways and metabolism play important roles in ion homeostasis and oxidation balance. Moreover, our studies revealed that alternative splicing also contributes to salt-stress responses at the posttranscriptional level, implying its functional role in response to salinity stress. This study not only provides a valuable resource for understanding the genetic control of salt stress in cotton, but also lays a substantial foundation for the genetic improvement of crop resistance to salt stress.
Abstract
Background
Adaptation to abiotic stresses is crucial for the survival of perennial plants in a natural environment. However, very little is known about the underlying mechanisms. Here, we adopted a liquid culture system to investigate plant adaptation to repeated salt stress in Populus trees.
Results
We first evaluated phenotypic responses and found that plants exhibit better stress tolerance after pre-treatment of salt stress. Time-course RNA sequencing (RNA-seq) was then performed to profile changes in gene expression over 12 h of salt treatments. Analysis of differentially expressed genes (DEGs) indicated that significant transcriptional reprogramming and adaptation to repeated salt treatment occurred. Clustering analysis identified two modules of co-expressed genes that were potentially critical for repeated salt stress adaptation, and one key module for salt stress response in general. Gene Ontology (GO) enrichment analysis identified pathways including hormone signaling, cell wall biosynthesis and modification, negative regulation of growth, and epigenetic regulation to be highly enriched in these gene modules.
Conclusions
This study illustrates phenotypic and transcriptional adaptation of Populus trees to salt stress, revealing novel gene modules which are potentially critical for responding and adapting to salt stress.
Adaptation to Temperature and Drought
Abstract
Rice (Oryza sativa L.) is one of the most important food crops, providing food for nearly half of the world population. Rice grain yields are affected by temperature changes. Temperature stresses, both low and high, affect male reproductive development, resulting in yield reduction. Thermosensitive genic male sterility (TGMS) rice is sterile at high temperature and fertile at low temperature conditions, facilitating hybrid production, and is a good model to study effects of temperatures on male development. Semithin sections of the anthers of a TGMS rice line under low (fertile) and high (sterile) temperature conditions showed differences starting from the dyad stage, suggesting that genes involved in male development play a role during postmeiotic microspore development. Using RNA sequencing (RNA-Seq), transcriptional profiling of TGMS rice panicles at the dyad stage revealed 232 genes showing differential expression (DEGs) in a sterile, compared to a fertile, condition. Using qRT-PCR to study expression of 20 selected DEGs using panicles of TGMS and wild type rice plants grown under low and high temperature conditions, revealed that six out of the 20 selected genes may be unique to TGMS, while the other 14 genes showed common responses to temperatures in both TGMS and wild-type rice plants. The results presented here would be useful for further investigation into molecular mechanisms controlling TGMS and rice responses to temperature alteration.
Abstract
Hops are valued for their secondary metabolites, including bitter acids, flavonoids, oils, and polyphenols, that impart flavor in beer. Previous studies have shown that hop yield and bitter acid content decline with increased temperatures and low-water stress. We looked at physiological traits and differential gene expression in leaf, stem, and root tissue from hop (Humulus lupulus) cv. USDA Cascade in plants exposed to high temperature stress, low-water stress, and a compound treatment of both high temperature and low-water stress for six weeks. The stress conditions imposed in these experiments caused substantial changes to the transcriptome, with significant reductions in the expression of numerous genes involved in secondary metabolite biosynthesis. Of the genes involved in bitter acid production, the critical gene valerophenone synthase (VPS) experienced significant reductions in expression levels across stress treatments, suggesting stress-induced lability in this gene and/or its regulatory elements may be at least partially responsible for previously reported declines in bitter acid content. We also identified a number of transcripts with homology to genes shown to affect abiotic stress tolerance in other plants that may be useful as markers for breeding improved abiotic stress tolerance in hop. Lastly, we provide the first transcriptome from hop root tissue.
Abstract
Plant roots integrate environmental signals with development using exquisite spatiotemporal control. This is apparent in the deposition of suberin, an apoplastic diffusion barrier, which regulates flow of water, solutes and gases, and is environmentally plastic. Suberin is considered a hallmark of endodermal differentiation but is absent in the tomato endodermis. Instead, suberin is present in the exodermis, a cell type that is absent in the model organism Arabidopsis thaliana. Here we demonstrate that the suberin regulatory network has the same parts driving suberin production in the tomato exodermis and the Arabidopsis endodermis. Despite this co-option of network components, the network has undergone rewiring to drive distinct spatial expression and with distinct contributions of specific genes. Functional genetic analyses of the tomato MYB92 transcription factor and ASFT enzyme demonstrate the importance of exodermal suberin for a plant water-deficit response and that the exodermal barrier serves an equivalent function to that of the endodermis and can act in its place.
Natural Elicitors as Sustainable Alternative to Chemical Pesticides and Primers for Abiotic Stress
Abstract
Biomass-derived oligo- and polysaccharides may act as elicitors, i.e., bioactive molecules that trigger plant immune responses. This is particularly important to increase the resistance of plants to abiotic and biotic stresses. In this study, cellulose nanofibrils (CNF) gels were obtained by TEMPO-mediated oxidation of unbleached and bleached kraft pulps. The molecular structures were characterized with ESI and MALDI MS. Analysis of the fine sequences was achieved by MS and MS/MS of the water-soluble oligosaccharides obtained by acid hydrolysis of the CNF gels. The analysis revealed the presence of two families: one corresponding to homoglucuronic acid sequences and the other composed by alternating glucose and glucuronic acid units. The CNF gels, alone or with the addition of the water-soluble oligosaccharides, were tested on Chili pepper (Capsicum annuum). Based on the characterization of the gene expression with Next Generation Sequencing (NGS) of the C. annuum‘s total messenger RNA, the differences in growth of the C. annuum seeds correlated well with the downregulation of the pathways regulating photosynthesis. A downregulation of the response to abiotic factors was detected, suggesting that these gels would improve the resistance of the C. annuum plants to abiotic stress due to, e.g., water deprivation and cold temperatures.
Regulation of Dormancy Unraveled with the aid of RNA sequencing
Dormancy is an adaptive mechanism in plants where metabolic activities are temporarily stopped to protect sensitive plant tissues from damage caused by e.g., extreme temperatures. Seed dormancy for example ensures that seeds of crops can survive unfavorable conditions in the soil even when the mature plants are eradicated, e.g., due to extreme heat or natural disasters. In agriculture, seed dormancy is of great importance to maintain the quality of seeds during storage. Bud dormancy ensures the survival of generative tissues during winter and continuous fruit production in orchards for years. In apple trees, bud dormancy is induced by a decrease in temperatures in fall, requires exposure to cold temperatures in winter and is reversed by warmer temperatures in spring which induce flowering. Through previous research, it is known that small RNAs are involved in modulation of temperature responses in many plant species, however, the role of sRNAs in bud dormancy remained elusive. A study from 2021 investigated functional small RNAs in apple buds isolated with TraPR and their role in dormancy.
Abstract
Winter dormancy is an adaptative mechanism that temperate and boreal trees have developed to protect their meristems against low temperatures. In apple trees (Malus domestica), cold temperatures induce bud dormancy at the end of summer/beginning of the fall. Apple buds stay dormant during winter until they are exposed to a period of cold, after which they can resume growth (budbreak) and initiate flowering in response to warmer temperatures in spring. It is well-known that small RNAs modulate temperature responses in many plant species, but however, how small RNAs are involved in genetic networks of temperature-mediated dormancy control in fruit tree species remains unclear. Here, we have made use of a recently developed ARGONAUTE (AGO)-purification technique to isolate small RNAs from apple buds. A small RNA-seq experiment resulted in the identification of 17 micro RNAs (miRNAs) that change their pattern of expression in apple buds during dormancy. Furthermore, the functional analysis of their predicted target genes suggests a main role of the 17 miRNAs in phenylpropanoid biosynthesis, gene regulation, plant development and growth, and response to stimulus. Finally, we studied the conservation of the Arabidopsis thaliana regulatory miR159-MYB module in apple in the context of the plant hormone abscisic acid homeostasis.
RNA-Seq in Plant Development and Chromatin Regulation
Plant developmental biology focuses on the regulation and coordination of processes in early plant embryos that lead to the development and differentiation of plant tissues, organs, and to the maturation of structures in the whole plant. Plants produce new tissues from undifferentiated (stem)cells, the meristem, throughout their life – a process that aids adaptation to environmental conditions. Understanding plant development is therefore becoming more and more important in agricultural biotechnology and applied crop sciences. Recent back-to-back publications address the impact of alternative 3′ ends (alternative poly(A) site usage) on chromatin regulation and development.
The Arabidopsis floral repressor FLOWERING LOCUS C (FLC) is a model system to detangle chromatin regulation, RNA processing, and underlying molecular mechanisms influencing transcriptional states and developmental timing in plants. Recent studies found that transcription termination events contribute to the definition of chromatin domains around genes and proximal termination aids in determining the rate of silencing. QuantSeq REV, a 3′ mRNA-Seq protocol designed to simplify alternative polyadenylation analysis, was used to determine poly(A) site usage and to identify further targets of the investigated transcription-coupled silencing mechanism.
Abstract
The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3′ processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3′ end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.
Abstract
The mechanisms and timescales controlling de novo establishment of chromatin-mediated transcriptional silencing by Polycomb repressive complex 2 (PRC2) are unclear. Here, we investigate PRC2 silencing at Arabidopsis FLOWERING LOCUS C (FLC), known to involve co-transcriptional RNA processing, histone demethylation activity, and PRC2 function, but so far not mechanistically connected. We develop and test a computational model describing proximal polyadenylation/termination mediated by the RNA-binding protein FCA that induces H3K4me1 removal by the histone demethylase FLD. H3K4me1 removal feeds back to reduce RNA polymerase II (RNA Pol II) processivity and thus enhance early termination, thereby repressing productive transcription. The model predicts that this transcription-coupled repression controls the level of transcriptional antagonism to PRC2 action. Thus, the effectiveness of this repression dictates the timescale for establishment of PRC2/H3K27me3 silencing. We experimentally validate these mechanistic model predictions, revealing that co-transcriptional processing sets the level of productive transcription at the locus, which then determines the rate of the ON-to-OFF switch to PRC2 silencing.
Outlook for Plant Transcriptomics
Even though RNA sequencing in plants can deliver this extremely important information, researchers still face substantial challenges, e.g., incomplete or missing annotations, insufficient resolution on untranslated regions and non-coding RNAs, or abundant sequences obscuring the view of lower expressed transcripts. We will explore common challenges when getting started with RNA-Seq from plants in our next blog.
Written by Dr. Yvonne Goepel
