Design, execution, and interpretation of plant RNA-seq analyses.

Genomics has transformed our understanding of the genetic architecture of traits and the genetic variation present in plants. Here, we present a review of how RNA-seq can be performed to tackle research challenges addressed by plant sciences. We discuss the importance of experimental design in RNA-seq, including considerations for sampling and replication, to avoid pitfalls and wasted resources. Approaches for processing RNA-seq data include quality control and counting features, and we describe common approaches and variations. Though differential gene expression analysis is the most common analysis of RNA-seq data, we review multiple methods for assessing gene expression, including detecting allele-specific gene expression and building co-expression networks. With the production of more RNA-seq data, strategies for integrating these data into genetic mapping pipelines is of increased interest. Finally, special considerations for RNA-seq analysis and interpretation in plants are needed, due to the high genome complexity common across plants. By incorporating informed decisions throughout an RNA-seq experiment, we can increase the knowledge gained.

Acute stress reduces population-level metabolic and proteomic variation.

BACKGROUND: Variation in omics data due to intrinsic biological stochasticity is often viewed as a challenging and undesirable feature of complex systems analyses. In fact, numerous statistical methods are utilized to minimize the variation among biological replicates. RESULTS: We demonstrate that the common statistics relative standard deviation (RSD) and coefficient of variation (CV), which are often used for quality control or part of a larger pipeline in omics analyses, can also be used as a metric of a physiological stress response. Using an approach we term Replicate Variation Analysis (RVA), we demonstrate that acute physiological stress leads to feature-wide canalization of CV profiles of metabolomes and proteomes across biological replicates. Canalization is the repression of variation between replicates, which increases phenotypic similarity. Multiple in-house mass spectrometry omics datasets in addition to publicly available data were analyzed to assess changes in CV profiles in plants, animals, and microorganisms. In addition, proteomics data sets were evaluated utilizing RVA to identify functionality of reduced CV proteins. CONCLUSIONS: RVA provides a foundation for understanding omics level shifts that occur in response to cellular stress. This approach to data analysis helps characterize stress response and recovery, and could be deployed to detect populations under stress, monitor health status, and conduct environmental monitoring.

Selection of favorable alleles of genes controlling flowering and senescence improves malt barley quality.

Malt barley (Hordeum vulgare L.) is an important cash crop with stringent grain quality standards. Timing of the switch from vegetative to reproductive growth and timing of whole-plant senescence and nutrient remobilization are critical for cereal grain yield and quality. Understanding the genetic variation in genes associated with these developmental traits can streamline genotypic selection of superior malt barley germplasm. Here, we determined the effects of allelic variation in three genes encoding a glycine-rich RNA-binding protein (HvGR-RBP1) and two NAC transcription factors (HvNAM1 and HvNAM2) on malt barley agronomics and quality using previously developed markers for HvGR-RBP1 and HvNAM1 and a novel marker for HvNAM2. Based on a single-nucleotide polymorphism (SNP) in the first intron, the utilized marker differentiates NAM2 alleles of low-grain protein variety 'Karl' and of higher protein variety 'Lewis'. We demonstrate that the selection of favorable alleles for each gene impacts heading date, senescence timing, grain size, grain protein concentration, and malt quality. Specifically, combining 'Karl' alleles for the two NAC genes with the 'Lewis' HvGR-RBP1 allele extends grain fill duration, increases the percentage of plump kernels, decreases grain protein, and provides malt quality stability. Molecular markers for these genes are therefore highly useful tools in malt barley breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01331-7.

Genetic dissection of natural variation in oilseed traits of camelina by whole-genome resequencing and QTL mapping.

Camelina [Camelina sativa (L.) Crantz] is an oilseed crop in the Brassicaceae family that is currently being developed as a source of bioenergy and healthy fatty acids. To facilitate modern breeding efforts through marker-assisted selection and biotechnology, we evaluated genetic variation among a worldwide collection of 222 camelina accessions. We performed whole-genome resequencing to obtain single nucleotide polymorphism (SNP) markers and to analyze genomic diversity. We also conducted phenotypic field evaluations in two consecutive seasons for variations in key agronomic traits related to oilseed production such as seed size, oil content (OC), fatty acid composition, and flowering time. We determined the population structure of the camelina accessions using 161,301 SNPs. Further, we identified quantitative trait loci (QTL) and candidate genes controlling the above field-evaluated traits by genome-wide association studies (GWAS) complemented with linkage mapping using a recombinant inbred line (RIL) population. Characterization of the natural variation at the genome and phenotypic levels provides valuable resources to camelina genetic studies and crop improvement. The QTL and candidate genes should assist in breeding of advanced camelina varieties that can be integrated into the cropping systems for the production of high yield of oils of desired fatty acid composition.

Roles of Brassinosteroids in Mitigating Heat Stress Damage in Cereal Crops.

Heat stress causes huge losses in the yield of cereal crops. Temperature influences the rate of plant metabolic and developmental processes that ultimately determine the production of grains, with high temperatures causing a reduction in grain yield and quality. To ensure continued food security, the tolerance of high temperature is rapidly becoming necessary. Brassinosteroids (BR) are a class of plant hormones that impact tolerance to various biotic and abiotic stresses and regulate cereal growth and fertility. Fine-tuning the action of BR has the potential to increase cereals' tolerance and acclimation to heat stress and maintain yields. Mechanistically, exogenous applications of BR protect yields through amplifying responses to heat stress and rescuing the expression of growth promoters. Varied BR compounds and differential signaling mechanisms across cereals point to a diversity of mechanisms that can be leveraged to mitigate heat stress. Further, hormone transport and BR interaction with other molecules in plants may be critical to utilizing BR as protective agrochemicals against heat stress. Understanding the interplay between heat stress responses, growth processes and hormone signaling may lead us to a comprehensive dogma of how to tune BR application for optimizing cereal growth under challenging environments in the field.

Combined effects of a glycine-rich RNA-binding protein and a NAC transcription factor extend grain fill duration and improve malt barley agronomic performance.

KEY MESSAGE: Two key barley genes independently control anthesis and senescence timing, enabling the manipulation of grain fill duration, grain size/plumpness, and grain protein concentration. Plant developmental processes such as flowering and senescence have direct effects on cereal yield and quality. Previous work highlighted the importance of two tightly linked genes encoding a glycine-rich RNA-binding protein (HvGR-RBP1) and a NAC transcription factor (HvNAM1), controlling barley anthesis timing, senescence, and percent grain protein. Varieties that differ in HvGR-RBP1 expression, 'Karl'(low) and 'Lewis'(high), also differ in sequence 1 KB upstream of translation start site, including an ~ 400 bp G rich insertion in the 5'-flanking region of the 'Karl' allele, which could disrupt gene expression. To improve malt quality, the (low-grain protein, delayed-senescence) 'Karl' HvNAM1 allele was introgressed into Montana germplasm. After several seasons of selection, the resulting germplasm was screened for the allelic combinations of HvGR-RBP1 and HvNAM1, finding lines combining 'Karl' alleles for both genes (-/-), lines combining 'Lewis' (functional, expressed) HvGR-RBP1 with 'Karl' HvNAM1 alleles ( ±), and lines combining 'Lewis' alleles for both genes (+ / +). Field experiments indicate that the functional ('Lewis,' +) HvGR-RBP1 allele is associated with earlier anthesis and with slightly shorter plants, while the 'Karl' (-) HvNAM1 allele delays maturation. Genotypes carrying the ± allele combination therefore had a significantly (3 days) extended grain fill duration, leading to a higher percentage of plump kernels, slightly enhanced test weight, and lower grain protein concentration when compared to the other allele combinations. Overall, our data suggest an important function for HvGR-RBP1 in the control of barley reproductive development and set the stage for a more detailed functional analysis of this gene.

Redundant and Cryptic Enhancer Activities of the Drosophilayellow Gene.

Cis-regulatory sequences known as enhancers play a key role in regulating gene expression. Evolutionary changes in these DNA sequences contribute to phenotypic evolution. The Drosophilayellow gene, which is required for pigmentation, has emerged as a model system for understanding how cis-regulatory sequences evolve, providing some of the most detailed insights available into how activities of orthologous enhancers have diverged between species. Here, we examine the evolution of yellow cis-regulatory sequences on a broader scale, by comparing the distribution and function of yellow enhancer activities throughout the 5' intergenic and intronic sequences of Drosophila melanogaster, D. pseudoobscura, and D. willistoni We find that cis-regulatory sequences driving expression in a particular tissue are not as modular as previously described, but rather have many redundant and cryptic enhancer activities distributed throughout the regions surveyed. Interestingly, cryptic enhancer activities of sequences from one species often drove patterns of expression observed in other species, suggesting that the frequent evolutionary changes in yellow expression observed among Drosophila species may be facilitated by gaining and losing repression of preexisting cis-regulatory sequences.

Redundancy, Feedback, and Robustness in the Arabidopsis thaliana BZR/BEH Gene Family.

Organismal development is remarkably robust, tolerating stochastic errors to produce consistent, so-called canalized adult phenotypes. The mechanistic underpinnings of developmental robustness are poorly understood, but recent studies implicate certain features of genetic networks such as functional redundancy, connectivity, and feedback. Here, we examine the BZR/BEH gene family, whose function contributes to embryonic stem development in the plant Arabidopsis thaliana, to test current assumptions on functional redundancy and trait robustness. Our analyses of BZR/BEH gene mutants and mutant combinations revealed that functional redundancy among these gene family members is not necessary for trait robustness. Connectivity is another commonly cited determinant of robustness; however, we found no correlation between connectivity among gene family members or their connectivity with other transcription factors and effects on developmental robustness. Instead, our data suggest that BEH4, the earliest diverged family member, modulates developmental robustness. We present evidence indicating that regulatory cross-talk among gene family members is integrated by BEH4 to promote wild-type levels of developmental robustness. Further, the chaperone HSP90, a known determinant of developmental robustness, appears to act via BEH4 in maintaining robustness of embryonic stem length. In summary, we demonstrate that even among closely related transcription factors, trait robustness can arise through the activity of a single gene family member, challenging common assumptions about the molecular underpinnings of robustness.

Molecular mechanisms governing differential robustness of development and environmental responses in plants.

BACKGROUND: Robustness to genetic and environmental perturbation is a salient feature of multicellular organisms. Loss of developmental robustness can lead to severe phenotypic defects and fitness loss. However, perfect robustness, i.e. no variation at all, is evolutionarily unfit as organisms must be able to change phenotype to properly respond to changing environments and biotic challenges. Plasticity is the ability to adjust phenotypes predictably in response to specific environmental stimuli, which can be considered a transient shift allowing an organism to move from one robust phenotypic state to another. Plants, as sessile organisms that undergo continuous development, are particularly dependent on an exquisite fine-tuning of the processes that balance robustness and plasticity to maximize fitness. SCOPE AND CONCLUSIONS: This paper reviews recently identified mechanisms, both systems-level and molecular, that modulate robustness, and discusses their implications for the optimization of plant fitness. Robustness in living systems arises from the structure of genetic networks, the specific molecular functions of the underlying genes, and their interactions. This very same network responsible for the robustness of specific developmental states also has to be built such that it enables plastic yet robust shifts in response to environmental changes. In plants, the interactions and functions of signal transduction pathways activated by phytohormones and the tendency for plants to tolerate whole-genome duplications, tandem gene duplication and hybridization are emerging as major regulators of robustness in development. Despite their obvious implications for plant evolution and plant breeding, the mechanistic underpinnings by which plants modulate precise levels of robustness, plasticity and evolvability in networks controlling different phenotypes are under-studied.

A Genome-Wide Association Analysis Reveals Epistatic Cancellation of Additive Genetic Variance for Root Length in Arabidopsis thaliana.

Efforts to identify loci underlying complex traits generally assume that most genetic variance is additive. Here, we examined the genetics of Arabidopsis thaliana root length and found that the genomic narrow-sense heritability for this trait in the examined population was statistically zero. The low amount of additive genetic variance that could be captured by the genome-wide genotypes likely explains why no associations to root length could be found using standard additive-model-based genome-wide association (GWA) approaches. However, as the broad-sense heritability for root length was significantly larger, and primarily due to epistasis, we also performed an epistatic GWA analysis to map loci contributing to the epistatic genetic variance. Four interacting pairs of loci were revealed, involving seven chromosomal loci that passed a standard multiple-testing corrected significance threshold. The genotype-phenotype maps for these pairs revealed epistasis that cancelled out the additive genetic variance, explaining why these loci were not detected in the additive GWA analysis. Small population sizes, such as in our experiment, increase the risk of identifying false epistatic interactions due to testing for associations with very large numbers of multi-marker genotypes in few phenotyped individuals. Therefore, we estimated the false-positive risk using a new statistical approach that suggested half of the associated pairs to be true positive associations. Our experimental evaluation of candidate genes within the seven associated loci suggests that this estimate is conservative; we identified functional candidate genes that affected root development in four loci that were part of three of the pairs. The statistical epistatic analyses were thus indispensable for confirming known, and identifying new, candidate genes for root length in this population of wild-collected A. thaliana accessions. We also illustrate how epistatic cancellation of the additive genetic variance explains the insignificant narrow-sense and significant broad-sense heritability by using a combination of careful statistical epistatic analyses and functional genetic experiments.

Hsp90 promotes kinase evolution.

Heat-shock protein 90 (Hsp90) promotes the maturation and stability of its client proteins, including many kinases. In doing so, Hsp90 may allow its clients to accumulate mutations as previously proposed by the capacitor hypothesis. If true, Hsp90 clients should show increased evolutionary rate compared with nonclients; however, other factors, such as gene expression and protein connectivity, may confound or obscure the chaperone's putative contribution. Here, we compared the evolutionary rates of many Hsp90 clients and nonclients in the human protein kinase superfamily. We show that Hsp90 client status promotes evolutionary rate independently of, but in a small magnitude similar to that of gene expression and protein connectivity. Hsp90's effect on kinase evolutionary rate was detected across mammals, specifically relaxing purifying selection. Hsp90 clients also showed increased nucleotide diversity and harbored more damaging variation than nonclient kinases across humans. These results are consistent with the central argument of the capacitor hypothesis that interaction with the chaperone allows its clients to harbor genetic variation. Hsp90 client status is thought to be highly dynamic with as few as one amino acid change rendering a protein dependent on the chaperone. Contrary to this expectation, we found that across protein kinase phylogeny Hsp90 client status tends to be gained, maintained, and shared among closely related kinases. We also infer that the ancestral protein kinase was not an Hsp90 client. Taken together, our results suggest that Hsp90 played an important role in shaping the kinase superfamily.

The protein chaperone HSP90 can facilitate the divergence of gene duplicates.

The heat-shock protein 90 (HSP90) acts as a chaperone by ensuring proper maturation and folding of its client proteins. The HSP90 capacitor hypothesis holds that interactions with HSP90 allow proteins to accumulate mutations while maintaining function. Following this logic, HSP90 clients would be predicted to show relaxed selection compared with nonclients. In this study, we identify a new HSP90 client in the plant steroid hormone pathway: the transcription factor BES1. Its closest paralog, BZR1, is not an HSP90 client. This difference in HSP90 client status in two highly similar proteins enabled a direct test of the capacitor hypothesis. We find that BES1 shows relaxed selection compared to BZR1, hallmarks of neo- and subfunctionalization, and dynamic HSP90 client status across independent evolutionary paths. These results suggested that HSP90's influence on gene evolution may be detectable if we compare gene duplicates because duplicates share most other properties influencing evolutionary rate that might otherwise conceal the chaperone's effect. We test this hypothesis using systematically identified HSP90 clients in yeast and observe a significant trend of HSP90 clients evolving faster than their nonclient paralogs. This trend was not detected when yeast clients and nonclients were compared without considering paralog status. Our data provide evidence that HSP90 influences selection on genes encoding its clients and facilitates divergence between gene duplicates.

Molecular mechanisms of robustness in plants.

Robustness, the ability of organisms to buffer phenotypes against perturbations, has drawn renewed interest among developmental biologists and geneticists. A growing body of research supports an important role of robustness in the genotype to phenotype translation, with far-reaching implications for evolutionary processes and disease susceptibility. Similar to animals and fungi, plant robustness is a function of genetic network architecture. Most perturbations are buffered; however, perturbation of network hubs destabilizes many traits. Here, we review recent advances in identifying molecular robustness mechanisms in plants that have been enabled by a combination of classical genetics and population genetics with genome-scale data.

Requirement for the ERI/DICER complex in endogenous RNA interference and sperm development in Caenorhabditis elegans.

Small regulatory RNAs are key regulators of gene expression. One class of small regulatory RNAs, termed the endogenous small interfering RNAs (endo siRNAs), is thought to negatively regulate cellular transcripts via an RNA interference (RNAi)-like mechanism termed endogenous RNAi (endo RNAi). A complex of proteins composed of ERI-1/3/5, RRF-3, and DICER (the ERI/DICER complex) mediates endo RNAi processes in Caenorhabditis elegans. We conducted a genetic screen to identify additional components of the endo RNAi machinery. Our screen recovered alleles of eri-9, which encodes a novel DICER-interacting protein, and a missense mutation within the helicase domain of DICER [DCR-1(G492R)]. ERI-9(-) and DCR-1(G492) animals exhibit defects in endo siRNA expression and a concomitant failure to regulate mRNAs that exhibit sequence homology to these endo siRNAs, indicating that ERI-9 and the DCR-1 helicase domain function in the C. elegans endo RNAi pathway. We define a subset of Eri mutant animals (including eri-1, rrf-3, eri-3, and dcr-1, but not eri-9 or ergo-1) that exhibit temperature-sensitive, sperm-specific sterility and defects in X chromosome segregation. Among these mutants we find multiple aberrations in sperm development beginning with cytokinesis and extending through terminal differentiation. These results identify novel components of the endo RNAi machinery, demonstrate differential requirements for the Eri factors in the sperm-producing germline, and begin to delineate the functional requirement for the ERI/DICER complex in sperm development.

An Argonaute transports siRNAs from the cytoplasm to the nucleus.

Ribonucleoprotein complexes consisting of Argonaute-like proteins and small regulatory RNAs function in a wide range of biological processes. Many of these small regulatory RNAs are predicted to act, at least in part, within the nucleus. We conducted a genetic screen to identify factors essential for RNA interference (RNAi) in nuclei of Caenorhabditis elegans and identified the Argonaute protein NRDE-3. In the absence of small interfering RNAs (siRNAs), NRDE-3 resides in the cytoplasm. NRDE-3 binds siRNAs generated by RNA-dependent RNA polymerases acting on messenger RNA templates in the cytoplasm and redistributes to the nucleus. Nuclear redistribution of NRDE-3 requires a functional nuclear localization signal, is required for nuclear RNAi, and results in NRDE-3 association with nuclear-localized nascent transcripts. Thus, specific Argonaute proteins can transport specific classes of small regulatory RNAs to distinct cellular compartments to regulate gene expression.