Physiological and Molecular Modulations to Drought Stress in the Brassica Species.

Climate change, particularly drought stress, significantly impacts plant growth and development, necessitating the development of resilient crops. This study investigated physiological and molecular modulations to drought stress between diploid parent species and their polyploid progeny in the Brassica species. While no significant phenotypic differences were observed among the six species, drought stress reduced growth parameters by 2.4% and increased oxidative stress markers by 1.4-fold. Drought also triggered the expression of genes related to stress responses and led to the accumulation of specific metabolites. We also conducted the first study of perfluorooctane sulfonic acid (PFOS) levels in leaves as a drought indicator. Lower levels of PFOS accumulation were linked to plants taking in less water under drought conditions. Both diploid and polyploid species responded to drought stress similarly, but there was a wide range of variation in their responses. In particular, responses were less variable in polyploid species than in diploid species. This suggests that their additional genomic components acquired through polyploidy may improve their flexibility to modulate stress responses. Despite the hybrid vigor common in polyploid species, Brassica polyploids demonstrated intermediate responses to drought stress. Overall, this study lays the framework for future omics-level research, including transcriptome and proteomic studies, to deepen our understanding of drought tolerance mechanisms in Brassica species.

Investigation of regulatory divergence between homoeologs in the recently formed allopolyploids, Tragopogon mirus and T. miscellus (Asteraceae).

Polyploidy is an important evolutionary process throughout eukaryotes, particularly in flowering plants. Duplicated gene pairs (homoeologs) in allopolyploids provide additional genetic resources for changes in molecular, biochemical, and physiological mechanisms that result in evolutionary novelty. Therefore, understanding how divergent genomes and their regulatory networks reconcile is vital for unraveling the role of polyploidy in plant evolution. Here, we compared the leaf transcriptomes of recently formed natural allotetraploids (Tragopogon mirus and T. miscellus) and their diploid parents (T. porrifolius X T. dubius and T. pratensis X T. dubius, respectively). Analysis of 35 400 expressed loci showed a significantly higher level of transcriptomic additivity compared to old polyploids; only 22% were non-additively expressed in the polyploids, with 5.9% exhibiting transgressive expression (lower or higher expression in the polyploids than in the diploid parents). Among approximately 7400 common orthologous regions (COREs), most loci in both allopolyploids exhibited expression patterns that were vertically inherited from their diploid parents. However, 18% and 20.3% of the loci showed novel expression bias patterns in T. mirus and T. miscellus, respectively. The expression changes of 1500 COREs were explained by cis-regulatory divergence (the condition in which the two parental subgenomes do not interact) between the diploid parents, whereas only about 423 and 461 of the gene expression changes represent trans-effects (the two parental subgenomes interact) in T. mirus and T. miscellus, respectively. The low degree of both non-additivity and trans-effects on gene expression may present the ongoing evolutionary processes of the newly formed Tragopogon polyploids (~80-90 years).

Secretome Analysis of Inductive Signals for BM-MSC Transdifferentiation into Salivary Gland Progenitors.

Severe dry mouth in patients with Sjögren's Syndrome, or radiation therapy for patients with head and neck cancer, significantly compromises their oral health and quality of life. The current clinical management of xerostomia is limited to palliative care as there are no clinically-proven treatments available. Previously, our studies demonstrated that mouse bone marrow-derived mesenchymal stem cells (mMSCs) can differentiate into salivary progenitors when co-cultured with primary salivary epithelial cells. Transcription factors that were upregulated in co-cultured mMSCs were identified concomitantly with morphological changes and the expression of acinar cell markers, such as α-amylase (AMY1), muscarinic-type-3-receptor(M3R), aquaporin-5(AQP5), and a ductal cell marker known as cytokeratin 19(CK19). In the present study, we further explored inductive molecules in the conditioned media that led to mMSC reprogramming by high-throughput liquid chromatography with tandem mass spectrometry and systems biology. Our approach identified ten differentially expressed proteins based on their putative roles in salivary gland embryogenesis and development. Additionally, systems biology analysis revealed six candidate proteins, namely insulin-like growth factor binding protein-7 (IGFBP7), cysteine-rich, angiogenetic inducer, 61(CYR61), agrin(AGRN), laminin, beta 2 (LAMB2), follistatin-like 1(FSTL1), and fibronectin 1(FN1), for their potential contribution to mMSC transdifferentiation during co-culture. To our knowledge, our study is the first in the field to identify soluble inductive molecules that drive mMSC into salivary progenitors, which crosses lineage boundaries.

A Robust Methodology for Assessing Differential Homeolog Contributions to the Transcriptomes of Allopolyploids.

Polyploidy has played a pivotal and recurring role in angiosperm evolution. Allotetraploids arise from hybridization between species and possess duplicated gene copies (homeologs) that serve redundant roles immediately after polyploidization. Although polyploidization is a major contributor to plant evolution, it remains poorly understood. We describe an analytical approach for assessing homeolog-specific expression that begins with de novo assembly of parental transcriptomes and effectively (i) reduces redundancy in de novo assemblies, (ii) identifies putative orthologs, (iii) isolates common regions between orthologs, and (iv) assesses homeolog-specific expression using a robust Bayesian Poisson-Gamma model to account for sequence bias when mapping polyploid reads back to parental references. Using this novel methodology, we examine differential homeolog contributions to the transcriptome in the recently formed allopolyploids Tragopogon mirus and T. miscellus (Compositae). Notably, we assess a larger Tragopogon gene set than previous studies of this system. Using carefully identified orthologous regions and filtering biased orthologs, we find in both allopolyploids largely balanced expression with no strong parental bias. These new methods can be used to examine homeolog expression in any tetrapolyploid system without requiring a reference genome.

Characterization of thiol-based redox modifications of Brassica napusSNF1-related protein kinase 2.6-2C.

Sucrose nonfermenting 1-related protein kinase 2.6 (SnRK2.6), also known as Open Stomata 1 (OST1) in Arabidopsis thaliana, plays a pivotal role in abscisic acid (ABA)-mediated stomatal closure. Four SnRK2.6 paralogs were identified in the Brassica napus genome in our previous work. Here we studied one of the paralogs, BnSnRK2.6-2C, which was transcriptionally induced by ABA in guard cells. Recombinant BnSnRK2.6-2C exhibited autophosphorylation activity and its phosphorylation sites were mapped. The autophosphorylation activity was inhibited by S-nitrosoglutathione (GSNO) and by oxidized glutathione (GSSG), and the inhibition was reversed by reductants. Using monobromobimane (mBBr) labeling, we demonstrated a dose-dependent modification of BnSnRK2.6-2C by GSNO. Furthermore, mass spectrometry analysis revealed previously uncharacterized thiol-based modifications including glutathionylation and sulfonic acid formation. Of the six cysteine residues in BnSnRK2.6-2C, C159 was found to have different types of thiol modifications, suggesting its high redox sensitivity and versatility. In addition, mBBr labeling on tyrosine residues was identified. Collectively, these data provide detailed biochemical characterization of redox-induced modifications and changes of the BnSnRK2.6-2C activity.

Genome-wide identification and homeolog-specific expression analysis of the SnRK2 genes in Brassica napus guard cells.

Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) proteins constitute a small plant-specific serine/threonine kinase family involved in abscisic acid (ABA) signaling and plant responses to biotic and abiotic stresses. Although SnRK2s have been well-studied in Arabidopsis thaliana, little is known about SnRK2s in Brassica napus. Here we identified 30 putative sequences encoding 10 SnRK2 proteins in the B. napus genome and the expression profiles of a subset of 14 SnRK2 genes in guard cells of B. napus. In agreement with its polyploid origin, B. napus maintains both homeologs from its diploid parents. The results of quantitative real-time PCR (qRT-PCR) and reanalysis of RNA-Seq data showed that certain BnSnRK2 genes were commonly expressed in leaf tissues in different varieties of B. napus. In particular, qRT-PCR results showed that 12 of the 14 BnSnRK2s responded to drought stress in leaves and in ABA-treated guard cells. Among them, BnSnRK2.4 and BnSnRK2.6 were of interest because of their robust responsiveness to ABA treatment and drought stress. Notably, BnSnRK2 genes exhibited up-regulation of different homeologs, particularly in response to abiotic stress. The homeolog expression bias in BnSnRK2 genes suggests that parental origin of genes might be responsible for efficient regulation of stress responses in polyploids. This work has laid a foundation for future functional characterization of the different BnSnKR2 homeologs in B. napus and its parents, especially their functions in guard cell signaling and stress responses.

Redox proteomics of tomato in response to Pseudomonas syringae infection.

Unlike mammals with adaptive immunity, plants rely on their innate immunity based on pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) for pathogen defense. Reactive oxygen species, known to play crucial roles in PTI and ETI, can perturb cellular redox homeostasis and lead to changes of redox-sensitive proteins through modification of cysteine sulfhydryl groups. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, little is known about redox proteins and how they function in PTI and ETI. In this study, cysTMT proteomics technology was used to identify similarities and differences of protein redox modifications in tomato resistant (PtoR) and susceptible (prf3) genotypes in response to Pseudomonas syringae pv tomato (Pst) infection. In addition, the results of the redox changes were compared and corrected with the protein level changes. A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation-reduction. This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses.

Gene-expression novelty in allopolyploid cotton: a proteomic perspective.

Allopolyploidization is accompanied by changes in gene expression that are thought to contribute to phenotypic diversification. Here we describe global changes in the single-celled cotton fiber proteome of two natural allopolyploid species (Gossypium hirsutum and G. barbadense) and living models of their diploid parents using two different proteomic approaches. In total, 1323 two-dimensional gel electrophoresis spots and 1652 identified proteins by isobaric tags for relative and absolute quantitation were quantitatively profiled during fiber elongation. Between allopolyploids and their diploid A- and D-genome progenitors, amounts of differential expression ranged from 4.4 to 12.8%. Over 80% of the allopolyploid proteome was additively expressed with respect to progenitor diploids. Interestingly, the fiber proteome of G. hirsutum resembles the parental A-genome more closely, where long, spinable fiber first evolved, than does the fiber proteome of G. barbadense. More protein expression patterns were A-dominant than D-dominant in G. hirsutum, but in G. barbadense, the direction of expression-level dominance switched from the D-genome to the A-genome during fiber development. Comparison of developmental changes between the two allopolyploid species revealed a high level of proteomic differentiation despite their shared ancestry, relatively recent evolutionary divergence, and similar gross morphology. These results suggest that the two allopolyploid species have achieved superficially similar modern fiber phenotypes through different evolutionary routes at the proteome level. We also detected homeolog-specific expression for 1001 proteins and present a novel approach to infer the relationship between homeolog-specific and duplicate expression patterns. Our study provides a proteomic perspective on understanding evolutionary consequences of allopolyploidization, showing how protein expression has been altered by polyploidization and subsequently has diversified among species.

Nonadditive gene expression in polyploids.

Allopolyploidy involves hybridization and duplication of divergent parental genomes and provides new avenues for gene expression. The expression levels of duplicated genes in polyploids can show deviation from parental additivity (the arithmetic average of the parental expression levels). Nonadditive expression has been widely observed in diverse polyploids and comprises at least three possible scenarios: (a) The total gene expression level in a polyploid is similar to that of one of its parents (expression-level dominance); (b) total gene expression is lower or higher than in both parents (transgressive expression); and (c) the relative contribution of the parental copies (homeologs) to the total gene expression is unequal (homeolog expression bias). Several factors may result in expression nonadditivity in polyploids, including maternal-paternal influence, gene dosage balance, cis- and/or trans-regulatory networks, and epigenetic regulation. As our understanding of nonadditive gene expression in polyploids remains limited, a new generation of investigators should explore additional phenomena (i.e., alternative splicing) and use other high-throughput "omics" technologies to measure the impact of nonadditive expression on phenotype, proteome, and metabolome.

Proteomics profiling of fiber development and domestication in upland cotton (Gossypium hirsutum L.).

Comparative proteomic analyses were performed to detail the evolutionary consequences of strong directional selection for enhanced fiber traits in modern upland cotton (Gossypium hirsutum L.). Using two complementary proteomic approaches, 2-DE and iTRAQ LC-MS/MS, fiber proteomes were examined for four representative stages of fiber development. Approximately 1,000 protein features were characterized using each strategy, collectively resulting in the identification and functional categorization of 1,223 proteins. Unequal contributions of homoeologous proteins were detected for over a third of the fiber proteome, but overall expression was balanced with respect to the genome-of-origin in the allopolyploid G. hirsutum. About 30% of the proteins were differentially expressed during fiber development within wild and domesticated cotton. Notably, domestication was accompanied by a doubling of protein developmental dynamics for the period between 10 and 20 days following pollination. Expression levels of 240 iTRAQ proteins and 293 2-DE spots were altered by domestication, collectively representing multiple cellular and metabolic processes, including metabolism, energy, protein synthesis and destination, defense and stress response. Analyses of homoeolog-specific expression indicate that duplicated gene products in cotton fibers can be differently regulated in response to selection. These results demonstrate the power of proteomics for the analysis of crop domestication and phenotypic evolution.

Proteomic profiling of developing cotton fibers from wild and domesticated Gossypium barbadense.

Pima cotton (Gossypium barbadense) is widely cultivated because of its long, strong seed trichomes ('fibers') used for premium textiles. These agronomically advanced fibers were derived following domestication and thousands of years of human-mediated crop improvement. To gain an insight into fiber development and evolution, we conducted comparative proteomic and transcriptomic profiling of developing fiber from an elite cultivar and a wild accession. Analyses using isobaric tag for relative and absolute quantification (iTRAQ) LC-MS/MS technology identified 1317 proteins in fiber. Of these, 205 were differentially expressed across developmental stages, and 190 showed differential expression between wild and cultivated forms, 14.4% of the proteome sampled. Human selection may have shifted the timing of developmental modules, such that some occur earlier in domesticated than in wild cotton. A novel approach was used to detect possible biased expression of homoeologous copies of proteins. Results indicate a significant partitioning of duplicate gene expression at the protein level, but an approximately equal degree of bias for each of the two constituent genomes of allopolyploid cotton. Our results demonstrate the power of complementary transcriptomic and proteomic approaches for the study of the domestication process. They also provide a rich database for mining for functional analyses of cotton improvement or evolution.

Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres.

Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments. Here we show that an abrupt five- to sixfold ploidy increase approximately 60 million years (Myr) ago, and allopolyploidy reuniting divergent Gossypium genomes approximately 1-2 Myr ago, conferred about 30-36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (Gossypium hirsutum and Gossypium barbadense), genetic complexity equalled only by Brassica among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred Gossypium herbaceum A and non-spinnable Gossypium longicalyx F genomes to one another and the outgroup D genome of non-spinnable Gossypium raimondii. The sequence of a G. hirsutum A(t)D(t) (in which 't' indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in G. hirsutum recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups.

The cytonuclear dimension of allopolyploid evolution: an example from cotton using rubisco.

During allopolyploid speciation, two divergent nuclear genomes merge, yet only one (usually the maternal) of the two sets of progenitor organellar genomes is maintained. Rubisco (1,5-bisphosphate carboxylase/oxygenase) is composed of nuclear-encoded small subunits (SSUs) and plastome-encoded large subunits (LSUs), providing an ideal system to explore the evolutionary process of cytonuclear accommodation. Here, we take initial steps in this direction, using Gossypium allopolyploids as our model. SSU copies from divergent (5-10 My) progenitor diploids ("A" and "D" genomes) were combined at the time of polyploid formation 1-2 Ma, with the LSU encoded by the maternal A-genome parent. LSU genes from A- and D-genome diploids and AD-genome allopolyploids were sequenced, revealing several nonsynonymous substitutions and suggesting the possibility of differential selection on the nuclear-encoded rbcS partner following allopolyploid formation. Sequence data for the rbcS gene family revealed nonreciprocal homoeologous recombination between A- and D-rbcS homoeologs in all polyploid species but not in a synthetic intergenomic F1 hybrid, demonstrating "gene conversion" during allopolyploid evolution. All progenitor rbcS genes are retained and expressed in the five extant allopolyploid species, but analysis of the leaf transcriptome showed that A-homoeologs are preferentially expressed in both the allopolyploid and hybrid, consistent with the maternal origin of rbcL. Although rbcS genes from both progenitor genomes are expressed, some appear to have experienced mutations that may represent cytonuclear coevolution.

An expressed sequence tag (EST) library from developing fruits of an Hawaiian endemic mint (Stenogyne rugosa, Lamiaceae): characterization and microsatellite markers.

BACKGROUND: The endemic Hawaiian mints represent a major island radiation that likely originated from hybridization between two North American polyploid lineages. In contrast with the extensive morphological and ecological diversity among taxa, ribosomal DNA sequence variation has been found to be remarkably low. In the past few years, expressed sequence tag (EST) projects on plant species have generated a vast amount of publicly available sequence data that can be mined for simple sequence repeats (SSRs). However, these EST projects have largely focused on crop or otherwise economically important plants, and so far only few studies have been published on the use of intragenic SSRs in natural plant populations. We constructed an EST library from developing fleshy nutlets of Stenogyne rugosa principally to identify genetic markers for the Hawaiian endemic mints. RESULTS: The Stenogyne fruit EST library consisted of 628 unique transcripts derived from 942 high quality ESTs, with 68% of unigenes matching Arabidopsis genes. Relative frequencies of Gene Ontology functional categories were broadly representative of the Arabidopsis proteome. Many unigenes were identified as putative homologs of genes that are active during plant reproductive development. A comparison between unigenes from Stenogyne and tomato (both asterid angiosperms) revealed many homologs that may be relevant for fruit development. Among the 628 unigenes, a total of 44 potentially useful microsatellite loci were predicted. Several of these were successfully tested for cross-transferability to other Hawaiian mint species, and at least five of these demonstrated interesting patterns of polymorphism across a large sample of Hawaiian mints as well as close North American relatives in the genus Stachys. CONCLUSION: Analysis of this relatively small EST library illustrated a broad GO functional representation. Many unigenes could be annotated to involvement in reproductive development. Furthermore, first tests of microsatellite primer pairs have proven promising for the use of Stenogyne rugosa EST SSRs for evolutionary and phylogeographic studies of the Hawaiian endemic mints and their close relatives. Given that allelic repeat length variation in developmental genes of other organisms has been linked with morphological evolution, these SSRs may also prove useful for analyses of phenotypic differences among Hawaiian mints.