Label-free quantitative proteomic analysis of alfalfa in response to microRNA156 under high temperature.

BACKGROUND: Abiotic stress, including heat, is one of the major factors that affect alfalfa growth and forage yield. The small RNA, microRNA156 (miR156), regulates multiple traits in alfalfa during abiotic stress. The aim of this study was to explore the role of miR156 in regulating heat response in alfalfa at the protein level. RESULTS: In this study, we compared an empty vector control and miR156 overexpressing (miR156OE) alfalfa plants after exposing them to heat stress (40 °C) for 24 h. We measured physiological parameters of control and miR156OE plants under heat stress, and collected leaf samples for protein analysis. A higher proline and antioxidant contents were detected in miR156OE plants than in controls under heat stress. Protein samples were analyzed by label-free quantification proteomics. Across all samples, a total of 1878 protein groups were detected. Under heat stress, 45 protein groups in the empty vector plants were significantly altered (P < 0.05; |log2FC| > 2). Conversely, 105 protein groups were significantly altered when miR156OE alfalfa was subjected to heat stress, of which 91 were unique to miR156OE plants. The identified protein groups unique to miR156OE plants were related to diverse functions including metabolism, photosynthesis, stress-response and plant defenses. Furthermore, we identified transcription factors in miR156OE plants, which belonged to squamosa promoter binding-like protein, MYB, ethylene responsive factors, AP2 domain, ABA response element binding factor and bZIP families of transcription factors. CONCLUSIONS: These results suggest a positive role for miR156 in heat stress response in alfalfa. They reveal a miR156-regulated network of mechanisms at the protein level to modulate heat responses in alfalfa.

The interplay between miR156/SPL13 and DFR/WD40-1 regulate drought tolerance in alfalfa.

BACKGROUND: Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop's sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. RESULTS: To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40-1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40-1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. CONCLUSIONS: Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40-1 expression, whereas higher miR156 overexpression results in drought susceptibility.

Corrigendum: miR156/SPL10 Modulates Lateral Root Development, Branching and Leaf Morphology in Arabidopsis by Silencing AGAMOUS-LIKE 79.

[This corrects the article DOI: 10.3389/fpls.2017.02226.].

Assessment of Antinutritional Compounds and Chemotaxonomic Relationships between Camelina sativa and Its Wild Relatives.

We compared the secondary metabolite composition in seeds of Camelina sativa and its wild relatives to identify potential germplasm with reduced levels of antinutritional compounds. Twenty Camelina accessions, from five different species, were analyzed by liquid chromatography mass spectrometry and subjected to principal component analysis, which revealed that Camelina spp. separated into distinct chemotaxonomic groups. Three major glucosinolates (GSs) were identified in our study, namely, 9-methylsulfinylnonyl GS (GS9), 10-methylsulfinyldecyl GS (GS10), and 11-methylsulfinylundecyl GS (GS11). While there were differences in total GS levels, species-specific patterns for GS9 and GS11 were noted. Sinapine content ranged between 1.4 and 5.6 mg/g FW, with the lowest levels observed in  C. laxa and C. sativa. Lignin levels were also lowest in C. sativa, with most accessions containing less than 6 mg/g FW. Our results show that wild Camelina spp. have distinct metabolomes, and based on their levels of major antinutritionals, some could be incorporated into breeding programs with C. sativa.

Transcriptome analysis of microRNA156 overexpression alfalfa roots under drought stress.

Drought is one of the major abiotic stresses that negatively impact alfalfa growth and productivity. The role of microRNA156 (miR156) in drought has been demonstrated in plants. To date, there are no published studies investigating the role of miR156 in regulating global gene expression in alfalfa under drought. In our study, alfalfa genotypes overexpressing miR156 (miR156OE) exhibited reduced water loss, and enhanced root growth under drought. Our RNA-seq data showed that in response to drought, a total of 415 genes were upregulated and 169 genes were downregulated specifically in miR156OE genotypes. Genotypic comparison revealed that 285 genes were upregulated and 253 genes were downregulated in miR156OE genotypes relative to corresponding WT under drought. Gene Ontology enrichment analysis revealed that the number of differentially expressed genes belonging to biological process, molecular function and cell component functional groups was decreased in miR156OE genotypes under drought. Furthermore, RNA-Seq data showed downregulation of a gene encoding WD40 repeat in a miR156-specific manner. 5' RACE experiments verified cleavage of WD40-2 transcript under drought. Moreover, alfalfa plants overexpressing WD40-2 showed drought sensitive, whereas those with silenced WD40-2 exhibited drought tolerant phenotypes. These findings suggest that miR156 improves drought tolerance in alfalfa by targeting WD40-2.

COP9 signalosome subunit 5A affects phenylpropanoid metabolism, trichome formation and transcription of key genes of a regulatory tri-protein complex in Arabidopsis.

BACKGROUND: Trichomes and phenylpropanoid-derived phenolics are structural and chemical protection against many adverse conditions. Their production is regulated by a network that includes a TTG1/bHLH/MYB tri-protein complex in Arabidopsis. CSN5a, encoding COP9 signalosome subunit 5a, has also been implicated in trichome and anthocyanin production; however, the regulatory roles of CSN5a in the processes through interaction with the tri-protein complex has yet to be investigated. RESULTS: In this study, a new csn5a mutant, sk372, was recovered based on its altered morphological and chemical phenotypes compared to wild-type control. Mutant characterization was conducted with an emphasis on trichome and phenylpropanoid production and possible involvement of the tri-protein complex using metabolite and gene transcription profiling and scanning electron microscopy. Seed metabolite analysis revealed that defective CSN5a led to an enhanced production of many compounds in addition to anthocyanin, most notably phenylpropanoids and carotenoids as well as a glycoside of zeatin. Consistent changes in carotenoids and anthocyanin were also found in the sk372 leaves. In addition, 370 genes were differentially expressed in 10-day old seedlings of sk372 compared to its wild type control. Real-time transcript quantitative analysis showed that in sk372, GL2 and tri-protein complex gene TT2 was significantly suppressed (p < 0.05) while complex genes EGL3 and GL3 slightly decreased (p > 0.05). Complex genes MYB75, GL1 and flavonoid biosynthetic genes TT3 and TT18 in sk372 were all significantly enhanced. Overexpression of GL3 driven by cauliflower mosaic virus 35S promotor increased the number of single pointed trichomes only, no other phenotypic recovery in sk372. CONCLUSIONS: Our results indicated clearly that COP9 signalosome subunit CSN5a affects trichome production and the metabolism of a wide range of phenylpropanoid and carotenoid compounds. Enhanced anthocyanin accumulation and reduced trichome production were related to the enhanced MYB75 and suppressed GL2 and some other differentially expressed genes associated with the TTG1/bHLH/MYB complexes.

Transcriptome profiling of Brassica napus stem sections in relation to differences in lignin content.

BACKGROUND: Brassica crops are cultivated widely for human consumption and animal feed purposes, and oilseed rape/canola (Brassica napus and rapa) is the second most important oilseed worldwide. Because of its natural diversity and genetic complexity, genomics studies on oilseed rape will be a useful resource base to modify the quantity and quality of biomass in various crops, and therefore, should have a positive impact on lignocellulosic biofuel production. The objective of this study was to perform microarray analysis on two variable lignin containing oilseed rape cultivars to target novel genes and transcription factors of importance in Brassica lignin regulation for applied research. RESULTS: To gain insight into the molecular networks controlling cell wall biosynthetic and regulatory events, we conducted lignin and microarray analysis of top and basal stem sections of brown seeded Brassica napus DH12075 and yellow seeded YN01-429 cultivars. A total of 9500 genes were differentially expressed 2-fold or higher in the stem between the cultivars, with a higher number of expressed genes in the basal section. Of the upregulated genes, many were transcription factors and a considerable number of these were associated with secondary wall synthesis and lignification in B. napus and other plant species. The three largest groups of transcription factors with differential expression were C2H2 and C3HC4 zinc fingers and bHLH. A significant number of genes related to lignin and carbohydrate metabolism also showed differential expression patterns between the stem sections of the two cultivars. Within the same cultivar, the number of upregulated genes was higher in the top section relative to the basal one. CONCLUSION: In this study, we identified and established expression patterns of many new genes likely involved in cell wall biosynthesis and regulation. Some genes with known roles in other biochemical pathways were also identified to have a potential role in cell wall biosynthesis. This stem transcriptome profiling will allow for selecting novel regulatory and structural genes for functional characterization, a strategy which may provide tools for modifying cell wall composition to facilitate fermentation for biofuel production.

Implementation of CsLIS/NES in linalool biosynthesis involves transcript splicing regulation in Camellia sinensis.

Volatile terpenoids produced in tea plants (Camellia sinensis) are airborne signals interacting against other ecosystem members, but also pleasant odorants of tea products. Transcription regulation (including transcript processing) is pivotal for plant volatile terpenoid production. In this study, a terpene synthase gene CsLIS/NES was recovered from tea plants (C. sinensis cv. "Long-Men Xiang"). CsLIS/NES transcription regulation resulted in 2 splicing forms: CsLIS/NES-1 and CsLIS/NES-2 lacking a 305 bp-fragment at N-terminus, both producing (E)-nerolidol and linalool in vitro. Transgenic tobacco studies and a gene-specific antisense oligo-deoxynucleotide suppression applied in tea leaves indicated that CsLIS/NES-1, localized in chloroplasts, acted as linalool synthase, whereas CsLIS/NES-2 localized in cytosol, functioned as a potential nerolidol synthase, but not linalool synthase. Expression patterns of the 2 transcript isoforms in tea were distinctly different and responded differentially to the application of stress signal molecule methyl jasmonate. Leaf expression of CsLIS/NES-1, but not CsLIS/NES-2, was significantly induced by methyl jasmonate. Our data indicated that distinct transcript splicing regulation patterns, together with subcellular compartmentation of CsLIS/NE-1 and CsLIS/NE-2 implemented the linalool biosynthesis regulation in tea plants in responding to endogenous and exogenous regulatory factors.

SPL13 regulates shoot branching and flowering time in Medicago sativa.

KEY MESSAGE: Our results show SPL13 plays a crucial role in regulating vegetative and reproductive development in Medicago sativa L. (alfalfa), and that MYB112 is targeted and downregulated by SPL13 in alfalfa. We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing microRNA156 (miR156) show a bushy phenotype, reduced internodal length, delayed flowering time, and enhanced biomass yield. In alfalfa, transcripts of seven SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, including SPL13, are targeted for cleavage by miR156. Thus, association of each target SPL gene to a trait or set of traits is essential for developing molecular markers for alfalfa breeding. In this study, we investigated SPL13 function using SPL13 overexpression and silenced alfalfa plants. Severe growth retardation, distorted branches and up-curled leaves were observed in miR156-impervious 35S::SPL13m over-expression plants. In contrast, more lateral branches and delayed flowering time were observed in SPL13 silenced plants. SPL13 transcripts were predominantly present in the plant meristems, indicating that SPL13 is involved in regulating shoot branch development. Accordingly, the shoot branching-related CAROTENOID CLEAVAGE DIOXYGENASE 8 gene was found to be significantly downregulated in SPL13 RNAi silencing plants. A R2R3-MYB gene MYB112 was also identified as being directly silenced by SPL13 based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays, suggesting that MYB112 may be involved in regulating alfalfa vegetative growth.

MsmiR156 affects global gene expression and promotes root regenerative capacity and nitrogen fixation activity in alfalfa.

MicroRNA156 (miR156) regulates a network of downstream genes to affect plant growth and development. We previously generated alfalfa (Medicago sativa) plants that overexpress homologous miR156 (MsmiR156OE), and identified three of its SPL target genes. These plants exhibited increased vegetative yield, delayed flowering and longer roots. In this study, we aimed to elucidate the effect of miR156 on the root system, including effect on nodulation and nitrogen fixation. We found that MsmiR156 overexpression increases root regeneration capacity in alfalfa, but with little effect on root biomass at the early stages of root development. MsmiR156 also promotes nitrogen fixation activity by upregulating expression of nitrogenase-related genes FixK, NifA and RpoH in roots inoculated with Sinorrhizobium meliloti. Furthermore, we conducted transcriptomics analysis of MsmiR156OE alfalfa roots and identified differentially expressed genes belonging to 132 different functional categories, including plant cell wall organization, peptidyl-hypusine synthesis, and response to water stress. Expression analysis also revealed miR156 effects on genes involved in nodulation, root development and phytohormone biosynthesis. The present findings suggest that miR156 regulates root development and nitrogen fixation activity. Taken together, these findings highlight the important role that miR156 may play as a tool in the biotechnological improvement of alfalfa, and potentially other crops.

Metabolic Flux Redirection and Transcriptomic Reprogramming in the Albino Tea Cultivar 'Yu-Jin-Xiang' with an Emphasis on Catechin Production.

In this study, shade-induced conversion from a young pale/yellow leaf phenotype to a green leaf phenotype was studied using metabolic and transcriptomic profiling and the albino cultivar 'Yu-Jin-Xiang' ('YJX') of Camellia sinensis for a better understanding of mechanisms underlying the phenotype shift and the altered catechin and theanine production. Shaded leaf greening resulted from an increase in leaf chlorophyll and carotenoid abundance and chloroplast development. A total of 1,196 differentially expressed genes (DEGs) were identified between the 'YJX' pale and shaded green leaves, and these DEGs affected 'chloroplast organization' and 'response to high light' besides many other biological processes and pathways. Metabolic flux redirection and transcriptomic reprogramming were found in flavonoid and carotenoid pathways of the 'YJX' pale leaves and shaded green leaves to different extents compared to the green cultivar 'Shu-Cha-Zao'. Enhanced production of the antioxidant quercetin rather than catechin biosynthesis was correlated positively with the enhanced transcription of FLAVONOL SYNTHASE and FLAVANONE/FLAVONOL HYDROXYLASES leading to quercetin accumulation and negatively correlated to suppressed LEUCOANTHOCYANIDIN REDUCTASE, ANTHOCYANIDIN REDUCTASE and SYNTHASE leading to catechin biosynthesis. The altered levels of quercetin and catechins in 'YJX' will impact on its tea flavor and health benefits.

An Insight into microRNA156 Role in Salinity Stress Responses of Alfalfa.

Salinity is one of the major abiotic stresses affecting alfalfa productivity. Developing salinity tolerant alfalfa genotypes could contribute to sustainable crop production. The functions of microRNA156 (miR156) have been investigated in several plant species, but so far, no studies have been published that explore the role of miR156 in alfalfa response to salinity stress. In this work, we studied the role of miR156 in modulating commercially important traits of alfalfa under salinity stress. Our results revealed that overexpression of miR156 increased biomass, number of branches and time to complete growth stages, while it reduced plant height under control and salinity stress conditions. We observed a miR156-related reduction in neutral detergent fiber under non-stress, and acid detergent fiber under mild salinity stress conditions. In addition, enhanced total Kjeldahl nitrogen content was recorded in miR156 overexpressing genotypes under severe salinity stress. Furthermore, alfalfa genotypes overexpressing miR156 exhibited an altered ion homeostasis under salinity conditions. Under severe salinity stress, miR156 downregulated SPL transcription factor family genes, modified expression of other important transcription factors, and downstream salt stress responsive genes. Taken together, our results reveal that miR156 plays a role in mediating physiological and transcriptional responses of alfalfa to salinity stress.

miR156/SPL10 Modulates Lateral Root Development, Branching and Leaf Morphology in Arabidopsis by Silencing AGAMOUS-LIKE 79.

The developmental functions of miR156-SPL regulatory network have been extensively studied in Arabidopsis, but the downstream genes regulated by each SPL have not been well characterized. In this study, Next Generation Sequencing-based transcriptome analysis was performed on roots of wild type (WT) and miR156 overexpression (miR156OE) plants. One of the SPL genes, SPL10, which represses lateral root growth in Arabidopsis, was significantly downregulated in miR156OE plants. A transcription factor, AGAMOUS-like MADS box protein 79 (AGL79), was also significantly downregulated in the miR156OE plants, but was upregulated in the SPL10 overexpression (SPL10OE) Arabidopsis plants. In addition, SPL10 was found to bind to the core consensus SPL binding sequences in AGL79 gene. Moreover, analyses of complementation lines revealed a linear relationship between SPL10 and AGL79 in regulating Arabidopsis plant development. In addition, it was observed that plant phenotypes are AGL79 dose-dependent, with higher expression causing narrow leaf shape, less number of leaves and early flowering time, whereas relatively lower AGL79 overexpression produce plants with more rosette leaves and more lateral branches. Our findings revealed direct binding of SPL10 to AGL79 promoter, which further suggests a role for miR156/SPL10 module in plant lateral root growth by directly regulating AGL79.

Hairy Canola (Brasssica napus) re-visited: Down-regulating TTG1 in an AtGL3-enhanced hairy leaf background improves growth, leaf trichome coverage, and metabolite gene expression diversity.

BACKGROUND: Through evolution, some plants have developed natural resistance to insects by having hairs (trichomes) on leaves and other tissues. The hairy trait has been neglected in Brassica breeding programs, which mainly focus on disease resistance, yield, and overall crop productivity. In Arabidopsis, a network of three classes of proteins consisting of TTG1 (a WD40 repeat protein), GL3 (a bHLH factor) and GL1 (a MYB transcription factor), activates trichome initiation and patterning. Introduction of a trichome regulatory gene AtGL3 from Arabidopsis into semi-glabrous Brassica napus resulted in hairy canola plants which showed tolerance to flea beetles and diamondback moths; however plant growth was negatively affected. In addition, the role of BnTTG1 transcription in the new germplasm was not understood. RESULTS: Here, we show that two ultra-hairy lines (K-5-8 and K-6-3) with BnTTG1 knock-down in the hairy AtGL3+ B. napus background showed stable enhancement of trichome coverage, density, and length and restored wild type growth similar to growth of the semi-glabrous Westar plant. In contrast, over-expression of BnTTG1 in the hairy AtGL3+ B. napus background gave consistently glabrous plants of very low fertility and poor stability, with only one glabrous plant (O-3-7) surviving to the T3 generation. Q-PCR trichome gene expression data in leaf samples combining several leaf stages for these lines suggested that BnGL2 controlled B. napus trichome length and out-growth and that strong BnTTG1 transcription together with strong GL3 expression inhibited this process. Weak expression of BnTRY in both glabrous and trichome-bearing leaves of B. napus in the latter Q-PCR experiment suggested that TRY may have functions other than as an inhibitor of trichome initiation in the Brassicas. A role for BnTTG1 in the lateral inhibition of trichome formation in neighbouring cells was also proposed for B. napus. RNA sequencing of first leaves identified a much larger array of genes with altered expression patterns in the K-5-8 line compared to the hairy AtGL3(+) B. napus background (relative to the Westar control plant). These genes particularly included transcription factors, protein degradation and modification genes, but also included pathways that coded for anthocyanins, flavonols, terpenes, glucosinolates, alkaloids, shikimates, cell wall biosynthesis, and hormones. A 2nd Q-PCR experiment was conducted on redox, cell wall carbohydrate, lignin, and trichome genes using young first leaves, including T4 O-3-7-5 plants that had partially reverted to yield two linked growth and trichome phenotypes. Most of the trichome genes tested showed to be consistant with leaf trichome phenotypes and with RNA sequencing data in three of the lines. Two redox genes showed highest overall expression in K-5-8 leaves and lowest in O-3-7-5 leaves, while one redox gene and three cell wall genes were consistently higher in the two less robust lines compared with the two robust lines. CONCLUSION: The data support the strong impact of BnTTG1 knockdown (in the presence of strong AtGL3 expression) at restoring growth, enhancing trichome coverage and length, and enhancing expression and diversity of growth, metabolic, and anti-oxidant genes important for stress tolerance and plant health in B. napus. Our data also suggests that the combination of strong (up-regulated) BnTTG1 expression in concert with strong AtGL3 expression is unstable and lethal to the plant.

SCARECROW-LIKE15 interacts with HISTONE DEACETYLASE19 and is essential for repressing the seed maturation programme.

Epigenetic regulation of gene expression is critical for controlling embryonic properties during the embryo-to-seedling phase transition. Here we report that a histone deacetylase19 (HDA19)-associated regulator, scarecrow-like15 (SCL15), is essential for repressing the seed maturation programme in vegetative tissues. SCL15 is expressed in and GFP-tagged SCL15 predominantly localizes to, the vascular bundles particularly in the phloem companion cells and neighbouring specialized cells. Mutation of SCL15 leads to a global shift in gene expression in seedlings to a profile resembling late embryogenesis in seeds. In scl15 seedlings, many genes involved in seed maturation are markedly derepressed with concomitant accumulation of seed 12S globulin; this is correlated with elevated levels of histone acetylation at a subset of seed-specific loci. SCL15 physically interacts with HDA19 and direct targets of HDA19-SCL15 association are identified. These studies reveal that SCL15 acts as an HDA19-associated regulator to repress embryonic traits in seedlings.

Ectopic expression of miR156 represses nodulation and causes morphological and developmental changes in Lotus japonicus.

The effects of microRNA156 overexpression on general plant architecture, branching, flowering time and nodulation were investigated in the model legume, Lotus japonicus. We cloned an miR156 homolog, LjmiR156a, from L. japonicus, and investigated its SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes and its biological function at enhancing vegetative biomass yield, extending flowering time, and its impact on nodulation. Thirteen potential targets for LjmiR156 were identified in vitro and their expression profiles were determined in aerial and underground parts of mature plants, including genes coding for eight SPLs, one WD-40, one RNA-directed DNA polymerase, two transport proteins, and one histidine-phosphotransfer protein. Two SPL and one WD-40 cleavage targets for LjmiR156-TC70253, AU089191, and TC57859-were identified. Transgenic plants with ectopic expression of LjmiR156a showed enhanced branching, dramatically delayed flowering, underdeveloped roots, and reduced nodulation. We also examined the transcript levels of key genes involved in nodule organogenesis and infection thread formation to determine the role of miR156 in regulating symbiosis. Overexpression of LjmiR156a led to repression of several nodulation genes during the early stages of root development such as three ENOD genes, SymPK, POLLUX, CYCLOPS, Cerberus, and Nsp1, and the stimulation of NFR1. Our results show that miR156 regulates vegetative biomass yield, flowering time and nodulation by silencing downstream target SPLs and other genes, suggesting that the miR156 regulatory network could be modified in forage legumes (such as alfalfa and trefoils) and in leafy vegetables (like lettuce and spinach) to positively impact economically valuable crop species.

MicroRNA156 as a promising tool for alfalfa improvement.

A precursor of miR156 (MsmiR156d) was cloned and overexpressed in alfalfa (Medicago sativa L.) as a means to enhance alfalfa biomass yield. Of the five predicted SPL genes encoded by the alfalfa genome, three (SPL6, SPL12 and SPL13) contain miR156 cleavage sites and their expression was down-regulated in transgenic alfalfa plants overexpressing miR156. These transgenic plants had reduced internode length and stem thickness, enhanced shoot branching, increased trichome density, a delay in flowering time and elevated biomass production. Minor effects on sugar, starch, lignin and cellulose contents were also observed. Moreover, transgenic alfalfa plants had increased root length, while nodulation was maintained. The multitude of traits affected by miR156 may be due to the network of genes regulated by the three target SPLs. Our results show that the miR156/SPL system has strong potential as a tool to substantially improve quality and yield traits in alfalfa.

Brassica villosa, a system for studying non-glandular trichomes and genes in the Brassicas.

Brassica villosa is a wild Brassica C genome species with very dense trichome coverage and strong resistance to many insect pests of Brassica oilseeds and vegetables. Transcriptome analysis of hairy B. villosa leaves indicated higher expression of several important trichome initiation genes compared with glabrous B. napus leaves and consistent with the Arabidopsis model of trichome development. However, transcripts of the TRY inhibitory gene in hairy B. villosa were surprisingly high relative to B. napus and relative transcript levels of SAD2, EGL3, and several XIX genes were low, suggesting potential ancillary or less important trichome-related roles for these genes in Brassica species compared with Arabidopsis. Several antioxidant, calcium, non-calcium metal and secondary metabolite genes also showed differential expression between these two species. These coincided with accumulation of two alkaloid-like compounds, high levels of calcium, and other metals in B. villosa trichomes that are correlated with the known tolerance of B. villosa to high salt and the calcium-rich natural habitat of this wild species. This first time report on the isolation of large amounts of pure B. villosa trichomes, on trichome content, and on relative gene expression differences in an exceptionally hairy Brassica species compared with a glabrous species opens doors for the scientific community to understand trichome gene function in the Brassicas and highlights the potential of B. villosa as a trichome research platform.

Comparison of five major trichome regulatory genes in Brassica villosa with orthologues within the Brassicaceae.

Coding sequences for major trichome regulatory genes, including the positive regulators GLABRA 1(GL1), GLABRA 2 (GL2), ENHANCER OF GLABRA 3 (EGL3), and TRANSPARENT TESTA GLABRA 1 (TTG1) and the negative regulator TRIPTYCHON (TRY), were cloned from wild Brassica villosa, which is characterized by dense trichome coverage over most of the plant. Transcript (FPKM) levels from RNA sequencing indicated much higher expression of the GL2 and TTG1 regulatory genes in B. villosa leaves compared with expression levels of GL1 and EGL3 genes in either B. villosa or the reference genome species, glabrous B. oleracea; however, cotyledon TTG1 expression was high in both species. RNA sequencing and Q-PCR also revealed an unusual expression pattern for the negative regulators TRY and CPC, which were much more highly expressed in trichome-rich B. villosa leaves than in glabrous B. oleracea leaves and in glabrous cotyledons from both species. The B. villosa TRY expression pattern also contrasted with TRY expression patterns in two diploid Brassica species, and with the Arabidopsis model for expression of negative regulators of trichome development. Further unique sequence polymorphisms, protein characteristics, and gene evolution studies highlighted specific amino acids in GL1 and GL2 coding sequences that distinguished glabrous species from hairy species and several variants that were specific for each B. villosa gene. Positive selection was observed for GL1 between hairy and non-hairy plants, and as expected the origin of the four expressed positive trichome regulatory genes in B. villosa was predicted to be from B. oleracea. In particular the unpredicted expression patterns for TRY and CPC in B. villosa suggest additional characterization is needed to determine the function of the expanded families of trichome regulatory genes in more complex polyploid species within the Brassicaceae.

Differential metabolite profiles and salinity tolerance between two genetically related brown-seeded and yellow-seeded Brassica carinata lines.

Brassica carinata (Ethiopian mustard) has previously been identified as a potential crop species suitable for marginal land in the North American prairies due to its relatively high salt tolerance. Two genetically related B. carinata lines with brown-seeded (BS) and yellow-seeded (YS) phenotypes were assessed for their tolerance to sodium sulfate. Specifically, each line was greenhouse-grown under 0, 50 and 100mM of salt, and analyzed after four weeks and eight weeks of treatment. Generally, the height of the BS line was greater than the YS line under both salt treatments, indicating enhanced salt tolerance of the BS line. NMR-based metabolite profiling and PCA analyses indicated a more pronounced shift in key stem metabolites after four weeks of treatment with the YS line compared to the BS line. For example, tryptophan and formate levels increased in the YS line after four weeks of 100mM salt treatment, while proline and threonine levels varied uniquely compared to other metabolites of the lines. Together, the data indicate that the brown-seeded line has greater sodium tolerance than the yellow-seed line, provide clues to the biochemical underpinnings for the phenotypic variation, and highlight the utility of B. carinata as a biorefinery crop for saline land.