Intragenic complementation at the Lotus japonicus CELLULOSE SYNTHASE-LIKE D1 locus rescues root hair defects.

Root hair cells form the primary interface of plants with the soil environment, playing key roles in nutrient uptake and plant defense. In legumes, they are typically the first cells to become infected by nitrogen-fixing soil bacteria during root nodule symbiosis. Here, we report a role for the CELLULOSE SYNTHASE-LIKE D1 (CSLD1) gene in root hair development in the legume species Lotus japonicus. CSLD1 belongs to the cellulose synthase protein family that includes cellulose synthases and cellulose synthase-like proteins, the latter thought to be involved in the biosynthesis of hemicellulose. We describe 11 Ljcsld1 mutant alleles that impose either short (Ljcsld1-1) or variable (Ljcsld1-2 to 11) root hair length phenotypes. Examination of Ljcsld1-1 and one variable-length root hair mutant, Ljcsld1-6, revealed increased root hair cell wall thickness, which in Ljcsld1-1 was significantly more pronounced and also associated with a strong defect in root nodule symbiosis. Lotus japonicus plants heterozygous for Ljcsld1-1 exhibited intermediate root hair lengths, suggesting incomplete dominance. Intragenic complementation was observed between alleles with mutations in different CSLD1 domains, suggesting CSLD1 function is modular and that the protein may operate as a homodimer or multimer during root hair development.

Involvement of the miR156/SPL module in flooding response in Medicago sativa.

The highly conserved plant microRNA, miR156, affects plant development, metabolite composition, and stress response. Our previous research revealed the role of miR156 in abiotic stress response in Medicago sativa exerted by downregulating SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE transcription factors. Here we investigated the involvement and possible mechanism of action of the miR156/SPL module in flooding tolerance in alfalfa. For that, we used miR156 overexpressing, SPL13RNAi, flood-tolerant (AAC-Trueman) and -sensitive (AC-Caribou) alfalfa cultivars exposed to flooding. We also used Arabidopsis ABA insensitive (abi1-2, abi5-8) mutants and transgenic lines with either overexpressed (KIN10-OX1, KIN10-OX2) or silenced (KIN10RNAi-1, KIN10RNAi-2) catalytic subunit of SnRK1 to investigate a possible role of ABA and SnRK1 in regulating miR156 expression under flooding. Physiological analysis, hormone profiling and global transcriptome changes revealed a role for miR156/SPL module in flooding tolerance. We also identified nine novel alfalfa SPLs (SPL1, SPL1a, SPL2a, SPL7, SPL7a, SPL8, SPL13a, SPL14, SPL16) responsive to flooding. Our results also showed a possible ABA-dependent SnRK1 upregulation to enhance miR156 expression, resulting in downregulation of SPL4, SPL7a, SPL8, SPL9, SPL13, and SPL13a. We conclude that these effects induce flooding adaptive responses in alfalfa and modulate stress physiology by affecting the transcriptome, ABA metabolites and secondary metabolism.

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.

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.

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.

MicroRNA156 improves drought stress tolerance in alfalfa (Medicago sativa) by silencing SPL13.

Alfalfa (Medicago sativa) is an important forage crop that is often grown in areas that frequently experience drought and water shortage. MicroRNA156 (miR156) is an emerging tool for improving various traits in plants. We tested the role of miR156d in drought response of alfalfa, and observed a significant improvement in drought tolerance of miR156 overexpression (miR156OE) alfalfa genotypes compared to the wild type control (WT). In addition to higher survival and reduced water loss, miR156OE genotypes also maintained higher stomatal conductance compared to WT during drought stress. Furthermore, we observed an enhanced accumulation of compatible solute (proline) and increased levels of abscisic acid (ABA) and antioxidants in miR156OE genotypes. Similarly, alfalfa plants with reduced expression of miR156-targeted SPL13 showed reduced water loss and enhanced stomatal conductance, chlorophyll content and photosynthetic assimilation. Several genes known to be involved in drought tolerance were differentially expressed in leaf and root of miR156 overexpression plants. Taken together, our findings reveal that miR156 improves drought tolerance in alfalfa at least partially by silencing SPL13.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 2 controls floral organ development and plant fertility by activating ASYMMETRIC LEAVES 2 in Arabidopsis thaliana.

A network of genes is coordinately expressed to ensure proper development of floral organs and fruits, which are essential for generating new offspring in flowering plants. In Arabidopsis thaliana, microRNA156 (miR156) plays a role in regulating the development of flowers and siliques by targeting members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family. Despite the important roles of the miR156/SPL network, our understanding of its downstream genes that are involved in floral organ and silique growth is still incomplete. Here, we report that the miR156/SPL2 regulatory pathway regulates pollen production, fertility rate, and the elongation of floral organs, including petals, sepals, and siliques in Arabidopsis. Transgenic plants exhibiting both overexpression of miR156 and dominant-negative alleles of SPL2 had reduced ASYMMETRIC LEAVES 2 (AS2) transcript levels in their siliques. Furthermore, their fertility phenotype was similar to that of the AS2 loss-of-function mutant. We also demonstrate that the SPL2 protein binds to the 5'UTR of the AS2 gene in vivo, indicating that AS2 is directly regulated by SPL2. Our results suggest that the miR156/SPL2 pathway affects floral organs, silique development and plant fertility, as well as directly regulates AS2 expression.

Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa.

BACKGROUND: Medicago sativa (alfalfa) is a low-input forage and potential bioenergy crop, and improving its yield and quality has always been a focus of the alfalfa breeding industry. Transgenic alfalfa plants overexpressing a precursor of alfalfa microRNA156 (MsmiR156) were recently generated by our group. These plants (miR156OE) showed enhanced biomass yield, reduced internodal length, increased shoot branching and trichome density, and a delay in flowering time. Transcripts of three SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes (MsSPL6, MsSPL12, and MsSPL13) were found to be targeted for cleavage by MsmiR156 in alfalfa. RESULTS: To further illustrate the molecular mechanisms underlying the effects of miR156 in alfalfa, two miR156OE genotypes (A11a and A17) were subjected to Next Generation RNA Sequencing with Illumina HiSeq. More than 1.11 billion clean reads were obtained from our available sequenced samples. A total of 160,472 transcripts were generated using Trinity de novo assembly and 4,985 significantly differentially expressed genes were detected in miR156OE plants A11a and A17 using the Medicago truncatula genome as reference. A total of 17 genes (including upregulated, downregulated, and unchanged) were selected for quantitative real-time PCR (qRT-PCR) validation, which showed that gene expression levels were largely consistent between qRT-PCR and RNA-Seq data. In addition to the established SPL genes MsSPL6, MsSPL12 and MsSPL13, four new SPLs; MsSPL2, MsSPL3, MsSPL4 and MsSPL9 were also down-regulated significantly in both miR156OE plants. These seven SPL genes belong to genes phylogeny clades VI, IV, VIII, V and VII, which have been reported to be targeted by miR156 in Arabidopsis thaliana. The gene ontology terms characterized electron transporter, starch synthase activity, sucrose transport, sucrose-phosphate synthase activity, chitin binding, sexual reproduction, flavonoid biosynthesis and lignin catabolism correlate well to the phenotypes of miR156OE alfalfa plants. CONCLUSIONS: This is the first report of changes in global gene expression in response to miR156 overexpression in alfalfa. The discovered miR156-targeted SPL genes belonging to different clades indicate miR156 plays fundamental and multifunctional roles in regulating alfalfa plant development.

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.

The translation elongation factor eEF-1Bß1 is involved in cell wall biosynthesis and plant development in Arabidopsis thaliana.

The eukaryotic translation elongation factor eEF-1Bß1 (EF1Bß) is a guanine nucleotide exchange factor that plays an important role in translation elongation. In this study, we show that the EF1Bß protein is localized in the plasma membrane and cytoplasm, and that the transcripts should be expressed in most tissue types in seedlings. Sectioning of the inflorescence stem revealed that EF1Bß predominantly localizes to the xylem vessels and in the interfascicular cambium. EF1Bß gene silencing in efß caused a dwarf phenotype with 38% and 20% reduction in total lignin and crystalline cellulose, respectively. This loss-of-function mutant also had a lower S/G lignin monomer ratio relative to wild type plants, but no changes were detected in a gain-of-function mutant transformed with the EF1Bß gene. Histochemical analysis showed a reduced vascular apparatus, including smaller xylem vessels in the inflorescence stem of the loss-of-function mutant. Over-expression of EF1Bß in an eli1 mutant background restored a WT phenotype and abolished ectopic lignin deposition as well as cell expansion defects in the mutant. Taken together, these data strongly suggest a role for EF1Bß in plant development and cell wall formation in Arabidopsis.

DIMINUTO 1 affects the lignin profile and secondary cell wall formation in Arabidopsis.

Brassinosteroids (BRs) play a crucial role in plant growth and development and DIMINUTO 1 (DIM1), a protein involved in BR biosynthesis, was previously identified as a cell elongation factor in Arabidopsis thaliana. Through promoter expression analysis, we showed that DIM1 was expressed in most of the tissue types in seedlings and sectioning of the inflorescence stem revealed that DIM1 predominantly localizes to the xylem vessels and in the interfascicular cambium. To investigate the role of DIM1 in cell wall formation, we generated loss-of-function and gain-of-function mutants. Disruption of the gene function caused a dwarf phenotype with up to 38 and 23% reductions in total lignin and cellulose, respectively. Metabolite analysis revealed a significant reduction in the levels of fructose, glucose and sucrose in the loss-of-function mutant compared to the wild type control. The loss-of-function mutant also had a lower S/G lignin monomer ratio relative to wild type, but no changes were detected in the gain-of-function mutant. Phloroglucinol and toluidine blue staining showed a size reduction of the vascular apparatus with smaller and disintegrated xylem vessels in the inflorescence stem of the loss-of-function mutant. Taken together, these data indicate a role for DIM1 in secondary cell wall formation. Moreover, this study demonstrated the potential role of BR hormones in modulating cell wall structure and composition.

Conservation of lotus and Arabidopsis basic helix-loop-helix proteins reveals new players in root hair development.

Basic helix-loop-helix (bHLH) proteins constitute a large family of transcriptional regulators in plants. Although they have been shown to play important roles in a wide variety of developmental processes, relatively few have been functionally characterized. Here, we describe the map-based cloning of the Lotus japonicus ROOTHAIRLESS1 (LjRHL1) locus. Deleterious mutations in this locus prevent root hair development, which also aborts root hair-dependent colonization of the host root by nitrogen-fixing bacteria. We show that the LjRHL1 gene encodes a presumed bHLH transcription factor that functions in a nonredundant manner to control root hair development in L. japonicus. Homology search and cross-species complementation experiments defined three members of the Arabidopsis (Arabidopsis thaliana) bHLH protein family, At2g24260, At4g30980, and At5g58010, as functionally equivalent to LjRHL1. Curiously, At2g24260 and At4g30980 mRNA species accumulate independently from the known positive regulators of root hair cell fate, while all three genes act in a partially redundant manner to regulate root hair development in Arabidopsis.

A cytokinin perception mutant colonized by Rhizobium in the absence of nodule organogenesis.

In legumes, Nod-factor signaling by rhizobia initiates the development of the nitrogen-fixing nodule symbiosis, but the direct cell division stimulus that brings about nodule primordia inception in the root cortex remains obscure. We showed that Lotus japonicus plants homozygous for a mutation in the HYPERINFECTED 1 (HIT1) locus exhibit abundant infection-thread formation but fail to initiate timely cortical cell divisions in response to rhizobial signaling. We demonstrated that the corresponding gene encodes a cytokinin receptor that is required for the activation of the nodule inception regulator Nin and nodule organogenesis.