Ji-Yi Zhang

Legume transcription factors: global regulators of plant development and response to the environment.

Transcription factors (TFs) are DNA-binding proteins that interact with other transcriptional regulators, including chromatin remodeling/modifying proteins, to recruit or block access of RNA polymerases to the DNA template. Plant genomes devote approximately 7% of their coding sequence to TFs, which

Overexpression of miR156 in switchgrass (Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production

Switchgrass (Panicum virgatum L.) has been developed into a dedicated herbaceous bioenergy crop. Biomass yield is a major target trait for genetic improvement of switchgrass. microRNAs have emerged as a prominent class of gene regulatory factors that has the potential to improve complex traits such as biomass yield. A miR156b precursor was overexpressed in switchgrass. The effects of miR156 overexpression on SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes were revealed by microarray and quantitative RT-PCR analyses. Morphological alterations, biomass yield, saccharification efficiency and forage digestibility of the transgenic plants were characterized. miR156 controls apical dominance and floral transition in switchgrass by suppressing its target SPL genes. Relatively low levels of miR156 overexpression were sufficient to increase biomass yield while producing plants with normal flowering time. Moderate levels of miR156 led to improved biomass but the plants were non-flowering. These two groups of plants produced 58%-101% more biomass yield compared with the control. However, high miR156 levels resulted in severely stunted growth. The degree of morphological alterations of the transgenic switchgrass depends on miR156 level. Compared with floral transition, a lower miR156 level is required to disrupt apical dominance. The improvement in biomass yield was mainly because of the increase in tiller number. Targeted overexpression of miR156 also improved solubilized sugar yield and forage digestibility, and offered an effective approach for transgene containment.

Heterologous expression of two Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response in freezing tolerance

Cuticular waxes are the major components of plant cuticle and play an important role in protecting aerial organs from damage caused by biotic and abiotic stresses. Here we report the functional characterization of two putative ERF transcription factor genes WXP1 and its paralog WXP2 from Medicago truncatula. Transgenic expression of WXP1 and WXP2 in Arabidopsis (ecotype Columbia) led to significantly increased cuticular wax deposition on leaves of 4-week-old and 6-week-old transgenic plants, assessed based on fresh weight or based on surface area. Differences in the accumulation of various wax components as well as their chain length distributions were found in the WXP1 and WXP2 plants. The major wax component in Arabidopsis, n-alkanes, increased substantially in both WXP1 and WXP2 transgenics, however, another wax component, primary alcohols, increased in WXP1 plants but decreased in WXP2 plants. Cuticle properties of the transgenic leaves were analyzed by chlorophyll leaching assay; while the WXP1 plants had no change, the WXP2 plants showed more chlorophyll leaching. Analysis of fresh weight loss from detached leaves revealed that the transgenic leaves tend to retain more water than the control. Both WXP1 and WXP2 transgenic plants showed significantly enhanced whole plant drought tolerance. Analysis of freezing tolerance at the whole plant level and measurement of electrolyte leakage from detached leaves revealed that the WXP1 plants had increased freezing tolerance while the WXP2 plants were more sensitive to low temperature when compared to the control. Transgenic expression of WXP1 had no obvious effects on plant growth and development, however, the expression of WXP2 led to slower plant growth. These results indicate that WXP1 is a useful candidate gene for improving plant drought and freezing tolerance by genetic transformation.

Global reprogramming of transcription and metabolism in Medicago truncatula during progressive drought and after rewatering

Medicago truncatula is a model legume forage crop native to the arid and semi-arid environments of the Mediterranean. Given its drought-adapted nature, it is an ideal candidate to study the molecular and biochemical mechanisms conferring drought resistance in plants. Medicago plants were subjected to a progressive drought stress over 14 d of water withholding followed by rewatering under controlled environmental conditions. Based on physiological measurements of plant water status and changes in morphology, plants experienced mild, moderate and severe water stress before rehydration. Transcriptome analysis of roots and shoots from control, mildly, moderately and severely stressed, and rewatered plants, identified many thousands of genes that were altered in expression in response to drought. Many genes with expression tightly coupled to the plant water potential (i.e. drought intensity) were identified suggesting an involvement in Medicago drought adaptation responses. Metabolite profiling of drought-stressed plants revealed the presence of 135 polar and 165 non-polar compounds in roots and shoots. Combining Medicago metabolomic data with transcriptomic data yielded insight into the regulation of metabolic pathways operating under drought stress. Among the metabolites detected in drought-stressed Medicago plants, myo-inositol and proline had striking regulatory profiles indicating involvement in Medicago drought tolerance. Global transcriptional and metabolic responses to drought and rewatering were investigated in Medicago truncatula, a naturally drought-adapted model legume species. Integration of metabolomic and transcriptomic data yielded insights into the regulation of metabolic pathways underlying drought-stress adaptation. Many genes and metabolites with expression tightly coupled to drought intensity were identified, suggesting active involvement in Medicago drought resistance. These could prove useful targets for future translational approaches to improve closely related crop plants such as common bean, soybean and pea.

Identification and overexpression of gibberellin 2‐oxidase (GA2ox) in switchgrass (Panicum virgatum L.) for improved plant architecture and reduced biomass recalcitrance

Gibberellin 2-oxidases (GA2oxs) are a group of 2-oxoglutarate-dependent dioxygenases that catalyse the deactivation of bioactive GA or its precursors through 2β-hydroxylation reaction. In this study, putatively novel switchgrass C20 GA2ox genes were identified with the aim of genetically engineering switchgrass for improved architecture and reduced biomass recalcitrance for biofuel. Three C20 GA2ox genes showed differential regulation patterns among tissues including roots, seedlings and reproductive parts. Using a transgenic approach, we showed that overexpression of two C20 GA2ox genes, that is PvGA2ox5 and PvGA2ox9, resulted in characteristic GA-deficient phenotypes with dark-green leaves and modified plant architecture. The changes in plant morphology appeared to be associated with GA2ox transcript abundance. Exogenous application of GA rescued the GA-deficient phenotypes in transgenic lines. Transgenic semi-dwarf lines displayed increased tillering and reduced lignin content, and the syringyl/guaiacyl lignin monomer ratio accompanied by the reduced expression of lignin biosynthetic genes compared to nontransgenic plants. A moderate increase in the level of glucose release in these transgenic lines might be attributed to reduced biomass recalcitrance as a result of reduced lignin content and lignin composition. Our results suggest that overexpression of GA2ox genes in switchgrass is a feasible strategy to improve plant architecture and reduce biomass recalcitrance for biofuel.

Overexpression of AtLOV1 in Switchgrass Alters Plant Architecture, Lignin Content, and Flowering Time

Background Switchgrass (Panicum virgatum L.) is a prime candidate crop for biofuel feedstock production in the United States. As it is a self-incompatible polyploid perennial species, breeding elite and stable switchgrass cultivars with traditional breeding methods is very challenging. Translational genomics may contribute significantly to the genetic improvement of switchgrass, especially for the incorporation of elite traits that are absent in natural switchgrass populations. Methodology/Principal Findings In this study, we constitutively expressed an Arabidopsis NAC transcriptional factor gene, LONG VEGETATIVE PHASE ONE (AtLOV1), in switchgrass. Overexpression of AtLOV1 in switchgrass caused the plants to have a smaller leaf angle by changing the morphology and organization of epidermal cells in the leaf collar region. Also, overexpression of AtLOV1 altered the lignin content and the monolignol composition of cell walls, and caused delayed flowering time. Global gene-expression analysis of the transgenic plants revealed an array of responding genes with predicted functions in plant development, cell wall biosynthesis, and flowering. Conclusions/Significance To our knowledge, this is the first report of a single ectopically expressed transcription factor altering the leaf angle, cell wall composition, and flowering time of switchgrass, therefore demonstrating the potential advantage of translational genomics for the genetic improvement of this crop.

Analysis of tall fescue ESTs representing different abiotic stresses, tissue types and developmental stages

BackgroundTall fescue (Festuca arundinacea Schreb) is a major cool season forage and turf grass species grown in the temperate regions of the world. In this paper we report the generation of a tall fescue expressed sequence tag (EST) database developed from nine cDNA libraries representing tissues from different plant organs, developmental stages, and abiotic stress factors. The results of inter-library and library-specific in silico expression analyses of these ESTs are also reported.ResultsA total of 41,516 ESTs were generated from nine cDNA libraries of tall fescue representing tissues from different plant organs, developmental stages, and abiotic stress conditions. The Festuca Gene Index (FaGI) has been established. To date, this represents the first publicly available tall fescue EST database. In silico gene expression studies using these ESTs were performed to understand stress responses in tall fescue. A large number of ESTs of known stress response gene were identified from stressed tissue libraries. These ESTs represent gene homologues of heat-shock and oxidative stress proteins, and various transcription factor protein families. Highly expressed ESTs representing genes of unknown functions were also identified in the stressed tissue libraries.ConclusionFaGI provides a useful resource for genomics studies of tall fescue and other closely related forage and turf grass species. Comparative genomic analyses between tall fescue and other grass species, including ryegrasses (Lolium sp.), meadow fescue (F. pratensis) and tetraploid fescue (F. arundinacea var glaucescens) will benefit from this database. These ESTs are an excellent resource for the development of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) PCR-based molecular markers.

Improvement of drought tolerance in white clover (Trifolium repens) by transgenic expression of a transcription factor gene WXP1

White clover (Trifolium repens L.) is an important pasture legume in many regions of the world. A commercial cultivar of white clover (cv. Patriot) was transformed with a Medicago truncatula L. transcription factor gene, WXP1, and a reporter gene, β-glucuronidase (GUS). The WXP1 gene and the GUS gene were placed under control of the Arabidopsis CER6 promoter. GUS staining and cross-section analysis revealed the CER6 promoter directed constitutive expression in leaves and epidermis preferential expression in petioles of white clover. Independent transgenic WXP1 lines, empty vector and wild-type controls were subjected to drought stress treatment. The plants were characterised by measuring several physiological parameters including gas exchange, chlorophyll fluorescence, relative water content and leaf water potential. The WXP1 transgenic lines had higher net photosynthetic rates, higher efficiency of PSII, higher relative water content and leaf water potential under drought-stressed conditions. Consistent with the results from physiological analyses, the transgenic white clover plants carrying WXP1 showed improved tolerance to drought stress.

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Global gene expression profiling of two switchgrass cultivars following inoculation with Burkholderia phytofirmans strain PsJN

Improvement and year-to-year stabilization of biomass yields are primary objectives for the development of a low-input switchgrass feedstock production system using microbial endophytes. An earlier investigation of the effect of Burkholderia phytofirmans strain PsJN on switchgrass germplasm demonstrated differential responses between genotypes. PsJN inoculation of cv. Alamo (lowland ecotype) increased the plant root system, shoot length, and biomass yields, whereas it had no beneficial effect on cv. Cave-in-Rock (upland ecotype). To understand the gene networks governing plant growth promotion responses triggered by PsJN, the gene expression profiles were analysed in these two hosts, following seedling inoculation. The Affymetrix platform switchgrass expressed sequence tag (EST) microarray chip representing 122 972 probe sets, developed by the DOE BioEnergy Science Center, was employed to assess transcript abundance at 0.5, 2, 4, and 8 DAI (days after PsJN inoculation). Approximately 20 000 switchgrass probe sets showed significant responses in either cultivar. Switchgrass identifiers were used to map 19 421 genes in MapMan software. There were apparent differences in gene expression profiling between responsive and non-responsive cultivars after PsJN inoculation. Overall, there were 14 984 and 9691 genes affected by PsJN inoculation in Alamo and Cave-in-Rock, respectively. Of these, 394 are annotated as pathogenesis-related genes. In the responsive cv. Alamo, 68 pathogenesis-related genes were affected, compared with only 10 in the non-responsive cv. Cave-in-Rock. At the very early stage at 0.5 DAI, both cultivars exhibited similar recognition and defence responses, such as genes in signalling and proteolysis, after which the defence reaction in the responsive cv. Alamo became weaker while it was sustained in non-responsive cv. Cave-in-Rock.