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"Wang, Zeng-Yu"
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Simultaneous regulation of F5H in COMT‐RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis
Summary Ferulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.
Journal Article
From model to crop: functional characterization of SPL8 in M. truncatula led to genetic improvement of biomass yield and abiotic stress tolerance in alfalfa
Summary Biomass yield, salt tolerance and drought tolerance are important targets for alfalfa (Medicago sativa L.) improvement. Medicago truncatula has been developed into a model plant for alfalfa and other legumes. By screening a Tnt1 retrotransposon‐tagged M. truncatula mutant population, we identified three mutants with enhanced branching. Branch development determines shoot architecture which affects important plant functions such as light acquisition, resource use and ultimately impacts biomass production. Molecular analyses revealed that the mutations were caused by Tnt1 insertions in the SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE 8 (SPL8) gene. The M. truncatula spl8 mutants had increased biomass yield, while overexpression of SPL8 in M. truncatula suppressed branching and reduced biomass yield. Scanning electron microscopy (SEM) analysis showed that SPL8 inhibited branching by directly suppressing axillary bud formation. Based on the M. truncatula SPL8 sequence, alfalfa SPL8 (MsSPL8) was cloned and transgenic alfalfa plants were produced. MsSPL8 down‐regulated or up‐regulated alfalfa plants exhibited similar phenotypes to the M. truncatula mutants or overexpression lines, respectively. Specifically, the MsSPL8 down‐regulated alfalfa plants showed up to 43% increase in biomass yield in the first harvest. The impact was even more prominent in the second harvest, with up to 86% increase in biomass production compared to the control. Furthermore, down‐regulation of MsSPL8 led to enhanced salt and drought tolerance in transgenic alfalfa. Results from this research offer a valuable approach to simultaneously improve biomass production and abiotic stress tolerance in legumes.
Journal Article
SPL7 and SPL8 represent a novel flowering regulation mechanism in switchgrass
The aging pathway in flowering regulation is controlled mainly by microRNA156 (miR156). Studies in Arabidopsis thaliana reveal that nine miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL) genes are involved in the control of flowering. However, the roles of SPLs in flowering remain elusive in grasses.
Inflorescence development in switchgrass was characterized using scanning electron microscopy (SEM). Microarray, quantitative reverse transcription polymerase chain reaction (qRT-PCR), chromatin immunoprecipitation (ChIP)-PCR and EMSA were used to identify regulators of phase transition and flowering. Gene function was characterized by downregulation and overexpression of the target genes.
Overexpression of SPL7 and SPL8 promotes flowering, whereas downregulation of individual genes moderately delays flowering. Simultaneous downregulation of SPL7/SPL8 results in extremely delayed or nonflowering plants. Furthermore, downregulation of both genes leads to a vegetative-to-reproductive reversion in the inflorescence, a phenomenon that has not been reported in any other grasses.
Detailed analyses demonstrate that SPL7 and SPL8 induce phase transition and flowering in grasses by directly upregulating SEPALLATA3 (SEP3) and MADS32. Thus, the SPL7/8 pathway represents a novel regulatory mechanism in grasses that is largely different from that in Arabidopsis. Additionally, genetic modification of SPL7 and SPL8 results in much taller plants with significantly increased biomass yield and sugar release.
Journal Article
RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis
Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9) offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF) activation domain to dCas9 bound with the VP64 (tetramer of VP16) activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1) and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1). The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.
Journal Article
Effects of autotoxicity and allelopathy on seed germination and seedling growth in Medicago truncatula
Autotoxicity is a form of intraspecific allelopathy, in which a plant species inhibits the establishment or growth of the same species through the release of toxic chemical compounds into the environment. The phenomenon of autotoxicity in crops is best traced in alfalfa ( Medicago sativa ). A close relative of alfalfa, M. truncatula, has been developed into an excellent model species for leguminous plants. However, it is not known whether M. truncatula has autotoxicity. In this study, M. truncatula root exudates showed a negative impact on the growth of M. truncatula seedlings, indicating autotoxicity. Detailed analyses with plant extracts from M. truncatula and alfalfa revealed varying degrees of suppression effects in the two species. The extracts negatively affected seed germination potential, germination rate, radicle length, hypocotyl length, synthetic allelopathic effect index, plant height, root growth, fresh weight, dry weight, net photosynthetic rate, transpiration rate, and stomatal conductance in both M. truncatula and alfalfa. The results demonstrated that autotoxicity and allelopathic effects exist in M. truncatula . This opens up a new way to use M. truncatula as a model species to carry out in-depth studies of autotoxicity and allelopathy to elucidate biochemical pathways of allelochemicals and molecular networks controlling biosynthesis of the chemicals.
Journal Article
The miR156-SPL4 module predominantly regulates aerial axillary bud formation and controls shoot architecture
Grasses possess basal and aerial axillary buds. Previous studies have largely focused on basal bud (tiller) formation but scarcely touched on aerial buds, which may lead to aerial branch development.
Genotypes with and without aerial buds were identified in switchgrass (Panicum virgatum), a dedicated bioenergy crop. Bud development was characterized using scanning electron microscopy. Microarray, RNA-seq and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to identify regulators of bud formation. Gene function was characterized by down-regulation and overexpression.
Overexpression of miR156 induced aerial bud formation in switchgrass. Various analyses revealed that SQUAMOSA PROMOTER BINDING PROTEIN LIKE4 (SPL4), one of the miR156 targets, directly regulated aerial axillary bud initiation. Down-regulation of SPL4 promoted aerial bud formation and increased basal buds, while overexpression of SPL4 seriously suppressed bud formation and tillering. RNA-seq and RT-qPCR identified potential downstream genes of SPL4.
Unlike all previously reported genes acting as activators of basal bud initiation, SPL4 acts as a suppressor for the formation of both aerial and basal buds. The miR156-SPL4 module predominantly regulates aerial bud initiation and partially controls basal bud formation. Genetic manipulation of SPL4 led to altered plant architecture with increased branching, enhanced regrowth after cutting and improved biomass yield.
Journal Article
Genome-Wide Identification and Characterization of the Msr Gene Family in Alfalfa under Abiotic Stress
Alfalfa (Medicago sativa) is an important leguminous forage, known as the “The Queen of Forages”. Abiotic stress seriously limits the growth and development of alfalfa, and improving the yield and quality has become an important research area. However, little is known about the Msr (methionine sulfoxide reductase) gene family in alfalfa. In this study, 15 Msr genes were identified through examining the genome of the alfalfa “Xinjiang DaYe”. The MsMsr genes differ in gene structure and conserved protein motifs. Many cis-acting regulatory elements related to the stress response were found in the promoter regions of these genes. In addition, a transcriptional analysis and qRT-PCR (quantitative reverse transcription PCR) showed that MsMsr genes show expression changes in response to abiotic stress in various tissues. Overall, our results suggest that MsMsr genes play an important role in the response to abiotic stress for alfalfa.
Journal Article
Comparative Transcriptome Analysis of Salt Stress-Induced Leaf Senescence in Medicago truncatula
Leaves are the most critical portion of forage crops such as alfalfa ( Medicago sativa ). Leaf senescence caused by environmental stresses significantly impacts the biomass and quality of forages. To understand the molecular mechanisms and identify the key regulator of the salt stress-induced leaf senescence process, we conducted a simple and effective salt stress-induced leaf senescence assay in Medicago truncatula , which was followed by RNA-Seq analysis coupled with physiological and biochemical characterization. By comparing the observed expression data with that derived from dark-induced leaf senescence at different time points, we identified 3,001, 3,787, and 4,419 senescence-associated genes (SAGs) for salt stress-induced leaf senescence on day 2, 4, and 6, respectively. There were 1546 SAGs shared by dark and salt stress treatment across the three time points. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that the 1546 SAGs were mainly related to protein and amino acids metabolism, photosynthesis, chlorophyll metabolism, and hormone signaling during leaf senescence. Strikingly, many different transcription factors (TFs) families out of the 1546 SAGs, including NAC, bHLH, MYB , and ERF , were associated with salt stress-induced leaf senescence processes. Using the transient expression system in Nicotiana benthamiana , we verified that three functional NAC TF genes from the 1546 SAGs were related to leaf senescence. These results clarify SAGs under salt stress in M. truncatula and provide new insights and additional genetic resources for further forage crop breeding.
Journal Article
Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa)
Alfalfa (
) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the
stay-green (
) gene. The replacement of CaMV35S promoter by the
ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.
Journal Article