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S -adenosyl- l -methionine (SAM) is the methyl donor involved in the biosynthesis of guaiacyl (G) and syringyl (S) lignins in vascular plants. SAM is synthesized from methionine through the catalysis of the enzyme S -adenosylmethionine synthase (SAMS). However, the detailed function of SAMS in lignin biosynthesis has not been widely investigated in plants, particularly in monocot species. In this study, we identified PvSAMS genes from switchgrass (Panicum virgatum L.), an important dual-purpose fodder and biofuel crop, and generated numerous transgenic switchgrass lines through PvSAMS RNA interference technology. Down-regulation of PvSAMS reduced the contents of SAM, G-lignins, and S-lignins in the transgenic switchgrass. The methionine and glucoside derivatives of caffeoyl alcohol were found to accumulate in the transgenic plants. Moreover, down-regulation of PvSAMS in switchgrass resulted in brownish stems associated with reduced lignin content and improved cell wall digestibility. Furthermore, transcriptomic analysis revealed that most sulfur deficiency-responsive genes were differentially expressed in the transgenic switchgrass, leading to a significant increase in total sulfur content; thus implying an important role of SAMS in the methionine cycle, lignin biosynthesis, and sulfur assimilation. Taken together, our results suggest that SAMS is a valuable target in lignin manipulation, and that manipulation of PvSAMS can simultaneously regulate the biosynthesis of SAM and methylated monolignols in switchgrass. [ABSTRACT FROM AUTHOR]

Switchgrass (Panicum virgatum L.) is an important perennial, noninvasive, tall ornamental grass that adds color and texture to gardens and landscapes. Moreover, switchgrass has been considered a forage and bioenergy crop because of its vigorous growth, low-input requirements, and broad geography. Here, we identified PvWOX3a from switchgrass, which encodes a WUSCHEL-related homeobox transcription factor. Transgenic overexpression of PvWOX3a in switchgrass increased stem length, internode diameter, and leaf blade length and width, all of which contributed to a 95% average increase in dry weight biomass compared with control plants. Yeast one-hybrid and transient dual-luciferase assays showed that PvWOX3a can repress the expression of gibberellin 2-oxidase and cytokinin oxidase/dehydrogenase through apparently direct interaction with their promoter sequences. These results suggested that overexpression of PvWOX3a could increase gibberellin and cytokinin levels in transgenic switchgrass plants, which promotes cell division, elongation, and vascular bundle development. We also overexpressed PvWOX3a in a transgenic miR156-overexpressing switchgrass line that characteristically exhibited more tillers, thinner internodes, and narrower leaf blades. Double transgenic switchgrass plants displayed significant increases in internode length and diameter, leaf blade width, and plant height but retained a tiller number comparable to that of plants expressing miR156 alone. Ultimately, the double transgenic switchgrass plants produced 174% more dry-weight biomass and 162% more solubilized sugars on average than control plants. These findings indicated that PvWOX3a is a viable potential genetic target for engineering improved shoot architecture and biomass yield of horticulture, fodder, and biofuel crops. [ABSTRACT FROM AUTHOR]

Medicago polymorpha is a nutritious and palatable forage and vegetable plant that also fixes nitrogen. Here, we reveal the chromosome-scale genome sequence of M. polymorpha using an integrated approach including Illumina, PacBio and Hi-C technologies. We combined PacBio full-length RNA-seq, metabolomic analysis, structural anatomy analysis and related physiological indexes to elucidate the important agronomic traits of M. polymorpha for forage and vegetable usage. The assembled M. polymorpha genome consisted of 457.53 Mb with a long scaffold N50 of 57.72 Mb, and 92.92% (441.83 Mb) of the assembly was assigned to seven pseudochromosomes. Comparative genomic analysis revealed that expansion and contraction of the photosynthesis and lignin biosynthetic gene families, respectively, led to enhancement of nutritious compounds and reduced lignin biosynthesis in M. polymorpha. In addition, we found that several positively selected nitrogen metabolism-related genes were responsible for crude protein biosynthesis. Notably, the metabolomic results revealed that a large number of flavonoids, vitamins, alkaloids, and terpenoids were enriched in M. polymorpha. These results imply that the decreased lignin content but relatively high nutrient content of M. polymorpha enhance its edibility and nutritional value as a forage and vegetable. Our genomic data provide a genetic basis that will accelerate functional genomic and breeding research on M. polymorpha as well as other Medicago and legume plants. [ABSTRACT FROM AUTHOR]

Anthocyanins biosynthesized from the flavonoid pathway are types of pigments that are involved in the protection of poplar from biotic and abiotic stresses. Previous researchers studying anthocyanin-related transcription factors and structural genes in poplar have made significant discoveries. However, little is known about the regulatory role of microRNAs in anthocyanin biosynthesis in poplar. Here, we overexpressed miR156 in poplar to study the comprehensive effects of the miR156-SPL module on the biosynthesis of anthocyanins. Small RNA sequencing analysis revealed 228 microRNAs differentially expressed in transgenic poplar plants with dramatically increased miR156 levels. Furthermore, integrated microRNAomic and transcriptomic analysis suggested that two microRNAs, miR160h, and miR858, have the potential to affect anthocyanin accumulation in poplar by regulating auxin response factors and MYB transcription factors, respectively. Additionally, the accumulation of miR160h and miR858 displayed a positive correlation with miR156 levels, suggesting a possible interaction between the miR156-SPL module and these microRNAs in poplar. Last, metabolomics analysis revealed that the levels of anthocyanins, flavones, and flavonols were substantially elevated in transgenic poplar plants overexpressing miR156 compared with the wild type, whereas the total lignin content was reduced in the transgenic plants. Taken together, our results indicate that miR156 can fine tune the anthocyanin biosynthetic pathway via multiple factors, including microRNAs, transcription factors, and the levels of structural genes, in poplar. This provides additional clues for understanding the complex regulatory network of anthocyanin biosynthesis in woody plants. [ABSTRACT FROM AUTHOR]

The brown midrib2 (bm2) mutant of maize, which has a modified lignin composition, contains a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. Here, we show that a MITE transposon insertion caused down-regulation of MTHFR , with an accompanying decrease in 5-methyl-tetrahydrofolate and an increase in 5, 10-methylene-tetrahydrofolate and tetrahydrofolate in the bm2 mutant. Furthermore, MTHFR mutation did not change the content of S -adenosyl methionine (SAM), the methyl group donor involved in the biosynthesis of guaiacyl and syringyl lignins, but increased the level of S -adenosyl homocysteine (SAH), the demethylation product of SAM. Moreover, competitive inhibition of the maize caffeoyl CoA O -methyltransferase (CCoAOMT) and caffeic acid O -methyltransferase (COMT) enzyme activities by SAH was found, suggesting that the SAH/SAM ratio, rather than the concentration of SAM, regulates the transmethylation reactions of lignin intermediates. Phenolic profiling revealed that caffeoyl alcohol glucose derivatives accumulated in the bm2 mutant, indicating impaired 3- O -methylation of monolignols. A remarkable increase in the unusual catechyl lignin in the mutant demonstrates that MTHFR down-regulation mainly affects guaiacyl lignin biosynthesis, consistent with the observation that CCoAOMT is more sensitive to SAH inhibition than COMT. This study uncovered a novel regulatory mechanism in lignin biosynthesis, which may offer an effective approach to utilizing lignocellulosic feedstocks in the future. [ABSTRACT FROM AUTHOR]

Class I KNOTTED -like homeobox (KNOXI) genes are critical for the maintenance of the shoot apical meristem. The expression domain of KNOXI is regulated by ASYMMETRIC LEAVES1 / ROUGHSHEATH2 / PHANTASTICA (ARP) genes, which are associated with leaf morphology. In the inverted repeat-lacking clade (IRLC) of Fabaceae, the orthologs of LEAFY (LFY) function in place of KNOXI to regulate compound leaf development. Here, we characterized loss-of-function mutants of ARP (PHAN) and SHOOTMERISTEMLESS (STM) - and BREVIPEDICELLUS (BP) -like KNOXI in the model IRLC legume species Medicago truncatula. The function of ARP genes is species specific. The repression of STM / BP-like KNOXI genes in leaves is not mediated by PHAN , and no suppression of PHAN by STM/BP-like KNOXI genes was observed either, indicating that STM/BP-like KNOXI genes are uncoupled from PHAN in M. truncatula. Furthermore, comparative analyses of phenotypic output in response to ectopic expression of KNOXI and the M. truncatula LFY ortholog, SINGLE LEAFLET1 (SGL1), reveal that KNOXI and SGL1 regulate parallel pathways in leaf development. We propose that SGL1 probably functions in a stage-specific manner in the regulation of the indeterminate state of developing leaves in M. truncatula. [ABSTRACT FROM AUTHOR]

Leaf shape elaboration and organ separation are critical for plant morphogenesis. We characterized the developmental roles of LOBED LEAFLET1 by analyzing a recessive mutant in the model legume Medicago truncatula. An ortholog of Arabidopsis thaliana ARGONAUTE7 (AGO7), Mt -AGO7/LOBED LEAFLET1, is required for the biogenesis of a trans -acting short interfering RNA (ta-siRNA) to negatively regulate the expression of AUXIN RESPONSE FACTORs in M. truncatula. Loss of function in AGO7 results in pleiotropic phenotypes in different organs. The prominent phenotype of the ago7 mutant is lobed leaf margins and more widely spaced lateral organs, suggesting that the trans -acting siRNA3 (TAS3) pathway negatively regulates the formation of boundaries and the separation of lateral organs in M. truncatula. Genetic interaction analysis with the smooth leaf margin1 (slm1) mutant revealed that leaf margin formation is cooperatively regulated by the auxin/SLM1 (ortholog of Arabidopsis PIN-FORMED1) module, which influences the initiation of leaf margin teeth, and the TAS3 ta-siRNA pathway, which determines the degree of margin indentation. Further investigations showed that the TAS3 ta-siRNA pathway and NO APICAL MERISTEM (ortholog of Arabidopsis CUP-SHAPED COTYLEDON) antagonistically regulate both leaf margin development and lateral organ separation, and the regulation is partially dependent on the auxin/SLM1 module. [ABSTRACT FROM AUTHOR]

It is necessary to overcome recalcitrance of the biomass to saccharification (sugar release) to make switchgrass (Panicum virgatum) economically viable as a feedstock for liquid biofuels. Lignin content correlates negatively with sugar release efficiency in switchgrass, but selecting the right gene candidates for engineering lignin biosynthesis in this tetraploid outcrossing species is not straightforward. To assist this endeavor, we have used an inducible switchgrass cell suspension system for studying lignin biosynthesis in response to exogenous brassinolide. By applying a combination of protein sequence phylogeny with whole-genome microarray analyses of induced cell cultures and developing stem internode sections, we have generated a list of candidate monolignol biosynthetic genes for switchgrass. Several genes that were strongly supported through our bioinformatics analysis as involved in lignin biosynthesis were confirmed by gene silencing studies, in which lignin levels were reduced as a result of targeting a single gene. However, candidate genes encoding enzymes involved in the early steps of the currently accepted monolignol biosynthesis pathway in dicots may have functionally redundant paralogues in switchgrass and therefore require further evaluation. This work provides a blueprint and resources for the systematic genome-wide study of the monolignol pathway in switchgrass, as well as other C4 monocot species. [ABSTRACT FROM AUTHOR]

The evolution of lignin biosynthesis was critical in the transition of plants from an aquatic to an upright terrestrial lifestyle. Lignin is assembled by oxidative polymerization of two major monomers, coniferyl alcohol and sinapyl alcohol. Although two recently discovered laccases, LAC4 and LAC17, have been shown to play a role in lignin polymerization in Arabidopsis thaliana , disruption of both genes only leads to a relatively small change in lignin content and only under continuous illumination. Simultaneous disruption of LAC11 along with LAC4 and LAC17 causes severe plant growth arrest, narrower root diameter, indehiscent anthers, and vascular development arrest with lack of lignification. Genome-wide transcript analysis revealed that all the putative lignin peroxidase genes are expressed at normal levels or even higher in the laccase triple mutant, suggesting that lignin laccase activity is necessary and nonredundant with peroxidase activity for monolignol polymerization during plant vascular development. Interestingly, even though lignin deposition in roots is almost completely abolished in the lac11   lac4   lac17 triple mutant, the Casparian strip, which is lignified through the activity of peroxidase, is still functional. Phylogenetic analysis revealed that lignin laccase genes have no orthologs in lower plant species, suggesting that the monolignol laccase genes diverged after the evolution of seed plants. [ABSTRACT FROM AUTHOR]