Understanding the responses of tillering to 2,4-D isooctyl ester in Setaria viridis L.

BACKGROUND: Green foxtail [Setaria viridis (L.)] is one of the most abundant and troublesome annual grass weeds in alfalfa fields in Northeast China. Synthetic auxin herbicide is widely used in agriculture, while how auxin herbicide affects tillering on perennial grass weeds is still unclear. A greenhouse experiment was conducted to examine the effects of auxin herbicide 2,4-D on green foxtail growth, especially on tillers. RESULTS: In the study, 2,4-D isooctyl ester was used. There was an inhibition of plant height and fresh weight on green foxtail after application. The photosynthetic rate of the leaves was dramatically reduced and there was an accumulation of malondialdehyde (MDA) content. Moreover, applying 2,4-D isooctyl ester significantly reduced the tillering buds at rates between 2100 and 8400 ga. i. /ha. Transcriptome results showed that applying 2,4-D isooctyl ester on leaves affected the phytohormone signal transduction pathways in plant tillers. Among them, there were significant effects on auxin, cytokinin, abscisic acid (ABA), gibberellin (GA), and brassinosteroid signaling. Indeed, external ABA and GA on leaves also limited tillering in green foxtail. CONCLUSIONS: These data will be helpful to further understand the responses of green foxtail to 2, 4-D isooctyl ester, which may provide a unique perspective for the development and identification of new target compounds that are effective against this weed species.

The Effects of Epicuticular Wax on Anthracnose Resistance of Sorghum bicolor.

Cuticular waxes are mixtures of hydrophobic compounds covering land plant surfaces and play key roles in plant resistance to abiotic and biotic stresses. However, it is still not clear whether the epicuticular wax could protect the plants from infection by anthracnose, one of the most important plant diseases worldwide, which seriously infects sorghum and causes great yield loss. In this study, Sorghum bicolor L., an important C4 crop with high wax coverage, was selected to analyze the relationship between epicuticular wax (EW) and anthracnose resistance. In vitro analysis indicated that the sorghum leaf wax significantly inhibited the anthracnose mycelium growth of anthracnose on potato dextrose agar (PDA) medium, with the plaque diameter smaller than that grown on medium without wax. Then, the EWs were removed from the intact leaf with gum acacia, followed by the inoculation of Colletotrichum sublineola. The results indicated that the disease lesion was remarkably aggravated on leaves without EW, which showed decreased net photosynthetic rate and increased intercellular CO2 concentrations and malonaldehyde content three days after inoculation. Transcriptome analysis further indicated that 1546 and 2843 differentially expressed genes (DEGs) were regulated by C. sublineola infection in plants with and without EW, respectively. Among the DEG encoded proteins and enriched pathways regulated by anthracnose infection, the cascade of the mitogen-activated protein kinases (MAPK) signaling pathway, ABC transporters, sulfur metabolism, benzoxazinoid biosynthesis, and photosynthesis were mainly regulated in plants without EW. Overall, the EW increases plant resistance to C. sublineola by affecting physiological and transcriptome responses through sorghum epicuticular wax, improving our understanding of its roles in defending plants from fungi and ultimately benefiting sorghum resistance breeding.

Down-regulation of PvSAMS impairs S-adenosyl-L-methionine and lignin biosynthesis, and improves cell wall digestibility in switchgrass.

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.

Microbial-type terpene synthase genes occur widely in nonseed land plants, but not in seed plants.

The vast abundance of terpene natural products in nature is due to enzymes known as terpene synthases (TPSs) that convert acyclic prenyl diphosphate precursors into a multitude of cyclic and acyclic carbon skeletons. Yet the evolution of TPSs is not well understood at higher levels of classification. Microbial TPSs from bacteria and fungi are only distantly related to typical plant TPSs, whereas genes similar to microbial TPS genes have been recently identified in the lycophyte Selaginella moellendorffii The goal of this study was to investigate the distribution, evolution, and biochemical functions of microbial terpene synthase-like (MTPSL) genes in other plants. By analyzing the transcriptomes of 1,103 plant species ranging from green algae to flowering plants, putative MTPSL genes were identified predominantly from nonseed plants, including liverworts, mosses, hornworts, lycophytes, and monilophytes. Directed searching for MTPSL genes in the sequenced genomes of a wide range of seed plants confirmed their general absence in this group. Among themselves, MTPSL proteins from nonseed plants form four major groups, with two of these more closely related to bacterial TPSs and the other two to fungal TPSs. Two of the four groups contain a canonical aspartate-rich "DDxxD" motif. The third group has a "DDxxxD" motif, and the fourth group has only the first two "DD" conserved in this motif. Upon heterologous expression, representative members from each of the four groups displayed diverse catalytic functions as monoterpene and sesquiterpene synthases, suggesting these are important for terpene formation in nonseed plants.

Emission and biosynthesis of volatile terpenoids from the plasmodial slime mold Physarum polycephalum.

Terpene synthases (TPSs) are pivotal enzymes for the production of diverse terpenes, including monoterpenes, sesquiterpenes, and diterpenes. In our recent studies, dictyostelid social amoebae, also known as cellular slime molds, were found to contain TPS genes for making volatile terpenes. For comparison, here we investigated Physarum polycephalum, a plasmodial slime mold also known as acellular amoeba. Plasmodia of P. polycephalum grown on agar plates were found to release a mixture of volatile terpenoids consisting of four major sesquiterpenes (α-muurolene, (E)-ß-caryophyllene, two unidentified sesquiterpenoids) and the monoterpene linalool. There were no qualitative differences in terpenoid composition at two stages of young plasmodia. To understand terpene biosynthesis, we analyzed the transcriptome and genome sequences of P. polycephalum and identified four TPS genes designated PpolyTPS1-PpolyTPS4. They share 28-73% of sequence identities. Full-length cDNAs for the four TPS genes were cloned and expressed in Escherichia coli to produce recombinant proteins, which were tested for sesquiterpene synthase and monoterpene synthase activities. While neither PpolyTPS2 nor PpolyTPS3 was active, PpolyTPS1 and PpolyTPS4 were able to produce sesquiterpenes and monoterpenes from the respective substrates farnesyl diphosphate and geranyl diphosphate. By comparing the volatile profile of P. polycephalum plasmodia and the in vitro products of PpolyTPS1 and PpolyTPS4, it was concluded that most sesquiterpenoids emitted from P. polycephalum were attributed to PpolyTPS4. Phylogenetic analysis placed the four PpolyTPSs genes into two groups: PpolyTPS1 and PpolyTPS4 being one group that was clustered with the TPSs from the dictyostelid social amoeba and PpolyTPS2 and PpolyTPS3 being the other group that showed closer relatedness to bacterial TPSs. The biological role of the volatile terpenoids produced by the plasmodia of P. polycephalum is discussed.

Methylenetetrahydrofolate reductase modulates methyl metabolism and lignin monomer methylation in maize.

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.

Dhurrin in Sorghum: Biosynthesis, Regulation, Biological Function and Challenges for Animal Production.

Sorghum (Sorghum bicolor) holds a significant position as the fifth most vital cereal crop globally. Its drought resistance and robust biomass production, coupled with commendable nutritional value, make sorghum a promising choice for animal feed. Nevertheless, the utilization of sorghum in animal production faces hurdles of dhurrin (a cyanogenic glycoside) poisoning. While dhurrin serves as a protective secondary metabolite during sorghum growth, the resulting highly toxic hydrogen cyanide poses a significant threat to animal safety. This review extensively examines the biometabolic processes of dhurrin, the pivotal genes involved in the regulation of dhurrin biosynthesis, and the factors influencing dhurrin content in sorghum. It delves into the impact of dhurrin on animal production and explores measures to mitigate its content, aiming to provide insights for advancing research on dhurrin metabolism regulation in sorghum and its rational utilization in animal production.

Expression of Foxtail Millet bZIP Transcription Factor SibZIP67 Enhances Drought Tolerance in Arabidopsis.

Foxtail millet is a drought-tolerant cereal and forage crop. The basic leucine zipper (bZIP) gene family plays important roles in regulating plant development and responding to stresses. However, the roles of bZIP genes in foxtail millet remain largely uninvestigated. In this study, 92 members of the bZIP transcription factors were identified in foxtail millet and clustered into ten clades. The expression levels of four SibZIP genes (SibZIP11, SibZIP12, SibZIP41, and SibZIP67) were significantly induced after PEG treatment, and SibZIP67 was chosen for further analysis. The studies showed that ectopic overexpression of SibZIP67 in Arabidopsis enhanced the plant drought tolerance. Detached leaves of SibZIP67 overexpressing plants had lower leaf water loss rates than those of wild-type plants. SibZIP67 overexpressing plants improved survival rates under drought conditions compared to wild-type plants. Additionally, overexpressing SibZIP67 in plants displayed reduced malondialdehyde (MDA) levels and enhanced activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) under drought stress. Furthermore, the drought-related genes, such as AtRD29A, AtRD22, AtNCED3, AtABF3, AtABI1, and AtABI5, were found to be regulated in SibZIP67 transgenic plants than in wild-type Arabidopsis under drought conditions. These data suggested that SibZIP67 conferred drought tolerance in transgenic Arabidopsis by regulating antioxidant enzyme activities and the expression of stress-related genes. The study reveals that SibZIP67 plays a beneficial role in drought response in plants, offering a valuable genetic resource for agricultural improvement in arid environments.

Influence of Cuticular Waxes from Triticale on Rumen Fermentation: A Metabolomic and Microbiome Profiling Study.

Cuticular wax, a critical defense layer for plants, remains a relatively unexplored factor in rumen fermentation. We investigated the impact of cuticular wax on rumen fermentation using triticale as a model. In total, six wax classes were identified, including fatty acids, aldehydes, alkane, primary alcohol, alkyresorcinol, and ß-diketone, with low-bloom lines predominated by 46.05% of primary alcohols and high-bloom lines by 35.64% of ß-diketone. Low-wax addition (2.5 g/kg DM) increased the gas production by 19.25% (P < 0.05) and total volatile fatty acids by 6.34% (P > 0.05), and enriched key carbohydrate-fermenting rumen microbes like Saccharofermentans, Ruminococcus, and Prevotellaceae, when compared to non-wax groups. Metabolites linked to nucleotide metabolism, purine metabolism, and protein/fat digestion in the rumen showed a positive correlation with low-wax, benefiting rumen microbes. This study highlights the intricate interplay among cuticular wax, rumen microbiota, fermentation, and metabolomics in forage digestion, providing insights into livestock nutrition and forage utilization.

Metabolomics analysis and membrane damage measurement reveal the antibacterial mechanism of lipoic acid against Yersinia enterocolitica.

Yersinia enterocolitica is a pathogenic microorganism that can cause food-borne diseases. Lipoic acid (LA) has been used as an antioxidant against bacteria, but its antibacterial mechanism is rarely reported. This study aims to explore the antibacterial mechanism of LA and its effect on the metabolites of Y. enterocolitica through membrane damage and metabolomics analysis. The results showed that the minimum inhibitory concentration (MIC) of LA against Y. enterocolitica was 2.5 mg mL-1. The membrane potential was depolarized, and intracellular pH (pHin) and ATP were significantly reduced, indicating that LA destroys the cell membrane structure. Confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM) further confirmed LA-induced cell membrane damage. The metabolic profile of Y. enterocolitica following LA treatment was analyzed by liquid chromatography-mass spectrometry (LC-MS). In the metabolome evaluation, 6209 differential metabolites were screened, including 3394 up-regulated and 2815 down-regulated metabolites. Fifteen metabolic pathways of Y. enterocolitica exhibited significant changes after LA treatment, including the pathways important for amino acid and nucleotide metabolism. The results show that LA is a bacteriostatic substance with potential application value in the food industry.

Genomic characterization and expression analysis of TCP transcription factors in Setaria italica and Setaria viridis.

The plant-specific TCP transcription factor plays important roles in plant development and environment adaptation. Setaria italica and Setaria viridis, the C4 model plants, can grow on drought or arid soils. However, there is no systematic information about the genomic dissection and the expression of Setaria TCP genes. A total of 22 TCP genes were both identified from S. italica and S. viridis genomes. They all contained bHLH domain and were grouped into three main clades (PCF, CIN, and CYC/TB1). The TCP genes in the same clades shared similar gene structures. Cis-element in the TCP promoter regions were analyzed and associated with hormones and stress responsiveness. Ten TCP genes were predicted to be targets of miRNA319. Moreover, gene ontology analysis indicated three SiTCP and three SvTCP genes were involved in the regulation of shoot development, and SiTCP16/SvTCP16 were clustered together with tillering controlling gene TB1. The TCP genes were differentially expressed in the organs, but SiTCP/SvTCP orthologs shared similar expression patterns. Ten SiTCP members were downregulated under drought or salinity stresses, indicating they may play regulatory roles in abiotic stresses. The study provides detailed information regarding Setaria TCP genes, providing the theoretical basis for agricultural applications.

Germination ecology of Chenopodium album L. and implications for weed management.

Chenopodium album L. is a troublesome annual species in various cropping systems, and a sound knowledge of the ecological response of C. album germination to environmental factors would suggest suitable management strategies for inhibiting its spread. Preliminary laboratory-based research was conducted to investigate germination and emergence requirements of C. album under various environmental conditions (e.g., photoperiods, constant temperature, salinity, moisture, soil pH, burial depth, and oat crop residue). Results showed C. album seeds were found to be photoblastic, with only 13% germination in darkness. The maximum germination (94%) of C. album occurred at an optimal temperature of 25°C, and the depressive effect of other temperatures on germination was more severe at lower rather than higher temperatures. Seed germination was suitably tolerant of salinity and osmotic potential, with germination observed at 200 mM NaCl (37.0%) and -0.8 MPa (20%), respectively. Germination was relatively uniform (88-92%) at pH levels ranging from 4 to 10. The maximum germination of C. album was observed on the soil surface, with no or rare emergence of seeds at a burial depth of 2 cm or under 7000 kg ha-1 oat straw cover, respectively. Information provided by this study will help to develop more sustainable and effective integrated weed management strategies for the control of C. album, including (i) a shallow-tillage procedures to bury weed seeds in conventional-tillage systems and (ii) oat residue retention or coverage on the soil surface in no-tillage systems.

Evaluation of the tolerance and forage quality of different ecotypes of seashore paspalum.

Seashore paspalum is a halophytic, warm-season grass with wide applications. It is noted for its superior salt tolerance in saline environments; however, the nutritive value of seashore paspalum and the effect of salinity remains to be determined. Therefore, this study aimed to evaluate the relationship between agronomic traits and forage quality and identified the effects of short-term high-salt stress (1 week, 700 mM NaCl) on the growth and forage nutritive value of 16 ecotypes of seashore paspalum. The salt and cold tolerances of the seashore paspalum ecotypes were assessed based on the survival rate following long-term high-salt stress (7 weeks, 700 mM NaCl) and exposure to natural low temperature stress. There were significant genetic (ecotype-specific) effects on plant height, leaf-stem ratio, and survival rate of seashore paspalum following salt or low temperature stress. Plant height was significantly negatively correlated with the leaf-stem ratio (r = -0.63, P<0.01), but the heights and leaf-stem ratios were not significantly correlated with the fresh weight (FW) and dry weight (DW) of the shoots. High salinity decreased the FW and DW of the shoots by 50.6% and 23.6%, respectively, on average. Seashore paspalum exhibited outstanding salt tolerance and forage quality at high salinity. The survival rate of the different ecotypes of seashore paspalum varied from 6.5% to 49.0% following treatment with 700 mM NaCl for 7 weeks. The crude protein (CP) content of the control and treatment groups (700 mM NaCl) was 17.4% and 19.3%, respectively, of the DW on average, and the CP content of most ecotypes was not significantly influenced by high salinity. The average ether extract (EE) content ranged from 4.6% to 4.4% of the DW under control and saline conditions, respectively, indicating that the influence was not significant. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents of the control group were 57.4% and 29.8%, respectively, of the DW on average. Salt stress reduced the content of NDF and ADF to 50.2% and 25.9%, respectively, of the DW on average. Altogether, the results demonstrated that stress did not have any significant effects on the CP and EE content of most ecotypes, but reduced the NDF and ADF content and improved relative feed value (RFV). The results obtained herein support the notion that seashore paspalum is a good candidate for improving the forage potential of saline soils and can provide useful guidelines for livestock producers.

Transcriptional and Metabolic Responses of Maize Shoots to Long-Term Potassium Deficiency.

Potassium is important for plant growth and crop yield. However, the effects of potassium (K+) deficiency on silage maize biomass yield and how maize shoot feedback mechanisms of K+ deficiency regulate whole plant growth remains largely unknown. Here, the study aims to explore the maize growth, transcriptional and metabolic responses of shoots to long-term potassium deficiency. Under the K+ insufficiency condition, the biomass yield of silage maize decreased. The transcriptome data showed that there were 922 and 1,107 differential expression genes in DH605 and Z58, respectively. In the two varieties, 390 differently expressed overlapping genes were similarly regulated. These genes were considered the fundamental responses to K+ deficiency in maize shoots. Many stress-induced genes are involved in transport, primary and secondary metabolism, regulation, and other processes, which are involved in K+ acquisition and homeostasis. Metabolic profiles indicated that most amino acids, phenolic acids, organic acids, and alkaloids were accumulated in shoots under K+ deficiency conditions and part of the sugars and sugar alcohols also increased. It revealed that putrescine and putrescine derivatives were specifically accumulated under the K+ deficiency condition, which may play a role in the feedback regulation of shoot growth. These results confirmed the importance of K+ on silage maize production and provided a deeper insight into the responses to K+ deficiency in maize shoots.

Effect of Light Intensity on Morphology, Photosynthesis and Carbon Metabolism of Alfalfa (Medicago sativa) Seedlings.

To understand how light intensity influences plant morphology and photosynthesis in the forage crop alfalfa (Medicago sativa L. cv. Zhongmu 1), we investigated changes in leaf angle orientation, chlorophyll fluorescence, parameters of photosynthesis and expression of genes related to enzymes involved in photosynthesis, the Calvin cycle and carbon metabolism in alfalfa seedlings exposed to five light intensities (100, 200, 300, 400 and 500 µmol m-2 s-1) under hydroponic conditions. Seedlings grown under low light intensities had significantly increased plant height, leaf hyponasty, specific leaf area, photosynthetic pigments, leaf nitrogen content and maximal PSII quantum yield, but the increased light-capturing capacity generated a carbon resource cost (e.g., decreased carbohydrates and biomass accumulation). Increased light intensity significantly improved leaf orientation toward the sun and upregulated the genes for Calvin cycle enzymes, thereby increasing photosynthetic capacity. Furthermore, high light (400 and 500 µmol m-2 s-1) significantly enhanced carbohydrate accumulation, accompanied by gene upregulation and increased activity of sucrose and starch-synthesis-related enzymes and those involved in carbon metabolism. Together, these results advance our understanding of morphological and physiological regulation in shade avoidance in alfalfa, which would guide the identification of suitable spatial planting patterns in the agricultural system.

Characterization of Two New brown midrib1 Mutations From an EMS-Mutagenic Maize Population for Lignocellulosic Biomass Utilization.

Gene mutations linked to lignin biosynthesis are responsible for the brown midrib (bm) phenotypes. The bm mutants have a brown-reddish midrib associated with changes in lignin content and composition. Maize bm1 is caused by a mutation of the cinnamyl alcohol dehydrogenase gene ZmCAD2. Here, we generated two new bm1 mutant alleles (bm1-E1 and bm1-E2) through EMS mutagenesis, which contained a single nucleotide mutation (Zmcad2-1 and Zmcad2-2). The corresponding proteins, ZmCAD2-1 and ZmCAD2-2 were modified with Cys103Ser and Gly185Asp, which resulted in no enzymatic activity in vitro. Sequence alignment showed that CAD proteins have high similarity across plants and that Cys103 and Gly185 are conserved in higher plants. The lack of enzymatic activity when Cys103 was replaced for other amino acids indicates that Cys103 is required for its enzyme activity. Enzymatic activity of proteins encoded by CAD genes in bm1-E plants is 23-98% lower than in the wild type, which leads to lower lignin content and different lignin composition. The bm1-E mutants have higher saccharification efficiency in maize and could therefore provide new and promising breeding resources in the future.

Genome-Wide Analysis of the TCP Gene Family in Switchgrass (Panicum virgatum L.).

The plant-specific transcription factor TCPs play multiple roles in plant growth, development, and stress responses. However, a genome-wide analysis of TCP proteins and their roles in salt stress has not been declared in switchgrass (Panicum virgatum L.). In this study, 42 PvTCP genes (PvTCPs) were identified from the switchgrass genome and 38 members can be anchored to its chromosomes unevenly. Nine PvTCPs were predicted to be microRNA319 (miR319) targets. Furthermore, PvTCPs can be divided into three clades according to the phylogeny and conserved domains. Members in the same clade have the similar gene structure and motif localization. Although all PvTCPs were expressed in tested tissues, their expression profiles were different under normal condition. The specific expression may indicate their different roles in plant growth and development. In addition, approximately 20 cis-acting elements were detected in the promoters of PvTCPs, and 40% were related to stress response. Moreover, the expression profiles of PvTCPs under salt stress were also analyzed and 29 PvTCPs were regulated after NaCl treatment. Taken together, the PvTCP gene family was analyzed at a genome-wide level and their possible functions in salt stress, which lay the basis for further functional analysis of PvTCPs in switchgrass.

Mutation of 4-coumarate: coenzyme A ligase 1 gene affects lignin biosynthesis and increases the cell wall digestibility in maize brown midrib5 mutants.

BACKGROUND: Maize brown midrib (bm) mutants associated with impaired lignin biosynthesis are a potential source for the breed of novel germplasms with improved cell wall digestibility. The spontaneous bm5 mutants had been identified since 2008. However, the gene responsible for the bm5 locus, and the comprehensive effects of bm5 mutation on lignin biosynthesis, soluble phenolics accumulation, and cell wall degradation have yet to be elucidated. RESULTS: The bm5 locus was identified to encode a major 4-coumarate: coenzyme A ligase (Zm4CL1) through analyzing MutMap-assisted gene mapping data. Two alleles of Zm4CL1 isolated from bm5 mutants contained two transposons inserted in the first exon and the second intron, respectively, and consequently, the activities of 4CLs in the crude enzyme extracts from bm5 midribs were reduced by 51-62% compared with the wild type. Furthermore, five 4CLs were retrieved from maize genome, and Zm4CL1 was the most highly expressed one in the lignified tissues. Mutation of Zm4CL1 mainly impeded the biosynthesis of guaiacyl (G) lignins and increased the level of soluble feruloyl derivatives without impacting maize growth and development. Moreover, both neutral detergent fiber digestibility and saccharification efficiency of cell walls were significantly elevated in the bm5 mutant. CONCLUSIONS: Zm4CL1 was identified as the Bm5 gene, since two independent alleles of Zm4CL1 were associated with the same mutant phenotype. Mutation of Zm4CL1 mainly affected G lignin biosynthesis and soluble feruloyl derivatives accumulation in maize lignified tissues. The reduced recalcitrance of the bm5 mutant suggests that Zm4CL1 is an elite target for cell wall engineering, and genetic manipulation of this gene will facilitate the utilization of crop straw and stover that have to be dealt with for environmental protection.

Terpene Synthase Genes Originated from Bacteria through Horizontal Gene Transfer Contribute to Terpenoid Diversity in Fungi.

Fungi are successful eukaryotes of wide distribution. They are known as rich producers of secondary metabolites, especially terpenoids, which are important for fungi-environment interactions. Horizontal gene transfer (HGT) is an important mechanism contributing to genetic innovation of fungi. However, it remains unclear whether HGT has played a role in creating the enormous chemical diversity of fungal terpenoids. Here we report that fungi have acquired terpene synthase genes (TPSs), which encode pivotal enzymes for terpenoid biosynthesis, from bacteria through HGT. Phylogenetic analysis placed the majority of fungal and bacterial TPS genes from diverse taxa into two clades, indicating ancient divergence. Nested in the bacterial TPS clade is a number of fungal TPS genes that are inferred as the outcome of HGT. These include a monophyletic clade of nine fungal TPS genes, designated as BTPSL for bacterial TPS-like genes, from eight species of related entomopathogenic fungi, including seven TPSs from six species in the genus Metarhizium. In vitro enzyme assays demonstrate that all seven BTPSL genes from the genus Metarhizium encode active enzymes with sesquiterpene synthase activities of two general product profiles. By analyzing the catalytic activity of two resurrected ancestral BTPSLs and one closely related bacterial TPS, the trajectory of functional evolution of BTPSLs after HGT from bacteria to fungi and functional divergence within Metarhizium could be traced. Using M. brunneum as a model species, both BTPSLs and typical fungal TPSs were demonstrated to be involved in the in vivo production of terpenoids, illustrating the general importance of HGT of TPS genes from bacteria as a mechanism contributing to terpenoid diversity in fungi.

Biochemical characterization of microbial type terpene synthases in two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis.

Microbial terpene synthase-like (MTPSL) genes are a type of terpene synthase genes only recently identified in plants. In contrast to typical plant terpene synthase genes, which are ubiquitous in land plants, MTPSL genes appear to occur only in nonseed plants. Our knowledge of catalytic functions of MTPSLs is very limited. Here we report biochemical characterization of the enzymes encoded by MTPSL genes from two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis. Seven full-length MTPSL genes were identified in A. punctatus (ApMTPSL1-7) based on the analysis of its genome sequence. Using homology-based cloning, the apparent orthologs for six of the ApMTPSL genes, except ApMTPSL2, were cloned from A. agrestis. They were designated AaMTPSL1, 3-7. The coding sequences for each of the 13 Anthoceros MTPSL genes were cloned into a protein expression vector. Escherichia coli-expressed recombinant MTPSLs from hornworts were assayed for terpene synthase activities. Six ApMTPSLs and five AaMTPSLs, except for ApMTPSL5 and AaMTPSL5, showed catalytic activities with one or more isoprenyl diphosphate substrates. All functional MTPSLs exhibited sesquiterpene synthase activities. In contrast, only ApMTPSL7 and AaMTPSL7 showed monoterpene synthase activity and only ApMTPSL2, ApMTPSL6 and AaMTPSL6 showed diterpene synthase activity. Most MTPSLs from Anthoceros contain uncanonical aspartate-rich motif in the form of either 'DDxxxD' or 'DDxxx'. Homology-based structural modeling analysis of ApMTPSL1 and ApMTPSL7, which contain 'DDxxxD' and 'DDxxx' motif, respectively, showed that 'DDxxxD' and 'DDxxx' motifs are localized in the similar positions as the canonical 'DDxxD' motif in known terpene synthases. To further understand the role of individual aspartate residues in the motifs, ApMTPSL1 and ApMTPSL7 were selected as two representatives for site-directed mutagenesis studies. No activities were detected when any of the conserved aspartic acid was mutated into alanine. This study provides new information about the catalytic functions of MTPSLs and the functionality of their uncanonical aspartate-rich motifs, and builds a knowledge base for studying the biological importance of MTPSL genes and their terpene products in nonseed plants.