VAMP726 from maize and Arabidopsis confers pollen resistance to heat and UV radiation by influencing lignin content of sporopollenin.

Sporopollenin in the pollen cell wall protects male gametophytes from stresses. Phenylpropanoid derivatives, including guaiacyl (G) lignin units, are known to be structural components of sporopollenin, but the exact composition of sporopollenin remains to be fully resolved. We analyzed the phenylpropanoid derivatives in sporopollenin from maize and Arabidopsis by thioacidolysis coupled with nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The NMR and GC-MS results confirmed the presence of p-hydroxyphenyl (H), G, and syringyl (S) lignin units in sporopollenin from maize and Arabidopsis. Strikingly, H units account for the majority of lignin monomers in sporopollenin from these species. We next performed a genome-wide association study to explore the genetic basis of maize sporopollenin composition and identified a vesicle-associated membrane protein (ZmVAMP726) that is strongly associated with lignin monomer composition of maize sporopollenin. Genetic manipulation of VAMP726 affected not only lignin monomer composition in sporopollenin but also pollen resistance to heat and UV radiation in maize and Arabidopsis, indicating that VAMP726 is functionally conserved in monocot and dicot plants. Our work provides new insight into the lignin monomers that serve as structural components of sporopollenin and characterizes VAMP726, which affects sporopollenin composition and stress resistance in pollen.

Characterization, expression, and functional analysis of the pathogenesis-related gene PtDIR11 in transgenic poplar.

Lignins and lignans are important for plant resistance to pathogens. Dirigent (DIR) proteins control the regio- and stereo-selectivity of coniferyl alcohol in lignan and lignin biosynthesis. DIR genes have been implicated in defense-related responses in several plant species, but their role in poplar immunity is unclear. We cloned PtDIR11 from Populus trichocarpa; we found that overexpression of PtDIR11 in poplar improved the lignan biosynthesis and enhanced the resistance of poplar to Septotis populiperda. PtDIR11 has a typical DIR domain; it belongs to the DIR-b/d family and is expressed in the cell membrane. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis showed that PtDIR11 expression was highest in stems, followed by leaves and roots. Furthermore, PtDIR11 expression was induced by S. populiperda, salicylic acid (SA), jasmonate (JA), and ethylene (ET) stresses. The recombinant PtDIR11 protein inhibited the growth of S. populiperda in vitro. Overexpressing (OE) PtDIR11 in "Nanlin 895" poplar enhanced growth. The OE lines exhibited minimal changes in lignin content, but their total lignan and flavonoid contents were significantly greater than in the wild-type (WT) lines. Overexpression of PtDIR11 affected multiple biological pathways of poplar, such as phenylpropanoid biosynthesis. The methanol extracts of OE-PtDIR11 lines showed greater anti-S. populiperda activity than did lignin extracts from the WT lines. Furthermore, OE-PtDIR11 lines upregulated genes that were related to phenylpropanoid biosynthesis and genes associated with the JA and ET signal transduction pathways; it downregulated genes that were related to SA signal transduction compared with the WT line under S. populiperda stress. Therefore, the OE transgenic plants analysis revealed that PtDIR11 is a good candidate gene for breeding of disease resistant poplar.

GhMYB4 downregulates lignin biosynthesis and enhances cotton resistance to Verticillium dahliae.

KEY MESSAGE: GhMYB4 acts as a negative regulator in lignin biosynthesis, which results in alteration of cell wall integrity and activation of cotton defense response. Verticillium wilt of cotton (Gossypium hirsutum) caused by the soil-borne fungus Verticillium dahliae (V. dahliae) represents one of the most important constraints of cotton production worldwide. Mining of the genes involved in disease resistance and illuminating the molecular mechanisms that underlie this resistance is of great importance in cotton breeding programs. Defense-induced lignification in plants is necessary for innate immunity, and there are reports of a correlation between increased lignification and disease resistance. In this study, we present an example in cotton whereby plants with reduced lignin content also exhibit enhanced disease resistance. We identified a negative regulator of lignin synthesis, in cotton encoded in GhMYB4. Overexpression of GhMYB4 in cotton and Arabidopsis enhanced resistance to V. dahliae  with reduced lignin deposition. Moreover, GhMYB4 could bind the promoters of several genes involved in lignin synthesis, such as GhC4H-1, GhC4H-2, Gh4CL-4, and GhCAD-3, and impair their expression. The reduction of lignin content in GhMYB4-overexpressing cotton led to alterations of cell wall integrity (CWI) and released more oligogalacturonides (OGs) which may act as damage-associated molecular patterns (DAMPs) to stimulate plant defense responses. In support of this hypothesis, exogenous application with polygalacturonic acid (PGA) in cotton activated biosynthesis of jasmonic acid (JA) and JA-mediated defense against V. dahliae, similar to that described for cotton plants overexpressing GhMYB4. This study provides a new candidate gene for cotton disease-resistant breeding and an increased understanding of the relationship between lignin synthesis, OG release, and plant immunity.

Ginseng-derived patatin-related phospholipase PgpPLAIIIß alters plant growth and lignification of xylem in hybrid poplars.

Patatin-liked phospholipase A (pPLAs) are major lipid acyl hydrolases that participate in various biological functions in plant growth and development. Previously, a ginseng-derived pPLAIII homolog was reported to reduce lignin content in Arabidopsis. This led us to evaluate its possible usefulness as a biomass source in wood plant. Herein, we report that there are six members in the pPLAIII gene family in poplar. Overexpression of pPLAIIIß derived from ginseng resulted in a reduced plant height with radially expanded stem growth in hybrid poplars. Compared with the wild type (WT), the chlorophyll content was increased in the overexpression poplar lines, whereas the leaf size was smaller. The secondary cell wall structure in overexpression lines was also altered, exhibiting reduced lignification in the xylem. Two transcription factors, MYB92 and MYB152, which control lignin biosynthesis, were downregulated in the overexpression lines. The middle xylem of the overexpression line showed heavy thickening, making it thicker than the other xylem parts and the WT xylem, which rather could have been contributed by the presence of more cellulose in the selected surface area. Taken together, the results suggest that PgpPLAIIIß plays a role not only in cell elongation patterns, but also in determining the secondary cell wall composition.

iTRAQ-Based Identification of Proteins Related to Lignin Synthesis in the Pear Pollinated with Pollen from Different Varieties.

Most pears in Anhui Province are a kind of self-incompatible fruit whose quality is strongly influenced by the male pollen. The proteomic variation of Dangshan Su pollinated by different varieties was analysed using the isobaric tag for relative and absolute quantitation (iTRAQ) to investigate the effect of pollination by different varieties on the pear lignin pathway. Among the 3980 proteins identified from the two samples, 139 proteins were identified as differentially expressed proteins (DEPs). Of these proteins, laccase-4 (LAC4), was found to be related with lignin synthesis, and ß-glucosidase 15 (BGLU15) and peroxidase 47 (PER47) were involved in the phenylpropanoid pathway. Moreover, the lignin and stone cell contents were lower in DW (Dangshan Su pollinated by Wonhwang) than those in DJ (Dangshan Su pollinated by Jingbaili). The effect of pollination on the synthesis of lignin through the regulation of the expression of PER47, BGLU15 and LAC4 ultimately affects the formation of stone cells and the fruit quality. We report for the first time that different pollinations influence the protein expression profile in the Dangshan Su pear, and this result provides some new epididymal targets for regulating the synthesis of lignin, regulating the content of stone cells and improving the quality of the pears.

Transcriptome analysis of secondary cell wall development in Medicago truncatula.

BACKGROUND: Legumes are important to humans by providing food, feed and raw materials for industrial utilizations. Some legumes, such as alfalfa, are potential bioenergy crops due to their high biomass productivity. Global transcriptional profiling has been successfully used to identify genes and regulatory pathways in secondary cell wall thickening in Arabidopsis, but such transcriptome data is lacking in legumes. RESULTS: A systematic microarray assay and high through-put real time PCR analysis of secondary cell wall development were performed along stem maturation in Medicago truncatula. More than 11,000 genes were differentially expressed during stem maturation, and were categorized into 10 expression clusters. Among these, 279 transcription factor genes were correlated with lignin/cellulose biosynthesis, therefore representing putative regulators of secondary wall development. The b-ZIP, NAC, WRKY, C2H2 zinc finger (ZF), homeobox, and HSF gene families were over-represented. Gene co-expression network analysis was employed to identify transcription factors that may regulate the biosynthesis of lignin, cellulose and hemicellulose. As a complementary approach to microarray, real-time PCR analysis was used to characterize the expression of 1,045 transcription factors in the stem samples, and 64 of these were upregulated more than 5-fold during stem maturation. Reverse genetics characterization of a cellulose synthase gene in cluster 10 confirmed its function in xylem development. CONCLUSIONS: This study provides a useful transcriptome and expression resource for understanding cell wall development, which is pivotal to enhance biomass production in legumes.

Structure, functionality and tuning up of laccases for lignocellulose and other industrial applications.

Laccases (EC 1.10.3.2) are copper-containing oxidoreductases that have a relatively high redox potential which enables them to catalyze oxidation of phenolic compounds, including lignin-derived phenolics. The laccase-catalyzed oxidation of phenolics is accompanied by concomitant reduction of dioxygen to water via copper catalysis and involves a series of electron transfer reactions balanced by a stepwise re-oxidation of copper ions in the active site of the enzyme. The reaction details of the catalytic four-copper mechanism of laccase-mediated catalysis are carefully re-examined and clarified. The substrate range for laccase catalysis can be expanded by means of supplementary mediators that essentially function as vehicles for electron transfer. Comparisons of amino acid sequences and structural traits of selected laccases reveal conservation of the active site trinuclear center geometry but differences in loop conformations. We also evaluate the features and regions of laccases in relation to modification and evolution of laccases for various industrial applications including lignocellulosic biomass processing.

A review of xylan and lignin biosynthesis: foundation for studying Arabidopsis irregular xylem mutants with pleiotropic phenotypes.

Plant cells are surrounded by a carbohydrate-rich extracellular matrix known as the cell wall. Primary cell walls are laid down around dividing and elongating cells and consist largely of the polysaccharides cellulose, hemicelluloses, and pectin along with approximately 10% protein. Specific cells such as xylem vessels and fibers lay down a secondary wall rich in cellulose, hemicellulose, and lignin, with lesser amounts of pectin. Most of the models depict the plant cell wall as a matrix of separate polysaccharides. However, the recent identification of a proteoglycan that contains covalently attached pectin and xylan indicates that at least some of these wall glycans exist as domains within a single glycopolymer and that current models of the wall need to be revised. Furthermore, several cell wall biosynthesis mutants, including the secondary cell wall mutant irregular xylem (irx) 8, are affected in multiple cell wall polymers making it challenging to define the biochemical function of the mutated gene. The goal of this review is to provide a background for studying genes which encode secondary cell wall biosynthetic proteins whose mutation affects multiple wall polymers including xylan and lignin. We first review the phenotypes of the irx mutants and then summarize the current understanding of the structure and synthesis of xylan and lignin along with a review of transcription factors known to affect secondary wall synthesis. This review is intended to serve as a resource for those studying genes that encode proteins involved in the synthesis of plant secondary wall lignin and xylan.

Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit.

To investigate the role of jasmonates (JAs) in the ripening of Fragaria chiloensis fruit, two concentrations of methyl jasmonate (MeJA, 10 and 100 µM) were evaluated at 2, 5 and 9 d using an in vitro ripening system. Fruit quality parameters; the contents of anthocyanin, lignin and cell wall polymers; and the transcriptional profiles of several ripening-related genes were analyzed. MeJA accelerated fruit ripening by means of a transitory increase in the soluble solid content/titratable acidity ratio, anthocyanin accumulation and an increase in softening at day 5. The expression of several phenylpropanoid-related genes, primarily those associated with anthocyanin biosynthesis, was increased under MeJA treatment, which correlated with an increased accumulation of anthocyanin. MeJA also altered the expression profiles of some cell wall-modifying genes, namely, EG1 and XTH1, and these changes correlated with a transient reduction in the firmness of MeJA-treated fruits. MeJA-responsive elements were observed in the promoter region of the EG1 gene. MeJA also increased the expression of LOX, AOS and OPR3, genes involved in the biosynthesis of JAs, and these changes correlated with the transient activation of fruit ripening observed. Conversely, the expression of ethylene and lignin biosynthesis genes (ACS, ACO, CAD and POD27) increased in MeJA-treated fruits at day 9. The present findings suggest that JAs promote the ripening of non-climacteric fruits through their involvement in anthocyanin accumulation, cell wall modification and the biosynthesis of ethylene and JAs.

Polyphenol distribution in plant organs of tomato introgression lines.

The content of total polyphenols, chlorogenic, caffeic (CaA) and ferulic acids, and rutin, was investigated in plant organs of three introgression lines (IL7-3, IL10-1 and IL12-4) of Solanum pennellii in Solanum lycopersicum cv M82 and compared with that of cropped parental. Such study aims to evidence factors associated to the introgressions that can affect polyphenol distribution in plant. Among genotypes few differences in polyphenols were recorded on fresh weight basis. IL7-3 showed higher total polyphenols in fruits and lower rutin in leaves than the other genotypes. IL12-4 showed an increasing trend of total polyphenol concentration in fresh vegetative organs; however, this seems to depend on the lower water content rather than on a higher polyphenol biosynthesis in the genotype. IL10-1 sowed higher CaA and lignin contents in leaves. Such differences agree with the morphological and physiological traits of the genotypes.

Ethylene-induced differential gene expression during abscission of citrus leaves.

The main objective of this work was to identify and classify genes involved in the process of leaf abscission in Clementina de Nules (Citrus clementina Hort. Ex Tan.). A 7 K unigene citrus cDNA microarray containing 12 K spots was used to characterize the transcriptome of the ethylene-induced abscission process in laminar abscission zone-enriched tissues and the petiole of debladed leaf explants. In these conditions, ethylene induced 100% leaf explant abscission in 72 h while, in air-treated samples, the abscission period started later and took 240 h. Gene expression monitored during the first 36 h of ethylene treatment showed that out of the 12 672 cDNA microarray probes, ethylene differentially induced 725 probes distributed as follows: 216 (29.8%) probes in the laminar abscission zone and 509 (70.2%) in the petiole. Functional MIPS classification and manual annotation of differentially expressed genes highlighted key processes regulating the activation and progress of the cell separation that brings about abscission. These included cell-wall modification, lipid transport, protein biosynthesis and degradation, and differential activation of signal transduction and transcription control pathways. Expression data associated with the petiole indicated the occurrence of a double defensive strategy mediated by the activation of a biochemical programme including scavenging ROS, defence and PR genes, and a physical response mostly based on lignin biosynthesis and deposition. This work identifies new genes probably involved in the onset and development of the leaf abscission process and suggests a different but co-ordinated and complementary role for the laminar abscission zone and the petiole during the process of abscission.

Transcriptional control of lignin biosynthesis by tobacco LIM protein.

Lignin is a complex phenolic plant polymer that is essential for mechanical support, defense, and water transport in higher plants. The AC-rich motif, Pal-box is an important cis-acting element for gene expression in phenylpropanoid biosynthesis. We isolated a cDNA clone (Ntlim1) encoding a Pal-box binding protein by Southwestern screening. The deduced amino acid sequence of Ntlim1 is highly similar to members of the LIM protein family that contain a zinc finger motif. Moreover, Ntlim1 had a specific DNA-binding ability and transiently activated transcription of a beta-glucuronidase reporter gene driven by the Pal-box sequence. The results of transient expression assays with tobacco cultured cells showed that fusion proteins between GFP and Ntlim1 can enter nuclei. Transgenic tobacco plants with antisense Ntlim1 showed low levels of transcripts from some key phenylpropanoid pathway genes such as phenylalanine ammonia-lyase, hydroxycinnamate CoA ligase and cinnamyl alcohol dehydrogenase. Furthermore, a greater than 20% reduction in lignin content was observed in transgenic tobacco with antisense Ntlim1.