A Genomewide Analysis of the Cinnamyl Alcohol Dehydrogenase Family in Sorghum [Sorghum bicolor (L.) Moench] Identifies SbCAD2 as the Brown midrib6 Gene

The content and composition of the plant cell wall polymer lignin affect plant fitness, carbon sequestration potential, and agro-industrial processing. These characteristics, are heavily influenced by the supply of hydroxycinnamyl alcohol precursors synthesized by the enzyme cinnamyl alcohol dehydrogenase (CAD). In angiosperms, CAD is encoded by a multigene family consisting of members thought to have distinct roles in different stages of plant development. Due to the high sequence similarity among CAD genes, it has been challenging to identify and study the role of the individual genes without a genome sequence. Analysis of the recently released sorghum genome revealed the existence of 14 CAD-like genes at seven genomic locations. Comparisons with maize and rice revealed subtle differences in gene number, arrangement, and expression patterns. Sorghum CAD2 is the predominant CAD involved in lignification based on the phylogenetic relationship with CADs from other species and genetic evidence showing that a set of three allelic brown midrib (bmr) lignin mutants contained mutations in this gene. The impact of the mutations on the structure of the protein was assessed using molecular modeling based on X-ray crystallography data of the closely related Arabidopsis CAD5. The modeling revealed unique changes in structure consistent with the observed phenotypes of the mutants.     

Evidence for a role of AtCAD 1 in lignification of elongating stems of Arabidopsis thaliana

The cinnamyl alcohol dehydrogenase (AtCAD) multigene family in Arabidopsis is composed of nine genes. Our previous studies focused on the two isoforms AtCAD C and AtCAD D which show a high homology to those related to lignification in other plants. This study focuses on the seven other Arabidopsis CAD for which functions are not yet elucidated. Their expression patterns were determined in different parts of Arabidopsis. Only CAD 1 protein can be detected in elongating stems, flowers, and siliques using Western-blot analysis. Tissue specific expression of CAD 1, B1, and G genes was determined using their promoters fused to the GUS reporter gene. CAD 1 expression was observed in primary xylem in accordance with a potential role in lignification. Arabidopsis T-DNA mutants knockout for the different genes CAD genes were characterized. Their stems displayed no substantial reduction of CAD activities for coniferyl and sinapyl alcohols as well as no modifications of lignin quantity and structure in mature inflorescence stems. Only a small reduction of lignin content could be observed in elongating stems of Atcad 1 mutant. These CAD genes in combination with the CAD D promoter were used to complement a CAD double mutant severely altered in lignification (cad c cad d). The expression of AtCAD A, B1, B2, F, and G had no effect on restoring a normal lignin profile of this mutant. In contrast, CAD 1 complemented partly this mutant as revealed by the partial restoration of conventional lignin units and by the decrease in the frequency of β-O-4 linked p-OH cinnamaldehydes.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Transcriptome-based identification of genes revealed differential expression profiles and lignin accumulation during root development in cultivated and wild carrots

Key message

Carrot root development associates lignin deposition and regulation.

Abstract

Carrot is consumed worldwide and is a good source of nutrients. However, excess lignin deposition may reduce the taste and quality of carrot root. Molecular mechanisms underlying lignin accumulation in carrot are still lacking. To address this problem, we collected taproots of wild and cultivated carrots at five developmental stages and analyzed the lignin content and characterized the lignin distribution using histochemical staining and autofluorescence microscopy. Genes involved in lignin biosynthesis were identified, and their expression profiles were determined. Results showed that lignin was mostly deposited in xylem vessels of carrot root. In addition, lignin content continuously decreased during root development, which was achieved possibly by reducing the expression of the genes involved in lignin biosynthesis. Carrot root may also prevent cell lignification to meet the demands of taproot growth. Our results will serve as reference for lignin biosynthesis in carrot and may also assist biologists to improve carrot quality.

     

Candidate genes and QTLs for fruit ripening and softening in melon

Different factors affect the quality of melon fruit and among them long shelf life is critical from the consumer’s point of view. In melon, cultivars showing both climacteric and non-climacteric ripening types are found. In this study we have investigated climacteric ripening and fruit softening using a collection of near-isogenic lines (NILs) derived from the non-climacteric melon parental lines PI 161375 (SC) and “Piel de Sapo” (PS). Surprisingly, we found that QTL eth3.5 in NIL SC3-5b induced a climacteric-ripening phenotype with increased respiration and ethylene levels. Data suggest that the non-climacteric phenotypes from PI 161375 and “Piel de Sapo” may be the result of mutations in different genes. Several QTLs for fruit flesh firmness were also detected. Candidate genes putatively involved in ethylene regulation, biosynthesis and perception and cell wall degradation were mapped and some colocations with QTLs were observed. These results may provide additional data towards understanding of non-climacteric ripening in melon.     

Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenyl- propanoids

Lignins are complex natural polymers resulting from oxidative coupling of, primarily, 4-hydroxyphenylpropanoids. An understanding of their nature is evolving as a result of detailed structural studies, recently aided by the availability of lignin-biosynthetic-pathway mutants and transgenics. The currently accepted theory is that the lignin polymer is formed by combinatorial-like phenolic coupling reactions, via radicals generated by peroxidase-H₂O₂, under simple chemical control where monolignols react endwise with the growing polymer. As a result, the actual structure of the lignin macromolecule is not absolutely defined or determined. The ``randomness'' of linkage generation (which is not truly statistically random but governed, as is any chemical reaction, by the supply of reactants, the matrix, etc.) and the astronomical number of possible isomers of even a simple polymer structure, suggest a low probability of two lignin macromolecules being identical. A recent challenge to the currently accepted theory of chemically controlled lignification, attempting to bring lignin into line with more organized biopolymers such as proteins, is logically inconsistent with the most basic details of lignin structure. Lignins may derive in part from monomers and conjugates other than the three primary monolignols (p-coumaryl, coniferyl, and sinapyl alcohols). The plasticity of the combinatorial polymerization reactions allows monomer substitution and significant variations in final structure which, in many cases, the plant appears to tolerate. As such, lignification is seen as a marvelously evolved process allowing plants considerable flexibility in dealing with various environmental stresses, and conferring on them a striking ability to remain viable even when humans or nature alter ``required'' lignin-biosynthetic-pathway genes/enzymes. The malleability offers significant opportunities to engineer the structures of lignins beyond the limits explored to date. Abbreviations: 4CL – 4-coumarate:CoA ligase; C3H –p-coumarate 3-hydroxylase; HCT –p-hydroxycinnamoyl-CoA: quinate shikimate p-hydroxycinnamoyltransferase; CCoAOMT – caffeoyl-CoA O-methyltransferase; CCR – cinnamoyl-CoA reductase; F5H – ferulate 5-hydroxylase; CAld5H – coniferaldehyde 5-hydroxylase; COMT – caffeic acid O-methyltransferase; AldOMT – (5-hydroxyconifer)aldehyde O-methyltransferase; CAD – cinnamyl alcohol dehydrogenase; NMR – nuclear magnetic resonance (spectroscopy); DFRC – derivatization followed by reductive cleavage; TIZ – tosylation, iodination, zinc (a DFRC method); DHP – dehydrogenation polymer.     

Structure of the cinnamyl-alcohol dehydrogenase gene family in rice and promoter activity of a member associated with lignification

Analysis of lignification in rice has been facilitated by the availability of the recently completed rice genome sequence, and rice will serve as an important model for understanding the relationship of grass lignin composition to cell wall digestibility. Cinnamyl-alcohol dehydrogenase (CAD) is an enzyme important in lignin biosynthesis. The rice genome contains 12 distinct genes present at nine different loci that encode products with significant similarity to CAD. The rice gene family is diverse with respect to other angiosperm and gymnosperm CAD genes isolated to date and includes one member (OsCAD6) that contains a peroxisomal targeting signal and is substantially diverged relative to other family members. Four closely related family members (OsCAD8A–D) are present at the same locus and represent the product of a localized gene duplication and inversion. Promoter-reporter gene fusions to OsCAD2, an orthologue of the CAD gene present at the bm1 (brown midrib 1) locus of maize, reveal that in rice expression is associated with vascular tissue in aerial parts of the plant and is correlated with the onset of lignification. In root tissue, expression is primarily in the cortical parenchyma adjacent to the exodermis and in vascular tissue.     

LtuCAD1 Is a Cinnamyl Alcohol Dehydrogenase Ortholog Involved in Lignin Biosynthesis in Liriodendron tulipifera L., a Basal Angiosperm Timber Species

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis and catalyzes the final step in the synthesis of monolignols. Seven CAD homologs (LtuCAD1 to LtuCAD7) have been previously identified from a basal angiosperm species Liriodendron tulipifera L., which is an important timber tree species with significant ecological and economic values. The phylogenetic analysis indicates that LtuCAD1 is the only Liriodendron CAD grouped with the bona fide CADs, the primary CAD genes involved in lignification. In this study, the predicted protein sequence of LtuCAD1 was found to have conserved domains and the same key determinant site with the bona fide CADs in other plant species. Additionally, LtuCAD1 had the highest expression level in xylem as revealed by quantitative RT-PCR analysis. The expression of beta-glucuronidase (GUS) driven by the LtuCAD1 promoter was largely localized in vascular tissues in Arabidopsis. In stem cross sections, GUS staining was found exclusively in xylem and phloem. When expressed in the Arabidopsis cad4 cad5 double mutant, LtuCAD1 was able to restore the total lignin content and decrease the S/G lignin ratio. Our data indicate that LtuCAD1 is a CAD ortholog involved in lignin biosynthesis in Liriodendron.