Zoran Minic

Physiological roles of plant glycoside hydrolases

The functions of plant glycoside hydrolases and transglycosidases have been studied using different biochemical and molecular genetic approaches. These enzymes are involved in the metabolism of various carbohydrates containing compounds present in the plant tissues. The structural and functional diversity of the carbohydrates implies a vast spectrum of enzymes involved in their metabolism. Complete genome sequence of Arabidopsis and rice has allowed the classification of glycoside hydrolases in different families based on amino acid sequence data. The genomes of these plants contain 29 families of glycoside hydrolases. This review summarizes the current research on plant glycoside hydrolases concerning their principal functional roles, which were attributed to different families. The majority of these plant glycoside hydrolases are involved in cell wall polysaccharide metabolism. Other functions include their participation in the biosynthesis and remodulation of glycans, mobilization of energy, defence, symbiosis, signalling, secondary plant metabolism and metabolism of glycolipids.

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

This work describes the purification and characterization of enzymes that exhibit β-d-xylosidase activity in stem tissues of Arabidopsis. This is the first detailed investigation that concerns the characterization of catalytic properties and sequence identity of enzymes with β-d-xylosidase activities in a dicotyledonous plant. Three different enzymes, ARAf, XYL4, and XYL1 with apparent molecular masses of 75, 67, and 64 kD, respectively, were purified to homogeneity. ARAf was identified as a putative α-l-arabinofuranosidase, and XYL4 and XYL1 as putative β-d-xylosidases using matrix-assisted laser-desorption ionization time of flight. ARAf belongs to family 51 and XYL4 and XYL1 to family 3 of glycoside hydrolases. ARAf and XYL1 have highest specificity for p-nitrophenyl-α-l-arabinofuranoside and XYL4 for p-nitrophenyl-β-d-xylopyranoside and natural substrates such as xylobiose and xylotetraose. XYL4 was shown to release mainly d-Xyl from oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, and oligoarabinoxylans. ARAf and XYL1 can also release d-Xyl from these substrates but less efficiently than XYL4. Moreover, they can also release l-Ara from arabinoxylans and arabinan. Overall, the results indicate that XYL4 possesses enzymatic specificity characteristic for a β-d-xylosidase, while ARAf and XYL1 act as bifunctional α-l-arabinofuranosidase/β-d-xylosidases. Analysis of the activity of these three enzymes in stem tissues at different stages of development has shown that young stems possess the highest activities for all three enzymes in comparison to the activities of the enzymes present in stems at older stages of development. High enzyme activities are most likely related to the necessary modifications of cell wall structure occurring during plant growth.

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes.

BackgroundDifferent strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins.ResultsTranscriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics.ConclusionAnalysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins.

Cell Wall Modifications in Arabidopsis Plants with Altered α-l-Arabinofuranosidase Activity

Although cell wall remodeling is an essential feature of plant growth and development, the underlying molecular mechanisms are poorly understood. This work describes the characterization of Arabidopsis (Arabidopsis thaliana) plants with altered expression of ARAF1, a bifunctional α-l-arabinofuranosidase/β-d-xylosidase (At3g10740) belonging to family 51 glycosyl-hydrolases. ARAF1 was localized in several cell types in the vascular system of roots and stems, including xylem vessels and parenchyma cells surrounding the vessels, the cambium, and the phloem. araf1 T-DNA insertional mutants showed no visible phenotype, whereas transgenic plants that overexpressed ARAF1 exhibited a delay in inflorescence emergence and altered stem architecture. Although global monosaccharide analysis indicated only slight differences in cell wall composition in both mutant and overexpressing lines, immunolocalization experiments using anti-arabinan (LM6) and anti-xylan (LM10) antibodies indicated cell type-specific alterations in cell wall structure. In araf1 mutants, an increase in LM6 signal intensity was observed in the phloem, cambium, and xylem parenchyma in stems and roots, largely coinciding with ARAF1 expression sites. The ectopic overexpression of ARAF1 resulted in an increase in LM10 labeling in the secondary walls of interfascicular fibers and xylem vessels. The combined ARAF1 gene expression and immunolocalization studies suggest that arabinan-containing pectins are potential in vivo substrates of ARAF1 in Arabidopsis.

Cell wall modifications in Arabidopsis thaliana plants with altered α-Larabinofuranosidase activity

Although cell wall remodeling is an essential feature of plant growth and development, the underlying molecular mechanisms are poorly understood. This work describes the characterization of Arabidopsis (Arabidopsis thaliana) plants with altered expression of ARAF1, a bifunctional α-l-arabinofuranosidase/β-d-xylosidase (At3g10740) belonging to family 51 glycosyl-hydrolases. ARAF1 was localized in several cell types in the vascular system of roots and stems, including xylem vessels and parenchyma cells surrounding the vessels, the cambium, and the phloem. araf1 T-DNA insertional mutants showed no visible phenotype, whereas transgenic plants that overexpressed ARAF1 exhibited a delay in inflorescence emergence and altered stem architecture. Although global monosaccharide analysis indicated only slight differences in cell wall composition in both mutant and overexpressing lines, immunolocalization experiments using anti-arabinan (LM6) and anti-xylan (LM10) antibodies indicated cell type-specific alterations in cell wall structure. In araf1 mutants, an increase in LM6 signal intensity was observed in the phloem, cambium, and xylem parenchyma in stems and roots, largely coinciding with ARAF1 expression sites. The ectopic overexpression of ARAF1 resulted in an increase in LM10 labeling in the secondary walls of interfascicular fibers and xylem vessels. The combined ARAF1 gene expression and immunolocalization studies suggest that arabinan-containing pectins are potential in vivo substrates of ARAF1 in Arabidopsis.

Cloning, purification and characterisation of brassinin glucosyltransferase, a phytoalexin-detoxifying enzyme from the plant pathogen Sclerotinia sclerotiorum

The plant-pathogenic fungus Sclerotinia sclerotiorum can detoxify cruciferous phytoalexins such as brassinin via glucosylation. Here we describe a multifaceted approach including genome mining, transcriptional induction, phytoalexin quantification, protein expression and enzyme purification that led to identification of a S. sclerotiorum glucosyltransferase that detoxifies brassinin. Transcription of this gene, denoted as brassinin glucosyltransferase 1 (SsBGT1), was induced significantly in response to the cruciferous phytoalexins camalexin, cyclobrassinin, brassilexin, brassinin and 3-phenylindole, a camalexin analogue. This gene was also up-regulated during infection of Brassica napus leaves. Levels of brassinin decreased significantly between 48 and 72h post-inoculation, with a concomitant increase in levels of 1-beta-d-glucopyranosylbrassinin, the product of the reaction catalysed by SsBGT1. These findings strongly implicate the involvement of this gene during infection of B. napus. This gene was cloned and expressed in Saccharomyces cerevisiae. The purified recombinant enzyme was able to glucosylate brassinin and two other phytoalexins, albeit much less effectively. This is the first report of a fungal gene involved in detoxification of plant defence molecules via glucosylation.

Brassinin oxidase, a fungal detoxifying enzyme to overcome a plant defense -- purification, characterization and inhibition.

Blackleg fungi [Leptosphaeria maculans (asexual stage Phoma lingam) and Leptosphaeria biglobosa] are devastating plant pathogens with well‐established stratagems to invade crucifers, including the production of enzymes that detoxify plant defenses such as phytoalexins. The significant roles of brassinin, both as a potent crucifer phytoalexin and a biosynthetic precursor of several other plant defenses, make it critical to plant fitness. Brassinin oxidase, a detoxifying enzyme produced by L. maculans both in vitro and in planta, catalyzes the detoxification of brassinin by the unusual oxidative transformation of a dithiocarbamate to an aldehyde. Purified brassinin oxidase has an apparent molecular mass of 57 kDa, is approximately 20% glycosylated, and accepts a wide range of cofactors, including quinones and flavins. Purified brassinin oxidase was used to screen a library of brassinin analogues and crucifer phytoalexins for potential inhibitory activity. Unexpectedly, it was determined that the crucifer phytoalexins camalexin and cyclobrassinin are competitive inhibitors of brassinin oxidase. This discovery suggests that camalexin could protect crucifers from attacks by L. maculans because camalexin is not metabolized by this pathogen and is a strong mycelial growth inhibitor.

Purification, Cloning and Functional Characterization of an Endogenous beta-Glucuronidase in Arabidopsis thaliana

Beta-glucuronidase (GUS) activities have been extensively characterized in bacteria, fungi, and animals, and the bacterial enzyme GUSA from Escherichia coli is commonly used as a reporter for gene expression studies in plants. Although endogenous GUS activity has been observed in plants, the nature and function of the enzymes involved remain elusive. Here we report on tissue-specific localization, partial purification and identification of AtGUS2, a GUS active under acidic conditions from Arabidopsis thaliana. This enzyme belongs to the GH79 family in the Carbohydrate-Active Enzymes database, which also includes mammalian heparanases that degrade the carbohydrate moieties of cell surface proteoglycans, and fungal enzymes active on arabinogalactan proteins (AGPs). We characterized a knockout insertion line (atgus2-1) and transgenic lines overexpressing AtGUS2 (Pro(35S):AtGUS2). Endogenous GUS activity assayed histochemically and biochemically was absent in atgus2-1 tissues and four times higher in Pro(35S):AtGUS2 lines. AGPs purified from atgus2-1 and Pro(35S):AtGUS2 seedlings showed higher and markedly lower glucuronic acid content, respectively. Our results suggest that endogenous GUS activity influences the sugar composition of the complex polysaccharide chains of AGPs. We also show that transgenics display hypocotyl and root growth defects compared to wild-type plants. Hypocotyl and root lengths are increased in Pro(35S):AtGUS2 seedlings, whereas hypocoyl length is reduced in atgus2-1 seedlings. These data are consistent with a role for the carbohydrate moieties of AGPs in cell growth.

Substrate specificity and inhibition of brassinin hydrolases, detoxifying enzymes from the plant pathogens Leptosphaeria maculans and Alternaria brassicicola.

Blackleg (Leptosphaeria maculans and Leptosphaeria biglobosa) and black spot (Alternaria brassicicola) fungi are devastating plant pathogens known to detoxify the plant defence metabolite, brassinin. The significant roles of brassinin as a crucifer phytoalexin and as a biosynthetic precursor of several other plant defences make it important in plant fitness. Brassinin detoxifying enzymes produced by L. maculans and A. brassicicola catalyse the detoxification of brassinin by hydrolysis of its dithiocarbamate group to indolyl‐3‐methanamine. The purification and characterization of brassinin hydrolases produced by L. maculans (BHLmL2) and A. brassicicola (BHAb) were accomplished: native BHLmL2 was found to be a tetrameric protein with a molecular mass of 220 kDa, whereas native BHAb was found to be a dimeric protein of 120 kDa. Protein characterization using LC‐MS/MS and sequence alignment analyses suggested that both enzymes belong to the family of amidases with the catalytic Ser/Ser/Lys triad. Furthermore, chemical modification of BHLmL2 and BHAb with selective reagents suggested that the amino acid serine was involved in the catalytic activity of both enzymes. The overall results indicated that BHs have new substrate specificities with a new catalytic activity that can be designated as dithiocarbamate hydrolase. Investigation of the effect of various phytoalexins on the activities of BHLmL2 and BHAb indicated that cyclobrassinin was a competitive inhibitor of both enzymes. On the basis of pH dependence, sequence analyses, chemical modifications of amino acid residues and identification of headspace volatiles, a chemical mechanism for hydrolysis of the dithiocarbamate group of brassinin catalysed by BHLmL2 and BHAb is proposed. The current information should facilitate the design of specific synthetic inhibitors of these enzymes for plant treatments against blackleg and black spot fungal infections.

Cell Wall Proteome

In this chapter, we will focus on the contribution of proteomics to the identification and determination of the structure and function of CWPs as well as discussing new perspectives in this area. The great variety of proteins found in the plant cell wall is described. Some families, such as glycoside hydrolases, proteases, lectins, and inhibitors of cell wall modifying enzymes, are discussed in detail. Examples of the use of proteomic techniques to elucidate the structure of various cell wall proteins, especially with post-translational modifications such as Nglycosylations, proline hydroxylation and O-glycosylations, addition of GPI anchors, and phosphorylation, are given. Finally, the emerging understanding of the functions of cell wall proteins is discussed, as well as proposals for future research.