High-throughput functional assessment of polysaccharide-active enzymes using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry as exemplified on plant cell wall polysaccharides.

Despite a wealth of sequence information on genes encoding carbohydrate-active enzymes (e.g., transferases, esterases, hydrolases), very few of these enzymes have been described in detail, particularly regarding substrate specificities. A facile and rapid method for the characterization of substrate specificities of polysaccharide-active enzymes that uses matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) has been developed. This method has been applied to characterize a xyloglucan fucosyltransferase and a pectin methyl-esterase. Reactions were performed in liquid phase, and aliquots of the reaction mixtures were spotted on a polyvinylidene fluoride (PVDF) membrane. Reaction products were precipitated onto the membrane and cleaned by treatment with an ethanol-water mixture. Subsequently, the reaction products were hydrolyzed by specific endoglycanases, and the resulting oligosaccharides were directly analyzed onto the PVDF membrane by MALDI-TOF MS. The new method is amenable to high-throughput analysis and, thus, constitutes an emerging avenue to rapidly fill the gap in our knowledge of the specificities of polysaccharide-active enzymes.

Biochemical and immunohistochemical analysis of pectic polysaccharides in the cell walls of Arabidopsis mutant QUASIMODO 1 suspension-cultured cells: implications for cell adhesion.

Mutation in the Arabidopsis thaliana QUASIMODO 1 gene (QUA1), which encodes a putative glycosyltransferase, reduces cell wall pectin content and cell adhesion. Suspension-cultured calli were generated from roots of wild-type (wt) and qua1-1 A. thaliana plants. The altered cell adhesion phenotype of the qua1-1 plant was also found with its suspension-cultured calli. Cell walls of both wt and qua1-1 calli were analysed by chemical, enzymatic and immunohistochemical techniques in order to assess the role of pectic polysaccharides in the mutant phenotype. Compared with the wt, qua1-1 calli cell walls contained more arabinose (23.6 versus 21.6 mol%), rhamnose (3.1 versus 2.7 mol%), and fucose (1.4 versus 1.2 mol%) and less uronic acid (24.2 versus 27.6 mol%), and they were less methyl-esterified (DM: 22.9% versus 30.3%). When sequential pectin extraction of calli cell walls was performed, qua1-1 water-soluble and chelator-soluble extracts contained more arabinose and less uronic acid than wt. Water-soluble pectins were less methyl-esterified in qua1-1 than in wt. Chelator-soluble pectins were more acetyl-esterified in qua1-1. Differences in the cell wall chemistry of wt and mutant calli were supported by a reduction in JIM7 labelling (methyl-esterified homogalacturonan) of the whole wall in small cells and particularly by a reduced labelling with 2F4 (calcium-associated homogalacturonan) in the middle lamella at tricellular junctions of large qua1-1 cells. Differences in the oligosaccharide profile obtained after endopolygalacturonase degradation of alkali extracts from qua1-1 and wt calli indicated variations in the structure of covalently bonded homogalacturonan. About 29% more extracellular polymers rich in pectins were recovered from the calli culture medium of qua1-1 compared with wt. These results show that perturbation of QUASIMODO 1-1 gene expression in calli resulted in alterations of homogalacturonan content and cell wall location. The consequences of these structural variations are discussed with regard to plant cell adhesion.

Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.).

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5' flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, (15)NH4(+) was incorporated into [5-(15)N]glutamine and [2-(15)N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2-(15)N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2-(15)N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15-20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO2 contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation.

Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths.

Changes in the composition and structure of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Arabidopsis thaliana microcalli. Three growth phases, namely the cell division phase, the cell expansion phase, and the stationary phase, were distinguished and associated with a decreasing cell cluster adhesion strength. Degradation of the homogalacturonan pectic backbone and of linear pectic side chains (1,4)-beta-D-galactan were observed concomitantly with the cell expansion and stationary phases and the decrease in cell adhesion. Also, in the stationary phase, branched (1,5)-alpha-L-arabinans were linearized. The AGP content of the culture medium increased while it decreased in the cell wall during cell growth and as cell adhesion decreased. These data suggest that, in addition to homogalacturonan, pectic side chains and AGP are involved in plant cell development and particularly in cell-cell attachment.

QUASIMODO1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion in Arabidopsis.

Pectins are a highly complex family of cell wall polysaccharides. As a result of a lack of specific mutants, it has been difficult to study the biosynthesis of pectins and their role in vivo. We have isolated two allelic mutants, named quasimodo1 (qua1-1 and qua1-2), that are dwarfed and show reduced cell adhesion. Mutant cell walls showed a 25% reduction in galacturonic acid levels compared with the wild type, indicating reduced pectin content, whereas neutral sugars remained unchanged. Immersion immunofluorescence with the JIM5 and JIM7 monoclonal antibodies that recognize homogalacturonan epitopes revealed less labeling of mutant roots compared with the wild type. Both mutants carry a T-DNA insertion in a gene (QUA1) that encodes a putative membrane-bound glycosyltransferase of family 8. We present evidence for the possible involvement of a glycosyltransferase of this family in the synthesis of pectic polysaccharides, suggesting that other members of this large multigene family in Arabidopsis also may be important for pectin biosynthesis. The mutant phenotype is consistent with a central role for pectins in cell adhesion.