SummaryXyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both`hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose ®bril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a signi®cant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-speci®c enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.

The Colorless non-ripening (Cnr) mutation in tomato (Solanum lycopersicum) results in mature fruits with colorless pericarp tissue showing an excessive loss of cell adhesion (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour [1999] Plant Physiol 120: 383-390). This pleiotropic mutation is an important tool for investigating the biochemical and molecular basis of cell separation during ripening. This study reports on the changes in enzyme activity associated with cell wall disassembly in Cnr and the effect of the mutation on the program of ripening-related gene expression. Real-time PCR and biochemical analysis demonstrated that the expression and activity of a range of cell wall-degrading enzymes was altered in Cnr during both development and ripening. These enzymes included polygalacturonase, pectinesterase (PE), galactanase, and xyloglucan endotransglycosylase. In the case of PE, the protein product of the ripening-related isoform PE2 was not detected in the mutant. In contrast with wild type, Cnr fruits were rich in basic chitinase and peroxidase activity. A microarray and differential screen were used to profile the pattern of gene expression in wild-type and Cnr fruits. They revealed a picture of the gene expression in the mutant that was largely consistent with the real-time PCR and biochemical experiments. Additionally, these experiments demonstrated that the Cnr mutation had a profound effect on many aspects of ripening-related gene expression. This included a severe reduction in the expression of ripening-related genes in mature fruits and indications of premature expression of some of these genes in immature fruits. The program of gene expression in Cnr resembles to some degree that found in dehiscence or abscission zones. We speculate that there is a link between events controlling cell separation in tomato, a fleshy fruit, and those involved in the formation of dehiscence zones in dry fruits.Colorless non-ripening (Cnr) is a pleiotropic dominant mutation of tomato (Solanum lycopersicum) that results in fruits with a white pericarp displaying much reduced cell-to-cell adhesion (Thompson et al., 1999;Fraser et al., 2001). The Cnr locus has been mapped to the middle of the long arm of chromosome 2 and is currently the subject of a map-based cloning exercise (Tor et al., 2002). We have recently isolated and sequenced a region of tomato chromosome 2 that cosegregates with the Cnr locus and are testing a candidate gene at this locus (K. Manning, J.J. Giovannoni, and G.B. Seymour, unpublished data).The loss of cell adhesion in Cnr appears to be due principally to modifications in cell wall structure. Sections of Cnr pericarp tissue show obvious changes in comparison with wild-type fruits, including larger intercellular spaces and thinner cell walls in ripe fruits (Orfila et al., 2001). Mechanical tests on pericarp tissue have revealed that the force required for cell wall failure is greater in Cnr, while tests on cell wall preparations showed ...

Acyl-carrier protein (ACP) is a key component involved in the regulation of fatty acid biosynthesis in plants. cDNA clones encoding ACP from Brassica napus (oil seed rape) embryos have been isolated using oligonucleotide probes derived from heterologous ACPs. Analysis of the DNA sequence data, in conjunction with N-terminal amino acid sequence data, revealed ACP to be synthesized from nuclear DNA as a precursor containing a 5 1 -amino-acid N-terminal extension.Immunocytochemical studies showed ACP to be localised solely within the plastids of B. nupus seed tissue and it would therefore appear that the N-terminal extension functions as a transit peptide to direct ACP into these organelles. Analysis of several cDNA clones revealed sequence heterogeneity and thus evidence for an ACP multigene family. From ten cDNA clones, six unique genes, encoding five different mature ACP polypeptides, were identified. Northern blot hybridisation studies provide evidence that the seed and leaf forms of rape ACP are encoded by structurally distinct gene sets.De novo synthesis of fatty acids is catalysed by fatty acid synthetase which consists of seven or eight catalytic domains. In animals [l] and yeast [2] the domains are present on one or two multifunctional polypeptide chains (type I fatty acid synthetase), which are localised within the cytoplasm. In contrast, in plants [3] the fatty acid synthetase domains exist as discreet, monofunctional activities (type 11) which are organellar in location.Some insight into the genetic regulation of type I1 fatty acid synthetase systems has recently been obtained through cloning of genes from both yeast [4] and mammals [5]. Our interest lies in understanding the genetic control of fatty acid biosynthesis in plants, in particular within developing oil seeds. As a first step towards that objective we report here the molecular cloning of cDNA encoding seed-expressed acylcarrier protein (ACP) from Brassica napus (oil seed rape).ACP is a key component of the plant Fatty acid biosynthetic machinery, serving both as a component of fatty acid synthetase and also as an acyl donor in desaturation and acyltransfer reactions [6]. Recent studies have shown two major ACP isoforms to be expressed in leaf tissue [7, 81, but apparently only one major isoform in seeds [8]. To date, characterisation of plant ACP has been largely confined to the leafexpressed forms. Thus, spinach [S] and barley [7] isoforms have been purified and N-terminal analysis suggests that, in both species, the isoforms are products of distinct genes. Using ACP as a representative marker protein, the site of fatty acid biosynthesis in leaves has been identified as the chloroplast [9]. In developing soybean seeds, ACP levels increase in close correlation with storage lipid synthesis [lo] suggestive of a regulatory role for ACP in this process. Despite this important role, ACP has not previously been localised within, or purified from, a seed source.This paper provides the first insight into the origin, structure and expression of gene...

Background and aims Tea is a strong accumulator of both aluminium (Al) and fluoride (F). We tested the hypothesis that Al helps detoxify F in tea plants by forming Al-F complexes. Methods Tea plants were grown hydroponically with a range of Al and F concentrations and in a soil pot experiment with amendments of NaF and acids. Growth and the uptake of F and Al were determined. Chemical species of F in the nutrient solutions and the cell saps of roots and leaves were determined by 19 F NMR. ResultsIn hydroponic experiments, F inhibited the growth of new roots and shoot tips in the absence of Al in the nutrient solutions, whereas Al stimulated root growth and alleviated the toxicity of F. Aluminium generally increased F concentration in roots but decreased F concentration in leaves. Geochem-PC calculations and 19 F NMR showed the presence of AlF 2+ , AlF 2 + and AlF 3 0 in the nutrient solutions when Al and F were present. Aluminium markedly decreased the NMR peak of free F in the cell saps from roots and leaves. An Al-F complex likely to be AlF 2+ was detected in the leaf cell sap from the plants treated with both F and Al. In the soil pot experiment, F caused leaf necrosis when the leaf Al to F molar ratio was smaller than 1. Conclusions Tea plants are sensitive to F toxicity in the absence of Al. Aluminium alleviates F toxicity in tea by forming Al-F complexes.