Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(1-->4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K(m) values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal=Galbeta(1-->4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.     

Transformation of antisense constructs of the chalcone synthase gene superfamily into Gerbera hybrida: differential effect on the expression of family members

Suppression of gene expression using antisense technology has been successful in various applications. In this paper we report differential inhibition of gene expression of the chalcone synthase (chs) gene superfamily members in transgenic Gerbera hybrida (Asteraceae) plants. We have transformed two different cDNAs of the chs gene family, gchs 1 [4] and gchs2, in antisense orientation under control of the CaMV 35S promoter into gerbera. Gchs1 codes for an enzyme with chalcone synthase activity while gchs2 is a more diverged member of the gene family having distinct structure and expression pattern. Furthermore, gchs2 is evidently not involved in anthocyanin synthesis and encodes an enzyme with novel catalytic properties. In both cases effective blocking of the resident sense gene expression was detected. In addition, the transformation affected differentially the expression of other members of the chs gene family. The degree of inhibition appeared to depend on the sequence homology between the antisense and the target genes. In the unevenly coloured inflorescences detected among anti-gchs1 transformants during their growth, relaxation of the antisense effect was here shown to start from the most distant member of the gene family, further demonstrating the influence of sequence homology in the stability of antisense inhibition.     

Interactions of Cd with Zn, Cu, Mn and Fe for lettuce (Lactuca sativa L.) in hydroponic culture

Abstract. The effect and accumulation of cadmium (Cd) in lettuce grown by means of the hydroponic technique was investigated by multivariate analysis, and was found to be affected by the concentration of other trace elements. Particularly iron acted in a strongly antagonistic way against Cd. Consequently, no absolute toxicity limits for Cd can be drawn without considering other trace elements.     

The distribution of C4 species of the Cyperaceae in North America in relation to climate

Summary The percent of species of the Cyperaceae possessing the C₄ pathway was calculated for twenty-five regions of North America. A stepwise multiple regression analysis was used to determine which climatic variables were most highly correlated with the percent C₄ species among the various regions. The square of the mean July minimum daily temperature exhibited the strongest correlation with the transformed percentage of C₄ species. It appears that the relationship to climatic variables of the C₄ species of the Cyperaceae is similar, but not identical to the relationship previously reported for C₄ species of the Gramineae.     

Radiation-induced improvement of the freeze-resistance of a cold-sensitive wheat variety: Freeze-tolerance and fatty acid patterns

Effects of 3–15 krad ⁶⁰Co gamma radiation on cereal seeds were investigated with regard to the frost hardiness of leaves of 5–7-day-old seedlings. Comparative studies were carried out on the gas-chromatographically determined distribution patterns of fatty acids in different pools (total fatty acid, total lipid, polar lipids) of a cold-resistant (cv. Mironowskaya 808) and a cold-sensitive (cv. Penjamo 62) cultivar of wheat (Triticum aestivum L.). Parallelism between fatty acid distribution pattern, empirical parameters suggested for quantitative measures of cold tolerance (the ratio unsat/sat and double-bond index), and the low-temperature behaviour of shoots grown from radiation treated seeds of ‘Penjamo 62’, was also examined. To monitor differences in the fatty acid syntheses of ‘Mironowskaya 808’and ‘Penjamo 62’, and to demonstrate radiation-induced changes in fatty acid turnover a [1–¹⁴C]-acetate incorporation technique was employed. The results of practical importance are: 1. A significant improvement in the frost-resistance of the cold-sensitive ‘Penjamo 62’variety could be achieved with 6–9 krad irradiation, the half-freeze-killing temperature dropping from ?6 to about ?18°C. 2. Freeze-hardiness, no matter whether inherited or gained, could be abolished by gamma irradiation with higher dose. The following conclusions of theoretical interest can be drawn: 1. Low-temperature behaviour of plant tissues is a dynamic property rather than a static one, not only the formation but also the breakdown of certain hydrocarbon chains may be of paramount relevance in cold-tolerance. 2. Not the linolenic (18:3) component, but rather the overall distribution pattern of the C₁₈ family seems to correlate with low-temperature-responses of shoots. 3. Empirical parameters investigated do not provide reliable quantitative measures of the susceptibility to freeze (and cold-) injuries. 4. Changes in the syntheses of some key proteins (e.g. peroxidase, water-soluble proteins, etc.) may also be important in adaptation to low-temperature conditions.     

Hardiness dependent accumulation of phospholipids in leaves of wheat cultivars

Phospholipid content and composition in the leaves of thirteen cultivars of wheat differing in frost hardiness, were compared before and after hardening in order to see whether phospholipids play a role in surviving at low temperatures. The content of phospholipids in the leaves at the end of the hardening could be related to the aquired hardiness, and the relationship between the two phenomena could be described by the regression equation y = (0.174 ± 0.0016) x + 1311. Accumulation of phospholipids represents a hardiness dependent augmentation of membranes. Phosphatidylcholine appeared to be preferentially accumulated in hardy cultivars during the hardening process. As there are no significant differences in phospholipid fatty acid compositions investigated earlier, these results suggest the importance of the polar head group composition of membranes in avoiding frost injury.