Rosa Casado

Overall Alteration of Circadian Clock Gene Expression in the Chestnut Cold Response

Cold acclimation in woody plants may have special features compared to similar processes in herbaceous plants. Recent studies have shown that circadian clock behavior in the chestnut tree (Castanea sativa) is disrupted by cold temperatures and that the primary oscillator feedback loop is not functional at 4°C or in winter. In these conditions, CsTOC1 and CsLHY genes are constantly expressed. Here, we show that this alteration also affects CsPRR5, CsPRR7 and CsPRR9. These genes are homologous to the corresponding Arabidopsis PSEUDO-RESPONSE REGULATOR genes, which are also components of the circadian oscillator feedback network. The practically constant presence of mRNAs of the 5 chestnut genes at low temperature reveals an unknown aspect of clock regulation and suggests a mechanism regulating the transcription of oscillator genes as a whole.

Purification and in vitro chaperone activity of a class I small heat-shock protein abundant in recalcitrant chestnut seeds.

A 20-kD protein has been purified from cotyledons of recalcitrant (desiccation-sensitive) chestnut (Castanea sativa) seeds, where it accumulates at levels comparable to those of major seed storage proteins. This protein, termed Cs smHSP 1, forms homododecameric complexes under nondenaturing conditions and appears to be homologous to cytosolic class I small heat-shock proteins (smHSPs) from plant sources. In vitro evidence has been obtained that the isolated protein can function as a molecular chaperone: it increases, at stoichiometric levels, the renaturation yields of chemically denatured citrate synthase and also prevents the irreversible thermal inactivation of this enzyme. Although a role in desiccation tolerance has been hypothesized for seed smHSPs, this does not seem to be the case for Cs smHSP 1. We have investigated the presence of immunologically related proteins in orthodox and recalcitrant seeds of 13 woody species. Our results indicate that the presence of Cs smHSP 1-like proteins, even at high levels, is not enough to confer desiccation tolerance, and that the amount of these proteins does not furnish a reliable criterion to identify desiccation-sensitive seeds. Additional proteins or mechanisms appear necessary to keep the viability of orthodox seeds upon shedding.

Seed storage protein from Pinus pinaster Ait.: homology of major components with 11S proteins from angiosperms

Abstract Pinus pinaster Ait. store glutelins as major seed proteins. Their structures are dimeric with two subunits of 21 and 34 kDa joined together by disulphide bridges. These subunits show considerable heterogeneity and are both basic. Monospecific polyclonal antibodies raised against purified 21-kDa subunits show cross-reactivity with 20–22-kDa proteins from pea and soybean globulins. The N-terminal sequence of the 21-kDa subunit is homologous with β subunits of angiosperm 11S globulins. The globulin fraction also represents a high percentage (26%) of the total protein content of P. pinaster seeds. Two classes of protein have been separated from this fraction: one class whose proteins have molecular weights of 150–200 kDa composed of 22–23, 27 and 47 kDa monomers that are not linked by disulphide bridges and another class of dimeric proteins of 14–18 kDa, whose monomers are joined by disulphide bridges.

BACTERIAL EXPRESSION OF AN ACTIVE CLASS IB CHITINASE FROM CASTANEA SATIVA COTYLEDONS

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.

Seed storage proteins in Fagaceae: similarity between Castanea globulins and Quercus glutelins

Abstract Castanea sativa Mill. and C. crenata Lieb. and Zucc. store 11S globulins as major seed proteins while Quercus ilex L. and Q. robur L., also members of the Fagaceae family, accumulate glutelins. The structure of the 11S Castanea globulins, which is similar to the accepted model for this class of proteins, has been studied by different two-dimensional electrophoretic methods. The oligomer has a M r of 240 000–260 000, and the pairs of acidic-basic subunits, which form it, show great heterogeneity. The Quercus glutelins are made up of dimers linked by disulphide bridges, and their characteristics indicate a similarity to the subunits of 11S Castanea globulins. We suggest that, as in the case of rice glutelins, Quercus glutelins can also be considered legumin-like proteins.

Identification of a homolog of Arabidopsis DSP4 (SEX4) in chestnut: its induction and accumulation in stem amyloplasts during winter or in response to the cold

Oligosaccharide synthesis is an important cryoprotection strategy used by woody plants during winter dormancy. At the onset of autumn, starch stored in the stem and buds is broken down in response to the shorter days and lower temperatures resulting in the buildup of oligosaccharides. Given that the enzyme DSP4 is necessary for diurnal starch degradation in Arabidopsis leaves, this study was designed to address the role of DSP4 in this seasonal process in Castanea sativa Mill. The expression pattern of the CsDSP4 gene in cells of the chestnut stem was found to parallel starch catabolism. In this organ, DSP4 protein levels started to rise at the start of autumn and elevated levels persisted until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsDSP4 gene. In dormant trees or cold-stressed plantlets, the CsDSP4 protein was immunolocalized both in the amyloplast stroma and nucleus of stem cells, whereas in the conditions of vegetative growth, immunofluorescence was only detected in the nucleus. The studies indicate a potential role for DSP4 in starch degradation and cold acclimation following low temperature exposure during activity-dormancy transition.