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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., (© 2011 Blackwell Publishing Ltd.)

Heat shock, and other stresses that cause protein misfolding and aggregation, trigger the accumulation of heat shock proteins (HSPs) in virtually all organisms. Among the HSPs of higher plants, those belonging to the small HSP (sHSP) family remain the least characterized in functional terms. We analyzed the occurrence of sHSPs in vegetative organs of Castanea sativa (sweet chestnut), a temperate woody species that exhibits remarkable freezing tolerance. A constitutive sHSP subject to seasonal periodic changes of abundance was immunodetected in stems. This protein was identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry and internal peptide sequencing as CsHSP17.5, a cytosolic class I sHSP previously described in cotyledons. Expression of the corresponding gene in stems was confirmed through cDNA cloning and reverse transcription-PCR. Stem protein and mRNA profiles indicated that CsHSP17.5 is significantly up-regulated in spring and fall, reaching maximal levels in late summer and, especially, in winter. In addition, cold exposure was found to quickly activate shsp gene expression in both stems and roots of chestnut seedlings kept in growth chambers. Our main finding is that purified CsHSP17.5 is very effective in protecting the cold-labile enzyme lactate dehydrogenase from freeze-induced inactivation (on a molar basis, CsHSP17.5 is about 400 times more effective as cryoprotectant than hen egg-white lysozyme). Consistent with these observations, repeated freezing/thawing did not affect appreciably the chaperone activity of diluted CsHSP17.5 nor its ability to form dodecameric complexes in vitro. Taken together, these results substantiate the hypothesis that sHSPs can play relevant roles in the acquisition of freezing tolerance.

Mature chestnut seeds, with one of the highest moisture contents described to date, accumulate certain defensive proteins at unusually elevated levels. In this work a major 23-kDa thaumatin-like protein, termed CsTL1, has been purified from mature chestnut (Castanea sativa) cotyledons. Amino acid sequencing and characterization of its full-length cDNA indicate that CsTL1 is synthesized as a preprotein with a signal peptide 22 amino acids in length. The mature protein contains 16 conserved cysteine residues presumably involved in disulfide bonding and has a high isoelectric point (ca. 9). Unlike most basic pathogenesis-related (PR) proteins, mature CsTL1 is localized to the extracellular matrix, as revealed by immunoelectron microscopy studies of cotyledonary cells. The isolated protein has in vitro antifungal activity against Trichoderma viride and Fusarium oxysporum and shows strong synergistic effects with CsCh1, the most abundant chestnut cotyledon endochitinase. Moreover, both CsTL1 and CsCh1 appear to be regulated in the same manner during seed development and germination. These observations, along with the recent finding of endoglucanase activity for some TL proteins, support the notion that CsTL1 and CsCh1 are part of a complex seed defensive system against microbial growth. Another possibility is that these, and probably other seed PR proteins, have antifreeze activity. Both functions would be particularly relevant for chestnut seeds given their remarkable moisture content at maturity. [ABSTRACT FROM AUTHOR]