Immunolocalization of freezing‐tolerance‐associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues

Abstract

SummaryA protein family associated with the development of freezing tolerance in wheat has been identified. This protein family is Gramineae-specific and coordinately regulated by low temperature. Antibodies directed against the 50 kDa (WCS120) protein recognize at least 5 members of this family. Using these antibodies, the cellular content and location of this protein family was determined in coldacclimated wheat seedlings. Western analyses of subcellular fractions indicated the presence of all members of the family in the cytosolic and purified nuclear fractions. These proteins accumulated to 0.9% of soluble proteins after 21 days of cold acclimation in winter wheat. This represents a cellular concentration of 1.34 pM. Imlhunohistochemical localization showed that these proteins are highly expressed in the vascular transition zone. No detectable expression was found in mature xylem, in the shoot apical meristem or lateral root primordia. This differential tissue expression suggests that the sensitive cells near the regions where water tends to freeze first require a higher amount of these proteins. This observation is consistent with the fact that regrowth after freezing stress is highly dependent on the viability of this region of the crown. Electron microscopy analysis using immunogold labelling showed that these proteins are present in the cytoplasm and in the nucleoplasm. They are not found in cell walls or other organelles. In vitro cryoprotective assays indicated that the WCS120 protein (PDs0 of 10 I~g ml'l or 0.2 IdVl) are as effective as BSA and sucrose (at 250 raM) against freezing denaturation of lactate dehydrogenese. These results suggest that this protein family may be involved in a general mechanism of protection in the soluble fraction of the cell. Their presence in the nucleoplesm may also suggest a possible protective function of the transcriptional machinery. The high hydrophilicity, the abundance and stability of these proteins to boiling suggest that they may provide a particular micro-environment needed for

Genetic approaches towards overcoming water deficit in plants - special emphasis on LEAs

“…DHNs localize primarily to the nucleus or cytoplasm (Houde et al, 1995;Bracale et al, 1997), but studies also corroborate their presence either in the plasma membrane (Danyluk et al, 1998), endoplasmic reticulum (Neven et al, 1993), chloroplast (Wisniewski et al, 1999), mitochondria (Borovskii et al, 2002) or even in the vacuole (Heyen et al, 2002). DHNs may thus be expressed either constitutively [e.g.…”

“…Arabidopsis DHNX] or confined to the root tip (ERD14 and LTI29), vascular tissues (ERD14, LTI29 and RAB18) and stomatal guard cells (RAB18). DHNs accumulate within vascular tissues (Godoy et al, 1994;Houde et al, 1995;Danyluk et al, 1998;Nylander et al, 2001), pollen sacs, guard cells and root meristems (Nylander et al, 2001), endosperm and zygotic embryos of mature seeds, developing root primordia (Godoy et al, 1994;Bracale et al, 1997), wheat crown tissues (Houde et al, 1995) and peach shoots (Wisniewski et al, 1999). DHNs found only in the cytosol include those of C. plantagineum DSP16 (Schneider et al, 1993) and rice RAB21 (Mundy and Chua, 1988).…”

A cold-responsive wheat (Triticum aestivum L.) gene wcor14 identified in a winter-hardy cultivar 'Mironovska 808'.

“…Some COR proteins, like Arabidopsis KIN1, show structural similarities to antifreeze proteins (Kurkela and Franck, 1990) and others to a HSP70 chaperon family which is known to have roles in protein transport, folding and assembly/disassembly processes (Neven et al, 1992). Although physiological function of these different classes of COR proteins remains largely unknown, some showed a positive correlation with the degree of freezing tolerance which is enhanced by cold acclimation (Monroy et al, 1993;Houde et al, 1995;Jaglo-Ottosen et al, 1998).…”

Isolation of a novel dehydrin gene from Codonopsis lanceolata and analysis of its response to abiotic stresses

“…DHNs have been found in the cytoplasm (12), nucleus (12,13), mitochondria (14), vacuole (15), and chloroplasts (16). They are known to associate with membranes (17,18), proteins (19) and excess salt ions (15,20).…”

“…Pekinensis (21). Many studies have reported a positive correlation between the accumulation of DHN transcripts or proteins and tolerance to freezing, drought, and salinity (12,17,(22)(23)(24). Moreover, modulation of transcripts by light has been reported for many DHN-encoding genes in drought-or cold-stressed plants (25)(26)(27)(28).…”