Molecular analysis of cold hardening in barley
Luigi Cattivelli, Cristina Crosatti, Maria Grossi,
Fulvia Rizza and A.Michele Stanca
Istituto sperimentale per la Cerealicoltura Sezione di
Fiorenzuola d'Arda -Via S. Protaso 302 - I-29017 Fiorenzuola
d'Arda (PC), Italy

In the past years we have described changes in gene expression induced by low temperatures (Cattivelli and Bartels, 1989) and isolated several Cold Regulated (COR) cDNA clones corresponding to mRNAs expressed in barley during cold acclimation (Cattivelli and Bartels, 1990). Recently the research work has been focused onto three genes or gene products characterized by peculiar features.

COR 75: a protein induced by ABA and low temperature
The hardening-triggered accumulation of a barley cold-regulated protein of 75 kDa (COR 75) has been investigated in several genotypes featuring contrasting frost tolerance capacity (Rizza et al., 1994; Murelli et al., in press). COR 75 is accumulated faster during the first days at low temperatures in frost resistant varieties when compared with extremely sensitive varieties, nevertheless after 7 days of hardening, COR 75 is expressed at the same level in all genotypes tested despite their differences in frost tolerance. Besides low temperature, COR 75 can also be induced by ABA or drought stress indicating that this protein may be a general response to variation in tissue moisture status (Crosatti et al., 1994).

paf93: a COR gene that responds rapidly to dehydration stress
The cDNA designed paf93 represents a gene inducible under cold condition and drought stress (Grossi et al., 1992) nevertheless, on the contrary of many other known genes induced by cold and drought, the induction of paf93 mRNA is not mediated by ABA. Sequencing of paf93 indicates that it is homologous to group II LEA proteins (Grossi et al., in press). paf93 is therefore a rare example of a gene homologous to ABA regulated genes without be under ABA control.

The accumulation of a COR chloroplastic protein is light dependent
The cDNA clone pt59 is homologous to a mRNA expressed only under low temperatures conditions in leaves. We have expressed in vitro the protein encoded by pt59 in order to raise the corresponding antibody (Crosatti et al., in press). In western blotting experiment the antibody cross-reacts with two protein bands of about 14 kDa (COR 14) localized in the stroma of chloroplasts. The synthesis and the accumulation of these polipeptides is induced by low temperature and it is light dependent. Etiolated plants exposed to low temperature do not synthesize COR 14, but 5 min of light exposure are

Table 1. Summarize several data concerning the main features of three cold-regulated genes/proteins with different expression behaviour.

_______________________________________________________________________
                                               Expression analysis
Cold-regulated  Corresponding  Molecular ______________________________
   protein          clone         mass       Treatment        Tissue 
                                  kDa    ________________  ____________
                                         Cold ABA Drought  Leaves Roots
_______________________________________________________________________

COR14                pt59         14.4     +   -     -        +      -

Homologous to
LEA proteins        paf93         27.6     +   -     +        +      +

COR75                 -           75 ca.   +   +     +        +      +
_______________________________________________________________________
+ = protein accumulation detected
- = protein accumulation not detected

enough to allow protein accumulation. COR 14 accumulates to the same extent in both cold tolerant and cold sensitive genotypes nevertheless cold resistant cultivars show an higher threshold temperature for protein accumulation when compared with cold sensitive one.

References

Cattivelli, L., and D. Bartels. 1989. Cold-induced mRNAs accumulate with different kinetics in barley coleoptiles. Planta 178: 184-188.

Cattivelli, L., and D. Bartels. 1990. Molecular cloning and characterization of cold-regulated genes in barley. Plant Physiol. 93: 1504-1510.

Crosatti, C., F. Rizza, and L. Cattivelli. 1994. Accumulation and characterization of the 75 kDa protein induced by low temperature in barley. Plant Sci. 97: 39-46.

Crosatti, C., C. Soncini, A.M. Stanca and L. Cattivelli. The accumulation of a cold-regulated chloroplastic proteins is light-dependent. Planta, in press.

Grossi, M., L. Cattivelli, V. Terzi, and A.M. Stanca. 1992. Modification of gene expression induced by ABA, in relation to drought and cold stress in barley shoots. Plant Physiol. Biochem. 30(1): 97-103.

Grossi, M., M. Gulli, A.M. Stanca and L. Cattivelli. Characterization of two barley cold-regulated genes that respond rapidly to dehydration stress. Plant Sci., in press.

Murelli, C., F. Rizza, F. Marinone Albini, A. Dulio, V. Terzi and L. Cattivelli. Metabolic changes associated with cold-acclimation in contrasting genotypes of barley. Physiol. Plant., in press.

Rizza, F., C. Crosatti, A.M. Stanca and L. Cattivelli. 1994. Studies for assessing the influence of hardening on cold tolerance of barley genotypes. Euphytica 75: 131-138.