The diversification of plant cytosolic small heat shock proteins preceded the divergence of mosses

A cDNA library was constructed with mRNA isolated from heat-stressed cell c ultures of Funaria hygrometrica (Bryophyta, Musci, Funariaceae). cDNA clone s encoding six cytosolic small heat shock proteins (sHSPs) were identified using differential screening. Phylogenetic analysis of these sHSP sequences with other known sHSPs identified them as members of the previously descri bed higher plant cytosolic class I and II families. Four of the F. hygromet rica sHSPs are members of the cytosolic class I family, and the other two a re members of the cytosolic class II family. The presence of members of the cytosolic I and II sHSP families in a bryophyte indicates that these gene families are ancient, and evolved at least 450 MYA. This result also indica tes that the plant sHSP gene families duplicated much earlier than did the well-studied phytochrome gene family. Members of the cytosolic I and II sHS P families are developmentally regulated in seeds and flowers in higher pla nts. Our findings show that the two cytosolic sHSP families evolved before the appearance of these specialized structures. Previous analysis of angios perm sHSPs had identified class- or family-specific amino acid consensus re gions and determined that rate heterogeneity exists among the different sHS P families. The analysis of the F. hygrometrica sHSP sequences reveals patt erns and rates of evolution distinct from those seen among angiosperm sHSPs . Some, but not all, of the amino acid consensus regions identified in seed plants are conserved in the F. hygrometrica sHSPs. Taken together, the res ults of this study illuminate the evolution of the sHSP gene families and i llustrate the importance of including representatives of basal land plant l ineages in plant molecular evolutionary studies.