Chloroplast small heat shock proteins: evidence for atypical evolution of an organelle-localized protein

Abstract

Knowledge of the origin and evolution of gene families is critical to our understanding of the evolution of protein function. To gain a detailed understanding of the evolution of the small heat shock proteins (sHSPs) in plants, we have examined the evolutionary history of the chloroplast (CP)-localized sHSPs. Previously, these nuclear-encoded CP proteins had been identified only from angiosperms. This study reveals the presence of the CP sHSPs in a moss, Funaria hygrometrica. Two clones for CP sHSPs were isolated from a F. hygrometrica heat shock cDNA library that represent two distinct CP sHSP genes. Our analysis of the CP sHSPs reveals unexpected evolutionary relationships and patterns of sequence conservation. Phylogenetic analysis of the CP sHSPs with other plant CP sHSPs and eukaryotic, archaeal, and bacterial sHSPs shows that the CP sHSPs are not closely related to the cyanobacterial sHSPs. Thus, they most likely evolved via gene duplication from a nuclear-encoded cytosolic sHSP and not via gene transfer from the CP endosymbiont. Previous sequence analysis had shown that all angiosperm CP sHSPs possess a methionine-rich region in the N-terminal domain. The primary sequence of this region is not highly conserved in the F. hygrometrica CP sHSPs. This lack of sequence conservation indicates that sometime in land plant evolution, after the divergence of mosses from the common ancestor of angiosperms but before the monocot-dicot divergence, there was a change in the selective constraints acting on the CP sHSPs.

Figure 1

Alignment of F. hygrometrica and angiosperm CP sHSPs. The alignment contains F. hygrometrica CP sHSPs, HSP 22, and HSP 21; monocot, Triticum aestivum (T.a.), Zea mays (Z.m.) and dicot A. thaliana (A.t.), Pisum sativum (P.s.) CP sHSPs, as well as HSP 16.6 from Synechocystis sp. The transit sequence consists of positions 1–51. The N-terminal domain consists of residues 52–146, and the C-terminal domain starts at residue 147. Gaps in the alignment are noted by a ⋅. The glutamine (Q) residue that is predicted to mark the start of the mature CP sHSPs is in bold italics. The methionine (M) residues conserved among angiosperm CP sHSPs are noted with *. Residues that are conserved among all sHSPs (eukaryotic, bacterial, and archaeal) are noted with #. A lack of a consensus residue is noted by –. Three consensus sequences are presented. The first is the angiosperm CP sHSPs consensus. The second is the land-plant CP sHSPs (including the F. hygrometrica sHSPs) consensus sequence. The third consensus sequence is based on all of the sequences, including Synechocystis HSP 16.6. The previously identified conserved consensus regions I, II, and III, discussed in the text, are in bold.

Figure 2

Amphipathic α-helix of A. thaliana HSP 21 and F. hygrometrica HSP 22. The predicted amphipathic α-helices and the corresponding amino acid sequences are shown for the F. hygrometrica and A. thaliana CP sHSPs. The numbers adjacent to the amino acid residues indicate the residue number for the region included in the helical structure. Amino acid residues in large bold text are conserved among all of the angiosperm CP sHSPs. The F. hygrometrica HSP 22 amino acid residues R₄ and K₁, marked by *, are the only residues that are not identical in HSP 21. However, the replacements in HSP 21 are conservative, with R₄ replaced by K and K₁ replaced by R.

Figure 3

Phylogenetic analysis of sHSP sequences. The tree presented is the result of parsimony analysis by using paup*. The tree has 2,299 steps. The numbers above the branches are bootstrap values. The branch that unites all of the land-plant sHSPs is noted. The CP sHSP clade is shaded and noted. The other land-plant sHSP clades, ER, cytosolic I and II, and MT are also noted and marked by brackets. All of the F. hygrometrica sHSPs sequences are in bold. The cyanobacterial sHSPs sequences are in bold italics.