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.

To understand the evolution of protein function, it is important to know both the history of a protein family and the patterns of sequence evolution of that family. This information can provide an understanding of when the family originated, how its members are related to other proteins, the extent to which functional constraints are acting on the protein family, and whether these constraints have been consistent across taxa and over long periods of time. Although progress is being made in the study of the evolution of protein function in animals, our understanding of the evolution of protein function in plants has been limited by a number of factors. First, although the vast majority of plant proteins are nuclear-encoded, most plant molecular evolutionary studies have focused on chloroplast (CP)-encoded proteins (1). Furthermore, much of what is known about the evolution of plant nuclear-encoded proteins is limited to events of the last 150 million years because of the paucity of sequence data from non-angiosperm lineages. Clearly, additional studies of nuclear-encoded proteins are needed for a fuller understanding of plant protein evolution.

One very important aspect of the history of a protein family is the timing of origin of members within the family (2). The origin of nuclear-encoded organelle-localized proteins has been the subject of recent study (3–5), and two models of nuclear-encoded organelle-localized protein evolution have been proposed. In the first model, the well established “gene transfer” (or functional specificity) model (6), organelle genes are transferred from the endosymbiont genome to the nuclear genome. Because the products of the transferred gene are required for the organelle function, the transferred gene is maintained in the nuclear genome and acquires a transit sequence for proper trafficking of the protein back into the organelle. This model is well supported by numerous examples of nuclear-encoded CP protein genes that are closely related to genes of cyanobacterial proteins (for examples, see refs. 7–9). In the second model, “functional redundancy,” the organelle and nuclear proteins were functionally equivalent or redundant in the early eukaryote (4). Therefore, it is not necessary to retain the endosymbiont protein. Furthermore, it may not matter which form (nuclear or organelle) is used in the cytosol or sent into the organelles. Recent evidence suggests that the functional redundancy model accurately describes the evolution of at least some organelle-localized proteins (3, 4). However, it is difficult to make definitive statements about the origin and evolution of many plant organelle-localized proteins because of both a lack of data and a lack of detailed analysis of available data. Most studies of the origin of plant organelle proteins have examined sequences from only one major plant lineage, angiosperms. More importantly, many of these studies did not include any cyanobacterial sequences, which are critical to evaluation of hypotheses concerning the origin of nuclear-encoded CP proteins. A full understanding of the evolution of protein function in plants requires more detailed studies of nuclear-encoded proteins in a diversity of plant lineages.

Our studies of the small heat shock proteins (sHSPs) in plants focus on protein evolution with the goals of elucidating the history and patterns of sHSP evolution and uncovering the general patterns of protein family evolution (10, 11). The sHSPs are a diverse group of proteins found in archaea, bacteria, and eukaryotes and include the vertebrate lens α-crystallin proteins. All of these proteins share an ≈100-aa C-terminal “heat shock” domain (12, 13). The sHSPs form homooligomers both in vivo and in vitro (14–18) and facilitate the folding and reactivation of misfolded or denatured proteins (14, 16, 19). It is interesting that although most organisms have just one to a few cytosolically localized sHSPs, plants have many diverse sHSPs. There are at least five families of nuclear-encoded plant sHSPs, and they make up a major portion of the protein produced during high-temperature stress in plants (20). The plant sHSP families are clearly distinguishable from one another based on sequence analysis, and different sHSPs localize to different parts of the cell (10, 21). Two of the plant sHSP families are cytosolically localized (I and II), one is localized to the CPs, another to the mitochondria (MT), and the fifth localizes to the endoplasmic reticulum (ER).

There are many unanswered questions concerning the origin of the plant sHSP families and the nature of the selective forces acting on them. For instance, it has previously been suggested that the CP sHSPs may not have originated via gene transfer to the nucleus from the cyanobacterial endosymbiont, but rather from the duplication of the nuclear-encoded cytosolic sHSPs (10, 22). However, this hypothesis has not been explicitly tested. If the CP sHSPs did evolve from the cytosolic sHSPs, then at some point in plant evolution, there must have been differential selection for structure and/or function among the sHSP families, because the angiosperm sHSP families are clearly distinct and display very different patterns and rates of sequence evolution (10). The angiosperm CP sHSPs possess a highly conserved methionine-rich region in the N-terminal domain that is not found in any other plant or nonplant sHSP. This region has a methionine content of at least 20% and is predicted to form an amphipathic α-helix (23). The origin, evolutionary history, and functional significance of this region are unknown, but it clearly illustrates the differing selective constraints acting on the plant sHSP families.

To address questions concerning the evolution of the sHSPs, we have chosen to study the sHSPs of Funaria hygrometrica, a moss. Mosses are one of the most basal land plant lineages, and fossil evidence suggests that moss divergence occurred at least 400–450 million years ago (24, 25). Land plants are monophyletic (24, 26); thus, mosses share a common ancestor with angiosperms. Inclusion of mosses in evolutionary studies can provide needed depth for understanding protein evolution in plants. To define the origin of the CP sHSPs and the selective forces acting on these proteins, we have examined the relationship of the F. hygrometrica sHSPs to angiosperm homologs, the relationship of the plant CP sHSPs to cyanobacterial sHSPs, and the patterns of sequence evolution among CP sHSPs.

The analyses suggest that the CP sHSPs most likely evolved through gene duplication from a nuclear-encoded cytosolic sHSP and not by gene transfer from the endosymbiont. In addition, whereas the methionine-rich region seen in angiosperm CP sHSPs is not conserved at the primary sequence level in F. hygrometrica, the α-helical structure conserved among the angiosperm CP sHSPs is present in the F. hygrometrica CP sHSPs. These changing patterns of sequence evolution may reflect shifts during land plant evolution in the selective constraints acting on the CP sHSPs and the functions of the CP sHSPs. The data suggest that the CP sHSPs fit neither the functional redundancy nor the functional specificity models of organelle protein evolution. Thus, there may be considerable diversity in the origin and evolution of organelle-localized proteins.