The plant proteome folding project: structure and positive selection in plant protein families.

Despite its importance, relatively little is known about the relationship between the structure, function, and evolution of proteins, particularly in land plant species. We have developed a database with predicted protein domains for five plant proteomes (http://pfp.bio.nyu.edu) and used both protein structural fold recognition and de novo Rosetta-based protein structure prediction to predict protein structure for Arabidopsis and rice proteins. Based on sequence similarity, we have identified ~15,000 orthologous/paralogous protein family clusters among these species and used codon-based models to predict positive selection in protein evolution within 175 of these sequence clusters. Our results show that codons that display positive selection appear to be less frequent in helical and strand regions and are overrepresented in amino acid residues that are associated with a change in protein secondary structure. Like in other organisms, disordered protein regions also appear to have more selected sites. Structural information provides new functional insights into specific plant proteins and allows us to map positively selected amino acid sites onto protein structures and view these sites in a structural and functional context.

Plant phenotypic plasticity in a changing climate.

Climate change is altering the availability of resources and the conditions that are crucial to plant performance. One way plants will respond to these changes is through environmentally induced shifts in phenotype (phenotypic plasticity). Understanding plastic responses is crucial for predicting and managing the effects of climate change on native species as well as crop plants. Here, we provide a toolbox with definitions of key theoretical elements and a synthesis of the current understanding of the molecular and genetic mechanisms underlying plasticity relevant to climate change. By bringing ecological, evolutionary, physiological and molecular perspectives together, we hope to provide clear directives for future research and stimulate cross-disciplinary dialogue on the relevance of phenotypic plasticity under climate change.

Diversity and divergence patterns in regulatory genes suggest differential gene flow in recently derived species of the Hawaiian silversword alliance adaptive radiation (Asteraceae).

The impact of gene flow and population size fluctuations in shaping genetic variation during adaptive radiation, at both the genome-wide and gene-specific levels, is very poorly understood. To examine how historical population size and gene flow patterns within and between loci have influenced lineage divergence in the Hawaiian silversword alliance, we have investigated the nucleotide sequence diversity and divergence patterns of four floral regulatory genes (ASAP1-A, ASAP1-B, ASAP3-A, ASAP3-B) and a structural gene (ASCAB9). Levels and patterns of molecular divergence across these five nuclear loci were estimated between two recently derived species (Dubautia ciliolata and Dubautia arborea) which are presumed to be sibling species. This multilocus analysis of genetic variation, haplotype divergence and historical demography indicates that population expansion and differential gene flow occurred subsequent to the divergence of these two lineages. Moreover, contrasting patterns of allele- sharing for regulatory loci vs. a structural locus between these two sibling species indicate alternative histories of genetic variation and partitioning among loci where alleles of the floral regulatory loci are shared primarily from D. arborea to D. ciliolata and alleles of the structural locus are shared in both directions. Taken together, these results suggest that adaptively radiating species can exhibit contrasting allele migration rates among loci such that allele movement at specific loci may supersede genetic divergence caused by drift and that lineage divergence during adaptive radiation can be associated with population expansion.

Accelerated regulatory gene evolution in an adaptive radiation.

The disparity between rates of morphological and molecular evolution remains a key paradox in evolutionary genetics. A proposed resolution to this paradox has been the conjecture that morphological evolution proceeds via diversification in regulatory loci, and that phenotypic evolution may correlate better with regulatory gene divergence. This conjecture can be tested by examining rates of regulatory gene evolution in species that display rapid morphological diversification within adaptive radiations. We have isolated homologues to the Arabidopsis APETALA3 (ASAP3/TM6) and APETALA1 (ASAP1) floral regulatory genes and the CHLOROPHYLL A/B BINDING PROTEIN9 (ASCAB9) photosynthetic structural gene from species in the Hawaiian silversword alliance, a premier example of plant adaptive radiation. We have compared rates of regulatory and structural gene evolution in the Hawaiian species to those in related species of North American tarweeds. Molecular evolutionary analyses indicate significant increases in nonsynonymous relative to synonymous nucleotide substitution rates in the ASAP3/TM6 and ASAP1 regulatory genes in the rapidly evolving Hawaiian species. By contrast, no general increase is evident in neutral mutation rates for these loci in the Hawaiian species. An increase in nonsynonymous relative to synonymous nucleotide substitution rate is also evident in the ASCAB9 structural gene in the Hawaiian species, but not to the extent displayed in the regulatory loci. The significantly accelerated rates of regulatory gene evolution in the Hawaiian species may reflect the influence of allopolyploidy or of selection and adaptive divergence. The analyses suggest that accelerated rates of regulatory gene evolution may accompany rapid morphological diversification in adaptive radiations.

Map-based cloning of quantitative trait loci: progress and prospects.

Map-based cloning has been considered problematic for isolating quantitative trait loci (QTLs) due to the confounding phenotypic effects of environment and other QTLs. However, five recent studies, all in plants, have succeeded in cloning QTLs using map-based methods. We review the important features of these studies and evaluate the prospects for broader application of the techniques. Successful map-based cloning requires that QTLs represent single genes that can be isolated in near-isogenic lines, and that genotypes can be unambiguously inferred by progeny testing. In plants or animals for which map-based cloning of genes with discrete phenotypes is feasible, the modified procedures required for QTLs should not be limiting in most cases. The choice between map-based cloning and alternative methods will depend on details of the species and traits being studied.

The molecular population genetics of regulatory genes.

Regulatory loci, which may encode both trans acting proteins as well as cis acting promoter regions, are crucial components of an organism's genetic architecture. Although evolution of these regulatory loci is believed to underlie the evolution of numerous adaptive traits, there is little information on natural variation of these genes. Recent molecular population genetic studies, however, have provided insights into the extent of natural variation at regulatory genes, the evolutionary forces that shape them and the phenotypic effects of molecular regulatory variants. These recent analyses suggest that it may be possible to study the molecular evolutionary ecology of regulatory diversification by examining both the extent and patterning of regulatory gene diversity, the phenotypic effects of molecular variation at these loci and their ecological consequences.

Variation and selection at the CAULIFLOWER floral homeotic gene accompanying the evolution of domesticated Brassica oleracea.

The evolution of plant morphologies during domestication events provides clues to the origin of crop species and the evolutionary genetics of structural diversification. The CAULIFLOWER gene, a floral regulatory locus, has been implicated in the cauliflower phenotype in both Arabidopsis thaliana and Brassica oleracea. Molecular population genetic analysis indicates that alleles carrying a nonsense mutation in exon 5 of the B. oleracea CAULIFLOWER (BoCAL) gene are segregating in both wild and domesticated B. oleracea subspecies. Alleles carrying this nonsense mutation are nearly fixed in B. oleracea ssp. botrytis (domestic cauliflower) and B. oleracea ssp. italica (broccoli), both of which show evolutionary modifications of inflorescence structures. Tests for selection indicate that the pattern of variation at this locus is consistent with positive selection at BoCAL in these two subspecies. This nonsense polymorphism, however, is also present in both B. oleracea ssp. acephala (kale) and B. oleracea ssp. oleracea (wild cabbage). These results indicate that specific alleles of BoCAL were selected by early farmers during the domestication of modified inflorescence structures in B. oleracea.

Duplication of the Brassica oleracea APETALA1 floral homeotic gene and the evolution of domesticated cauliflower.

Development of the cauliflower phenotype in Arabidopsis thaliana requires mutations at both the CAULIFLOWER and APETALA1 loci. BoAP1 is the Brassica oleracea orthologue to the Arabidopsis AP1 gene, and is present in two copies in Brassica genomes. The BoAP1-A gene appears to encode a full-length protein, but BoAP1-B alleles in B. oleracea contain insertions that lead to premature translation termination. The BoAP1-B allele found in most B. oleracea subspecies, including B. oleracea ssp. botrytis (domesticated cauliflower) contains a 9 bp insertion in exon 4. This insertion leads to the formation of an in-frame translation termination codon, and these alleles can encode a protein that is truncated at the K domain of this MADS-box transcriptional activator. The allele in B. oleracea ssp. oleracea (wild cabbage) lacks this insertion and instead contains a downstream 4 bp frameshift mutation resulting in the formation of a nonsense mutation. The structure of the BoAP1-B alleles suggests that they are impaired in their ability to perform their floral meristem identity function. These mutations, in conjunction with mutations at the BoCAULIFLOWER (BoCAL) locus, may be associated with the evolution of domesticated cauliflower.

Patterns of molecular evolution among paralogous floral homeotic genes.

The plant MADS-box regulatory gene family includes several loci that control different aspects of inflorescence and floral development. Orthologs to the Arabidopsis thaliana MADS-box floral meristem genes APETALA1 and CAULIFLOWER and the floral organ identity genes APETALA3 and PISTILLATA were isolated from the congeneric species Arabidopsis lyrata. Analysis of these loci between these two Arabidopsis species, as well as three other more distantly related taxa, reveal contrasting dynamics of molecular evolution between these paralogous floral regulatory genes. Among the four loci, the CAL locus evolves at a significantly faster rate, which may be associated with the evolution of genetic redundancy between CAL and AP1. Moreover, there are significant differences in the distribution of replacement and synonymous substitutions between the functional gene domains of different floral homeotic loci. These results indicate that divergence in developmental function among paralogous members of regulatory gene families is accompanied by changes in rate and pattern of sequence evolution among loci.

Interspecific hybrid ancestry of a plant adaptive radiation: allopolyploidy of the Hawaiian silversword alliance (Asteraceae) inferred from floral homeotic gene duplications.

The polyploid Hawaiian silversword alliance (Asteraceae), a spectacular example of adaptive radiation in plants, was shown previously to have descended from North American tarweeds of the Madia/Raillardiopsis group, a primarily diploid assemblage. The origin of the polyploid condition in the silversword alliance was not resolved in earlier biosystematic, cytogenetic, and molecular studies, apart from the determination that polyploidy in modern species of Madia/Raillardiopsis arose independent of that of the Hawaiian group. We determined that two floral homeotic genes, ASAP3/TM6 and ASAP1, are found in duplicate copies within members of the Hawaiian silversword alliance and appear to have arisen as a result of interspecific hybridization between two North American tarweed species. Our molecular phylogenetic analyses of the ASAP3/TM6 loci suggest that the interspecific hybridization event in the ancestry of the Hawaiian silversword alliance involved members of lineages that include Raillardiopsis muirii (and perhaps Madia nutans) and Raillardiopsis scabrida. The ASAP1 analysis also indicates that the two species of Raillardiopsis are among the closest North American relatives of the Hawaiian silversword alliance. Previous biosystematic evidence demonstrates the potential for allopolyploid formation between members of the two North American tarweed lineages; a vigorous hybrid between R. muirii and R. scabrida has been produced that formed viable, mostly tetraporate (diploid) pollen, in keeping with observed meiotic failure. Various genetic consequences of allopolyploidy may help to explain the phenomenal evolutionary diversification of the silversword alliance.

Molecular population genetics of floral homeotic loci. Departures from the equilibrium-neutral model at the APETALA3 and PISTILLATA genes of Arabidopsis thaliana.

Molecular variation in genes that regulate development provides insights into the evolutionary processes that shape the diversification of morphogenetic pathways. Intraspecific sequence variation at the APETALA3 and PISTILLATA floral homeotic genes of Arabidopsis thaliana was analyzed to infer the extent and nature of diversity at these regulatory loci. Comparison of AP3 and PI diversity with three previously studied genes revealed several features in the patterning of nucleotide polymorphisms common between Arabidopsis nuclear loci, including an excess of low-frequency nucleotide polymorphisms and significantly elevated levels of intraspecific replacement variation. This pattern suggests that A. thaliana has undergone recent, rapid population expansion and now exists in small, inbred subpopulations. The elevated intraspecific replacement levels may thus represent slightly deleterious polymorphisms that differentiate distinct ecotypes. The distribution of replacement and synonymous changes in AP3 and PI core and noncore functional domains also indicates differences in the patterns of molecular evolution between these interacting floral regulatory genes.

The molecular evolution of development.

Morphological differences between species, from simple single-character differences to large-scale variation in body plans, can be traced to changes in the timing and location of developmental events. This has led to a growing interest in understanding the genetic basis behind the evolution of developmental systems. Molecular evolutionary genetics provides one of several approaches to dissecting the evolution of developmental systems, by allowing us to reconstruct the history of developmental genetic pathways, infer the origin and diversification of developmental gene functions, and assess the relative contributions of various evolutionary forces in shaping regulatory gene evolution.

Molecular population genetics of the Arabidopsis CAULIFLOWER regulatory gene: nonneutral evolution and naturally occurring variation in floral homeotic function.

The evolution of interspecies differences in morphology requires sufficient within-species variation in developmental regulatory systems on which evolutionary forces can act. Molecular analyses of naturally occurring alleles of the Arabidopsis thaliana CAULIFLOWER locus reveal considerable intraspecific diversity at this floral homeotic gene, and the McDonald-Kreitman test suggests that this gene is evolving in a nonneutral fashion, with an excess of intraspecific replacement polymorphisms. The naturally occurring molecular variation within this floral regulatory gene is associated with functionally different alleles, which can be distinguished phenotypically by their differential ability to direct floral meristem development.

The evolution of the conserved ATPase domain (CAD): reconstructing the history of an ancient protein module.

The AAA proteins (ATPases Associated with a variety of cellular Activities) are found in eubacterial, archaebacterial, and eukaryotic species and participate in a large number of cellular processes, including protein degradation, vesicle fusion, cell cycle control, and cellular secretory processes. The AAA proteins are characterized by the presence of a 230 to 250-amino acid ATPase domain referred to as the Conserved ATPase Domain or CAD. Phylogenetic analysis of 133 CAD sequences from 38 species reveal that AAA CADs are organized into discrete groups that are related not only in structure but in cellular function. Evolutionary analyses also indicate that the CAD was present in the last common ancestor of eubacteria, archaebacteria, and eukaryotes. The eubacterial CADs are found in metalloproteases, while CAD-containing proteins in the archaebacterial and eukaryotic lineages appear to have diversified by a series of gene duplication events that lead to the establishment of different functional AAA proteins, including proteasomal regulatory, NSF/Sec, and Pas proteins. The phylogeny of the CADs provides the basis for establishing the patterns of evolutionary change that characterize the AAA proteins.

The MADS-box floral homeotic gene lineages predate the origin of seed plants: phylogenetic and molecular clock estimates.

Flower development in angiosperms is controlled in part by floral homeotic genes, many of which are members of the plant MADS-box regulatory gene family. The evolutionary history of these developmental genes was reconstructed using 74 loci from 15 dicot, three monocot, and one conifer species. Molecular clock estimates suggest that the different floral homeotic gene lineages began to diverge from one another about 450-500 mya, around the time of the origin of land plants themselves.

Molecular evolution of flower development: diversification of the plant MADS-box regulatory gene family.

Floral homeotic genes that control the specification of meristem and organ identity in developing flowers have been isolated from both Arabidopsis thaliana and Antirrhinum majus. Most of these genes belong to a large family of regulatory genes and possess a characteristic DNA binding domain known as the MADS-box. Members of this gene family display primarily floral-specific expression and are homologous to transcription factors found in several animal and fungal species. Molecular evolutionary analyses reveal that there are appreciable differences in the substitution rates between different domains of these plant MADS-box genes. Phylogenetic analyses also demonstrate that members of the plant MADS-box gene family are organized into several distinct gene groups: the AGAMOUS, APETALA3/PISTILLATA and APETALA1/AGL9 groups. The shared evolutionary history of members of a gene group appear to reflect the distinct functional roles these MADS-box genes play in flower development. Molecular evolutionary analyses also suggest that these different gene groups were established in a relatively short span of evolutionary time and that the various floral homeotic loci originated even before the appearance of the flowering plants.

Transposon signatures: species-specific molecular markers that utilize a class of multiple-copy nuclear DNA.

Transposable elements are mobile sequences found in nuclear genomes and can potentially serve as molecular markers in various phylogenetic and population genetic investigations. A PCR-based method that utilizes restriction site variation of element copies within a genome is developed. These patterns of site variation, referred to as transposon signatures, are useful in differentiating between closely related groups. Signature data using the magellan retrotransposon, for example, is useful in examining relationships within the genus Zea and Tripsacum. This method allows transposable elements, or even other multiple-copy nuclear DNA sequences, to be generally utilized as molecular markers in discriminating between other closely related species and subspecies.

Molecular evolution of the plant R regulatory gene family.

Anthocyanin pigmentation patterns in different plant species are controlled in part by members of the myc-like R regulatory gene family. We have examined the molecular evolution of this gene family in seven plant species. Three regions of the R protein show sequence conservation between monocot and dicot R genes. These regions encode the basic helix-loop-helix domain, as well as conserved N-terminal and C-terminal domains; mean replacement rates for these conserved regions are 1.02 x 10(-9) nonsynonymous nucleotide substitutions per site per year. More than one-half of the protein, however, is diverging rapidly, with nonsynonymous substitution rates of 4.08 x 10(-9) substitutions per site per year. Detailed analysis of R homologs within the grasses (Poaceae) confirm that these variable regions are indeed evolving faster than the flanking conserved domains. Both nucleotide substitutions and small insertion/deletions contribute to the diversification of the variable regions within these regulatory genes. These results demonstrate that large tracts of sequence in these regulatory loci are evolving at a fairly rapid rate.

Molecular evolution of magellan, a maize Ty3/gypsy-like retrotransposon.

The magellan transposable element is responsible for a spontaneous 5.7-kb insertion in the maize wx-M allele. This element has the sequence and structural characteristics of a Ty3/gypsy-like retrotransposon. The magellan element is present in all Zea species and Tripsacum andersonii; it is absent, however, in the genomes of all other Tripsacum species analyzed. The genetic distances between magellan elements suggest that this retrotransposon is evolving faster than other Zea nuclear loci. The phylogeny of magellan within Zea and T. andersonii also reveals a pattern of interspecies transfers, resulting in the movement of magellan subfamilies between different species genomes. Interspecific hybridization may be a major mechanism by which this retrotransposon invades and establishes itself in new taxa.