Hongzhi Kong

The Evolution of the SEPALLATA Subfamily of MADS-Box Genes: A Preangiosperm Origin With Multiple Duplications Throughout Angiosperm History

Members of the SEPALLATA (SEP) MADS-box subfamily are required for specifying the “floral state” by contributing to floral organ and meristem identity. SEP genes have not been detected in gymnosperms and seem to have originated since the lineage leading to extant angiosperms diverged from extant gymnosperms. Therefore, both functional and evolutionary studies suggest that SEP genes may have been critical for the origin of the flower. To gain insights into the evolution of SEP genes, we isolated nine genes from plants that occupy phylogenetically important positions. Phylogenetic analyses of SEP sequences show that several gene duplications occurred during the evolution of this subfamily, providing potential opportunities for functional divergence. The first duplication occurred prior to the origin of the extant angiosperms, resulting in the AGL2/3/4 and AGL9 clades. Subsequent duplications occurred within these clades in the eudicots and monocots. The timing of the first SEP duplication approximately coincides with duplications in the DEFICIENS/GLOBOSA and AGAMOUS MADS-box subfamilies, which may have resulted from either a proposed genome-wide duplication in the ancestor of extant angiosperms or multiple independent duplication events. Regardless of the mechanism of gene duplication, these pairs of duplicate transcription factors provided new possibilities of genetic interactions that may have been important in the origin of the flower.

Genome-wide analysis of the cyclin family in arabidopsis and comparative phylogenetic analysis of plant cyclin-like proteins

Cyclins are primary regulators of the activity of cyclin-dependent kinases, which are known to play critical roles in controlling eukaryotic cell cycle progression. While there has been extensive research on cell cycle mechanisms and cyclin function in animals and yeasts, only a small number of plant cyclins have been characterized functionally. In this paper, we describe an exhaustive search for cyclin genes in the Arabidopsis genome and among available sequences from other vascular plants. Based on phylogenetic analysis, we define 10 classes of plant cyclins, four of which are plant-specific, and a fifth is shared between plants and protists but not animals. Microarray and reverse transcriptase-polymerase chain reaction analyses further provide expression profiles of cyclin genes in different tissues of wild-type Arabidopsis plants. Comparative phylogenetic studies of 174 plant cyclins were also performed. The phylogenetic results imply that the cyclin gene family in plants has experienced more gene duplication events than in animals. Expression patterns and phylogenetic analyses of Arabidopsis cyclin genes suggest potential gene redundancy among members belonging to the same group. We discuss possible divergence and conservation of some plant cyclins. Our study provides an opportunity to rapidly assess the position of plant cyclin genes in terms of evolution and classification, serving as a guide for further functional study of plant cyclins.

Evolution of F-box genes in plants: Different modes of sequence divergence and their relationships with functional diversification

F-box proteins are substrate-recognition components of the Skp1-Rbx1-Cul1-F-box protein (SCF) ubiquitin ligases. In plants, F-box genes form one of the largest multigene superfamilies and control many important biological functions. However, it is unclear how and why plants have acquired a large number of F-box genes. Here we identified 692, 337, and 779 F-box genes in Arabidopsis, poplar and rice, respectively, and studied their phylogenetic relationships and evolutionary patterns. We found that the plant F-box superfamily can be divided into 42 families, each of which has a distinct domain organization. We also estimated the number of ancestral genes for each family and identified highly conservative versus divergent families. In conservative families, there has been little or no change in the number of genes since the divergence between eudicots and monocots ≈145 million years ago. In divergent families, however, the numbers have increased dramatically during the same period. In two cases, the numbers of genes in extant species are >100 times greater than that in the most recent common ancestor (MRCA) of the three species. Proteins encoded by highly conservative genes always have the same domain organization, suggesting that they interact with the same or similar substrates. In contrast, proteins of rapidly duplicating genes sometimes have quite different domain structures, mainly caused by unusually frequent shifts of exon-intron boundaries and/or frameshift mutations. Our results indicate that different F-box families, or different clusters of the same family, have experienced dramatically different modes of sequence divergence, apparently having resulted in adaptive changes in function.

Origins and evolution of the recA/RAD51 gene family: Evidence for ancient gene duplication and endosymbiotic gene transfer

The bacterial recA gene and its eukaryotic homolog RAD51 are important for DNA repair, homologous recombination, and genome stability. Members of the recA/RAD51 family have functions that have differentiated during evolution. However, the evolutionary history and relationships of these members remains unclear. Homolog searches in prokaryotes and eukaryotes indicated that most eubacteria contain only one recA. However, many archaeal species have two recA/RAD51 homologs (RADA and RADB), and eukaryotes possess multiple members (RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCC2, XRCC3, and recA). Phylogenetic analyses indicated that the recA/RAD51 family can be divided into three subfamilies: (i) RADα, with highly conserved functions; (ii) RADβ, with relatively divergent functions; and (iii) recA, functioning in eubacteria and eukaryotic organelles. The RADα and RADβ subfamilies each contain archaeal and eukaryotic members, suggesting that a gene duplication occurred before the archaea/eukaryote split. In the RADα subfamily, eukaryotic RAD51 and DMC1 genes formed two separate monophyletic groups when archaeal RADA genes were used as an outgroup. This result suggests that another duplication event occurred in the early stage of eukaryotic evolution, producing the DMC1 clade with meiosis-specific genes. The RADβ subfamily has a basal archaeal clade and five eukaryotic clades, suggesting that four eukaryotic duplication events occurred before animals and plants diverged. The eukaryotic recA genes were detected in plants and protists and showed strikingly high levels of sequence similarity to recA genes from proteobacteria or cyanobacteria. These results suggest that endosymbiotic transfer of recA genes occurred from mitochondria and chloroplasts to nuclear genomes of ancestral eukaryotes.

Divergence of duplicate genes in exon–intron structure

Gene duplication plays key roles in organismal evolution. Duplicate genes, if they survive, tend to diverge in regulatory and coding regions. Divergences in coding regions, especially those that can change the function of the gene, can be caused by amino acid-altering substitutions and/or alterations in exon–intron structure. Much has been learned about the mode, tempo, and consequences of nucleotide substitutions, yet relatively little is known about structural divergences. In this study, by analyzing 612 pairs of sibling paralogs from seven representative gene families and 300 pairs of one-to-one orthologs from different species, we investigated the occurrence and relative importance of structural divergences during the evolution of duplicate and nonduplicate genes. We found that structural divergences have been very prevalent in duplicate genes and, in many cases, have led to the generation of functionally distinct paralogs. Comparisons of the genomic sequences of these genes further indicated that the differences in exon–intron structure were actually accomplished by three main types of mechanisms (exon/intron gain/loss, exonization/pseudoexonization, and insertion/deletion), each of which contributed differently to structural divergence. Like nucleotide substitutions, insertion/deletion and exonization/pseudoexonization occurred more or less randomly, with the number of observable mutational events per gene pair being largely proportional to evolutionary time. Notably, however, compared with paralogs with similar evolutionary times, orthologs have accumulated significantly fewer structural changes, whereas the amounts of amino acid replacements accumulated did not show clear differences. This finding suggests that structural divergences have played a more important role during the evolution of duplicate than nonduplicate genes.

Resolution of deep angiosperm phylogeny using conserved nuclear genes and estimates of early divergence times

Angiosperms are the most successful plants and support human livelihood and ecosystems. Angiosperm phylogeny is the foundation of studies of gene function and phenotypic evolution, divergence time estimation and biogeography. The relationship of the five divergent groups of the Mesangiospermae (~99.95% of extant angiosperms) remains uncertain, with multiple hypotheses reported in the literature. Here transcriptome data sets are obtained from 26 species lacking sequenced genomes, representing each of the five groups: eudicots, monocots, magnoliids, Chloranthaceae and Ceratophyllaceae. Phylogenetic analyses using 59 carefully selected low-copy nuclear genes resulted in highly supported relationships: sisterhood of eudicots and a clade containing Chloranthaceae and Ceratophyllaceae, with magnoliids being the next sister group, followed by monocots. Our topology allows a re-examination of the evolutionary patterns of 110 morphological characters. The molecular clock estimates of Mesangiospermae diversification during the late to middle Jurassic correspond well to the origins of some insects, which may have been a factor facilitating early angiosperm radiation.

Evolution of Plant MADS Box Transcription Factors: Evidence for Shifts in Selection Associated with Early Angiosperm Diversification and Concerted Gene Duplications

Phylogenomic analyses show that gene and genome duplication events have led to the diversification of transcription factor gene families throughout the evolutionary history of land plants and that gene duplications have played an important role in shaping regulatory networks influencing key phenotypic characters including floral development and flowering time. A molecular evolutionary investigation of the mode and tempo of selection acting on the angiosperm MADS box AP1/SQUA, AP3/PI, AG/AGL11, and SEP gene subfamilies revealed site-specific patterns of shifting evolutionary constraint throughout angiosperm history. Specific positions in the four canonical MADS box gene regions, especially K domains and C-terminal regions of all four of these MADS box gene subfamilies exhibited clade-specific shifts in selective constraint following concerted duplication events. Moreover, the frequency of site-specific shifts in constraint was correlated with gene duplications and early angiosperm diversification. We hypothesize that coevolution among interacting MADS box proteins may be responsible for simultaneous increases in the ratio of nonsynonymous to synonymous substitutions (d(N)/d(S) = omega) early in angiosperm history and following concerted duplication events.

Disruption of the petal identity gene APETALA3-3 is highly correlated with loss of petals within the buttercup family (Ranunculaceae)

Absence of petals, or being apetalous, is usually one of the most important features that characterizes a group of flowering plants at high taxonomic ranks (i.e., family and above). The apetalous condition, however, appears to be the result of parallel or convergent evolution with unknown genetic causes. Here we show that within the buttercup family (Ranunculaceae), apetalous genera in at least seven different lineages were all derived from petalous ancestors, indicative of parallel petal losses. We also show that independent petal losses within this family were strongly associated with decreased or eliminated expression of a single floral organ identity gene, APETALA3-3 (AP3-3), apparently owing to species-specific molecular lesions. In an apetalous mutant of Nigella, insertion of a transposable element into the second intron has led to silencing of the gene and transformation of petals into sepals. In several naturally occurring apetalous genera, such as Thalictrum, Beesia, and Enemion, the gene has either been lost altogether or disrupted by deletions in coding or regulatory regions. In Clematis, a large genus in which petalous species evolved secondarily from apetalous ones, the gene exhibits hallmarks of a pseudogene. These results suggest that, as a petal identity gene, AP3-3 has been silenced or down-regulated by different mechanisms in different evolutionary lineages. This also suggests that petal identity did not evolve many times independently across the Ranunculaceae but was lost in numerous instances. The genetic mechanisms underlying the independent petal losses, however, may be complex, with disruption of AP3-3 being either cause or effect.

Petal-specific subfunctionalization of an APETALA3 paralog in the Ranunculales and its implications for petal evolution

• The petals of the lower eudicot family Ranunculaceae are thought to have been derived many times independently from stamens. However, investigation of the genetic basis of their identity has suggested an alternative hypothesis: that they share a commonly inherited petal identity program. This theory is based on the fact that an ancient paralogous lineage of APETALA3 (AP3) in the Ranunculaceae appears to have a conserved, petal-specific expression pattern. • Here, we have used a combination of approaches, including RNAi, comparative gene expression and molecular evolutionary studies, to understand the function of this petal-specific AP3 lineage. • Functional analysis of the Aquilegia locus AqAP3-3 has demonstrated that the paralog is required for petal identity with little contribution to the identity of the other floral organs. Expanded expression studies and analyses of molecular evolutionary patterns provide further evidence that orthologs of AqAP3-3 are primarily expressed in petals and are under higher purifying selection across the family than the other AP3 paralogs. • Taken together, these findings suggest that the AqAP3-3 lineage underwent progressive subfunctionalization within the order Ranunculales, ultimately yielding a specific role in petal identity that has probably been conserved, in stark contrast with the multiple independent origins predicted by botanical theories.

Characterization of candidate class A, B and E floral homeotic genes from the perianthless basal angiosperm Chloranthus spicatus (Chloranthaceae)

The classic ABC model explains the activities of each class of floral homeotic genes in specifying the identity of floral organs. Thus, changes in these genes may underlay the origin of floral diversity during evolution. In this study, three MADS-box genes were isolated from the perianthless basal angiosperm Chloranthus spicatus. Sequence and phylogenetic analyses revealed that they are AP1-like, AP3-like and SEP3-like genes, and hence these genes were termed CsAP1, CsAP3 and CsSEP3, respectively. Due to these assignments, they represent candidate class A, class B and class E genes, respectively. Expression patterns suggest that the CsAP1, CsAP3 and CsSEP3 genes function during flower development of C. spicatus. CsAP1 is expressed broadly in the flower, which may reflect the ancestral function of SQUA-like genes in the specification of inflorescence and floral meristems rather than in patterning of the flower. CsAP3 is exclusively expressed in male floral organs, providing the evidence that AP3-like genes have ancestral function in differentiation between male and female reproductive organs. CsSEP3 expression is not detectable in spike meristems, but its mRNA accumulates throughout the flower, supporting the view that SEP-like genes have conserved expression pattern and function throughout angiosperm. Studies of synonymous vs nonsynonymous nucleotide substitutions indicate that these genes have not evolved under changes in evolutionary forces. All the data above suggest that the genes may have maintained at least some ancestral functions despite the lack of perianth in the flowers of C. spicatus.