Patterns of gene duplication and functional diversification during the evolution of the AP1/SQUA subfamily of plant MADS-box genes

Members of the AP1/SQUA subfamily of plant MADS-box genes play broad roles in the regulation of reproductive meristems, the specification of sepal and petal identities, and the development of leaves and fruits. It has been shown that AP1/SQUA-like genes are angiosperm-specific, and have experienced several major duplication events. However, the evolutionary history of this subfamily is still uncertain. Here, we report the isolation of 14 new AP1/SQUA-like genes from seven early-diverging eudicots and the identification of 11 previously uncharacterized ESTs and genomic sequences from public databases. Sequence comparisons of these and other published sequences reveal a conserved C-terminal region, the FUL motif, in addition to the known euAP1/paleoAP1 motif, in AP1/SQUA-like proteins. Phylogenetic analyses further suggest that there are three major lineages (euAP1, euFUL, and AGL79) in core eudicots, likely resulting from two close duplication events that predated the divergence of core eudicots. Among the three lineages, euFUL is structurally very similar to FUL-like genes from early-diverging eudicots and basal angiosperms, whereas euAP1 might have originally been generated through a 1-bp deletion in the exon 8 of an ancestral euFUL- or FUL-like gene. Because euFUL- and FUL-like genes usually have broad expression patterns, we speculate that AP1/SQUA-like genes initially had broad functions. Based on these observations, the evolutionary fates of duplicate genes and the contributions of the frameshift mutation and alternative splicing to functional diversity are discussed.     

Expression of floral MADS-box genes in basal angiosperms: implications for the evolution of floral regulators

The ABC model of floral organ identity is based on studies of Arabidopsis and Antirrhinum, both of which are highly derived eudicots. Most of the genes required for the ABC functions in Arabidopsis and Antirrhinum are members of the MADS-box gene family, and their orthologs are present in all major angiosperm lineages. Although the eudicots comprise 75% of all angiosperms, most of the diversity in arrangement and number of floral parts is actually found among basal angiosperm lineages, for which little is known about the genes that control floral development. To investigate the conservation and divergence of expression patterns of floral MADS-box genes in basal angiosperms relative to eudicot model systems, we isolated several floral MADS-box genes and examined their expression patterns in representative species, including Amborella (Amborellaceae), Nuphar (Nymphaeaceae) and Illicium (Austrobaileyales), the successive sister groups to all other extant angiosperms, plus Magnolia and Asimina, members of the large magnoliid clade. Our results from multiple methods (relative-quantitative RT-PCR, real-time PCR and RNA in situ hybridization) revealed that expression patterns of floral MADS-box genes in basal angiosperms are broader than those of their counterparts in eudicots and monocots. In particular, (i) AP1 homologs are generally expressed in all floral organs and leaves, (ii) AP3/PI homologs are generally expressed in all floral organs and (iii) AG homologs are expressed in stamens and carpels of most basal angiosperms, in agreement with the expectations of the ABC model; however, an AG homolog is also expressed in the tepals of Illicium. The broader range of strong expression of AP3/PI homologs is inferred to be the ancestral pattern for all angiosperms and is also consistent with the gradual morphological intergradations often observed between adjacent floral organs in basal angiosperms.     

Function and evolution of the plant MADS-box gene family

The function of MADS-box genes in flower and fruit development has been uncovered at a rapid pace over the past decade. Evolutionary biologists can now analyse the expression pattern of MADS-box genes during the development of different plant species, and study the homology of body parts and the evolution of body plans. These studies have shown that floral development is conserved among divergent species, and indicate that the basic mechanism of floral patterning might have evolved in an ancient flowering plant.     

Two frameshift mutations in the cystic fibrosis gene

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified in exon 7 two frameshift mutations, one caused by a two-nucleotide insertion and the other caused by a one-nucleotide deletion; these mutations--CF1154insTC and CF1213delT, respectively, are predicted to shift the reading frame of the protein and to introduce UAA(ochre) termination codons at residues 369 and 368.     

C1 inhibitor gene sequence facilitates frameshift mutations.

Mutations disrupting the function or production of C1 inhibitor cause the disease hereditary angioneurotic edema. Patient mutations identified an imperfect inverted repeat sequence that was postulated to play a mechanistic role in the mutations. To test this hypothesis, the inverted repeat was cloned into the chloramphenicol acetyltransferase gene in pBR325 and its mutation rate was studied in four bacterial strains. These strains were selected to assay the effects of recombination and superhelical tension on mutation frequency. Mutations that revert bacteria to chloramphenicol resistance (Cmr) were scored. Both pairs of isogenic strains had reversion frequencies of approximately 10(-8). These rare reversion events in bacteria were most often a frameshift that involved the imperfect inverted repeat with a deletion or a tandem duplication, an event very similar to the human mutations. Increased DNA superhelical tension, which would be expected to enhance cruciform extrusion, did not accentuate mutagenesis. This finding suggests that the imperfect inverted repeat may form a stem-loop structure in the single-stranded DNA created by the duplex DNA melting prior to replication. Models explaining the slippage can be drawn using the lagging strand of the replication fork. In this model, the formation of a stem-loop structure is responsible for bringing the end of the deletion or duplication into close proximity.     

Structural evolution and functional diversification analyses of argonaute protein

Argonaute (AGO) proteins are highly specialized small-RNA-binding modules and small RNAs are anchored to their specific binding pockets guiding AGO proteins to target mRNA molecules for silencing or destruction. The 135 full-length AGO protein sequences derived from 36 species covering prokaryote, archaea, and eukaryote are chosen for structural and functional analyses. The results show that bacteria and archaeal AGO proteins are clustered in the same clade and there exist multiple AGO proteins in most eukaryotic species, demonstrating that the increase of AGO gene copy number and horizontal gene transfer (HGT) have been the main evolutionary driving forces for adaptability and biodiversity. And the emergence of PAZ domain in AGO proteins is the unique evolutionary event. The analysis of middle domain (MID)-nucleotide contaction shows that either the position of sulfate I bond in Nc_QDE2 or the site of phosphate I bond in Hs_AGO2 represents the 5'-nucleotide binding site of miRNA. Also, H334, T335, and Y336 of Hs_AGO1 can form hydrogen bonds with 3'-overhanging ends of miRNAs and the same situation exists in Hs_AGO2, Hs_AGO3, Hs_AGO4, Dm_AGO1, and Ce_Alg1. Some PIWI domains containing conserved DDH motif have no slicer activity, and post-translational modifications may be associated with the endonucleolytic activities of AGOs. With the numbers of AGO genes increasing and fewer crystal structures available, the evolutionary and functional analyses of AGO proteins can help clarify the molecular mechanism of function diversification in response to environmental changes, and solve major issues including host defense mechanism against virus infection and molecular basis of disease.     

Involvement of the MADS-box gene ZMM4 in floral induction and inflorescence development in maize

The switch from vegetative to reproductive growth is marked by the termination of vegetative development and the adoption of floral identity by the shoot apical meristem (SAM). This process is called the floral transition. To elucidate the molecular determinants involved in this process, we performed genome-wide RNA expression profiling on maize (Zea mays) shoot apices at vegetative and early reproductive stages using massively parallel signature sequencing technology. Profiling revealed significant up-regulation of two maize MADS-box (ZMM) genes, ZMM4 and ZMM15, after the floral transition. ZMM4 and ZMM15 map to duplicated regions on chromosomes 1 and 5 and are linked to neighboring MADS-box genes ZMM24 and ZMM31, respectively. This gene order is syntenic with the vernalization1 locus responsible for floral induction in winter wheat (Triticum monococcum) and similar loci in other cereals. Analyses of temporal and spatial expression patterns indicated that the duplicated pairs ZMM4-ZMM24 and ZMM15-ZMM31 are coordinately activated after the floral transition in early developing inflorescences. More detailed analyses revealed ZMM4 expression initiates in leaf primordia of vegetative shoot apices and later increases within elongating meristems acquiring inflorescence identity. Expression analysis in late flowering mutants positioned all four genes downstream of the floral activators indeterminate1 (id1) and delayed flowering1 (dlf1). Overexpression of ZMM4 leads to early flowering in transgenic maize and suppresses the late flowering phenotype of both the id1 and dlf1 mutations. Our results suggest ZMM4 may play roles in both floral induction and inflorescence development.     

PROLIFERATING INFLORESCENCE MERISTEM,a MADS-box gene that regulates floral meristem identity in pea

SQUAMOSA and APETALA1 are floral meristem identity genes from snapdragon (Antirrhinum majus) and Arabidopsis, respectively. Here, we characterize the floral meristem identity mutation proliferating inflorescence meristem (pim) from pea (Pisum sativum) and show that it corresponds to a defect in the PEAM4 gene, a homolog of SQUAMOSA and APETALA1. The PEAM4 coding region was deleted in the pim-1 allele, and this deletion cosegregated with the pim-1 mutant phenotype. The pim-2 allele carried a nucleotide substitution at a predicted 5' splice site that resulted in mis-splicing of pim-2 mRNA. PCR products corresponding to unspliced and exon-skipped mRNA species were observed. The pim-1 and pim-2 mutations delayed floral meristem specification and altered floral morphology significantly but had no observable effect on vegetative development. These floral-specific mutant phenotypes and the restriction of PIM gene expression to flowers contrast with other known floral meristem genes in pea that additionally affect vegetative development. The identification of PIM provides an opportunity to compare pathways to flowering in species with different inflorescence architectures.     

Structural changes of D1 C-terminal alpha-carboxylate during S-state cycling in photosynthetic oxygen evolution

Changes in the chemical structure of alpha-carboxylate of the D1 C-terminal Ala-344 during S-state cycling of photosynthetic oxygen-evolving complex were selectively measured using light-induced Fourier transform infrared (FTIR) difference spectroscopy in combination with specific [(13)C]alanine labeling and site-directed mutagenesis in photosystem II core particles from Synechocystis sp. PCC 6803. Several bands for carboxylate symmetric stretching modes in an S(2)/S(1) FTIR difference spectrum were affected by selective (13)C labeling of the alpha-carboxylate of Ala with l-[1-(13)C]alanine, whereas most of the isotopic effects failed to be induced in a site-directed mutant in which Ala-344 was replaced with Gly. Labeling of the alpha-methyl of Ala with l-[3-(13)C]alanine had much smaller effects on the spectrum to induce isotopic bands due to a symmetric CH(3) deformation coupled with the alpha-carboxylate. The isotopic bands for the alpha-carboxylate of Ala-344 showed characteristic changes during S-state cycling. The bands appeared prominently upon the S(1)-to-S(2) transition and to a lesser extent upon the S(2)-to-S(3) transition but reappeared at slightly upshifted frequencies with the opposite sign upon the S(3)-to-S(0) transition. No obvious isotopic band appeared upon the S(0)-to-S(1) transition. These results indicate that the alpha-carboxylate of C-terminal Ala-344 is structurally associated with a manganese ion that becomes oxidized upon the S(1)-to-S(2) transition and reduced reversely upon the S(3)-to-S(0) transition but is not associated with manganese ion(s) oxidized during the S(0)-to-S(1) (and S(2)-to-S(3)) transition(s). Consistently, l-[1-(13)C]alanine labeling also induced spectral changes in the low frequency (670-350 cm(-1)) S(2)/S(1) FTIR difference spectrum.     

Functional diversification of B MADS-box homeotic regulators of flower development: Adaptive evolution in protein-protein interaction domains after major gene duplication events

B-class MADS-box genes have been shown to be the key regulators of petal and stamen specification in several eudicot model species such as Arabidopsis thaliana, Antirrhinum majus, and Petunia hybrida. Orthologs of these genes have been found across angiosperms and gymnosperms, and it is thought that the basic regulatory function of B proteins is conserved in seed plant lineages. The evolution of B genes is characterized by numerous duplications that might represent key elements fostering the functional diversification of duplicates with a deep impact on their role in the evolution of the floral developmental program. To evaluate this, we performed a rigorous statistical analysis with B gene sequences. Using maximum likelihood and Bayesian methods, we estimated molecular substitution rates and determined the selective regimes operating at each residue of B proteins. We implemented tests that rely on phylogenetic hypotheses and codon substitution models to detect significant differences in substitution rates (DSRs) and sites under positive adaptive selection (PS) in specific lineages before and after duplication events. With these methods, we identified several protein residues fixed by PS shortly after the origin of PISTILLATA-like and APETALA3-like lineages in angiosperms and shortly after the origin of the euAP3-like lineage in core eudicots, the 2 main B gene duplications. The residues inferred to have been fixed by positive selection lie mostly within the K domain of the protein, which is key to promote heterodimerization. Additionally, we used a likelihood method that accommodates DSRs among lineages to estimate duplication dates for AP3-PI and euAP3-TM6, calibrating with data from the fossil record. The dates obtained are consistent with angiosperm origins and diversification of core eudicots. Our results strongly suggest that novel multimer formation with other MADS proteins could have been crucial for the functional divergence of B MADS-box genes. We thus propose a mechanism of functional diversification and persistence of gene duplicates by the appearance of novel multimerization capabilities after duplications. Multimer formation in different combinations of regulatory proteins can be a mechanistic basis for the origin of novel regulatory functions and a gene regulatory mechanism for the appearance of morphological innovations.     

Globin gene family evolution and functional diversification in annelids

Globins are the most common type of oxygen-binding protein in annelids. In this paper, we show that circulating intracellular globin (Alvinella pompejana and Glycera dibranchiata), noncirculating intracellular globin (Arenicola marina myoglobin) and extracellular globin from various annelids share a similar gene structure, with two conserved introns at canonical positions B12.2 and G7.0. Despite sequence divergence between intracellular and extracellular globins, these data strongly suggest that these three globin types are derived from a common ancestral globin-like gene and evolved by duplication events leading to diversification of globin types and derived functions. A phylogenetic analysis shows a distinct evolutionary history of annelid extracellular hemoglobins with respect to intracellular annelid hemoglobins and mollusc and arthropod extracellular hemoglobins. In addition, dehaloperoxidase (DHP) from the annelid, Amphitrite ornata, surprisingly exhibits close phylogenetic relationships to some annelid intracellular globins. We have characterized the gene structure of A. ornata DHP to confirm assumptions about its homology with globins. It appears that it has the same intron position as in globin genes, suggesting a common ancestry with globins. In A. ornata, DHP may be a derived globin with an unusual enzymatic function.     

Towards a global perspective for Salvia L.: Phylogeny,diversification and floral evolution

Salvia is the most species-rich genus in Lamiaceae, encompassing approximately 1000 species distributed all over the world. We sought a new evolutionary perspective for Salvia by employing macroevolutionary analyses to address the tempo and mode of diversification. To study the association of floral traits with speciation and extinction, we modelled and explored the evolution of corolla length and the lever-mechanism pollination system across our Salvia phylogeny. We reconstructed a multigene phylogeny for 366 species of Salvia in the broad sense including all major recognized lineages and 50 species from Iran, a region previously overlooked in studies of the genus. Our comprehensive sampling of Iranian species of Salvia provides higher phylogenetic resolution for southwestern Asian species than obtained in previous studies. Our phylogenetic data in combination with divergence time estimates were used to examine the evolution of corolla length, woody versus herbaceous habit, and presence versus absence of a lever mechanism. We investigated the timing and dependence of Salvia diversification related to corolla length evolution through a disparity test and BAMM analysis. A HiSSE model was used to evaluate the dependency of diversification on the lever-mechanism pollination system in Salvia. A medium corolla length (15–18 mm) was reconstructed as the ancestral state for Salvia with multiple shifts to shorter and longer corollas. Macroevolutionary model analyses indicate that corolla length disparity is high throughout Salvia evolution, significantly different from expectations under a Brownian motion model during the last 28 million years of evolution. Our analyses show evidence of a higher diversification rate of corolla length for some Andean species of Salvia compared to other members of the genus. Based on our tests of diversification models, we reject the hypothesis of a direct effect of the lever mechanism on Salvia diversification. Therefore, we suggest caution in considering the lever-mechanism pollination system as one of the main drivers of speciation in Salvia.     

A novel MADS-box gene subfamily with a sister-group relationship to class B floral homeotic genes

Class B floral homeotic genes specify the identity of petals and stamens during the development of angiosperm flowers. Recently, putative orthologs of these genes have been identified in different gymnosperms. Together, these genes constitute a clade, termed B genes. Here we report that diverse seed plants also contain members of a hitherto unknown sister clade of the B genes, termed B(sister) (B(s)) genes. We have isolated members of the B(s) clade from the gymnosperm Gnetum gnemon, the monocotyledonous angiosperm Zea mays and the eudicots Arabidopsis thaliana and Antirrhinum majus. In addition, MADS-box genes from the basal angiosperm Asarum europaeum and the eudicot Petunia hybrida were identified as B(s) genes. Comprehensive expression studies revealed that B(s) genes are mainly transcribed in female reproductive organs (ovules and carpel walls). This is in clear contrast to the B genes, which are predominantly expressed in male reproductive organs (and in angiosperm petals). Our data suggest that the B(s) genes played an important role during the evolution of the reproductive structures in seed plants. The establishment of distinct B and B(s) gene lineages after duplication of an ancestral gene may have accompanied the evolution of male microsporophylls and female megasporophylls 400-300 million years ago. During flower evolution, expression of B(s) genes diversified, but the focus of expression remained in female reproductive organs. Our findings imply that a clade of highly conserved close relatives of class B floral homeotic genes has been completely overlooked until recently and awaits further evaluation of its developmental and evolutionary importance. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00438-001-0615-8.     

Dramatic expansion and developmental expression diversification of the methuselah gene family during recent Drosophila evolution

Functional studies of the methuselah/methuselah-like (mth/mthl) gene family have focused on the founding member mth, but little is known regarding the developmental functions of this receptor or any of its paralogs. We undertook a comprehensive analysis of developmental expression and sequence divergence in the mth/mthl gene family. Using in situ hybridization techniques, we detect expression of six genes (mthl1, 5, 9, 11, 13, and 14) in the embryo during gastrulation and development of the gut, heart, and lymph glands. Four receptors (mthl3, 4, 6, and 8) are expressed in the larval central nervous system, imaginal discs, or both, and two receptors (mthl10 and mth) are expressed in both embryos and larvae. Phylogenetic analysis of all mth/mthl genes in five Drosophila species, mosquito and flour beetle structured the mth/mthl family into several subclades. mthl1, 5, and 14 are present in most species, each forming a separate clade. A newly identified Drosophila mthl gene (CG31720; herein mthl15) formed another ancient clade. The remaining Drosophila receptors, including mth, are members of a large "superclade" that diversified relatively recently during dipteran evolution, in many cases within the melanogaster subgroup. Comparing the expression patterns of the mth/mthl "superclade" paralogs to the embryonic expression of the singleton ortholog in Tribolium suggests both subfunctionalization and acquisition of novel functionalities. Taken together, our findings shed novel light on mth as a young member of an adaptively evolving developmental gene family.