Divergence Of Monocots Research Articles

Identification of MYC genes in four Cucurbitaceae species and their roles in the response to temperature stress

BackgroundMyelocytomatosis (MYC) transcription factors are crucial mediators of the response of plants to environmental stresses through via binding to DNA regulatory regions. However, few systematic characterizations of MYC genes are available in Cucurbitaceae species.ResultsIn this study, we identified 10, 8, 12, and 10 MYC genes in Cucumis sativus, Cucumis melo, Citrullus lanatus, and Benincasa hispida, respectively. Characterization revealed that all of the MYC proteins contain a highly conserved H4-V5-E6-E8-R9-R11-R12 sequence, which is essential for the binding of DNA regulatory regions. Evolutionary analysis enabled us to categorize 40 predicted MYC proteins from seven species into five distinct groups and revealed that the expansion of the MYC genes occurred before the divergence of monocots and dicots. The upstream promoter regions of the MYC genes contain a variety of developmental, stress, and hormone-responsive regulatory elements. The expression of cucumber MYC genes varies significantly across organs, with particularly high expression of CsaV3_3G001710 observed across all organs. Transcriptomic analysis revealed that certain cucumber MYC genes undergo specific upregulation or downregulation in response to both biotic and abiotic stressors. In particular, under temperature stress, the cucumber genes CsaV3_3G007980 and CsaV3_3G001710 were significantly upregulated. Interestingly, the homologs of these two genes in C. lanatus presented a similar expression pattern to that in C. sativus, whereas in B. hispida, they presented the opposite pattern, i.e., significant downregulation. These findings indicated that these two genes indeed respond to temperature stress but with different expression patterns, highlighting the divergent functions of homologous genes across different species.ConclusionsThis study analyzed the size and composition of the MYC gene family in four Cucurbitaceae species and investigated stress-responsive expression profiles, especially under temperature stress. All the results showed that MYC genes play important roles in development and stress responses, laying a theoretical foundation for further investigations of these response mechanisms.

Genome-Wide Analysis of the TCP Gene Family and Their Expression Pattern Analysis in Tea Plant (Camellia sinensis)

TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors TEOSINTE BRANCHED1/CYCLOIDEA/PCF have been suggested to control the cell growth and proliferation in meristems and lateral organs. A total of 37 CsTCP genes were identified and divided into two classes, class I (PCF, group 1) and class II (CIN CYC/TB1, groups 2, and 3). The residues of TEOSINTE BRANCHED1/CYCLOIDEA/PCF of Camellia sinensis (Tea plant) (CsTCP) proteins between class I and class II were definitely different in the loop, helix I, and helix II regions; however, eighteen conserved tandem was found in bHLH. There are a large number of CsTCP homologous gene pairs in three groups. Additionally, most CsTCP proteins have obvious differences in motif composition. The results illuminated that CsTCP proteins in different groups are supposed to have complementary functions, whereas those in the same class seem to display function redundancies. There is no relationship between the number of CsTCP gene members and genome size, and the CsTCP gene family has only expanded since the divergence of monocots and eudicots. WGD/segmental duplication played a vital role in the expansion of the CsTCP gene family in tea plant, and the CsTCP gene family has expanded a lot. Most CsTCP genes of group 1 are more widely and non-specifically expressed, and the CsTCP genes of group 2 are mainly expressed in buds, flowers, and leaves. Most genes of group 1 and some genes of group 2 were up-/downregulated in varying degrees under different stress, CsTCP genes of group 3 basically do not respond to stress. TCP genes involved in abiotic stress response mostly belong to PCF group. Some CsTCP genes may have the same function as the homologous genes in Arabidopsis, but there is functional differentiation.

The Litsea genome and the evolution of the laurel family

The laurel family within the Magnoliids has attracted attentions owing to its scents, variable inflorescences, and controversial phylogenetic position. Here, we present a chromosome-level assembly of the Litsea cubeba genome, together with low-coverage genomic and transcriptomic data for many other Lauraceae. Phylogenomic analyses show phylogenetic discordance at the position of Magnoliids, suggesting incomplete lineage sorting during the divergence of monocots, eudicots, and Magnoliids. An ancient whole-genome duplication (WGD) event occurred just before the divergence of Laurales and Magnoliales; subsequently, independent WGDs occurred almost simultaneously in the three Lauralean lineages. The phylogenetic relationships within Lauraceae correspond to the divergence of inflorescences, as evidenced by the phylogeny of FUWA, a conserved gene involved in determining panicle architecture in Lauraceae. Monoterpene synthases responsible for production of specific volatile compounds in Lauraceae are functionally verified. Our work sheds light on the evolution of the Lauraceae, the genetic basis for floral evolution and specific scents.

The evolutionary history of the sucrose synthase gene family in higher plants

BackgroundSucrose synthase (SUS) is widely considered a key enzyme participating in sucrose metabolism in higher plants and regarded as a biochemical marker for sink strength in crops. However, despite significant progress in characterizing the physiological functions of the SUS gene family, knowledge of the trajectory of evolutionary processes and significance of the family in higher plants remains incomplete.ResultsIn this study, we identified over 100 SUS genes in 19 plant species and reconstructed their phylogenies, presenting a potential framework of SUS gene family evolution in higher plants. Three anciently diverged SUS gene subfamilies (SUS I, II and III) were distinguished based on their phylogenetic relationships and unique intron/exon structures in angiosperms, and they were found to have evolved independently in monocots and dicots. Each subfamily of SUS genes exhibited distinct expression patterns in a wide range of plants, implying that their functional differentiation occurred before the divergence of monocots and dicots. Furthermore, SUS III genes evolved under relaxed purifying selection in dicots and displayed narrowed expression profiles. In addition, for all three subfamilies of SUS genes, the GT-B domain was more conserved than the “regulatory” domain.ConclusionsThe present study reveals the evolution of the SUS gene family in higher plants and provides new insights into the evolutionary conservation and functional divergence of angiosperm SUS genes.

The roles of Aux/IAA gene family in development of Dendrocalamus sinicus (Poaceae: Bambusoideae) inferred by comprehensive analysis and expression profiling.

Auxin is an important hormone in many plant developmental processes. In this study, the auxin/indole acetic acid (Aux/IAA) gene family was comprehensively identified using Dendrocalamus sinicus transcriptome data. A total of 26 Aux/IAA genes (DsIAA1-DsIAA26) were mined using four conserved Aux/IAA family motifs (PF02309). They encoded hydrophilic proteins, including one or two nuclear localisation signals. The D. sinicus Aux/IAA proteins were classified into two groups, including seven sister-gene pairs based on their phylogenetic relationships. A phylogenetic tree generated by aligning 108 predicted protein sequences of 26 DsIAAs, 43 PhIAAs (Phyllostachys heterocycla), 29 AtIAAs (Arabidopsis), 31 OsIAAs (Oryza sativa) and 22 PtIAAs (Populus) revealed nine major groups. Among them, four groups, including 96 IAA proteins of all five species, suggested that the genes originated before divergence of monocots and dicots. The expression profiling in different tissues showed that most of the DsIAAs preferentially expressed in leaves and shoots, suggesting their important roles in the development of leaves and shoots in D. sinicus. Continuously high expression of DsIAA3, DsIAA4, DsIAA15, and DsIAA20 may be important for regulating shoot development in D. sinicus. These results provide useful information for further research into the function of Aux/IAA genes in woody sympodial bamboos.

Efficient Replication of the Plastid Genome Requires an Organellar Thymidine Kinase.

Thymidine kinase (TK) is a key enzyme of the salvage pathway that recycles thymidine nucleosides to produce deoxythymidine triphosphate. Here, we identified the single TK of maize (Zea mays), denoted CPTK1, as necessary in the replication of the plastidial genome (cpDNA), demonstrating the essential function of the salvage pathway during chloroplast biogenesis. CPTK1 localized to both plastids and mitochondria, and its absence resulted in an albino phenotype, reduced cpDNA copy number and a severe deficiency in plastidial ribosomes. Mitochondria were not affected, indicating they are less reliant on the salvage pathway. Arabidopsis (Arabidopsis thaliana) TKs, TK1A and TK1B, apparently resulted from a gene duplication after the divergence of monocots and dicots. Similar but less-severe effects were observed for Arabidopsis tk1a tk1b double mutants in comparison to those in maize cptk1 TK1B was important for cpDNA replication and repair in conditions of replicative stress but had little impact on the mitochondrial phenotype. In the maize cptk1 mutant, the DNA from the small single-copy region of the plastidial genome was reduced to a greater extent than other regions, suggesting preferential abortion of replication in this region. This was accompanied by the accumulation of truncated genomes that resulted, at least in part, from unfaithful microhomology-mediated repair. These and other results suggest that the loss of normal cpDNA replication elicits the mobilization of new replication origins around the rpoB (beta subunit of plastid-encoded RNA polymerase) transcription unit and imply that increased transcription at rpoB is associated with the initiation of cpDNA replication.

Identification and analysis of copine/BONZAI proteins among evolutionarily diverse plant species.

Copines are evolutionarily conserved calcium-dependent membrane-binding proteins with potentially critical biological functions. In plants, the function of these proteins has not been analyzed except for in Arabidopsis thaliana where they play critical roles in development and disease resistance. To facilitate functional studies of copine proteins in crop plants, genome-wide identification, curation, and phylogeny analysis of copines in 16 selected plant species were conducted. All the identified 32 plant copines have conserved features of the two C2 domains (C2A and C2B) and the von Willebrand factor A (vWA) domain. Different from animal and protozoa copines, plant copines have glycine at the second residue potentially acquiring a unique protein myristoylation modification. Phylogenetic analysis suggests that copine was present as one copy when evolving from green algae to basal flowering plants, and duplicated before the divergence of monocots and dicots. In addition, gene expression and protein localization study of rice copines suggests both conserved and different properties of copines in dicots and monocots. This study will contribute to uncovering the role of copine genes in different plant species.

Genomic identification, phylogeny, and expression analysis of MLO genes involved in susceptibility to powdery mildew in Fragaria vesca.

The MLO (powdery mildew locus O) gene family is important in resistance to powdery mildew (PM). In this study, all of the members of the MLO family were identified and analyzed in the strawberry (Fragaria vesca) genome. The strawberry contains at least 20 members of the MLO family, and the protein sequence contained between 171 and 1485 amino acids, with 0-34 introns. Chromosomal localization showed that the MLOs were unevenly distributed on each of the chromosomes, except for chromosome 4. The greatest number of MLOs (seven) was found on chromosome 3. A phylogenetic tree showed that the MLOs were divided into seven groups (I-VII), four of which consisted of MLOs from strawberry, Arabidopsis thaliana, rice, and maize, suggesting that these genes may have evolved after the divergence of monocots and dicots. Multiple sequence alignment showed that strawberry MLO candidates related to powdery mildew resistance possessed seven highly conserved transmembrane domains, a calmodulin-binding domain, and two conserved regions, all of which are important domains for powdery mildew resistance genes. Expressed sequence tag analysis revealed that the MLOs were induced by multiple abiotic stressors, including low and high temperature, drought, and high salinity. These findings will contribute to the functional characterization of MLOs related to PM susceptibility, and will assist in the development of disease resistance in strawberries.

Role of Plant Somatic Embryogenesis Receptor Kinases (SERKs) in Cell-to-Embryo Transitional Activity: Key at Novel Assorted Structural Subunits

Somatic Embryogenesis Receptor Kinase (SERK) family of receptor kinases is functionally diverse, involved in cell-to-embryo transition and controlling a number of other fundamental aspects of plant development. The morphological transformation of somatic to embryonic cells has drawn scientific attention utmost due to remarkable genetic-switch system evolved across species. Receptor kinases having direct role in somatic embryogenesis (SE) and involved in other functions are designated as “SERK” and “SERK-like” genes, respectively. We aim for phylogenetic reconstruction to reveal major SERK groups across plant species (angiosperm to gymnosperm) for their functional diversification. Data indicate that the development of SERK proteins occurred prior to the divergence of monocots and eudicots. Also, the SERK orthology is not directly proportional to their functions. Structure prediction results identified novel transmembrane topologies, short linear motifs and O-glycosylation sites exclusively in SERK proteins than SERK-like proteins. Comparative temporal expression analyses of SERK and SERK-like genes provided significant accordance with their physiological function. The identification of intrinsic disordered regions (IDRs) exclusively in SERK proteins was assumed to perceive external stress-induced signals that may lead to rapid protein folding. In a result it switches-on the precise cellular signals essential for the acquisition of SE. Moreover, the regulatory sequences of SERK genes are evolved with unique cellular fate deciding AP2-like ethylene responsive transcription factor AINTEGUMENTA binding sites for their spatial expression in SE. Based on these analyses we suggest future avenues of research that may be imperative for elucidating the importance of SERK gene evolution in SE.

Functional Conservation of MIKC*-Type MADS Box Genes in Arabidopsis and Rice Pollen Maturation

There are two groups of MADS intervening keratin-like and C-terminal (MIKC)-type MADS box genes, MIKC(C) type and MIKC* type. In seed plants, the MIKC(C) type shows considerable diversity, but the MIKC* type has only two subgroups, P- and S-clade, which show conserved expression in the gametophyte. To examine the functional conservation of MIKC*-type genes, we characterized all three rice (Oryza sativa) MIKC*-type genes. All three genes are specifically expressed late in pollen development. The single knockdown or knockout lines, respectively, of the S-clade MADS62 and MADS63 did not show a mutant phenotype, but lines in which both S-clade genes were affected showed severe defects in pollen maturation and germination, as did knockdown lines of MADS68, the only P-clade gene in rice. The rice MIKC*-type proteins form strong heterodimeric complexes solely with partners from the other subclade; these complexes specifically bind to N10-type C-A-rich-G-boxes in vitro and regulate downstream gene expression by binding to N10-type promoter motifs. The rice MIKC* genes have a much lower degree of functional redundancy than the Arabidopsis thaliana MIKC* genes. Nevertheless, our data indicate that the function of heterodimeric MIKC*-type protein complexes in pollen development has been conserved since the divergence of monocots and eudicots, roughly 150 million years ago.

Genome-wide identification, characterization, and expression analysis of the MLO gene family in Cucumis sativus

Mildew resistance locus o (MLO) is a plant-specific seven-transmembrane (TM) gene family. Several studies have revealed that certain members of the MLO gene family mediate powdery mildew susceptibility in three plant species, namely, Arabidopsis, barley, and tomato. The sequenced cucumber genome provides an opportunity to conduct a comprehensive overview of the MLO gene family. Fourteen genes (designated CsMLO01 through CsMLO14) have been identified within the Cucumis sativus genome by using an in silico cloning method with the MLO amino acid sequences of Arabidopsis thaliana and rice as probes. Sequence alignment revealed that numerous features of the gene family, such as TMs, a calmodulin-binding domain, peptide domains I and II, and 30 important amino acid residues for MLO function, are well conserved. Phylogenetic analysis of the MLO genes from cucumber and other plant species reveals seven different clades (I through VII). Three of these clades comprised MLO genes from A. thaliana, rice, maize, and cucumber, suggesting that these genes may have evolved after the divergence of monocots and dicots. In silico mapping showed that these CsMLOs were located on chromosomes 1, 2, 3, 4, 5, and 6 without any obvious clustering, except CsMLO01. To our knowledge, this paper is the first comprehensive report on MLO genes in C. sativus. These findings will facilitate the functional characterization of the MLOs related to powdery mildew susceptibility and assist in the development of disease resistance in cucumber.

A functional phylogenomic view of the seed plants.

A novel result of the current research is the development and implementation of a unique functional phylogenomic approach that explores the genomic origins of seed plant diversification. We first use 22,833 sets of orthologs from the nuclear genomes of 101 genera across land plants to reconstruct their phylogenetic relationships. One of the more salient results is the resolution of some enigmatic relationships in seed plant phylogeny, such as the placement of Gnetales as sister to the rest of the gymnosperms. In using this novel phylogenomic approach, we were also able to identify overrepresented functional gene ontology categories in genes that provide positive branch support for major nodes prompting new hypotheses for genes associated with the diversification of angiosperms. For example, RNA interference (RNAi) has played a significant role in the divergence of monocots from other angiosperms, which has experimental support in Arabidopsis and rice. This analysis also implied that the second largest subunit of RNA polymerase IV and V (NRPD2) played a prominent role in the divergence of gymnosperms. This hypothesis is supported by the lack of 24nt siRNA in conifers, the maternal control of small RNA in the seeds of flowering plants, and the emergence of double fertilization in angiosperms. Our approach takes advantage of genomic data to define orthologs, reconstruct relationships, and narrow down candidate genes involved in plant evolution within a phylogenomic view of species' diversification.

A rice high-affinity potassium transporter (HKT) conceals a calcium-permeable cation channel

Plant high-affinity K(+) transport (HKT) proteins are so named because of their relation to bacterial and fungal transporters that mediate high-affinity K(+) uptake. The view that HKT family members are sodium-selective uniporters or sodium-potassium symporters is widely held. We have found that one of the rice HKT proteins also functions as a Ca(2+)-permeable cation channel that conducts current carried by a wide range of monovalent and divalent cations. The HKT rice gene, named OsHKT2;4, is expressed in several cell types, including root hairs and vascular parenchyma cells. The protein is localized to the plasma membrane, thereby providing a mechanism for cation uptake and extrusion. This finding goes against firmly entrenched dogma in showing that HKT proteins can function as both ion carriers and channels. The study further extends the function of HKT proteins to Ca(2+)-linked processes and, in so doing, defines a previously undescribed type of Ca(2+)-permeable cation channels in plants. The work also raises questions about the evolutionary changes in this protein family following the divergence of monocots and dicots.

Evolution of serine carboxypeptidase-like acyltransferases in the monocots

The serine carboxypeptidases are a large family of proteases. In higher plants some members of this family have diversified and adopted new functions as acyltransferases required for the synthesis of natural products. We recently reported the first serine carboxypeptidase-like (SCPL) acyltransferase enzyme to be characterised from monocotyledonous plants.1 This enzyme, AsSCPL1, is required for acylation of antimicrobial terpenes (avenacins) that are produced in the roots of oat (Avena spp.) and that provide protection against soil-borne pathogens. The SCPL enzyme family has undergone substantial expansion following the divergence of monocots and dicots. Here we discuss the evolution of the SCPL enzyme family in monocots, their contribution to metabolic diversity, and the roles of these enzymes in biotic and abiotic stress tolerance.

Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom

BackgroundAs a major component of plant cell wall, lignin plays important roles in mechanical support, water transport, and stress responses. As the main cause for the recalcitrance of plant cell wall, lignin modification has been a major task for bioenergy feedstock improvement. The study of the evolution and function of lignin biosynthesis genes thus has two-fold implications. First, the lignin biosynthesis pathway provides an excellent model to study the coordinative evolution of a biochemical pathway in plants. Second, understanding the function and evolution of lignin biosynthesis genes will guide us to develop better strategies for bioenergy feedstock improvement.ResultsWe analyzed lignin biosynthesis genes from fourteen plant species and one symbiotic fungal species. Comprehensive comparative genome analysis was carried out to study the distribution, relatedness, and family expansion of the lignin biosynthesis genes across the plant kingdom. In addition, we also analyzed the comparative synteny map between rice and sorghum to study the evolution of lignin biosynthesis genes within the Poaceae family and the chromosome evolution between the two species. Comprehensive lignin biosynthesis gene expression analysis was performed in rice, poplar and Arabidopsis. The representative data from rice indicates that different fates of gene duplications exist for lignin biosynthesis genes. In addition, we also carried out the biomass composition analysis of nine Arabidopsis mutants with both MBMS analysis and traditional wet chemistry methods. The results were analyzed together with the genomics analysis.ConclusionThe research revealed that, among the species analyzed, the complete lignin biosynthesis pathway first appeared in moss; the pathway is absent in green algae. The expansion of lignin biosynthesis gene families correlates with substrate diversity. In addition, we found that the expansion of the gene families mostly occurred after the divergence of monocots and dicots, with the exception of the C4H gene family. Gene expression analysis revealed different fates of gene duplications, largely confirming plants are tolerant to gene dosage effects. The rapid expansion of lignin biosynthesis genes indicated that the translation of transgenic lignin modification strategies from model species to bioenergy feedstock might only be successful between the closely relevant species within the same family.

Composition and phylogenetic analysis of wheat cryptochrome gene family

Cryptochrome (CRY) gene family encodes photoreceptors mediating developmental responses to blue light throughout the life of plants. We report here the characterization of CRY gene family in hexaploid wheat. Degenerate PCR amplification of the regions encoding the conserved flavin-binding domain of CRY proteins yielded seven bands, resulting from amplification of CRY1a, CRY1b and CRY2 homologous genes. Assignment of individual amplicons to subgenomes was accomplished by comparing their sequence compositions with those from the ancestor species of wheat. ESTs coding for CRY-DASH like proteins were identified in wheat EST database in GenBank. Southern blot showed that TaCRY1a, TaCRY1b and TaCRY2 are single copy genes. We mapped TaCRY1a and TaCRY2 to chromosomes of homoeologous group 6, TaCRY1b to group 2, and TaCRY-DASH to group 7. Phylogenetic analysis showed that CRY subfamily diversification occurred before the divergence of monocots and dicots. The regulatory and functional changes of CRY members within subfamily are discussed.

Phytochrome gene expression and phylogenetic analysis in the short‐day plant Pharbitis nil (Convolvulaceae): Differential regulation by light and an endogenous clock

To investigate the role of distinct phytochrome pools in photoperiodic timekeeping, we characterized four phytochrome genes in the short-day plant Pharbitis nil. Each PHY gene had different photosensory properties and sensitivity to night break that inhibits flowering. During extended dark periods, PHYE, PHYB, and PHYC mRNA accumulation exhibited a circadian rhythmicity indicative of control by an endogenous clock. Phylogenetic analysis recovered four clades of angiosperm phytochrome genes, phyA, phyB, phyC, and phyE. All except the phyE clade included sequences from both monocots and eudicots. In addition, phyA is sister to phyC and phyE sister to phyB, with gymnosperm sequences sister to either the phyA-phyC clade or to the phyB-phyE clade. These results suggest that a single duplication occurred in an ancestral seed plant before the divergence of extant gymnosperms from angiosperms and that two subsequent duplications occurred in an ancestral angiosperm before the divergence of monocots from eudicots. Thus in P. nil, a multigene family with different patterns of mRNA abundance in light and darkness contributes to the total phytochrome pool: one pool is light labile (phyA), whereas the other is light stable (phyB and phyE). In addition, PHYC mRNA represents a third phytochrome pool with intermediate photosensory properties.

Phylogeny and evolution of Rab7 and Rab9 proteins

BackgroundAn important role in the evolution of intracellular trafficking machinery in eukaryotes played small GTPases belonging to the Rab family known as pivotal regulators of vesicle docking, fusion and transport. The Rab family is very diversified and divided into several specialized subfamilies. We focused on the VII functional group comprising Rab7 and Rab9, two related subfamilies, and analysed 210 sequences of these proteins. Rab7 regulates traffic from early to late endosomes and from late endosome to vacuole/lysosome, whereas Rab9 participates in transport from late endosomes to the trans-Golgi network.ResultsAlthough Rab7 and Rab9 proteins are quite small and show heterogeneous rates of substitution in different lineages, we found a phylogenetic signal and inferred evolutionary relationships between them. Rab7 proteins evolved before radiation of main eukaryotic supergroups while Rab9 GTPases diverged from Rab7 before split of choanoflagellates and metazoans. Additional duplication of Rab9 and Rab7 proteins resulting in several isoforms occurred in the early evolution of vertebrates and next in teleost fishes and tetrapods. Three Rab7 lineages emerged before divergence of monocots and eudicots and subsequent duplications of Rab7 genes occurred in particular angiosperm clades. Interestingly, several Rab7 copies were identified in some representatives of excavates, ciliates and amoebozoans. The presence of many Rab copies is correlated with significant differences in their expression level. The diversification of analysed Rab subfamilies is also manifested by non-conserved sequences and structural features, many of which are involved in the interaction with regulators and effectors. Individual sites discriminating different subgroups of Rab7 and Rab9 GTPases have been identified.ConclusionPhylogenetic reconstructions of Rab7 and Rab9 proteins were performed by a variety of methods. These Rab GTPases show diversification both at the phylogenetic, expression and structural levels. The presence of many Rab7 and Rab9 isoforms suggests their functional specialization and complexity of subcellular trafficking even in unicellular eukaryotes. The identified less conserved regions in analysed Rab sequences may directly contribute to such a differentiation.

Duplications and functional divergence of ADP-glucose pyrophosphorylase genes in plants

BackgroundADP-glucose pyrophosphorylase (AGPase), which catalyses a rate limiting step in starch synthesis, is a heterotetramer comprised of two identical large and two identical small subunits in plants. Although the large and small subunits are equally sensitive to activity-altering amino acid changes when expressed in a bacterial system, the overall rate of non-synonymous evolution is ~2.7-fold greater for the large subunit than for the small subunit. Herein, we examine the basis for their different rates of evolution, the number of duplications in both large and small subunit genes and document changes in the patterns of AGPase evolution over time.ResultsWe found that the first duplication in the AGPase large subunit family occurred early in the history of land plants, while the earliest small subunit duplication occurred after the divergence of monocots and eudicots. The large subunit also had a larger number of gene duplications than did the small subunit. The ancient duplications in the large subunit family raise concern about the saturation of synonymous substitutions, but estimates of the absolute rate of AGPase evolution were highly correlated with estimates of ω (the non-synonymous to synonymous rate ratio). Both subunits showed evidence for positive selection and relaxation of purifying selection after duplication, but these phenomena could not explain the different evolutionary rates of the two subunits. Instead, evolutionary constraints appear to be permanently relaxed for the large subunit relative to the small subunit. Both subunits exhibit branch-specific patterns of rate variation among sites.ConclusionThese analyses indicate that the higher evolutionary rate of the plant AGPase large subunit reflects permanent relaxation of constraints relative to the small subunit and they show that the large subunit genes have undergone more gene duplications than small subunit genes. Candidate sites potentially responsible for functional divergence within each of the AGPase subunits were investigated by examining branch-specific patterns of rate variation. We discuss the phenotypes of mutants that alter some candidate sites and strategies for examining candidate sites of presently unknown function.

Substitution of the Gene for Chloroplast RPS16 Was Assisted by Generation of a Dual Targeting Signal

Organelle (mitochondria and chloroplasts in plants) genomes lost a large number of genes after endosymbiosis occurred. Even after this major gene loss, organelle genomes still lose their own genes, even those that are essential, via gene transfer to the nucleus and gene substitution of either different organelle origin or de novo genes. Gene transfer and substitution events are important processes in the evolution of the eukaryotic cell. Gene loss is an ongoing process in the mitochondria and chloroplasts of higher plants. The gene for ribosomal protein S16 (rps16) is encoded in the chloroplast genome of most higher plants but not in Medicago truncatula and Populus alba. Here, we show that these 2 species have compensated for loss of the rps16 from the chloroplast genome by having a mitochondrial rps16 that can target the chloroplasts as well as mitochondria. Furthermore, in Arabidopsis thaliana, Lycopersicon esculentum, and Oryza sativa, whose chloroplast genomes encode the rps16, we show that the product of the mitochondrial rps16 has dual targeting ability. These results suggest that the dual targeting of RPS16 to the mitochondria and chloroplasts emerged before the divergence of monocots and dicots (140-150 MYA). The gene substitution of the chloroplast rps16 by the nuclear-encoded rps16 in higher plants is the first report about ongoing gene substitution by dual targeting and provides evidence for an intermediate stage in the formation of this heterogeneous organelle.