Molecular Phylogeny and Evolution of the Plant-Specific Seven-Transmembrane MLO Family

Abstract Homologues of barley Mlo encode the only family of seven-transmembrane (TM) proteins in plants. Their topology, subcellular localization, and sequence diversification are reminiscent of those of G-protein coupled receptors (GPCRs) from animals and fungi. We present a computational analysis of MLO family members based on 31 full-size and 3 partial sequences, which originate from several monocot species, the dicot Arabidopsis thaliana, and the moss Ceratodon purpureus. This enabled us to date the origin of the Mlo gene family back at least to the early stages of land plant evolution. The genomic organization of the corresponding genes supports a monophyletic origin of the Mlo gene family. Phylogenetic analysis revealed five clades, of which three contain both monocot and dicot members, while two indicate class-specific diversification. Analysis of the ratio of nonsynonymous-to-synonymous changes in coding sequences provided evidence for functional constraint on the evolution of the DNA sequences and purifying selection, which appears to be reduced in the first extracellular loop of 12 closely related orthologues. The 31 full-size sequences were examined for potential domain-specific intramolecular coevolution. This revealed evidence for concerted evolution of all three cytoplasmic domains with each other and the C-terminal cytoplasmic tail, suggesting interplay of all intracellular domains for MLO function.     

木薯Mlo基因家族成员的鉴定及其序列特征分析

M／o基因家族是植物重要的抗病基因。本文通过系统分析木薯基因组数据库,从中共鉴定出21个M／o成员,其中20个具有完整序列,1个只有部分序列。对其中20个具有完整序列的基因与其他物种的Mlo基因进行聚类关系分析,结果显示,可将木薯Mlo基因家族分为6类（I～VI）,其中4类都包括有来自拟南芥的Mlo基因,第vI类只包括2个木薯Mlo基因,可能是木薯中特有的一类Mlo;6个木薯Mlo与已知的抗病Mlo基因分别聚在第1V和第V类,这6个基因可能是木薯基因组中具有抗病功能的Mlo。对所有的木薯Mlo蛋白进行结构分析发现,除了MeMl020外,其他蛋白均具有6～8个跨膜结构,其中3个蛋白具有N端信号肽。     

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. Received: 31 January 1997 / Accepted: 9 April 1997     

Genome-scale identification of MLO domain-containing genes in soybean (Glycine max L. Merr.)

In plants, powdery-mildew-resistance locus o (Mlo) genes encode proteins that are calmodulin-binding proteins involved in a variety of cellular processes. However, systematic characterization of this gene family in soybean (Glycine max L. Merr.) has not been yet reported. In this study, we identified MLO domain-contained members in soybean and examined their expression under phytohormone treatment and abiotic stress conditions. A total of 20 soybean Mlo genes were identified (GmMlo1-20), which are distributed on 13 chromosomes, and display diverse exon-intron structures. Phylogenetic analysis indicated that the Mlo family can be classified into four subfamilies. Sequence comparison was used to reveal the conserved calmodulin-binding domain (CaMBD) in GmMLO proteins. The expression of GmMlo genes was influenced by various phytohormone treatments and abiotic stresses, suggesting that these Mlo genes have various roles in the response of soybean to environmental stimuli. Promoter sequence analysis revealed an overabundance of stress and/or phytohormone-related cis-elements in GmMlo genes. These data provide important clues for elucidating the functions of genes of the Mlo gene family.     

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 Phylogeny,Evolution, and Functional Divergence of the LSD1-Like Gene Family: Inference from the Rice Genome

The identification of LSD1-like genes in parasite, green algae, moss, pine, and monocot and dicot species allowed us to trace the phylogenetic history of this gene family. Computational analysis showed that the diversification of members of this family could be dated back to the early stage of plant evolution. The evolution of plant LSD1-like genes was possibly shaped by two duplication events. These proteins, which contain three copies of the LSD1 zinc finger (zf-LSD1) domain within their entire polypeptides and play crucial roles in modulating disease defense and cell death, resulted from the second duplication. A gain of zf-LSD1 domain model was reasonable for explaining the origination of three-zf-LSD1 domain-containing proteins. The zf-LSD1 domain phylogeny showed that the middle (M) and C-terminal (C) domains originated from a common ancestor; the N-terminal (N) domain might be more ancient than the former two. The divergence of the N, M, and C domains was well before the monocot-dicot split. Coevolution analysis revealed that four intramolecular domain pairs, including the N domain and the interregion between the M and the C domains (INTER2), the M and C domain, the N- and C-terminus, and the M domain and C-terminus, possibly coevolved during the evolution of three-zf-LSD1 domain-containing proteins. The three zf-LSD1 domains are evolutionary conserved. Thus, the differences at the N- and C-terminus would be crucial for functional specificity of LSD1 genes. Strong functional constraints should work on the zf-LSD1 domains, whereas reduced functional constraint was found in the INTER2 region. Functional divergence analysis showed that three-zf-LSD1 domain-containing proteins were significantly functionally divergent from those proteins containing only one zf-LSD1 domain, a result demonstrating that shifted evolutionary rates between the two clusters were significantly different from each other. [Reviewing Editor: Dr. Joshua Plotkin]     

Molecular evolution of the zinc-containing long-chain alcohol dehydrogenase genes

Phylogenetic relationships and rates of nucleotide substitution were studied for alcohol dehydrogenase (ADH) genes by using DNA sequences from mammals and plants. Mammalian ADH sequences include the three class I genes and a class II gene from humans and one gene each from baboon, rat, and mouse. Plant sequences include two ADH genes each from maize and rice, three genes from barley, and one gene each from wheat and two dicots, Arabidopsis and pea. Phylogenetic trees show that relationships among ADH genes are generally consistent with taxonomic relationships: mammalian and plant ADH genes are classified into two distinct groups; primate class I genes are clustered; and two dicot sequences are clustered separately from monocot sequences. Accelerated evolution has been detected among the duplicated ADH genes in plants, in which synonymous substitutions occurred more often within the coenzyme-binding domain than within the catalytic domains.     

The Egg apparatus 1 gene from maize is a member of a large gene family found in both monocots and dicots

The maize ZmEA1 protein was recently postulated to be involved in short-range pollen tube guidance from the embryo sac. To date, EA1-like sequences had only been identified in monocot species. Using a more conserved C-terminal motif found in the monocot species, numerous ZmEA1-like sequences were retrieved in EST databases from dicot species, as well as from unannotated genomic sequences of Arabidopsis thaliana. RT-PCR analyses were produced for these unannotated genes and showed that these were indeed expressed genes. Further structural and phylogenetic analyses revealed that all members of the EA1-like (EAL) gene family shared a conserved 27–29 amino acid motif, termed the EA box near the C-terminal end, and appear to be secretory proteins. Therefore, the EA box proteins defines a new class of small secretory proteins, some of which being possibly involved in pollen tube guidance. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Maize U2 snRNAs: gene sequence and expression.

The complexity of plant U-type small nuclear ribonucleoprotein particles (UsnRNPs) may represent one level at which differences in splicing between animals and plants and between monocotyledonous and dicotyledonous plants could be effected. The maize (monocot.) U2snRNA multigene family consists of some 25 to 40 genes which from RNA blot and RNase protection analyses produce U2snRNAs varying in both size and sequence. The first 77 nucleotides of the maize U2-27 snRNA gene are identical to U2snRNA genes of Arabidopsis (dicot). Despite much lower sequence homology in the remaining 120 nucleotides the secondary structure of the RNA is conserved. The difference in splicing between monocot. and dicot. plants cannot be explained on the basis of sequence differences between monocot, and dicot. U2snRNAs in the region which may interact with intron branch point sequences.     

Divergent Evolution of Plant NBS-LRR Resistance Gene Homologues in Dicot and Cereal Genomes

The majority of plant disease resistance genes are members of very large multigene families. They encode structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine rich repeats (LRR). The N-terminal region of some resistance genes contain a short sequence called TIR with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes. Only a few plant resistance genes have been functionally analyzed and the origin and evolution of plant resistance genes remain obscure. We have reconstructed gene phylogeny by exhaustive analysis of available genome and amplified NBS domain sequences. Our study shows that NBS domains faithfully predict whole gene structure and can be divided into two major groups. Group I NBS domains contain group-specific motifs that are always linked with the TIR sequence in the N terminus. Significantly, Group I NBS domains and their associated TIR domains are widely distributed in dicot species but were not detected in cereal databases. Furthermore, Group I specific NBS sequences were readily amplified from dicot genomic DNA but could not be amplified from cereal genomic DNA. In contrast, Group II NBS domains are always associated with putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. These results suggest that the two main groups of resistance genes underwent divergent evolution in cereal and dicot genomes and imply that their cognate signaling pathways have diverged as well. Received: 17 May 1999 / Accepted: 25 September 1999     

Stepwise origin and functional diversification of the AFL subfamily B3 genes during land plant evolution

The AFL genes (ABI3/VP1, FUS3 and LEC2) belong to the plant-specific B3 superfamily, playing important roles in regulating seed development and maturation. It is unclear, however, whether these genes appeared at the same time as the origin of seed plants and if all these genes are necessary and sufficient for seed development for all seed plants. By conducting a genome-wide comparative analysis of the putative AFL genes in various plant species, we found that the ABI3 homologous genes existed in all land plant genomes, but the FUS3 homologous were present only in seed plant genomes and the LEC2-like sequences only in dicot genomes. Phylogenetic analysis indicated that the AFL genes had undergone successive rounds of gene duplication and subsequent diversification during land plant evolution, resulting in the stepwise origin of the ABI3, FUS3 and LEC2 genes. Comparison of gene structure of the AFL genes revealed a trend of decreasing in the number of conserved domains from ABI3 to FUS3 and LEC2.     

Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1

Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea.     

Identification of novel Mlo family members in wheat and their genetic characterization

Mlo is a plant-specific gene family, which is known to show stress responses in various plants. To reveal the genetic characteristics of the Mlo family in wheat, we isolated wheat Mlo members from a database and studied their expression in young shoots and roots under salt and osmotic stress conditions. In an in silico investigation, we identified seven Mlo members in wheat and named them TaMlo-1~TaMlo-7. None of the wheat Mlo showed significant induction or reduction of their expression under salt or osmotic stress, but organ-specific expression was observed in several TaMlo members. TaMlo-1, TaMlo-2, and TaMlo-5 were constitutively expressed in both shoots and roots, but TaMlo-3 and TaMlo-4 showed root-specific expression, and TaMlo-7 showed dominant expression in shoots. TaMlo-6 was weakly expressed in both shoots and roots. Phylogenetic analysis classified the plant Mlo members into six classes; four of them were comprised of angiosperm Mlo members, and the remaining two consisted of fern and moss Mlo members. The seven wheat Mlo members were classified into four angiosperm Mlo classes, similar to those of Arabidopsis and rice, indicating that the formation of each of the Mlo classes preceded the divergence of dicots and monocots. The differentiation of the expressional patterns among the seven TaMlo members was not related to their phylogenetic classification. This result suggested that the organ specific expression of individual Mlo members occurred relatively recently in their evolution.     

Comprehensive Evolutionary and Expression Analysis of FCS-Like Zinc finger Gene Family Yields Insights into Their Origin,Expansion and Divergence

Plant evolution is characterized by frequent genome duplication events. Expansion of habitat resulted in the origin of many novel genes and genome duplication events which in turn resulted in the expansion of many regulatory gene families. The plant-specific FCS-Like Zinc finger (FLZ) gene family is characterized by the presence of a FCS-Like Zinc finger (FLZ) domain which mediates the protein-protein interaction. In this study, we identified that the expansion of FLZ gene family size in different species is correlated with ancestral and lineage-specific whole genome duplication events. The subsequent gene loss found to have a greater role in determining the size of this gene family in many species. However, genomic block duplications played the significant role in the expansion of FLZ gene family in some species. Comparison of Arabidopsis thaliana and Oryza sativa FLZ gene family revealed monocot and dicot specific evolutionary trends. The FLZ genes were found to be under high purifying selection. The spatiotemporal expression analyses of Arabidopsis thaliana FLZ gene family revealed that majority of the members are highly expressed in reproductive organs. FLZ genes were also found to be highly expressed during vegetative-to-reproductive phase transition which is correlated with the proposed role of this gene family in sugar signaling. The comparison of sequence, structural and expression features of duplicated genes identified lineage-specific redundancy and divergence. This extensive evolutionary analysis and expression analysis of Arabidopsis thaliana FLZ genes will pave the way for further functional analysis of FLZ genes.     

Evolutionary analyses of non‐family genes in plants

There are a large number of ‘non‐family’ (NF) genes that do not cluster into families with three or more members per genome. While gene families have been extensively studied, a systematic analysis of NF genes has not been reported. We performed comparative studies on NF genes in 14 plant species. Based on the clustering of protein sequences, we identified ~94 000 NF genes across these species that were divided into five evolutionary groups: Viridiplantae wide, angiosperm specific, monocot specific, dicot specific, and those that were species specific. Our analysis revealed that the NF genes resulted largely from less frequent gene duplications and/or a higher rate of gene loss after segmental duplication relative to genes in both low‐copy‐number families (LF; 3–10 copies per genome) and high‐copy‐number families (HF; >10 copies). Furthermore, we identified functions enriched in the NF gene set as compared with the HF genes. We found that NF genes were involved in essential biological processes shared by all plant lineages (e.g. photosynthesis and translation), as well as gene regulation and stress responses associated with phylogenetic diversification. In particular, our analysis of an Arabidopsis protein–protein interaction network revealed that hub proteins with the top 10% most connections were over‐represented in the NF set relative to the HF set. This research highlights the roles that NF genes may play in evolutionary and functional genomics research.