Evolutionary dynamics of leucine-rich repeat receptor-like kinases and related genes in plants：A phylogenomic approach

Leucine-rich repeat （LRR） receptor-like kinases （RLKs）, evolutionarily related LRR receptor-like proteins （RLPs） and receptor-like cytoplasmic kinases （RLCKs） have important roles in plant signaling, and their gene subfamilies are large with a complicated history of gene duplication and loss. In three pairs of closely related lineages, including Arabidopsis thaliana and A. lyrata （Arabidopsis）, Lotus japonicus, and Medicago truncatula （Legumes）, Oryza sativa ssp. japonica, and O. sativa ssp. indica （Rice）, we find that LRR RLKs comprise the largest group of these LRR-related subfamilies, while the related RLCKs represent the smal est group. In addition, comparison of orthologs indicates a high frequency of reciprocal gene loss of the LRR RLK/LRR RLP/RLCK subfamilies. Furthermore, pairwise comparisons show that reciprocal gene loss is often associated with lineage-specific duplication（s） in the alternative lineage. Last, analysis of genes in A. thaliana involved in development revealed that most are highly conserved orthologs without species-specific duplication in the two Arabidopsis species and originated from older Arabidopsis-specific or rosid-specific duplications. We discuss＆amp;nbsp;potential pitfal s related to functional prediction for genes that have undergone frequent turnover （duplications, losses, and domain architecture changes）, and conclude that prediction based on phylogenetic relationships wil likely outperform that based on sequence similarity alone.     

Crystal structure of a plant leucine rich repeat protein with two island domains

Leucine rich repeat(LRR)domain,characterized by a repetitive sequence pattern rich in leucine residues,is a universal protein-protein interaction motif present in all life forms.LRR repeats interrupted by sequences of 30 70 residues(termed island domain,ID)have been found in some plant LRR receptor-like kinases(RLKs)and animal Toll-like receptors(TLR7-9).Recent studies provide insight into how a single ID is structurally integrated into an LRR protein.However,structural information on an LRR protein with two IDs is lacking.The receptor-like protein kinase 2(RPK2)is an LRR-RLK and has important roles in controlling plant growth and development by perception and transduction of hormone signal.Here we present the crystal structure of the extracellular LRR domain of RPK2(RPK2-LRR)containing two IDs from Arabidopsis.The structure reveals that both of the IDs are helical and located at the central region of the single RPK2-LRR solenoid.One of them binds to the inner surface of the solenoid,whereas the other one mainly interacts with the lateral side.Unexpectedly,a long loop immediately following the N-terminal capping domain of RPK2-LRR is presented toward and sandwiched between the two IDs,further stabilizing their embedding to the LRR solenoid.A potential ligand binding site formed by the two IDs and the solenoid is located at the C-terminal side of RPK2-LRR.The structural information of RPK2-LRR broadens our understanding toward the large family of LRR proteins and provides insight into RPK2-mediated signaling.     

Analysis of expressed receptor-like kinases （RLKs） in soybean

Receptor-like kinases （RLKs） play crucial roles in cellular signal perception and propagation. To study the evolutionary relationships among RLKs in soybean, a large-scale expressed sequence tags （ESTs） survey for RLKs-related sequences was conducted. By doing BLAST analysis using our database and The Gene Index Database, 605 putative RLK genes were identified. Based on the phylogeny of the kinase domain, these soybean RLKs were classified into 58 different small subfamilies. The phylogenetic analysis of RLKs in soybean, rice and Arabidopsis showed that different subfamilies of RLKs had different functions and could have experienced different selective pressures.     

Receptor-Like Kinases in Plant Innate Immunity

Plants employ a highly effective surveillance system to detect potential pathogens, which is critical for the success of land plants in an environment surrounded by numerous microbes. Recent efforts have led to the identification of a number of immune receptors and components of immune receptor complexes. It is now clear that receptor-like kinases （RLKs） and receptor-like proteins （RLPs） are key pattern-recognition receptors （PRRs） for microbe- and plant-derived molecular patterns that are associated with pathogen invasion. RLKs and RLPs involved in immune signaling belong to large gene families in plants and have undergone lineage specific expansion. Molecular evolution and population studies on phytopathogenic molecular signatures and their receptors have provided crucial insight into the co-evolution between plants and pathogens.     

A Microarray Based Genomic Hybridization Method for Identification of New Genes in Plants：Case Analyses of Arabidopsis and Oryza

To systematically estimate the gene duplication events In closely related species, we have to use comparative genomlc approaches, either through genomlc sequence comparison or comparative genomlc hybridization （CGH）. Given the scarcity of complete genomlc sequences of plant species, in the present study we adopted an array based CGH to Investigate gene duplications In the genus Arabldopsls. Fragment genomlc DNA from four species, namely Arabidopsls thallana, A. lyrata subsp, lyrata, A. lyrata subsp, petraea, and A. halleri, was hybridized to Affymetrlx （Santa Clara, CA, USA） tiling arrays that are designed from the genomlc sequences of A. thallana. Pairwlse comparisons of signal intensity were made to infer the potential duplicated candidates along each phylogenetic branch. Ninety-four potential candidates of gene duplication along the genus were Identified. Among them, the majority （69 of 94） were A. thallana lineage specific. This result indicates that the array based CGH approach may be used to Identify candidates of duplication In other plant genera containing closely related species, such as Oryza, particularly for the AA genome species. We compared the degree of gene duplication through retrotransposon between O. satlva and A. thallana and found a strikingly higher number of chimera retroposed genes In rice. The higher rate of gene duplication through retroposltlon and other mechanisms may Indicate that the grass species Is able to adapt to more diverse environments.     

SbHKT1;4, a member of the high‐affinity potassium transporter gene family from Sorghum bicolor,functions to maintain optimal Na~+/K~+ balance under Na~+ stress

In halophytic plants, the high‐affinity potassium transporter HKT gene family can selectively uptake Kt in the presence of toxic concentrations of Nat. This has so far not been well examined in glycophytic crops. Here, we report the characterization of SbHKT1;4, a member of the HKT gene family from Sorghum bicolor. Upon Nat stress, SbHKT1;4 expression was more strongly upregulated in salt‐tolerant sorghum accession, correlating with a better balanced Nat/ Kt ratio and enhanced plant growth. Heterogeneous expression analyses in mutants of Saccharomyces cerevisiae and Arabidopsis thaliana indicated that overexpressing SbHKT1;4 resulted in hypersensitivity to Nat stress, and such hypersensitivity could be alleviated with the supply of elevated levels of Kt, implicating that SbHKT1;4 may mediate Kt uptake in the presence of excessive Nat. Further electrophysiological evidence demonstrated that SbHKT1;4 could transport Nat and Kt when expressed in Xenopus laevis oocytes. The relevance of the finding that SbHKT1;4 functions to maintain optimal Nat/Kt balance under Nat stress to the breeding of salt‐tolerant glycophytic crops is discussed.     

Big Roles of Small Kinases： The Complex Functions of Receptor-Like Cytoplasmic Kinases in Plant Immunity and Development

Plants have evolved a large number of receptor-like cytoplasmic kinases （RLCKs） that often functionally and physically associate with receptor-like kinases （RLKs） to modulate plant growth, development and immune responses. Without any apparent extracellular domain, RLCKs relay intracellular signaling often via RLK complex-mediated transphosphorylation events. Recent advances have suggested essential roles of diverse RLCKs in concert with RLKs in regulating various cellular and physiological responses. We summarize here the complex roles of RLCKs in mediating plant immune responses and growth regulation, and discuss specific and overlapping functions of RLCKs in transducing diverse signaling pathways.     

Meta‐analysis and candidate gene mining of low‐phosphorus tolerance in maize

Plants with tolerance to low‐phosphorus(P) can grow better under low‐P conditions, and understanding of genetic mechanisms of low‐P tolerance can not only facilitate identifying relevant genes but also help to develop low‐P tolerant cultivars. QTL meta‐analysis was conducted after a comprehensive review of the reports on QTL mapping for low‐P tolerance‐related traits in maize. Meta‐analysis produced 23 consensus QTL(cQTL), 17 of which located in similar chromosome regions to those previously reported to influence root traits. Meanwhile, candidate gene mining yielded 215 genes, 22 of which located in the cQTL regions.These 22 genes are homologous to 14 functionally characterized genes that were found to participate in plant low‐P tolerance, including genes encoding miR399s, Pi transporters and purple acid phosphatases. Four cQTL loci(cQTL2‐1,cQTL5‐3, cQTL6‐2, and cQTL10‐2) may play important roles for low‐P tolerance because each contains more original QTL and has better consistency across previous reports.     

A Brassinosteroid-Signaling Kinase Interacts withMultiple Receptor-Like Kinases in Arabidopsis

Dear Editor, Higher plants have evolved hundreds of genes encodingreceptor-like kinases （RLKs）, which function as cell surfacereceptors perceiving developmental and environmental sig-nals （Shiu et al., 2004）. Many RLKs have been shown to playspecific roles in hormone responses, developmental regula-tion, defense against pathogen infection, and adaptationto abiotic stresses （Chae et al., 2009; Antolin-Llovera et al.,2012）. The mechanisms that ensure specific signal transduc-tion from each RLK to target cellular responses remain poorlyunderstood. Recent studies revealed that many RLKs trans-duce signals by phosphorylating receptor-like cytoplasmickinases （RLCKs）, which lack the transmembrane domainsbut are anchored at the plasma membrane through lipidmodification （Tang et al., 2008; Zhang et al., 2010; Shi et al.,2013）. There are over 400 RLKs and only about 150 RLCKs inArabidopsis （Shiu et al., 2004）. One outstanding question iswhether each RLCK mediates signaling downstream of a spe-cific RLK, participates in multiple RLK pathways, or mediatescrosstalk between RLK pathways.     

Arabidopsis ADC1 functions as an N~δ-acetylornithine decarboxylase

Polyamines are small aliphatic amines found in almost all organisms, ranging from bacteria to plants and animals. In most plants, putrescine, the metabolic precursor for longer polyamines, such as spermidine and spermine, is produced from arginine, with either agmatine or ornithine as intermediates. Here we show that Arabidopsis thaliana(Arabidopsis) arginine decarboxylase 1(ADC1), one of the two known arginine decarboxylases in Arabidopsis, not only synthesizes agmatine from arginine, but also converts N~δ-acetylornithine to N-acetylputrescine. Phylogenetic analyses indicate that duplication and neofunctionalization of ADC1 and NATA1, the enzymes that synthesize N~δ-acetylornithine in Arabidopsis, co-occur in a small number of related species in the Brassicaceae. Unlike ADC2, which is localized in the chloroplasts, ADC1 is in the endoplasmic reticulum together with NATA1, an indication that these two enzymes have access to the same substrate pool. Together, these results are consistent with a model whereby NATA1 and ADC1 together provide a pathway for the synthesis of N-acetylputrescine in Arabidopsis.     

Genome-wide Analysis of Kelch Repeat-containing F-box Family

The ubiquitin-dependent protein degradation pathway plays diverse roles in eukaryotes. Previous studies indicate that both F-box and Kelch motifs are common in a variety of organisms. F-box proteins are subunits of E3 ubiquitin ligase complexes called SCFs （SKP1, Cullinl, F-box protein, and Rbxl）; they have an N-terminal F-box motif that binds to SKP1 （S-phase kinase associated protein）, and often have C-terminal protein-protein interaction domains, which specify the protein substrates for degradation via the ubiquitin pathway. One of the most frequently found protein interaction domains in F-box proteins is the Kelch repeat domain. Although both the F-box and Kelch repeats are ancient motifs, Kelch repeats-containing F-box proteins （KFB） have only been reported for human and Arabidopsis previously. The recent sequencing of the rice genome and other plant genomes provides an opportunity to examine the possible evolution history of KFB. We carried out extensive BLAST searches to identify putative KFBs in selected organisms, and analyzed their relationships phylogenetically. We also carried out the analysis of both gene duplication and gene expression of the KFBs in rice and Arabidopsis. Our study indicates that the origin of KFBs occurs before the divergence of animals and plants, and plant KFBs underwent rapid gene duplications.     

EMBRYONIC FACTOR 19 encodes a pentatricopeptide repeat protein that is essential for the initiation of zygotic embryogenesis in Arabidopsis

Early embryogenesis is the most fundamental developmental process in biology.Screening of ethyl methanesulfonate(EMS)-mutagenized populations of Arabidopsis thaliana led to the identification of a zygote-lethal mutant embryonic factor 19(fac19)in which embryo development was arrested at the elongated zygote to octant stage.The number of endosperm nuclei decreased significantly in fac19 embryos.Genetic analysis showed fac19 was caused by a single recessive mutation with typical mendelian segregation,suggesting equal maternal and paternal contributions of FAC19 towards zygotic embryogenesis.Positional cloning showed that FAC19 encodes a putative mitochondrial protein with 16 conserved pentatricopeptide repeat(PPR)motifs.The fac19 mutation caused a conversion from hydrophilic serine located in a previously unknown domain to hydrophobic leucine.Crosses between FAC19/fac19 and the T-DNA insertion mutants in the same gene failed to complement the fac19 defects,confirming the identity of the gene.This study revealed the critical importance of a PPR protein-mediated mitochondrial function in early embryogenesis.     

Knockdown of LjALD1, AGD2‐like defense response protein 1, influences plant growth and nodulation in Lotus japonicus

The discovery of the enzyme L,L‐diaminopimelate aminotransferase(LL‐DAP‐AT, EC 2.6.1.83) uncovered a unique step in the L‐lysine biosynthesis pathway in plants. In Arabidopsis thaliana, LL‐DAP‐AT has been shown to play a key role in plant‐pathogen interactions by regulation of the salicylic acid(SA) signaling pathway. Here, a full‐length cDNA of LL‐DAP‐AT named as LjALD1 from Lotus japonicus(Regel)Larsen was isolated. The deduced amino acid sequence shares 67% identity with the Arabidopsis aminotransferase AGD2‐LIKE DEFENSE RESPONSE PROTEIN1(AtALD1) and is predicted to contain the same key elements: a conserved aminotransferase domain and a pyridoxal‐5'‐phosphate cofactor binding site.Quantitative real‐time PCR analysis showed that LjALD1 was expressed in all L. japonicus tissues tested, being strongest in nodules. Expression was induced in roots that had been infected with the symbiotic rhizobium Mesorhizobium loti or treated with SA agonist benzo‐(1, 2, 3)‐thiadiazole‐7‐carbothioic Researchacid. LjALD1 Knockdown exhibited a lower SA content, an increased number of infection threads and nodules, and a slight reduction in nodule size. In addition, compared with wild‐type,root growth was increased and shoot growth was suppressed in LjALD1 RNAi plant lines. These results indicate that LjALD1 may play important roles in plant development and nodulation via SA signaling in L. japonicus.     

Expression of two soybean resistance gene candidates shows divergence of paralogous single-copy genes

The cloning of several plant genes directly involved in triggering a disease resistance response has shown that numerous resistance genes in the nucleotide binding site (NBS)/leucine-rich repeat (LRR) class have similar conserved amino acid sequences. In this study, we used a short soybean DNA sequence, previously cloned based on its conserved NBS, as a probe to identify full-length resistance gene candidates. Two homologous, but genetically independent genes were identified. One gene maps to the soybean molecular linkage group (MLG) F and a second is coded on MLG E. The first gene contains a 3,279 nucleotide open reading frame (ORF) sequence and possesses all the functional motifs characteristic of previously cloned NBS/LRR resistance genes. The N-terminal sequence of the deduced gene product is highly characteristic of other resistance genes in the subgroup of NBS/LRR genes which show homology to the Toll/Interleukin-1 receptor genes. The C-terminal region is somewhat more divergent as seen in other cloned disease resistance genes. This region of the F-linked gene contains an LRR region that is characterized by two alternatively spliced products which produce gene products with either a four-repeat or a ten-repeat LRR. The second cloned gene that maps to soybean MLG E contains 1,565 nucleotides of ORF in the N-terminal domain. Despite strong homology, however, the 3′ region of this gene contains several in-frame stop codons and apparent frame shifts compared to the F-linked gene, suggesting that its functionality as a disease resistance gene is questionable. These two disease resistance gene candidates are shown to be closely related to one another and to the members of the NBS/LRR class of disease resistance genes. Received: 29 November 1999 / Accepted: 22 December 1999     

Disruption of the autism-related gene Pak1 causes stereocilia disorganization,hair cell loss,and deafness in mice

Several clinical studies have reported that hearing loss is correlated with autism in children. However, little is known about the underlying mechanism between hearing loss and autism. p21-activated kinases(PAKs)are a family of serine/threonine kinases that can be activated by multiple signaling molecules, particularly the Rho family of small GTPases. Previous studies have shown that Pak1 mutations are associated with autism. In the present study, we take advantage of Pak1 knockout(Pak1à/à) mice to investigate the role of PAK1 in hearing function. We find that PAK1 is highly expressed in the postnatal mouse cochlea and that PAK1 deficiency leads to hair cell(HC) apoptosis and severe hearing loss. Further investigation indicates that PAK1 deficiency downregulates the phosphorylation of cofilin and ezrin-radixin-moesin and the expression of b II-spectrin, which further decreases the HC synapse density in the basal turn of cochlea and disorganized the HC stereocilia in all three turns of cochlea in Pak1à/àmice. Overall, our work demonstrates that the autism-related gene Pak1 plays a crucial role in hearing function. As the first candidate gene linking autism and hearing loss, Pak1 may serve as a potential target for the clinical diagnosis of autism-related hearing loss.