Comparative phylogenetic analysis of genome-wide Mlo gene family members from Glycine max and Arabidopsis thaliana

Powdery mildew locus O (Mlo) gene family is one of the largest seven transmembrane protein-encoding gene families. The Mlo proteins act as negative regulators of powdery mildew resistance and a loss-of-function mutation in Mlo is known to confer broad-spectrum resistance to powdery mildew. In addition, the Mlo gene family members are known to participate in various developmental and biotic and abiotic stress response-related pathways. Therefore, a genome-wide similarity search using the characterized Mlo protein sequences of Arabidopsis thaliana was carried out to identify putative Mlo genes in soybean (Glycine max) genome. This search identified 39 Mlo domain containing protein-encoding genes that were distributed on 15 of the 20 G. max chromosomes. The putative promoter regions of these Mlo genes contained response elements for different external stimuli, including different hormones and abiotic stresses. Of the 39 GmMlo proteins, 35 were rich (8.7–13.1 %) in leucine, while five were serine-rich (9.2–11.9 %). Furthermore, all the GmMlo members were localized in the plasma membrane. Phylogenetic analysis of the GmMlo and the AtMlo proteins classified them into three main clusters, and the cluster I comprised two sub-clusters. Multiple sequence alignment visualized the location of seven transmembrane domains, and a conserved CaM-binding domain. Some of the GmMlo proteins (GmMlo10, 20, 22, 23, 32, 36, 37) contained less than seven transmembrane domains. The motif analysis yielded 27 motifs; out of these, motif 2, the only motif present in all the GmMlos, was highly conserved and three amino acid residues were essentially invariant. Five of the GmMlo members were much smaller in size; presumably they originated through deletion following a gene duplication event. The presence of a large number of GmMlo members in the G. max genome may be due to its paleopolyploid nature and the large genome size as compared to that of Arabidopsis. The findings of this study may further help in characterization and isolation of individual GmMlo members.     

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。     

Genome-wide identification and characterisation of F-box family in maize

F-box-containing proteins, as the key components of the protein degradation machinery, are widely distributed in higher plants and are considered as one of the largest known families of regulatory proteins. The F-box protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, systematic analysis of the F-box family in maize (Zea mays) has not been reported yet. In this paper, we identified and characterised the maize F-box genes in a genome-wide scale, including phylogenetic analysis, chromosome distribution, gene structure, promoter analysis and gene expression profiles. A total of 359 F-box genes were identified and divided into 15 subgroups by phylogenetic analysis. The F-box domain was relatively conserved, whereas additional motifs outside the F-box domain may indicate the functional diversification of maize F-box genes. These genes were unevenly distributed in ten maize chromosomes, suggesting that they expanded in the maize genome because of tandem and segmental duplication events. The expression profiles suggested that the maize F-box genes had temporal and spatial expression patterns. Putative cis-acting regulatory DNA elements involved in abiotic stresses were observed in maize F-box gene promoters. The gene expression profiles under abiotic stresses also suggested that some genes participated in stress responsive pathways. Furthermore, ten genes were chosen for quantitative real-time PCR analysis under drought stress and the results were consistent with the microarray data. This study has produced a comparative genomics analysis of the maize ZmFBX gene family that can be used in further studies to uncover their roles in maize growth and development.     

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 expression analysis of rice phospholipase D gene family during abiotic stresses and development

Phospholipase D is one of the crucial enzymes involved in lipid mediated signaling, triggered during various developmental and physiological processes. Different members of PLD gene family have been known to be induced under different abiotic stresses and during developmental processes in various plant species. In this report, we are presenting a detailed microarray based expression analysis and expression profiles of entire set of PLD genes in rice genome, under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages). Seven and nine PLD genes were identified, which were expressed differentially under abiotic stresses and during reproductive developmental stages, respectively. PLD genes, which were expressed significantly under abiotic stresses exhibited an overlapping expression pattern and were also differentially expressed during developmental stages. Moreover, expression pattern for a set of stress induced genes was validated by real time PCR and it supported the microarray expression data. These findings emphasize the role of PLDs in abiotic stress signaling and development in rice. In addition, expression profiling for duplicated PLD genes revealed a functional divergence between the duplicated genes and signify the role of gene duplication in the evolution of this gene family in rice. This expressional study will provide an important platform in future for the functional characterization of PLDs in crop plants.     

Genome-wide analysis of the stress associated protein (SAP) gene family containing A20/AN1 zinc-finger(s) in rice and their phylogenetic relationship with Arabidopsis

Proteins with the A20/AN1 zinc-finger domain are present in all eukaryotes and are well characterized in animals, but little is known about their function in plants. Earlier, we have identified an A20/AN1 zinc-finger containing stress associated protein 1 gene (SAP1) in rice and validated its function in abiotic stress tolerance. In this study, genome-wide survey of genes encoding proteins possessing A20/AN1 zinc-finger, named SAP gene family, has been carried out in rice and Arabidopsis. The genomic distribution and gene architecture as well as domain structure and phylogenetic relationship of encoded proteins numbering 18 and 14 in rice and Arabidopsis, respectively, have been studied. Expression analysis of the rice SAP family was done to investigate their response under abiotic stress conditions. All the genes were inducible by one or the other abiotic stresses indicating that the OsSAP gene family is an important component of stress response in rice. Manipulation of their expression and identification of their superior alleles should help confer stress tolerance in target crops.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses

Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals.     

Genome-wide survey of the RIP domain family in Oryza sativa and their expression profiles under various abiotic and biotic stresses

Ribosome-inactivating proteins (RIPs) are N-glycosidases that inhibit protein synthesis by depurinating rRNA. Despite their identification more than 25 years ago, little is known about their biological functions. Here, we report a genome-wide identification of the RIP family in rice based on the complete genome sequence analysis. Our data show that rice genome encodes at least 31 members of this family and they all belong to type 1 RIP genes. This family might have evolved in parallel to species evolution and genome-wide duplications represent the major mechanism for this family expansion. Subsequently, we analyzed their expression under biotic (bacteria and fungus infection), abiotic (cold, drought and salinity) and the phytohormone ABA treatment. These data showed that some members of this family were expressed in various tissues with differentiated expression abundances whereas several members showed no expression under normal growth conditions or various environmental stresses. On the other hand, the expression of many RIP members was regulated by various abiotic and biotic stresses. All these data suggested that specific members of the RIP family in rice might play important roles in biotic and abiotic stress-related biological processes and function as a regulator of various environmental cues and hormone signaling. They may be potentially useful in improving plant tolerance to various abiotic and biotic stresses by over-expressing or suppressing these genes.     

Kinase-dead mutation: A novel strategy for improving soybean resistance to soybean cyst nematode Heterodera glycines

Protein kinases phosphorylate proteins for functional changes and are involved in nearly all cellular processes, thereby regulating almost all aspects of plant growth and development, and responses to biotic and abiotic stresses. We generated two independent co-expression networks of soybean genes using control and stress response gene expression data and identified 392 differentially highly interconnected kinase hub genes among the two networks. Of these 392 kinases, 90 genes were identified as “syncytium highly connected hubs”, potentially essential for activating kinase signalling pathways in the nematode feeding site. Overexpression of wild-type coding sequences of five syncytium highly connected kinase hub genes using transgenic soybean hairy roots enhanced plant susceptibility to soybean cyst nematode (SCN; Heterodera glycines) Hg Type 0 (race 3). In contrast, overexpression of kinase-dead variants of these five syncytium kinase hub genes significantly enhanced soybean resistance to SCN. Additionally, three of the five tested kinase hub genes enhanced soybean resistance to SCN Hg Type 1.2.5.7 (race 2), highlighting the potential of the kinase-dead approach to generate effective and durable resistance against a wide range of SCN Hg types. Subcellular localization analysis revealed that kinase-dead mutations do not alter protein cellular localization, confirming the structure–function of the kinase-inactive variants in producing loss-of-function phenotypes causing significant decrease in nematode susceptibility. Because many protein kinases are highly conserved and are involved in plant responses to various biotic and abiotic stresses, our approach of identifying kinase hub genes and their inactivation using kinase-dead mutation could be translated for biotic and abiotic stress tolerance.