Genome-Wide Identification and Analysis of MAPK and MAPKK Gene Families in Brachypodium distachyon

MAPK cascades are universal signal transduction modules and play important roles in plant growth, development and in response to a variety of biotic and abiotic stresses. Although MAPKs and MAPKKs have been systematically investigated in several plant species including Arabidopsis, rice and poplar, no systematic analysis has been conducted in the emerging monocot model plant Brachypodium distachyon. In the present study, a total of 16 MAPK genes and 12 MAPKK genes were identified from B. distachyon. An analysis of the genomic evolution showed that both tandem and segment duplications contributed significantly to the expansion of MAPK and MAPKK families. Evolutionary relationships within subfamilies were supported by exon-intron organizations and the architectures of conserved protein motifs. Synteny analysis between B. distachyon and the other two plant species of rice and Arabidopsis showed that only one homolog of B. distachyon MAPKs was found in the corresponding syntenic blocks of Arabidopsis, while 13 homologs of B. distachyon MAPKs and MAPKKs were found in that of rice, which was consistent with the speciation process of the three species. In addition, several interactive protein pairs between the two families in B. distachyon were found through yeast two hybrid assay, whereas their orthologs of a pair in Arabidopsis and other plant species were not found to interact with each other. Finally, expression studies of closely related family members among B. distachyon, Arabidopsis and rice showed that even recently duplicated representatives may fulfill different functions and be involved in different signal pathways. Taken together, our data would provide a foundation for evolutionary and functional characterization of MAPK and MAPKK gene families in B. distachyon and other plant species to unravel their biological roles.     

A TILLING Platform for Functional Genomics in Brachypodium distachyon

The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called “BRACHYTIL”, for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6, a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to assess the Genetically Effective Cell Number that was shown to be at least equal to 4 cells in Brachypodium distachyon. The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.     

Genome-Wide Identification of the Invertase Gene Family in Populus

Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.     

Comparative Genome Analysis between Agrostis stolonifera and Members of the Pooideae Subfamily,including Brachypodium distachyon

Creeping bentgrass (Agrostis stolonifera, allotetraploid 2n = 4x = 28) is one of the major cool-season turfgrasses. It is widely used on golf courses due to its tolerance to low mowing and aggressive growth habit. In this study, we investigated genome relationships of creeping bentgrass relative to the Triticeae (a consensus map of Triticum aestivum, T. tauschii, Hordeum vulgare, and H. spontaneum), oat, rice, and ryegrass maps using a common set of 229 EST-RFLP markers. The genome comparisons based on the RFLP markers revealed large-scale chromosomal rearrangements on different numbers of linkage groups (LGs) of creeping bentgrass relative to the Triticeae (3 LGs), oat (4 LGs), and rice (8 LGs). However, we detected no chromosomal rearrangement between creeping bentgrass and ryegrass, suggesting that these recently domesticated species might be closely related, despite their memberships to different Pooideae tribes. In addition, the genome of creeping bentgrass was compared with the complete genome sequence of Brachypodium distachyon in Pooideae subfamily using both sequences of the above-mentioned mapped EST-RFLP markers and sequences of 8,470 publicly available A. stolonifera ESTs (AgEST). We discovered large-scale chromosomal rearrangements on six LGs of creeping bentgrass relative to B. distachyon. Also, a total of 24 syntenic blocks based on 678 orthologus loci were identified between these two grass species. The EST orthologs can be utilized in further comparative mapping of Pooideae species. These results will be useful for genetic improvement of Agrostis species and will provide a better understanding of evolution within Pooideae species.     

Genome-Wide Analysis of the Sus Gene Family in Cotton

Sucrose synthase (Sus) is a key enzyme in plant sucrose metabolism. In cotton, Sus (EC 2.4.1.13) is the main enzyme that degrades sucrose imported into cotton fibers from the phloem of the seed coat. This study demonstrated that the genomes of Gossypium arboreum L., G. raimondii Ulbr., and G. hirsutum L., contained 8, 8, and 15 Sus genes, respectively. Their structural organizations, phylogenetic relationships, and expression profiles were characterized. Comparisons of genomic and coding sequences identified multiple introns, the number and positions of which were highly conserved between diploid and allotetraploid cotton species. Most of the phylogenetic clades contained sequences from all three species, suggesting that the Sus genes of tetraploid G. hirsutum derived from those of its diploid ancestors. One Sus group (Sus I) underwent expansion during cotton evolution. Expression analyses indicated that most Sus genes were differentially expressed in various tissues and had development-dependent expression profiles in cotton fiber cells. Members of the same orthologous group had very similar expression patterns in all three species. These results provide new insights into the evolution of the cotton Sus gene family, and insight into its members' physiological functions during fiber growth and development.     

Evaluation of the Use of the Polyubiquitin Genes,Ubi4 and Ubi10 as Reference Genes for Expression Studies in Brachypodium distachyon

Background

Brachypodium distachyon is emerging as the model plant for temperate grass research and the genome of the community line Bd21 has been sequenced. Additionally, techniques have been developed for Agrobacterium-mediated transformation for the generation of T-DNA insertional lines. Recently, it was reported that expression of the polyubiquitin genes, Ubi4 and Ubi10 are stable in different tissues and growth hormone-treated plant samples, leading to the conclusion that both Ubi4 and Ubi10 are good reference genes for normalization of gene expression data using real-time, quantitative PCR (qPCR).

Principal Findings

Mining of the Joint Genome Institute (JGI) 8X Brachypodium distachyon genome assembly showed that Ubi4 and Ubi10 share a high level of sequence identity (89%), and in silico analyses of the sequences of Ubi4 (Bradi3g04730) and Ubi10 (Bradi1g32860) showed that the primers used previously exhibit multiple binding sites within the coding sequences arising from the presence of tandem repeats of the coding regions. This can potentially result in over-estimation of steady-state levels of Ubi4 and Ubi10. Additionally, due to the high level of sequence identity between both genes, primers used previously for amplification of Ubi4 can bind to Ubi10 and vice versa, resulting in the formation of non-specific amplification products.

Conclusions

The results from this study indicate that the primers used previously were not sufficiently robust and specific. Additionally, their use would result in over-estimation of the steady-state expression levels of Ubi4. Our results question the validity of using the previously proposed primer sets for qPCR amplification of Ubi4 and Ubi10. We demonstrate that primers designed to target the 3′-UTRs of Ubi4 and Ubi10 are better suited for real-time normalization of steady-state expression levels in Brachypodium distachyon.     

Genome-Wide Identification and Analysis of the TIFY Gene Family in Grape

Background

The TIFY gene family constitutes a plant-specific group of genes with a broad range of functions. This family encodes four subfamilies of proteins, including ZML, TIFY, PPD and JASMONATE ZIM-Domain (JAZ) proteins. JAZ proteins are targets of the SCF^COI1 complex, and function as negative regulators in the JA signaling pathway. Recently, it has been reported in both Arabidopsis and rice that TIFY genes, and especially JAZ genes, may be involved in plant defense against insect feeding, wounding, pathogens and abiotic stresses. Nonetheless, knowledge concerning the specific expression patterns and evolutionary history of plant TIFY family members is limited, especially in a woody species such as grape.

Methodology/Principal Findings

A total of two TIFY, four ZML, two PPD and 11 JAZ genes were identified in the Vitis vinifera genome. Phylogenetic analysis of TIFY protein sequences from grape, Arabidopsis and rice indicated that the grape TIFY proteins are more closely related to those of Arabidopsis than those of rice. Both segmental and tandem duplication events have been major contributors to the expansion of the grape TIFY family. In addition, synteny analysis between grape and Arabidopsis demonstrated that homologues of several grape TIFY genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of lineages that led to grape and Arabidopsis. Analyses of microarray and quantitative real-time RT-PCR expression data revealed that grape TIFY genes are not a major player in the defense against biotrophic pathogens or viruses. However, many of these genes were responsive to JA and ABA, but not SA or ET.

Conclusion

The genome-wide identification, evolutionary and expression analyses of grape TIFY genes should facilitate further research of this gene family and provide new insights regarding their evolutionary history and regulatory control.     

Genome-Wide Analysis of Mitogen-Activated Protein Kinase Gene Family in Maize

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in plants. As the last component of the MAPK cascade (MAPKKK–MAPKK–MAPK), MAPK plays important roles in linking upstream kinases and downstream substrates. The MAPK proteins belong to a complex gene family in plants, with 20 MAPK genes in the Arabidopsis genome, 17 in the rice genome, and 21 in the poplar genome. Although the maize genome sequencing has been completed, no comprehensive study has been reported thus far for the MAPK gene family in maize. In this study, we identified 19 MAPK genes in maize. These ZmMPK genes belong to four groups (A–D) found in other plants. The phylogeny, chromosomal location, gene structure, and the functional relevancy of ZmMPK genes were analyzed. Moreover, we discuss the evolutionary divergence of MAPK genes in maize. Furthermore, we analyzed the expression profiles of ZmMPKs using the public microarray data and performed expression analyses in maize seedlings and adult plants. The data obtained from our study contribute to a better understanding of the complexity of MAPKs in plants and provide a useful reference for further functional analysis of MAPK genes in maize.     

Genome-Wide Analysis of LAZ1 Gene Family from Maize

Journal of Plant Growth Regulation - Lazarus 1 (LAZ1) is a six transmembrane protein with DUF300 domain, and functions as organic solute transporter in vertebrates. However, there was no any...     

Genome-Wide Identification,Classification, and Expression Analysis of 14-3-3 Gene Family in Populus

Background

In plants, 14-3-3 proteins are encoded by a large multigene family and are involved in signaling pathways to regulate plant development and protection from stress. Although twelve Populus 14-3-3s were identified based on the Populus trichocarpa genome V1.1 in a previous study, no systematic analysis including genome organization, gene structure, duplication relationship, evolutionary analysis and expression compendium has been conducted in Populus based on the latest P. trichocarpa genome V3.0.

Principal Findings

Here, a comprehensive analysis of Populus 14-3-3 family is presented. Two new 14-3-3 genes were identified based on the latest P. trichocarpa genome. In P. trichocarpa, fourteen 14-3-3 genes were grouped into ε and non-ε group. Exon-intron organizations of Populus 14-3-3s are highly conserved within the same group. Genomic organization analysis indicated that purifying selection plays a pivotal role in the retention and maintenance of Populus 14-3-3 family. Protein conformational analysis indicated that Populus 14-3-3 consists of a bundle of nine α-helices (α1-α9); the first four are essential for formation of the dimer, while α3, α5, α7, and α9 form a conserved peptide-binding groove. In addition, α1, α3, α5, α7, and α9 were evolving at a lower rate, while α2, α4, and α6 were evolving at a relatively faster rate. Microarray analyses showed that most Populus 14-3-3s are differentially expressed across tissues and upon exposure to various stresses.

Conclusions

The gene structures and their coding protein structures of Populus 14-3-3s are highly conserved among group members, suggesting that members of the same group might also have conserved functions. Microarray and qRT-PCR analyses showed that most Populus 14-3-3s were differentially expressed in various tissues and were induced by various stresses. Our investigation provided a better understanding of the complexity of the 14-3-3 gene family in poplars.     

番茄NAC转录因子家族的鉴定及生物信息学分析

NAC转录因子家族是植物特有的一类转录因子,在植物的生长发育、器官建成及逆境胁迫和激素信号应答中均发挥重要作用。本研究在基因组范围内,利用生物信息学方法对番茄的NAC转录因子家族成员、分布及结构和功能等进行分析。预测结果显示番茄NAC家族包含102个蛋白质,分为12亚族,其中茄属特有的TNAC亚族中成员最多,具有25个,其他NAC转录因子与拟南芥NAc家族具有相似分类。保守基序分析,在番茄NAC结构域中包含7个保守的NAM基序,主要分布在序列的N端,表明这些基序的存在对NAC蛋白质功能的执行是必需的。理化性质和结构分析表明,番茄NAC蛋白质绝大多数是亲水蛋白质,主要以无规则卷曲构成,而α-螺旋、β-折叠和β-转角则散布于整个蛋白质中,在各亚族中没有规律。     

谷子ARF基因家族的鉴定与生物信息学分析

生长素应答因子(ARF,auxin response factors)是一类可以结合在生长素应答基因启动子部位的转录因子,在植物的生长发育中起至关重要的作用。本研究以谷子为材料,共鉴定出24个ARF基因,命名为Si ARFs。利用生物信息学对谷子Si ARFs基因的结构、染色体分布、基因倍增模式、系统进化以及基因的表达模式进行分析。结果表明,Si ARF基因家族在染色体上呈不均匀分布,除2号染色体外,其他染色体上都有该家族基因,基因的扩增模式为分散复制与片段复制。Si ARFs基因家族具有相对保守的结构,即包含1个保守的B3 DNA结构域、ARF结构域和Aux/IAA结构域,ARF蛋白的3D结构含有3个α螺旋和7个β折叠结构。进化树分析表明谷子ARF蛋白和物种相近的高粱、玉米聚在一起。大多数ARF基因在谷子根、茎、叶和穗中都有表达,且不同基因表达量有较大差异。