Molecular Cloning and Characterization of WRKY12, A Pathogen Induced WRKY Transcription Factor from Akebia trifoliata.

WRKY transcription factors (TFs), which are plant-specific TFs, play significant roles in plant defense. Here, a pathogen-induced WRKY gene, named AktWRKY12, which was the homologous gene of AtWRKY12, was isolated from Akebia trifoliata. The AktWRKY12 gene has a total length of 645 nucleotides and an open reading frame (ORF) encoding 214 amino acid polypeptides. The characterizations of AktWRKY12 were subsequently performed with the ExPASy online tool Compute pI/Mw, PSIPRED and SWISS-MODEL softwares. The AktWRKY12 could be classified as a member of WRKY group II-c TFs based on sequence alignment and phylogenetic analysis. The results of tissue-specific expression analysis revealed that the AktWRKY12 gene was expressed in all the tested tissues, and the highest expression level was detected in A. trifoliata leaves. Subcellular localization analysis showed that AktWRKY12 was a nuclear protein. Results showed that the expression level of AktWRKY12 significantly increased in A. trifoliata leaves with pathogen infection. Furthermore, heterologous over-expression of AktWRKY12 in tobacco resulted in suppressed expression of lignin synthesis key enzyme genes. Based on our results, we speculate that AktWRKY12 might play a negative role in A. trifoliata responding to biotic stress by regulating the expression of lignin synthesis key enzyme genes during pathogen infection.

Germplasm resources and genetic improvement of Akebia: A new fruit crop in China.

Akebia species, belonging to Lardizabalaceae, are widespread from subtropical to temperate environments of China, Japan, and Korea. All known Akebia species have medicinal and dietary value and have been widely cultivated as a new fruit crop in many areas of China. However, compared with other crop species, the breeding improvement and commercial cultivation of Akebia remain in their infancy. This review systematically introduces the present germplasm resources, geographical distribution, biological characteristics, interspecific and intraspecific cross compatibility, molecular biology, and breeding progress in Akebia species. Akebia plants are widely distributed in Shanxi, Henan, Sichuan, Chongqing, Hunan, Hubei, Jiangxi, Zhejiang, and Fujian provinces of China, and wild Akebia plants exhibit abundant phenotypic and genetic diversity due to their wide range of geographical distribution and high adaptability in different habitats. Interspecific artificial hybridization experiments have been conducted in our Akebia germplasm resources nursery. The results showed that there was no reproductive isolation between Akebia species, and fertile progeny could be produced. The synthesis of knowledge on these species provides insights for the rational development and utilization of these germplasm resources, and can facilitate the development of new breeding lines or varieties for commercial cultivation or production. Finally, perspectives on Akebia breeding research are discussed and conclusions are provided. This review provided breeders with new insights into Akebia domestication and breeding, and we also proposed five basic steps in the domestication of new fruit crops.

Characterizing the complete chloroplast genome of the Impatiens davidii (Balsaminaceae).

Impatiens davidii Franch, 1886 is a rare ornamental flower used in gardens and has high economic value. In this study, we characterized the chloroplast genome of I. davidii and analyzed its phylogenetic relationship with other Impatiens species. The length of the complete chloroplast genome sequence of I. davidii is 152,214 bp, with a GC content of 36.9%. The chloroplast genome shows a typical quadripartite structure with a pair of inverted repeats (IRs) of 25,634 bp, separated by one large single copy (LSC) region of 83,128 bp and one small single copy (SSC) region of 17,818 bp. We annotated 125 genes, of which there were 85 protein-coding genes, 32 tRNA genes, and 8 rRNA genes. The Bayesian phylogenetic tree strongly supports that I. davidii has a close phylogenetic relationship with a group including I. piufanensis and I. alpicola.

Characterization of the WRKY gene family in Akebia trifoliata and their response to Colletotrichum acutatum.

BACKGROUND: Akebia trifoliata, belonging to the Lardizabalaceae family, is a well-known Chinese traditional medicinal plant, susceptible to many diseases, such as anthracnose and powdery mildew. WRKY is one of the largest plant-specific transcription factor families and plays important roles in plant growth, development and stress response, especially in disease resistance. However, little was known about the numbers, characters, evolutionary relationship and expression of WRKY genes in A. trifoliata in response to plant disease due to lacking of A. trifoliata genome. RESULTS: A total of 42 putative AktWRKY genes were identified based on the full-length transcriptome-sequencing data of A. trifoliata. Then 42 AktWRKY genes were divided into three major groups (Group I-III) based on the WRKY domains. Motif analysis showed members within same group shared a similar motif composition, implying a functional conservation. Tissue-specific expression analysis showed that AktWRKY genes could be detected in all tissues, while few AktWRKY genes were tissue specific. We further evaluated the expression of AktWRKY genes in three varieties in response to Colletotrichum acutatum by qRT-PCR. The expression patterns of AktWRKY genes were similar between C01 and susceptible variety I02, but distinctly different in resistant variety H05. In addition, it showed that more than 64 percentages of AktWRKY genes were differentially expressed during fungal infection in I02 and H05. Furthermore, Gene ontology (GO) analysis showed that AktWRKY genes were categorized into 26 functional groups under cellular components, molecular functions and biological processes, and a predicted protein interaction network was also constructed. CONCLUSIONS: Results of bioinformation analysis and expression patterns implied that AktWRKYs might play multiple function in response to biotic stresses. Our study could facilitate to further investigate the function and regulatory mechanism of the WRKY in A. trifoliata during pathogen response.

The complete chloroplast genome of Akebia longeracemosa (Lardizabalaceae).

The complete chloroplast genome of Akebia longeracemosa, a monoecious woody vine endemic to China, was determined. The total genome size is 158,020 bp, containing a large single copy region of 86,659 bp, a small single copy region of 19,059 bp, and a pair of inverted repeats of 26,151 bp. The chloroplast genome encodes 113 unique genes, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Among them, fifteen genes have one intron each, and three genes contain two introns. The overall GC content is 38.7%, while the corresponding values of LSC, SSC, and IR regions are 37.1, 33.6, and 43.1%, respectively. Phylogenetic analysis showed that A. longeracemosa was closely related to A. trifoliata.

The complete chloroplast genome of Stauntonia chinensis and compared analysis revealed adaptive evolution of subfamily Lardizabaloideae species in China.

BACKGROUND: Stauntonia chinensis DC. belongs to subfamily Lardizabaloideae, which is widely grown throughout southern China. It has been used as a traditional herbal medicinal plant, which could synthesize a number of triterpenoid saponins with anticancer and anti-inflammatory activities. However, the wild resources of this species and its relatives were threatened by over-exploitation before the genetic diversity and evolutionary analysis were uncovered. Thus, the complete chloroplast genome sequences of Stauntonia chinensis and comparative analysis of chloroplast genomes of Lardizabaloideae species are necessary and crucial to understand the plastome evolution of this subfamily. RESULTS: A series of analyses including genome structure, GC content, repeat structure, SSR component, nucleotide diversity and codon usage were performed by comparing chloroplast genomes of Stauntonia chinensis and its relatives. Although the chloroplast genomes of eight Lardizabaloideae plants were evolutionary conserved, the comparative analysis also showed several variation hotspots, which were considered as highly variable regions. Additionally, pairwise Ka/Ks analysis showed that most of the chloroplast genes of Lardizabaloideae species underwent purifying selection, whereas 25 chloroplast protein coding genes were identified with positive selection in this subfamily species by using branch-site model. Bayesian and ML phylogeny on CCG (complete chloroplast genome) and CDs (coding DNA sequences) produced a well-resolved phylogeny of Lardizabaloideae plastid lineages. CONCLUSIONS: This study enhanced the understanding of the evolution of Lardizabaloideae and its relatives. All the obtained genetic resources will facilitate future studies in DNA barcode, species discrimination, the intraspecific and interspecific variability and the phylogenetic relationships of subfamily Lardizabaloideae.

Genome-wide survey and expression analysis of calcium-dependent protein kinase (CDPK) in grass Brachypodium distachyon.

BACKGROUND: Ca2+ played as a ubiquitous secondary messenger involved in plant growth, development, and responses to various environmental stimuli. Calcium-dependent protein kinases (CDPK) were important Ca2+ sensors, which could directly translate Ca2+ signals into downstream phosphorylation signals. Considering the importance of CDPKs as Ca2+ effectors for regulation of plant stress tolerance and few studies on Brachypodium distachyon were available, it was of interest for us to isolate CDPKs from B. distachyon. RESULTS: A systemic analysis of 30 CDPK family genes in B. distachyon was performed. Results showed that all BdCDPK family members contained conserved catalytic Ser/Thr protein kinase domain, autoinhibitory domain, and EF-hand domain, and a variable N-terminal domain, could be divided into four subgroup (I-IV), based upon sequence homology. Most BdCDPKs had four EF-hands, in which EF2 and EF4 revealed high variability and strong divergence from EF-hand in AtCDPKs. Synteny results indicated that large number of syntenic relationship events existed between rice and B. distachyon, implying their high conservation. Expression profiles indicated that most of BdCDPK genes were involved in phytohormones signal transduction pathways and regulated physiological process in responding to multiple environmental stresses. Moreover, the co-expression network implied that BdCDPKs might be both the activator and the repressor involved in WRKY transcription factors or MAPK cascade genes mediated stress response processes, base on their complex regulatory network. CONCLUSIONS: BdCDPKs might play multiple function in WRKY or MAPK mediated abiotic stresses response and phytohormone signaling transduction in B. distachyon. Our genomics analysis of BdCDPKs could provide fundamental information for further investigation the functions of CDPKs in integrating Ca2+ signalling pathways in response to environments stresses in B. distachyon.

Identification and in Silico Characterization of GT Factors Involved in Phytohormone and Abiotic Stresses Responses in Brachypodium distachyon.

GT factors play critical roles in plant growth and development and in response to various environmental stimuli. Considering the new functions of GT factors on the regulation of plant stress tolerance and seeing as few studies on Brachypodium distachyon were available, we identified GT genes in B. distachyon, and the gene characterizations and phylogenies were systematically analyzed. Thirty-one members of BdGT genes were distributed on all five chromosomes with different densities. All the BdGTs could be divided into five subfamilies, including GT-1, GT-2, GTγ, SH4, and SIP1, based upon their sequence homology. BdGTs exhibited considerably divergent structures among each subfamily according to gene structure and conserved functional domain analysis, but the members within the same subfamily were relatively structure-conserved. Synteny results indicated that a large number of syntenic relationship events existed between rice and B. distachyon. Expression profiles indicated that the expression levels of most of BdGT genes were changed under abiotic stresses and hormone treatments. Moreover, the co-expression network exhibited a complex regulatory network between BdGTs and BdWRKYs as well as that between BdGTs and BdMAPK cascade gene. Results showed that GT factors might play multiple functions in responding to multiple environmental stresses in B. distachyon and participate in both the positive and negative regulation of WRKY- or MAPK-mediated stress response processes. The genome-wide analysis of BdGTs and the co-regulation network under multiple stresses provide valuable information for the further investigation of the functions of BdGTs in response to environment stresses.

The complete chloroplast genome of Holboellia angustifolia (Ranales: Lardizabalaceae), a traditional herbal species.

The complete chloroplast genome of Holboellia  angustifolia was 157,797 bp in length, displayed a typical quadripartite structure, composed of a LSC region of 86,543 bp and a SSC region of 18,972 bp, separated by a pair of IRs of 26,141 bp each. The chloroplast genome contains 130 genes, consisting of 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Fifteen genes have one intron, and 3 genes contain two introns. The overall A/T content in the chloroplast genome of H. angustifolia was 61.31%. Phylogenetic analysis showed that H. angustifolia was closely related to Holboellia latifolia.

Identification, characterization, and expression analysis of auxin response factor (ARF) gene family in Brachypodium distachyon.

Auxin response factors (ARFs) are one type of essential family of transcription factors that bind with auxin response elements (AuxRE), and play vital roles in variety of plant development and physiological processes. Brachypodium distachyon, related to the major cereal grain species, were recently developed to be a good model organism for functional genomics research. So far, genome-wide overview of the ARF gene family in B. distachyon was not available. Here, a systemic analysis of ARF gene family members in B. distachyon was performed. A comprehensive overview of the characterization of the BdARFs was obtained by multiple bioinformatics analyses, including the gene and protein structure, chromosome locations, conserved motifs of proteins, phylogenetic analysis, and cis-elements in promoters of BdARF. Results showed that all BdARFs contained conserved DBD, MR, and CTD could be divided into four classes, Ia, IIa, IIb, and III. Expression profiles of BdARF genes indicated that they were expressed across various tissues and organs, which could be clustered into three main expression groups, and most of BdARF genes were involved in phytohormone signal transduction pathways and regulated physiological process in responding to multiple environmental stresses. And predicted regulatory network between B. distachyon ARFs and IAAs was also discussed. Our genomics analysis of BdARFs could yield new insights into the complexity of the control of BdARF genes and lead to potential applications in the investigation of the accurate regulatory mechanisms of ARFs in herbaceous plants.

Genome-wide survey of heat shock factors and heat shock protein 70s and their regulatory network under abiotic stresses in Brachypodium distachyon.

The heat shock protein 70s (Hsp70s) and heat shock factors (Hsfs) play key roles in protecting plant cells or tissues from various abiotic stresses. Brachypodium distachyon, recently developed an excellent model organism for functional genomics research, is related to the major cereal grain species. Although B. distachyon genome has been fully sequenced, the information of Hsf and Hsp70 genes and especially the regulatory network between Hsfs and Hsp70s remains incomplete. Here, a total of 24 BdHsfs and 29 BdHsp70s were identified in the genome by bioinformatics analysis and the regulatory network between Hsfs and Hsp70s were performed in this study. Based on highly conserved domain and motif analysis, BdHsfs were grouped into three classes, and BdHsp70s divided into six groups, respectively. Most of Hsf proteins contain five conserved domains: DBD, HR-A/B region, NLS and NES motifs and AHA domain, while Hsp70 proteins have three conserved domains: N-terminal nucleotide binding domain, peptide binding domain and a variable C-terminal lid region. Expression data revealed a large number of BdHsfs and BdHsp70s were induced by HS challenge, and a previous heat acclimation could induce the acquired thermotolerance to help seedling suffer the severe HS challenge, suggesting that the BdHsfs and BdHsp70s played a role in alleviating the damage by HS. The comparison revealed that, most BdHsfs and BdHsp70s genes responded to multiple abiotic stresses in an overlapping relationship, while some of them were stress specific response genes. Moreover, co-expression relationships and predicted protein-protein interaction network implied that class A and B Hsfs played as activator and repressors, respectively, suggesting that BdHsp70s might be regulated by both the activation and the repression mechanisms under stress condition. Our genomics analysis of BdHsfs and BdHsp70s provides important evolutionary and functional characterization for further investigation of the accurate regulatory mechanisms among Hsfs and Hsp70s in herbaceous plants.