Liangsheng Zhang

The Evolutionary History and Diverse Physiological Roles of the Grapevine Calcium-Dependent Protein Kinase Gene Family

Calcium-dependent protein kinases (CDPKs) are molecular switches that bind Ca2+, ATP, and protein substrates, acting as sensor relays and responders that convert Ca2+ signals, created by developmental processes and environmental stresses, into phosphorylation events. The precise functions of the CDPKs in grapevine (Vitis vinifera) are largely unknown. We therefore investigated the phylogenetic relationships and expression profiles of the 17 CDPK genes identified in the 12x grapevine genome sequence, resolving them into four subfamilies based on phylogenetic tree topology and gene structures. The origins of the CDPKs during grapevine evolution were characterized, involving 13 expansion events. Transcriptomic analysis using 54 tissues and developmental stages revealed three types of CDPK gene expression profiles: constitutive (housekeeping CDPKs), partitioned functions, and prevalent in pollen/stamen. We identified two duplicated CDPK genes that had evolved from housekeeping to pollen-prevalent functions and whose origin correlated with that of seed plants, suggesting neofunctionalization with an important role in pollen development and also potential value in the breeding of seedless varieties. We also found that CDPKs were involved in three abiotic stress signaling pathways and could therefore be used to investigate the crosstalk between stress responses.

The Dendrobium catenatum Lindl. genome sequence provides insights into polysaccharide synthase, floral development and adaptive evolution

Orchids make up about 10% of all seed plant species, have great economical value, and are of specific scientific interest because of their renowned flowers and ecological adaptations. Here, we report the first draft genome sequence of a lithophytic orchid, Dendrobium catenatum. We predict 28,910 protein-coding genes, and find evidence of a whole genome duplication shared with Phalaenopsis. We observed the expansion of many resistance-related genes, suggesting a powerful immune system responsible for adaptation to a wide range of ecological niches. We also discovered extensive duplication of genes involved in glucomannan synthase activities, likely related to the synthesis of medicinal polysaccharides. Expansion of MADS-box gene clades ANR1, StMADS11, and MIKC*, involved in the regulation of development and growth, suggests that these expansions are associated with the astonishing diversity of plant architecture in the genus Dendrobium. On the contrary, members of the type I MADS box gene family are missing, which might explain the loss of the endospermous seed. The findings reported here will be important for future studies into polysaccharide synthesis, adaptations to diverse environments and flower architecture of Orchidaceae.

Complex evolutionary history and diverse domain organization of SET proteins suggest divergent regulatory interactions

• Plants and animals possess very different developmental processes, yet share conserved epigenetic regulatory mechanisms, such as histone modifications. One of the most important forms of histone modification is methylation on lysine residues of the tails, carried out by members of the SET protein family, which are widespread in eukaryotes. • We analyzed molecular evolution by comparative genomics and phylogenetics of the SET genes from plant and animal genomes, grouping SET genes into several subfamilies and uncovering numerous gene duplications, particularly in the Suv, Ash, Trx and E(z) subfamilies. • Domain organizations differ between different subfamilies and between plant and animal SET proteins in some subfamilies, and support the grouping of SET genes into seven main subfamilies, suggesting that SET proteins have acquired distinctive regulatory interactions during evolution. We detected evidence for independent evolution of domain organization in different lineages, including recruitment of new domains following some duplications. • More recent duplications in both vertebrates and land plants are probably the result of whole-genome or segmental duplications. The evolution of the SET gene family shows that gene duplications caused by segmental duplications and other mechanisms have probably contributed to the complexity of epigenetic regulation, providing insights into the evolution of the regulation of chromatin structure.

SynFind: compiling syntenic regions across any set of genomes on demand

The identification of conserved syntenic regions enables discovery of predicted locations for orthologous and homeologous genes, even when no such gene is present. This capability means that synteny-based methods are far more effective than sequence similarity-based methods in identifying true-negatives, a necessity for studying gene loss and gene transposition. However, the identification of syntenic regions requires complex analyses which must be repeated for pairwise comparisons between any two species. Therefore, as the number of published genomes increases, there is a growing demand for scalable, simple-to-use applications to perform comparative genomic analyses that cater to both gene family studies and genome-scale studies. We implemented SynFind, a web-based tool that addresses this need. Given one query genome, SynFind is capable of identifying conserved syntenic regions in any set of target genomes. SynFind is capable of reporting per-gene information, useful for researchers studying specific gene families, as well as genome-wide data sets of syntenic gene and predicted gene locations, critical for researchers focused on large-scale genomic analyses. Inference of syntenic homologs provides the basis for correlation of functional changes around genes of interests between related organisms. Deployed on the CoGe online platform, SynFind is connected to the genomic data from over 15,000 organisms from all domains of life as well as supporting multiple releases of the same organism. SynFind makes use of a powerful job execution framework that promises scalability and reproducibility. SynFind can be accessed at http://genomevolution.org/CoGe/SynFind.pl. A video tutorial of SynFind using Phytophthrora as an example is available at http://www.youtube.com/watch?v=2Agczny9Nyc.

Rapid Reassortment of Internal Genes in Avian Influenza A(H7N9) Virus

TO THE EDITOR—H7N9 is a novel avian virus recently first reported from Shanghai in China. Very recently an isolate from Taiwan was also reported [1]. H7N9 is able to infect humans and has caused many fatal cases [2, 3]. Determining the origin and evolution of these disease-causing viruses is important for surveillance and prevention of the influenza epidemics. With more and more reported pathogenic H7N9 isolates reported, it is important to reexamine whether these disease-causing H7N9 isolates all came from the same common ancestor and whether they have undergone further reassortments. Previous studies indicated that the 6 H7N9 internal genes were from a single avian H9N2 strain [3] or from a reassortment of 2 separate H9N2 strains: the NS gene was from an H9N2 strain in the Jiangsu area, whereas the other 5 genes, M, NP, PA, PB1, and PB2, were from the other H9N2 strain in the Zhejiang area [4]. Our results are consistent with the previous results regarding the origins of HA, NA, and NS; however, we found that the remaining 5 internal genes (M, NP, PA, PB1, and PB2) appeared to come from multiple sources as they were grouped into 2 or 3 clusters in the phylogenetic trees (Figure 1). However, for M, PA, PB1, and PB2, each has a cluster together with the A/brambling/Beijing/16/2012, suggesting that they may have originated from the brambling, and each of them has another cluster due to rapid assortments. Based on these different clusters, the current H7N9 isolates can be grouped into 9 lineages or genotypes (Figure 1I). The phylogenetic tree of the H7N9 M gene has 2 separate clusters, with Shanghai/Patient4, Hangzhou/3, Shanghai/ Patient2, Shanghai/Patient5, and Taiwan/ 1 in one cluster closing the A/brambling/ Beijing/16/2012, and the remaining H7N9 strains forming the other cluster (Figure 1D). This observation indicates that the M genes in these 2 clusters may have originated from 2 different H9N2 strains. We also calculated the nucleotide substitution rate for each gene of H7N9. The nucleotide substitution rates for the 3 single-origin genes HA, NA, and NS (approximately 0.0068) are lower than the

Expansion and Functional Divergence of Jumonji C-Containing Histone Demethylases: Significance of Duplications in Ancestral Angiosperms and Vertebrates

Duplication and sequence divergence of a gene family for histone demethylases likely contributed to the enhanced, chromatin-based regulation in angiosperms and vertebrates. Histone modifications, such as methylation and demethylation, are crucial mechanisms altering chromatin structure and gene expression. Recent biochemical and molecular studies have uncovered a group of histone demethylases called Jumonji C (JmjC) domain proteins. However, their evolutionary history and patterns have not been examined systematically. Here, we report extensive analyses of eukaryotic JmjC genes and define 14 subfamilies, including the Lysine-Specific Demethylase3 (KDM3), KDM5, JMJD6, Putative-Lysine-Specific Demethylase11 (PKDM11), and PKDM13 subfamilies, shared by plants, animals, and fungi. Other subfamilies are detected in plants and animals but not in fungi (PKDM12) or in animals and fungi but not in plants (KDM2 and KDM4). PKDM7, PKDM8, and PKDM9 are plant-specific groups, whereas Jumonji, AT-Rich Interactive Domain2, KDM6, and PKDM10 are animal specific. In addition to known domains, most subfamilies have characteristic conserved amino acid motifs. Whole-genome duplication (WGD) was likely an important mechanism for JmjC duplications, with four pairs from an angiosperm-wide WGD and others from subsequent WGDs. Vertebrates also experienced JmjC duplications associated with the vertebrate ancestral WGDs, with additional mammalian paralogs from tandem duplication and possible transposition. The sequences of paralogs have diverged in both known functional domains and other regions, showing evidence of selection pressure. The increases of JmjC copy number and the divergences in sequence and expression might have contributed to the divergent functions of JmjC genes, allowing the angiosperms and vertebrates to adapt to a great number of ecological niches and contributing to their evolutionary successes.

Expansion and functional divergence of the JmjC gene family: significance of duplications in ancestral angiosperms and vertebrates

Duplication and sequence divergence of a gene family for histone demethylases likely contributed to the enhanced, chromatin-based regulation in angiosperms and vertebrates. Histone modifications, such as methylation and demethylation, are crucial mechanisms altering chromatin structure and gene expression. Recent biochemical and molecular studies have uncovered a group of histone demethylases called Jumonji C (JmjC) domain proteins. However, their evolutionary history and patterns have not been examined systematically. Here, we report extensive analyses of eukaryotic JmjC genes and define 14 subfamilies, including the Lysine-Specific Demethylase3 (KDM3), KDM5, JMJD6, Putative-Lysine-Specific Demethylase11 (PKDM11), and PKDM13 subfamilies, shared by plants, animals, and fungi. Other subfamilies are detected in plants and animals but not in fungi (PKDM12) or in animals and fungi but not in plants (KDM2 and KDM4). PKDM7, PKDM8, and PKDM9 are plant-specific groups, whereas Jumonji, AT-Rich Interactive Domain2, KDM6, and PKDM10 are animal specific. In addition to known domains, most subfamilies have characteristic conserved amino acid motifs. Whole-genome duplication (WGD) was likely an important mechanism for JmjC duplications, with four pairs from an angiosperm-wide WGD and others from subsequent WGDs. Vertebrates also experienced JmjC duplications associated with the vertebrate ancestral WGDs, with additional mammalian paralogs from tandem duplication and possible transposition. The sequences of paralogs have diverged in both known functional domains and other regions, showing evidence of selection pressure. The increases of JmjC copy number and the divergences in sequence and expression might have contributed to the divergent functions of JmjC genes, allowing the angiosperms and vertebrates to adapt to a great number of ecological niches and contributing to their evolutionary successes.

Analysis of Arabidopsis floral transcriptome: detection of new florally expressed genes and expansion of Brassicaceae-specific gene families

The flower is essential for sexual reproduction of flowering plants and has been extensively studied. However, it is still not clear how many genes are expressed in the flower. Here, we performed RNA-seq analysis as a highly sensitive approach to investigate the Arabidopsis floral transcriptome at three developmental stages. We provide evidence that at least 23, 961 genes are active in the Arabidopsis flower, including 8512 genes that have not been reported as florally expressed previously. We compared gene expression at different stages and found that many genes encoding transcription factors are preferentially expressed in early flower development. Other genes with expression at distinct developmental stages included DUF577 in meiotic cells and DUF220, DUF1216, and Oleosin in stage 12 flowers. DUF1216 and DUF577 are Brassicaceae specific, and together with other families experienced expansion within the Brassicaceae lineage, suggesting novel/greater roles in Brassicaceae floral development than other plants. The large dataset from this study can serve as a resource for expression analysis of genes involved in flower development in Arabidopsis and for comparison with other species. Together, this work provides clues regarding molecular networks underlying flower development.

Identification and characterization of OsEBS, a gene involved in enhanced plant biomass and spikelet number in rice

Common wild rice (Oryza rufipogon Griff.) is an important genetic reservoir for rice improvement. We investigated a quantitative trait locus (QTL), qGP5-1, which is related to plant height, leaf size and panicle architecture, using a set of introgression lines of O. rufipogon in the background of the Indica cultivar Guichao2 (Oryza sativa L.). We cloned and characterized qGP5-1 and confirmed that the newly identified gene OsEBS (enhancing biomass and spikelet number) increased plant height, leaf size and spikelet number per panicle, leading to an increase in total grain yield per plant. Our results showed that the increased size of vegetative organs in OsEBS-expressed plants was enormously caused by increasing cell number. Sequence alignment showed that OsEBS protein contains a region with high similarity to the N-terminal conserved ATPase domain of Hsp70, but it lacks the C-terminal regions of the peptide-binding domain and the C-terminal lid. More results indicated that OsEBS gene did not have typical characteristics of Hsp70 in this study. Furthermore, Arabidopsis (Arabidopsis thaliana) transformed with OsEBS showed a similar phenotype to OsEBS-transgenic rice, indicating a conserved function of OsEBS among plant species. Together, we report the cloning and characterization of OsEBS, a new QTL that controls rice biomass and spikelet number, through map-based cloning, and it may have utility in improving grain yield in rice.

Differential evolution of members of the rhomboid gene family with conservative and divergent patterns

Rhomboid proteins are intramembrane serine proteases that are involved in a plethora of biological functions, but the evolutionary history of the rhomboid gene family is not clear. We performed a comprehensive molecular evolutionary analysis of the rhomboid gene family and also investigated the organization and sequence features of plant rhomboids in different subfamilies. Our results showed that eukaryotic rhomboids could be divided into five subfamilies (RhoA-RhoD and PARL). Most orthology groups appeared to be conserved only as single or low-copy genes in all lineages in RhoB-RhoD and PARL, whereas RhoA genes underwent several duplication events, resulting in multiple gene copies. These duplication events were due to whole genome duplications in plants and animals and the duplicates might have experienced functional divergence. We also identified a novel group of plant rhomboid (RhoB1) that might have lost their enzymatic activity; their existence suggests that they might have evolved new mechanisms. Plant and animal rhomboids have similar evolutionary patterns. In addition, there are mutations affecting key active sites in RBL8, RBL9 and one of the Brassicaceae PARL duplicates. This study delineates a possible evolutionary scheme for intramembrane proteins and illustrates distinct fates and a mechanism of evolution of gene duplicates.