Evolutionary Analysis of the YABBY Gene Family in Brassicaceae.

The YABBY gene family is one of the plant transcription factors present in all seed plants. The family members were extensively studied in various plants and shown to play important roles in plant growth and development, such as the polarity establishment in lateral organs, the formation and development of leaves and flowers, and the response to internal plant hormone and external environmental stress signals. In this study, a total of 364 YABBY genes were identified from 37 Brassicaceae genomes, of which 15 were incomplete due to sequence gaps, and nine were imperfect (missing C2C2 zinc-finger or YABBY domain) due to sequence mutations. Phylogenetic analyses resolved these YABBY genes into six compact clades except for a YAB3-like gene identified in Aethionema arabicum. Seventeen Brassicaceae species each contained a complete set of six basic YABBY genes (i.e., 1 FIL, 1 YAB2, 1 YAB3, 1 YAB5, 1 INO and 1 CRC), while 20 others each contained a variable number of YABBY genes (5-25) caused mainly by whole-genome duplication/triplication followed by gene losses, and occasionally by tandem duplications. The fate of duplicate YABBY genes changed considerably according to plant species, as well as to YABBY gene type. These YABBY genes were shown to be syntenically conserved across most of the Brassicaceae species, but their functions might be considerably diverged between species, as well as between paralogous copies, as demonstrated by the promoter and expression analysis of YABBY genes in two Brassica species (B. rapa and B. oleracea). Our study provides valuable insights for understanding the evolutionary story of YABBY genes in Brassicaceae and for further functional characterization of each YABBY gene across the Brassicaceae species.

Genome wide survey, evolution and expression analysis of PHD finger genes reveal their diverse roles during the development and abiotic stress responses in Brassica rapa L.

BACKGROUND: Plant homeodomain (PHD) finger proteins are widely present in all eukaryotes and play important roles in chromatin remodeling and transcriptional regulation. The PHD finger can specifically bind a number of histone modifications as an "epigenome reader", and mediate the activation or repression of underlying genes. Many PHD finger genes have been characterized in animals, but only few studies were conducted on plant PHD finger genes to this day. Brassica rapa (AA, 2n = 20) is an economically important vegetal, oilseed and fodder crop, and also a good model crop for functional and evolutionary studies of important gene families among Brassica species due to its close relationship to Arabidopsis thaliana. RESULTS: We identified a total of 145 putative PHD finger proteins containing 233 PHD domains from the current version of B. rapa genome database. Gene ontology analysis showed that 67.7% of them were predicted to be located in nucleus, and 91.3% were predicted to be involved in protein binding activity. Phylogenetic, gene structure, and additional domain analyses clustered them into different groups and subgroups, reflecting their diverse functional roles during plant growth and development. Chromosomal location analysis showed that they were unevenly distributed on the 10 B. rapa chromosomes. Expression analysis from RNA-Seq data showed that 55.7% of them were constitutively expressed in all the tested tissues or organs with relatively higher expression levels reflecting their important housekeeping roles in plant growth and development, while several other members were identified as preferentially expressed in specific tissues or organs. Expression analysis of a subset of 18 B. rapa PHD finger genes under drought and salt stresses showed that all these tested members were responsive to the two abiotic stress treatments. CONCLUSIONS: Our results reveal that the PHD finger genes play diverse roles in plant growth and development, and can serve as a source of candidate genes for genetic engineering and improvement of Brassica crops against abiotic stresses. This study provides valuable information and lays the foundation for further functional determination of PHD finger genes across the Brassica species.

Comprehensive Genomic Survey, Characterization and Expression Analysis of the HECT Gene Family in Brassica rapa L. and Brassica oleracea L.

The HECT-domain protein family is one of the most important classes of E3 ligases. While the roles of this family in human diseases have been intensively studied, the information for plant HECTs is limited. In the present study, we performed the identification of HECT genes in Brassica rapa and Brassica oleracea, followed by analysis of phylogeny, gene structure, additional domains, putative cis-regulatory elements, chromosomal location, synteny, and expression. Ten and 13 HECT genes were respectively identified in B. rapa and B. oleracea and then resolved into seven groups along with their Arabidopsis orthologs by phylogenetic analysis. This classification is well supported by analyses of gene structure, motif composition within the HECT domain and additional protein domains. Ka/Ks ratio analysis showed that these HECT genes primarily underwent purifying selection with varied selection pressures resulting in different rates of evolution. RNA-Seq data analysis showed that the overwhelming majority of them were constitutively expressed in all tested tissues. qRT-PCR based expression analysis of the 10 B. rapa HECT genes under salt and drought stress conditions showed that all of them were responsive to the two stress treatments, which was consistent with their promoter sequence analysis revealing the presence of an important number of phytohormone-responsive and stress-related cis-regulatory elements. Our study provides useful information and lays the foundation for further functional determination of each HECT gene across the Brassica species.

Comprehensive genomic survey, structural classification and expression analysis of C2H2 zinc finger protein gene family in Brassica rapa L.

C2H2 zinc finger protein (ZFP) genes have been extensively studied in many organisms and can function as transcription factors and be involved in many biological processes including plant growth and development and stress responses. In the current study, a comprehensive genomics analysis of the C2H2-ZFP genes in B. rapa was performed. A total of 301 B. rapa putative C2H2-ZFP (BrC2H2-ZFP) genes were identified from the available Brassica genome databases, and further characterized through analysis of conserved amino acid residues in C2H2-ZF domains and their organization, subcellular localization, phylogeny, additional domain, chromosomal location, synteny relationship, Ka/Ks ratio, and expression pattern. We also analyzed the expression patterns of eight B. rapa C2H2-ZFP genes under salt and drought stress conditions by using qRT-PCR technique. Our results showed that about one-third of these B. rapa C2H2-ZFP genes were originated from segmental duplication caused by the WGT around 13 to 17 MYA, one-third of them were highly and consecutively expressed in all tested tissues, and 92% of them were located in nucleus by prediction supporting then their functional roles as transcription factors, of which some may play important roles in plant growth and development. The Ka/Ks ratios of 264 orthologous C2H2-ZFP gene pairs between A. thaliana and B. rapa were all, except two, inferior to 1 (varied from 0.0116 to 1.4919, with an average value of 0.3082), implying that these genes had mainly experienced purifying selection during species evolution. The estimated divergence times of the same set of gene pairs ranged from 6.23 to 38.60 MY, with an average value of 18.29 MY, indicating that these gene members have undergone different selective pressures resulting in different evolutionary rates during species evolution. In addition, a few of these B. rapa C2H2-ZFPs were shown to be involved in stress responses in a similar way as their orthologs in A. thaliana. Comparison between A. thaliana and B. rapa orthologous C2H2-ZFP genes showed that the majority of these C2H2-ZFP gene members encodes proteins with conserved subcellular localization and functional domains between the two species but differed in their expression patterns in five tissues or organs. Thus, our study provides valuable information for further functional determination of each C2H2-ZFP gene across the Brassica species, and may help to select the appropriate gene targets for further in-depth studies, and genetic engineering and improvement of Brassica crops.

Genome-wide survey, characterization, and expression analysis of RING finger protein genes in Brassica oleracea and their syntenic comparison to Brassica rapa and Arabidopsis thaliana.

The ubiquitin-mediated post-translational regulatory pathway regulates a broad range of cell functions in all eukaryotes. It requires the involvement of a large number of E3 ligases, of which more than one third belongs to the RING protein family as in Arabidopsis thaliana. In this study, a total of 756 RING domains in 734 predicted proteins were identified in Brassica oleracea. Their encoding genes were characterized by RING domain type, additional domain, and expression pattern, and mapped on the nine chromosomes of B. oleracea. Comparison of these results with B. rapa and A. thaliana revealed some common as well as species-specific features. Our results showed that the differential gene loss following the whole genome triplication has largely contributed to the RING protein gene number variation among these species, although other factors such as tandem duplication, RING domain loss, or modification had also contributed to this variation. Analysis of RNA-seq data showed that these RING protein genes were functionally diversified and involved in all the stages of plant growth and development, and that the triplicated members were also diverged in expression with one member often more dominantly expressed over the two others in the majority of cases. Our study lays the foundation for further functional determination of each RING protein gene among species of the genus Brassica.

Molecular insights into the origin of the brown rust resistance gene Bru1 among Saccharum species.

KEY MESSAGE: Analysis of 387 sugarcane clones using Bru 1 diagnostic markers revealed two possible sources of Bru 1 in Chinese cultivars: one from Saccharum spontaneum and another from Saccharum robustum of New Guinea. Sugarcane brown rust (SBR) is an important fungal disease in many sugarcane production areas around the world, and can cause considerable yield losses in susceptible sugarcane cultivars. One major SBR resistance gene, named Bru1, initially identified from cultivar R570, was shown to be a major SBR resistance source in most of the sugarcane producing areas of the world. In this study, by using the two Bru1-associated markers, R12H16 and 9O20-F4, we surveyed the presence of Bru1 in a Chinese sugarcane germplasm collection of 387 clones, consisting of 228 hybrid cultivars bred by different Chinese sugarcane breeding establishments, 54 exotic hybrid cultivars introduced from other countries and 105 clones of sugarcane ancestral species. The Bru1-bearing haplotype was detected in 43.4% of Chinese sugarcane cultivars, 20.4% of exotic hybrid cultivars, and only 3.8% of ancestral species. Among the 33 Chinese cultivars for which phenotypes of resistance to SBR were available, Bru1 was present in 69.2% (18/26) of the resistant clones. Analyses of the allelic sequence variations of R12H16 and 9O20-F4 suggested two possible sources of Bru1 in Chinese cultivars: one from S. spontaneum and another from S. robustum of New Guinea. In addition, we developed an improved Bru1 diagnostic marker, 9O20-F4-HaeIII, which can eliminate all the false results of 9O20-F4-RsaI observed among S. spontaneum, as well as a new dominant Bru1 diagnostic marker, R12E03-2, from the BAC ShCIR12E03. Our results provide valuable information for further efforts of breeding SBR-resistant varieties, searching new SBR resistance sources and cloning of Bru1 in sugarcane.

Genome-wide identification, evolution and expression analysis of RING finger protein genes in Brassica rapa.

More and more RING finger genes were found to be implicated in various important biological processes. In the present study, a total of 731 RING domains in 715 predicted proteins were identified in Brassica rapa genome (AA, 2n = 20), which were further divided into eight types: RING-H2 (371), RING-HCa (215), RING-HCb (47), RING-v (44), RING-C2 (38), RING-D (10), RING-S/T (5) and RING-G (1). The 715 RING finger proteins were further classified into 51 groups according to the presence of additional domains. 700 RING finger protein genes were mapped to the 10 chromosomes of B. rapa with a range of 47 to 111 genes for each chromosome. 667 RING finger protein genes were expressed in at least one of the six tissues examined, indicating their involvement in various physiological and developmental processes in B. rapa. Hierarchical clustering analysis of RNA-seq data divided them into seven major groups, one of which includes 231 members preferentially expressed in leaf, and constitutes then a panel of gene candidates for studying the genetic and molecular mechanisms of leafy head traits in Brassica crops. Our results lay the foundation for further studies on the classification, evolution and putative functions of RING finger protein genes in Brassica species.

Overexpression of a Triticum aestivum Calreticulin gene (TaCRT1) Improves Salinity Tolerance in Tobacco.

Calreticulin (CRT) is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants. However, the roles played by this protein in salt stress responses in wheat (Triticum aestivum) remain obscure. In this study, three TaCRT genes were identified in wheat and named TaCRT1, TaCRT2 and TaCRT3-1 based on their sequence characteristics and their high homology to other known CRT genes. Quantitative real-time PCR expression data revealed that these three genes exhibit different expression patterns in different tissues and are strongly induced under salt stress in wheat. The calcium-binding properties of the purified recombinant TaCRT1 protein were determined using a PIPES/Arsenazo III analysis. TaCRT1 gene overexpression in Nicotiana tabacum decreased salt stress damage in transgenic tobacco plants. Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than non-transgenic tobacco under normal growth conditions. Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in plants.

A dominant point mutation in a RINGv E3 ubiquitin ligase homoeologous gene leads to cleistogamy in Brassica napus.

In the allopolyploid Brassica napus, we obtained a petal-closed flower mutation by ethyl methanesulfonate mutagenesis. Here, we report cloning and characterization of the Bn-CLG1A (CLG for cleistogamy) gene and the Bn-clg1A-1D mutant allele responsible for the cleistogamy phenotype. Bn-CLG1A encodes a RINGv E3 ubiquitin ligase that is highly conserved across eukaryotes. In the Bn-clg1A-1D mutant allele, a C-to-T transition converts a Pro at position 325 to a Leu (P325L), causing a dominant mutation leading to cleistogamy. B. napus and Arabidopsis thaliana plants transformed with a Bn-clg1A-1D allele show cleistogamous flowers, and characterization of these flowers suggests that the Bn-clg1A-1D mutation causes a pronounced negative regulation of cutin biosynthesis or loading and affects elongation or differentiation of petal and sepal cells. This results in an inhibition or a delay of petal development, leading to folded petals. A homoeologous gene (Bn-CLG1C), which shows 99.5% amino acid identity and is also constitutively and equally expressed to the wild-type Bn-CLG1A gene, was also identified. We showed that P325L is not a loss-of-function mutation and did not affect expression of Bn-clg1A-1D or Bn-CLG1C. Our findings suggest that P325L is a gain-of-function semidominant mutation, which led to either hyper- or neofunctionalization of a redundant homoeologous gene.