Comparative Genomics and Synteny Analysis of <Emphasis Type="BoldItalic">KCS17</Emphasis>-<Emphasis Type="BoldItalic">KCS18</Emphasis> Cluster Across Different Genomes and Sub-genomes of Brassicaceae for Analysis of Its Evolutionary History

Comparative genomics-based synteny analysis has proved to be an effective strategy to understand evolution of genomic regions spanning a single gene (micro-unit) to large segments encompassing hundreds of kilobases to megabases. Brassicaceae is in a unique position to contribute to understanding genome and trait evolution through comparative genomics because whole genome sequences from as many as nine species have been completed and are available for analysis. In the present work, we compared genomic loci surrounding the KCS17-KCS18 cluster across these nine genomes. KCS18 or FAE 1 gene encodes beta-ketoacyl synthase, (β-KCS) a membrane-bound enzyme that catalyses the key rate-limiting step during synthesis of VLCFAs such as erucic acid (C22) present in seed oil in Brassicaceae by elongating carbon chain from C18 to C22; knowledge on role of KCS17 in plant development is however lacking. Synteny across the genomic segments harbouring FAE1 showed variable levels of gene retention ranging between 26% (Arabidopsis thaliana and Brassica napus C03) and 89% (between A. thaliana and Brassica rapa A01), and gene density ranged between 1 gene/2.86 kb and 1 gene/4.88 kb. Interestingly, in diploid Brassica species, FAE1 was retained in only one of the sub-genomes in spite of the presence of three sub-genomes created as a result of genome triplication; in contrast, FAE1 was present at three loci, with four copies in Camellina sativa which is also known to have experienced a recent genome triplication revealing contrasting fates upon duplication. The organization of KCS17 and KCS18 as head-to-tail cluster was conserved across most of the species, except the C genome containing Brassicas, namely B. oleracea and B. napus, where disruptions because of other genes were observed. Even in the conserved blocks, the distance between KCS17 and KCS18 varied; the functional implication of the organization of KCS17-KCS18 as a cluster vis-à-vis fatty acid biosynthesis needs to be dissected, as the cis-regulatory region is expected to be present in the intergenic space. Phylogenetic analysis of KCS gene family along with KCS17-KCS18 from members of Brassicaceae reveals their ancestral relationship with KCS8-KCS9 block. Further comparative functional analysis between KCS8, KCS9, KCS16, KCS17 and KCS18 across evolutionary time-scale will be required to understand the conservation or diversification of roles of these members of KCS family in fatty acid biosynthesis during course of evolution.     

Identification and Characterization of New Members of Vacuolar H<Superscript>+</Superscript>-Pyrophosphatase Family from <Emphasis Type="Italic">Oryza sativa</Emphasis> Genome

The vacuolar H⁺-pyrophosphatase (V-PPase) is an electrogenic H⁺ pump localized in the plant vacuolar membrane. V-PPase from many species has been characterized previously and the corresponding genes/cDNAs have been cloned. Cloning of the V-PPase genes from many plant species has revealed conserved motifs that may correspond to catalytic sites. The completion of the entire DNA sequence of Oryza sativa (430 Mb) presented an opportunity to study the structure and function of V-PPase proteins, and also to identify new members of this family in Oryza sativa. Our analysis identified three novel V-PPase proteins in the Oryza sativa genome that contain functional domains typical of V-PPase. We have designated them as OVP3 to OVP5. The new predicted OVPs have chromosomal locations different from previously characterized V-PPases (OVP1 and OVP2) located on chromosome 6. They all contain three characteristic motifs of V-PPase and also a conserved motif [DE]YYTS, specific to type I V-PPases and involved in coupling PP_i hydrolysis to H⁺ translocation.     

Polymorphism of the <Emphasis Type="Italic">LEAFY</Emphasis> Gene in <Emphasis Type="Italic">Brassica</Emphasis> Plants

The arabidopsis gene LEAFY controls the induction of flowering and maintenance of the floral meristem identity. By comparing the primary structure of LEAFY and its homologs in other Brassicaceae species and beyond this family, we singled out four clusters corresponding to three systematically remote families of angiosperms, Brassicaceae, Solanaceae, and Poaceae, and to gymnosperms. Both structural and functional distinctions of LEAFY homologs from their arabidopsis prototype expanded in the range Brassicaceae—Solanaceae—Poaceae. A LEAFY homolog from B. juncea cloned in our laboratory was used as a hybridization probe to analyze the restriction fragment length polymorphism in six Brassica species comprising diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) genomes. In this way we recognized LEAFY fragments specific of genomes A, B, and C; in contrast, the variations of the length and structure of the LEAFY intron 2 were not genome-specific. LEAFY polymorphism in the Brassica accessions comprising genome B was related to their geographic origin and apparently to the adaptation to day length.     

Genome-Wide Analysis of Potassium Transport-Related Genes in Chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) and Their Role in Abiotic Stress Responses

Potassium is the most abundant inorganic cation that constitutes up to 10% of the total plant dry weight and plays a prominent role in plant growth and development. Plants exhibit a complex but highly organized system of channels and transporters, which are involved in absorption and distribution of K⁺ from soil to different parts of plants. In this study, we explored the K⁺ transport system in chickpea genome and identified 36 genes encoding potassium channels and transporters. The identified genes were further classified on the basis of their domain structure and conserved motifs. It includes K⁺ transporters (23 genes: 2 HKTs, 6 KEAs, and 15 KUP/HAK/KTs) and K⁺ channels (13 genes: 8 Shakers and 5 TPKs). Chromosomal localization of these genes demonstrated that various K⁺ transporters and channels are randomly distributed across all the eight chromosomes. Comparative phylogenetic analysis of K⁺ transport system genes from Arabidopsis thaliana, Glycine max, Medicago truncatula, and Oryza sativa revealed their strong conservation in different plant species. Similarly, gene structure analysis displayed conservation of family-specific intron/exon organization in the K⁺ transport system genes. Evolutionary analysis of these genes suggested the segmental duplication as principal route of expansion for this family in chickpea. Several abiotic stress-related cis-regulatory elements were also identified in promoter regions suggesting their role in abiotic stress tolerance. Expression analysis of selected genes under drought, heat, osmotic, and salt stress demonstrated their differential expression in response to these stresses. This signifies the importance of these genes in the modulation of stress response in chickpea. Present study provides the first insight into K⁺ transport system in chickpea and can serve as a basis for their functional analysis.     

Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

Biosynthesis and therapeutic implications of iridoid glycosides from <Emphasis Type="Italic">Picrorhiza</Emphasis> genus: the road ahead

Picrorhiza genus is emerging as an important paradigm for herbal drug formulations due to its versatile iridoid glycosides exhibition and robustness in the treatment of diverse infections including hepatic amoebiasis, cancer, malaria, ulcerative colitis and cerebral ischemia reperfusion injury. Owing to the superiority of these bioactivities, iridoid glycosides from Picrorhiza have become a hot research area over the years. A metabolic pathway for the formation of iridoid glycosides has been proposed. However, some enzymes and genes of this route are still unidentified and demand the enumeration of facilitating pathways contributing to the biosynthesis of iridoid glycosides. This review summarizes the current knowledge of all naturally occurring iridoid glycosides from Picrorhiza, their biosynthesis and pharmacological capabilities which could provide the insight into metabolic regulation and the basis for the development of new drugs.     

Identification and functional analysis of anthocyanin biosynthesis genes in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

Phalaenopsis species are among the most popular potted flowers for their fascinating flowers. When their whole-genome sequencing was completed, they have become useful for studying the molecular mechanism of anthocyanin biosynthesis. Here, we identified 49 candidate anthocyanin synthetic genes in the Phalaenopsis genome. Our results showed that duplication events might contribute to the expansion of some gene families, such as the genes encoding chalcone synthase (PeCHS), flavonoid 3′-hydroxylase (PeF3′H), and myeloblastosis (PeMYB). To elucidate their functions in anthocyanin biosynthesis, we conducted a global expression analysis. We found that anthocyanin synthesis occurred during the very early flower development stage and that the flavanone 3-hydroxylase (F3H), F3′H, and dihydroflavonol 4-reductase (DFR) genes played key roles in this process. Over-expression of Phalaenopsis flavonoid 3′,5′-hydroxylase (F3′5′H) in petunia showed that it had no function in anthocyanin production. Furthermore, global analysis of sequences and expression patterns show that the regulatory genes are relatively conserved and might be important in regulating anthocyanin synthesis through different combined expression patterns. To determine the functions of MYB2, 11, and 12, we over-expressed them in petunia and performed yeast two-hybrid analysis with anthocyanin (AN)1 and AN11. The MYB2 protein had strong activity in regulating anthocyanin biosynthesis and induced significant pigment accumulation in transgenic plant petals, whereas MYB11 and MYB12 had lower activities. Our work provided important improvement in the understanding of anthocyanin biosynthesis and established a foundation for floral colour breeding in Phalaenopsis through genetic engineering.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

Controlling Soil Factor in Plant Growth and Salt Tolerance of Leguminous Plant <Emphasis Type="Italic">Alhagi sparsifolia</Emphasis> Shap. in Saline Deserts,Northwest China

The understanding of relationships between the plants and environmental variables is important for ecological restoration. The objective of this research was to investigate the controlling soil factor in plant growth and salt tolerance of leguminous plant Alhagi sparsifolia Shap. (A. sparsifolia) in a saline desert ecosystem of western China. Results showed that coverage of A. sparsifolia was positively linked to soil total K. A. sparsifolia accumulated the toxic Na⁺ in either roots or stems and thus reduced their allocation in the leaves to adapt the salt stress. The most important factor that determined Na⁺ uptake of A. sparsifolia was soil total K content, which suggests that total K can alleviate toxic Na⁺ accumulation in A. sparsifolia. In addition, there was a significant negative correlation among toxic Cl^? concentration in A. sparsifolia, soil total K and pH. Overall, soil total K accounted for 18.2% of plant growth and salt tolerance of A. sparsifolia, followed by soil pH 13.1%, and soil total P 11.6%. In order to restore A. sparsifolia in the degraded desert ecosystems on salinized soil, first we need to consider the effects of soil total K, and then synergistically consider the soil pH and total P.     

Comparative genomic analysis of <Emphasis Type="Italic">Geosporobacter ferrireducens</Emphasis> and its versatility of anaerobic energy metabolism

Members of the family Clostridiaceae within phylum Firmicutes are ubiquitous in various iron-reducing environments. However, genomic data on iron-reducing bacteria of the family Clostridiaceae, particularly regarding their environmental distribution, are limited. Here, we report the analysis and comparison of the genomic properties of Geosporobacter ferrireducens IRF9, a strict anaerobe that ferments sugars and degrades toluene under iron-reducing conditions, with those of the closely related species, Geosporobacter subterraneus DSM 17957. Putative alkyl succinate synthase-encoding genes were observed in the genome of strain IRF9 instead of the typical benzyl succinate synthase-encoding genes. Canonical genes associated with iron reduction were not observed in either genome. The genomes of strains IRF9 and DMS 17957 harbored genes for acetogenesis, that encode two types of Rnf complexes mediating the translocation of H⁺ and Na⁺ ions, respectively. Strain IRF9 harbored two different types of ATPases (Na⁺-dependent F-type ATPase and H⁺-dependent V-type ATPase), which enable full exploitation of ion gradients. The versatile energy conservation potential of strain IRF9 promotes its survival in various environmental conditions.     

<Emphasis Type="Italic">BcMF11</Emphasis> and its homologous sequences may form a lncRNA family in <Emphasis Type="Italic">Brassica</Emphasis> diploids

BcMF11 is a long non-coding RNA that has been identified in Brassica rapa and shown to be involved in pollen development. Here, when re-cloned the gene sequence, multiple paralogous copies of BcMF11 were identified in B. rapa (A genome). Multiple paralogous copies of BcMF11 were also found in B. nigra (B genome) and Brassica oleracea (C genome), the other two primary diploids of Brassica U triangle. While in the early diverging Brassicaceae lineage including Arabidopsis thaliana, no BcMF11 homolog was found. Phylogenetic analysis showed that the BcMF11 homologous sequences cloned from A genome or C genome could be clustered into a separate branch, respectively. However, there was no distinct cluster defined for BcMF11 homologous sequences cloned from B genome. The expression of BcMF11 in B. rapa was investigated and revealed a different result in the previous study. In addition, 12 expressed sequence tags from B. napus and B. rapa showing high similarities with BcMF11 were identified in the NCBI database, which further verified that rather than the useless repeat fragments in the genome, the BcMF11 homologous genes could transcribe. It is possible that BcMF11 and its homologous sequences may form a large gene family which might be originated in the recent ancestral lineage of Brassica.