Phylogeny and evolution of plant cyclic nucleotide-gated ion channel (CNGC) gene family and functional analyses of tomato CNGCs

Cyclic nucleotide-gated ion channels (CNGCs) are calcium-permeable channels that are involved in various biological functions. Nevertheless, phylogeny and function of plant CNGCs are not well understood. In this study, 333 CNGC genes from 15 plant species were identified using comprehensive bioinformatics approaches. Extensive bioinformatics analyses demonstrated that CNGCs of Group IVa were distinct to those of other groups in gene structure and amino acid sequence of cyclic nucleotide-binding domain. A CNGC-specific motif that recognizes all identified plant CNGCs was generated. Phylogenetic analysis indicated that CNGC proteins of flowering plant species formed five groups. However, CNGCs of the non-vascular plant Physcomitrella patens clustered only in two groups (IVa and IVb), while those of the vascular non-flowering plant Selaginella moellendorffii gathered in four (IVa, IVb, I and II). These data suggest that Group IV CNGCs are most ancient and Group III CNGCs are most recently evolved in flowering plants. Furthermore, silencing analyses revealed that a set of CNGC genes might be involved in disease resistance and abiotic stress responses in tomato and function of SlCNGCs does not correlate with the group that they are belonging to. Our results indicate that Group IVa CNGCs are structurally but not functionally unique among plant CNGCs.     

Gene Expression Profiles Deciphering Rice Phenotypic Variation between Nipponbare (Japonica) and 93-11 (Indica) during Oxidative Stress

Rice is a very important food staple that feeds more than half the world''s population. Two major Asian cultivated rice (Oryza sativa L.) subspecies, japonica and indica, show significant phenotypic variation in their stress responses. However, the molecular mechanisms underlying this phenotypic variation are still largely unknown. A common link among different stresses is that they produce an oxidative burst and result in an increase of reactive oxygen species (ROS). In this study, methyl viologen (MV) as a ROS agent was applied to investigate the rice oxidative stress response. We observed that 93-11 (indica) seedlings exhibited leaf senescence with severe lesions under MV treatment compared to Nipponbare (japonica). Whole-genome microarray experiments were conducted, and 1,062 probe sets were identified with gene expression level polymorphisms between the two rice cultivars in addition to differential expression under MV treatment, which were assigned as Core Intersectional Probesets (CIPs). These CIPs were analyzed by gene ontology (GO) and highlighted with enrichment GO terms related to toxin and oxidative stress responses as well as other responses. These GO term-enriched genes of the CIPs include glutathine S-transferases (GSTs), P450, plant defense genes, and secondary metabolism related genes such as chalcone synthase (CHS). Further insertion/deletion (InDel) and regulatory element analyses for these identified CIPs suggested that there may be some eQTL hotspots related to oxidative stress in the rice genome, such as GST genes encoded on chromosome 10. In addition, we identified a group of marker genes individuating the japonica and indica subspecies. In summary, we developed a new strategy combining biological experiments and data mining to study the possible molecular mechanism of phenotypic variation during oxidative stress between Nipponbare and 93-11. This study will aid in the analysis of the molecular basis of quantitative traits.     

Pathogenesis related protein-1 (PR-1) genes in tomato (Solanum lycopersicum L.): Bioinformatics analyses and expression profiles in response to drought stress

The pathogenesis-related protein 1 (PR-1) gene family play important roles in the plant metabolism in response to biotic and abiotic stresses. The present study aimed genome-wide identification and bioinformatics analyses of PR-1 genes in tomato (Solanum lycopersicum L.). The analyses resulted in the identification of 13 novel SlPR-1 genes, each of which produce a protein belonging to the CAP superfamily (PF00188). The KEGG annotation analyses revealed that the SlPR-1 proteins functioned in the environmental information processing (09130). The expression patterns of the PR-1 genes and some stress-related physiological parameters were investigated in Fusarium oxysporum sensitive and tolerant tomato varieties under drought stress. The drought stress leaded upregulation of all SlPR-1 genes, reaching up to 50 folds. The results indicate that the SlPR-1 genes play active roles in response to drought. This is the first study exhibiting the expression profiles of SlPR-1 genes under an abiotic stress, drought, in tomato.     

Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.     

Identification and Evaluation of Reference Genes for Accurate Transcription Normalization in Safflower under Different Experimental Conditions

Safflower (Carthamus tinctorius L.) has received a significant amount of attention as a medicinal plant and oilseed crop. Gene expression studies provide a theoretical molecular biology foundation for improving new traits and developing new cultivars. Real-time quantitative PCR (RT-qPCR) has become a crucial approach for gene expression analysis. In addition, appropriate reference genes (RGs) are essential for accurate and rapid relative quantification analysis of gene expression. In this study, fifteen candidate RGs involved in multiple metabolic pathways of plants were finally selected and validated under different experimental treatments, at different seed development stages and in different cultivars and tissues for real-time PCR experiments. These genes were ABCS, 60SRPL10, RANBP1, UBCL, MFC, UBCE2, EIF5A, COA, EF1-β, EF1, GAPDH, ATPS, MBF1, GTPB and GST. The suitability evaluation was executed by the geNorm and NormFinder programs. Overall, EF1, UBCE2, EIF5A, ATPS and 60SRPL10 were the most stable genes, and MBF1, as well as MFC, were the most unstable genes by geNorm and NormFinder software in all experimental samples. To verify the validation of RGs selected by the two programs, the expression analysis of 7 CtFAD2 genes in safflower seeds at different developmental stages under cold stress was executed using different RGs in RT-qPCR experiments for normalization. The results showed similar expression patterns when the most stable RGs selected by geNorm or NormFinder software were used. However, the differences were detected using the most unstable reference genes. The most stable combination of genes selected in this study will help to achieve more accurate and reliable results in a wide variety of samples in safflower.     

The Evolutionary Fate of the Horizontally Transferred Agrobacterial Mikimopine Synthase Gene in the Genera Nicotiana and Linaria

Few cases of spontaneously horizontally transferred bacterial genes into plant genomes have been described to date. The occurrence of horizontally transferred genes from the T-DNA of Agrobacterium rhizogenes into the plant genome has been reported in the genus Nicotiana and in the species Linaria vulgaris. Here we compare patterns of evolution in one of these genes (a gene encoding mikimopine synthase, mis) following three different events of horizontal gene transfer (HGT). As this gene plays an important role in Agrobacterium, and there are known cases showing that genes from pathogens can acquire plant protection function, we hypothesised that in at least some of the studied species we will find signs of selective pressures influencing mis sequence. The mikimopine synthase (mis) gene evolved in a different manner in the branch leading to Nicotiana tabacum and N. tomentosiformis, in the branch leading to N. glauca and in the genus Linaria. Our analyses of the genus Linaria suggest that the mis gene began to degenerate soon after the HGT. In contrast, in the case of N. glauca, the mis gene evolved under significant selective pressures. This suggests a possible role of mikimopine synthase in current N. glauca and its ancestor(s). In N. tabacum and N. tomentosiformis, the mis gene has a common frameshift mutation that disrupted its open reading frame. Interestingly, our results suggest that in spite of the frameshift, the mis gene could evolve under selective pressures. This sequence may still have some regulatory role at the RNA level as suggested by coverage of this sequence by small RNAs in N. tabacum.     

Association of jacalin-related lectins with wheat responses to stresses revealed by transcriptional profiling

Jacalin-related lectins (JRLs) are carbohydrate-binding proteins widely present in plants and have one or more jacalin domains in common. However, JRLs’ structural types and functions are still poorly understood. In the present study, a total of 67 wheat (Triticum aestivum) JRL genes were identified through an exhausted search of EST database coupling with genome walking using published 454 sequence reads of Chinese Spring. A comparison of the translated wheat JRL proteins with those from other plants showed plant JRLs generally had low sequence similarity within and between species but exhibited conserved modular domain structures. More JRL genes encoded multiple jacalin domains in Arabidopsis thaliana, whereas more genes encoded chimeric JRLs in cereal plants. Dirigent domain-containing JRL genes were Poaceae-specific and accounted for nearly half of the identified wheat JRL genes. The dirigent domains were evolutionarily significantly correlated with the covalently linked jacalin domains. A phylogenetic analysis showed JRL proteins have experienced a substantial diversification after speciation. Moreover, new structural features conserved across the taxa were identified. Digital expression analysis and RT-PCR assays showed the expression of wheat JRL genes was largely tissue specific, typically low, and mostly inducible by biotic and abiotic stresses and stress hormones. These results suggest plant JRLs are critical for plant adaptation to stressful environments.     

Analysis of biochemical variations and microRNA expression in wild (Ipomoea campanulata) and cultivated (Jacquemontia pentantha) species exposed to in vivo water stress

The current study analyses few important biochemical parameters and microRNA expression in two closely related species (wild but tolerant Ipomoea campanulata L. and cultivated but sensitive Jacquemontia pentantha Jacq.G.Don) exposed to water deficit conditions naturally occurring in the field. Under soil water deficit, both the species showed reduction in their leaf area and SLA as compared to well-watered condition. A greater decrease in chlorophyll was noticed in J. pentantha (~50 %) as compared to I. campanulata (20 %) under stress. By contrast, anthocyanin and MDA accumulation was greater in J. pentantha as compared to I. campanulata. Multiple isoforms of superoxide dismutases (SODs) with differing activities were observed under stress in these two plant species. CuZnSOD isoforms showed comparatively higher induction (~10–40 %) in I. campanulata than J. pentantha. MicroRNAs, miR398, miR319, miR395 miR172, and miR408 showed opposing expression under water deficit in these two plant species. Expression of miR156, miR168, miR171, miR172, miR393, miR319, miR396, miR397 and miR408 from either I. campanulata or J. pentantha or both demonstrated opposite pattern of expression to that of drought stressed Arabidopsis. The better tolerance of the wild species (I. campanulata) to water deficit could be attributed to lesser variations in chlorophyll and anthocyanin levels; and relatively higher levels of SODs than J. pentantha. miRNA expression was different in I. campanulata than J. pentantha.     

CLAVATA3-like genes are differentially expressed in grape vine (Vitis vinifera) tissues

The CLAVATA3 (CLV3)/endosperm surrounding region [(ESR) CLE] peptides function as intercellular signaling molecules that regulate various physiological and developmental processes in diverse plant species. We identified five CLV3-like genes from grape vine (Vitis vinifera var. Pinot Noir): VvCLE 6, VvCLE 25-1, VvCLE 25-2, VvCLE 43 and VvCLE TDIF. These CLV3-like genes encode short proteins containing 43–128 amino acids. Except VvCLE TDIF, grape vine CLV3-like proteins possess a consensus amino acid sequence known as the CLE domain. Phylogenic analysis suggests that the VvCLE 6, VvCLE25-1, VvCLE25-2 and VvCLE43 genes have evolved from a single common ancestor to the Arabidopsis CLV3 gene. Expression analyses showed that the five grape CLV3-like genes are expressed in leaves, stems, roots and axillary buds with significant differences in their levels of expression. For example, while all of them were strongly expressed in axillary buds, VvCLE6 and VvCLE43 expression prevailed in roots, and VvCLE25-1, VvCLE25-2 and VvCLE TDIF expression in stems. The differential expression of the five grape CLV3-like peptides suggests that they play different roles in different organs and developmental stages.