Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower

Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs represented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previously reported for sunflower. Several potentially interesting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The libraries described here represent an original and valuable resource for the discovery of yet unknown genes putatively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos.Communicated by R. Hagemann     

Insect feeding-induced differential expression of <Emphasis Type="Italic">Beta vulgaris</Emphasis> root genes and their regulation by defense-associated signals

Root responses to insect pests are an area of plant defense research that lacks much information. We have identified more than 150 sugar beet root ESTs enriched for genes responding to sugar beet root maggot feeding from both moderately resistant, F1016, and susceptible, F1010, genotypes using suppressive subtractive hybridization. The largest number of identified F1016 genes grouped into the defense/stress response (28%) and secondary metabolism (10%) categories with a polyphenol oxidase gene, from F1016, identified most often from the subtractive libraries. The differential expression of the root ESTs was confirmed with RT-PCR. The ESTs were further characterized using macroarray-generated expression profiles from F1016 sugar beet roots following mechanical wounding and treatment of roots with the signaling molecules methyl jasmonate, salicylic acid and ethylene. Of the examined root ESTs, 20, 17 and 11% were regulated by methyl jasmonate, salicylic acid and ethylene, respectively, suggesting these signaling pathways are involved in sugar beet root defense responses to insects. Identification of these sugar beet root ESTs provides knowledge in the field of plant root defense and will lead to the development of novel control strategies for control of the sugar beet root maggot.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users     

Expressed sequence tag-based gene expression analysis under aluminum stress in rye

To understand the mechanisms responsible for aluminum (Al) toxicity and tolerance in plants, an expressed sequence tag (EST) approach was used to analyze changes in gene expression in roots of rye (Secale cereale L. cv Blanco) under Al stress. Two cDNA libraries were constructed (Al stressed and unstressed), and a total of 1,194 and 774 ESTs were generated, respectively. The putative proteins encoded by these cDNAs were uncovered by Basic Local Alignment Search Tool searches, and those ESTs showing similarity to proteins of known function were classified according to 13 different functional categories. A total of 671 known function genes were used to analyze the gene expression patterns in rye cv Blanco root tips under Al stress. Many of the previously identified Al-responsive genes showed expression differences between the libraries within 6 h of Al stress. Certain genes were selected, and their expression profiles were studied during a 48-h period using northern analysis. A total of 13 novel genes involved in cell elongation and division (tonoplast aquaporin and ubiquitin-like protein SMT3), oxidative stress (glutathione peroxidase, glucose-6-phosphate-dehydrogenase, and ascorbate peroxidase), iron metabolism (iron deficiency-specific proteins IDS3a, IDS3b, and IDS1; S-adenosyl methionine synthase; and methionine synthase), and other cellular mechanisms (pathogenesis-related protein 1.2, heme oxygenase, and epoxide hydrolase) were demonstrated to be regulated by Al stress. These genes provide new insights about the response of Al-tolerant plants to toxic levels of Al.     

Paenibacillus lentimorbus Enhanced Abiotic Stress Tolerance Through Lateral Root Formation and Phytohormone Regulation

Under the stressed conditions plant growth-promoting rhizobacteria (PGPR) are able to stimulate plant growth through several mechanisms, including antioxidants alleviation, regulation of stress responsive genes and phytohormones etc. Present study is conducted to investigate the impact of Paenibacillus lentimorbus B-30488 inoculation on salinity and drought stress mitigation in Arabidopsis thaliana through modulation in defense enzymes, phyto-hormones and root system architecture associated gene expression profiling. In vitro experiments clearly demonstrated the role of B-30488 in stimulating the root length, branches, lateral root formation and biomass under salinity and drought stress. The inoculation of B-30488 modulated the phytohormones levels to protect the plants from salinity and drought stress. Similarly, defence enzymes were also activated under the stressed conditions, but B-30488 inoculation reduced the antioxidants content during salinity and drought stress as compared to their respective controls. Microscopy results showed decrease in lateral roots hair formation under both stresses and B-30488 inoculation not only mitigate but also enhanced the lateral root formation. Gene expression analysis through real time polymerase chain reaction (RT-PCR) showed modulated expression of several genes related to root development, stress and lateral root formation in B-30488 inoculated seedlings. Results based on the present study, B-30488 is also involved in alteration root architecture, its growth regulation via modulation in phytohormones and genes expression and overall significant improvement in plant growth under stress conditions.

     

Construction of a haustorium development associated SSH library in Thesium chinense and analysis of specific ESTs included by Imperata cylindrica

Thesium chinense Turcz. is a facultative hemiparasite that can invade host plants by inducing haustoria on neighboring host roots to access the water and nutrients of the host. Suppression subtractive hybridization (SSH) was performed to identify the differentially expressed genes during haustorial development. A total of 395 recombinants were randomly selected for sequencing. Two hundred seventy-six ESTs were retained after this screening procedure, and after DNA sequencing, 217 ESTs (78.6%) exhibited significant homology with previously identified genes in GenBank. Based on GO annotation, we selected 9 ESTs that may be associated with the development of haustorium and be induced by Imperata cylindrica (L.) Beauv.var.major (Nees)C．E．Hubb. for qRT-PCR. The results revealed that the relative quantities of the 4 ESTs from among the 9 ESTs that involved cell-wall modification (pectin methylesterase, TcPME), signal transduction (auxin-responsive aux/iaa gene, TcAux/IAA) and metabolism (class III peroxidase, TcPrx; phenylalanine ammonia-lyase, TcPAL) all increased simultaneously with the development of the roots of T. chinense and I. cylindrica. These results indicated that the relative quantities of TcPME, TcAux/IAA, TcPrx and TcPAL were affected by the secretions of the I. cylindrica root that T. chinense accreted and that these effects were meaningful to the development of the haustorium of T. chinense.     

A unigene catalogue of 5700 expressed genes in cassava

Two economically important characters, starch content and cassava bacterial blight resistance, were targeted to generate a large collection of cassava ESTs. Two libraries were constructed from cassava root tissues of varieties with high and low starch contents. Other libraries were constructed from plant tissues challenged by the pathogen Xanthomonas axonopodis pv.manihotis. We report here the single pass sequencing of 11 954 cDNA clones from the 5’ ends, including 111 from the 3’ ends. Cluster analysis permitted the identification of a unigene set of 5700 sequences. Sequence analyses permitted the assignment of a putative functional category for 37% of sequences whereas ~ 16% sequences did not show any significant similarity with other proteins present in the database and therefore can be considered as cassava specific genes. A group of genes belonging to a large multigene family was identified. We characterize a set of genes detected only in infected libraries putatively involved in the defense response to pathogen infection. By comparing two libraries obtained from cultivars contrasting in their starch content a group of genes associated to starch biosynthesis and differentially expressed was identified. This is the first large cassava EST resource developed today and publicly available thus making a significant contribution to genomic knowledge of cassava.     

Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.