Identification of the genes and pathways associated with pigment gland morphogenesis in cotton by transcriptome profiling of near-isogenic lines

Cottonseed protein is underutilized due to the presence of pigment gland containing a toxic compound called gossypol. Cotton produces gossypol and related compounds in various tissues to protect itself against microbial, insect, and rodent attacks. Understanding the mechanism of cotton pigment gland formation and regulation of gossypol biosynthesis will greatly facilitate the research efforts in developing a cotton variety with a gossypol free seed and normally glanded foliage. In this study we make use of near-isogenic lines of cotton pigment gland to screen the genes related to gland morphogenesis applying both GeneChip and suppression subtractive hybridization (SSH) methods.We identified 880 differentially expressed genes associated with gland morphogenesis in cotton by comparing transcriptome profiles of cotton from glandless and glanded near-isogenic lines using a GeneChip. Gene ontology (GO) analysis showed that 880 genes were distributed mainly among the following GO categories: cellular process (14.45%), physiological process (14.23%), catalytic activity (9.21%), metabolism (8.99%), and cell parts (5.24%). Molecular pathway analysis revealed that these differentially expressed genes were involved in 58 KEGG pathways. Differentially expressed genes were also identified and isolated using suppression subtractive hybridization (SSH) with the same near-isogenic lines. A total of 147 ESTs were identified whose expression was either up- or down-regulated. Sequencing and BLAST analysis indicated that some of these genes were novel, while others were related to energy metabolism, transcription factors, and biotic responses. 13 genes were found to be differentially expressed both in SSH and GeneChip analysis. The expression pattern of these genes was verified by real-time PCR. The gene expression profiles produced in this study provide useful information on the molecular mechanism and regulation of gland formation and the related process in cotton. Of particular interest for future study are the genes identified by both SSH and GeneChip analysis. The outcomes are helping for our understanding of the development of specialised structures such as trichomes in plant species, from an applied and basic science perspective and promoting the application in molecular breeding.

Dual Affymetrix GeneChip analysis of the perennial ryegrass-endophyte symbiosis

Grass associations with Epichloë/Neotyphodium endophytes display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. To gain a comprehensive understanding of the complex biological interactions that occur between a plant host and fungal symbiont, a transcriptomics approach using custom designed Affymetrix GeneChip®s was employed. We are currently comparing and analysing symbiotic interactions of perennial ryegrass (Lolium perenne) with endophytes N. lolii and E. festucae as well as comparing it against endophyte-free perennial ryegrass. Both N. lolii and E. festucae have been grown in culture in order to compare in planta versus in vitro gene expression. Additionally, targeted gene replacements in E. festucae have been performed and a comparative analysis of the knock-outs with wild-type E. festucae infected plants is in progress. These comparative analyses have revealed changes in gene expression which may lead to the identification of gene pathways/networks and the roles of these genes in symbiosis. Keywords: Neotyphodium, Epichloë, transcriptomics, symbiosis