Comparative transcriptional profiling-based identification of raphanusanin-inducible genes

Abstract

Background: Raphanusanin (Ra) is a light-induced growth inhibitor involved in the inhibition of hypocotyl growth in response to unilateral blue-light illumination in radish seedlings. Knowledge of the roles of Ra still remains elusive. To understand the roles of Ra and its functional coupling to light signalling, we constructed the Ra-induced gene library using the Suppression Subtractive Hybridisation (SSH) technique and present a comparative investigation of gene regulation in radish seedlings in response to short-term Ra and blue-light exposure.

Results: The predicted gene ontology (GO) term revealed that 55% of the clones in the Ra-induced gene library were associated with genes involved in common defence mechanisms, including thirty four genes homologous to Arabidopsis genes implicated in R-gene-triggered resistance in the programmed cell death (PCD) pathway. Overall, the library was enriched with transporters, hydrolases, protein kinases, and signal transducers. The transcriptome analysis revealed that, among the fifty genes from various functional categories selected from 88 independent genes of the Ra-induced library, 44 genes were up-regulated and 4 were down-regulated. The comparative analysis showed that, among the transcriptional profiles of 33 highly Ra-inducible genes, 25 ESTs were commonly regulated by different intensities and duration of blue-light irradiation. The transcriptional profiles, coupled with the transcriptional regulation of early blue light, have provided the functional roles of many genes expected to be involved in the light-mediated defence mechanism.

Conclusions: This study is the first comprehensive survey of transcriptional regulation in response to Ra. The results described herein suggest a link between Ra and cellular defence and light signalling, and thereby contribute to further our understanding of how Ra is involved in light-mediated mechanisms of plant defence.

Figure 1

Formation of raphanusanin growth inhibitor by hydrolysis of 4-MTBG by myrosinase. Phototropic stimulation promotes myrosinase activity on the illuminated side of radish hypocotyls, releasing bioactive, 4-MTGI from inactive 4-MTBG and simultaneously producing bioactive raphanusanin (modified from Yamada et al., 2003).

Figure 2

Functional classification of raphanusanin-induced cDNA clones. A total of 88 genes were classified in to their physiological functions, A, and biochemical functions, B, based on the BLASTX homology search.

Figure 3

Genes differentially regulated in a time dependent manner in 4-d-old etiolated seedlings subjected to 50 ng of raphanusanin (Ra) treatment over time. Number of genes up-and down-regulated by Ra after 15 min and/or 30 min time points. To clarify, the genes expression levels (≤ 1.5 or ≥ 0.6) were excluded from the total number of differentially regulated genes.

Figure 4

Response of raphanusanin (Ra)-induced genes to three different blue light (BL) intensities (0.45 μmol m^-2 s^-1), BL (0.1 μmol m^-2 s^-1), BL (30 μmol m^-2 s^-1, pulse for 15 sec). Venn diagram indicates the differential expression of genes upon the three different treatments of BL at different time points.

Figure 5

BL and Ra induce the genes related to growth inhibition and cellular defence. Time courses of transcript levels of seven genes involved in growth inhibition pathways relative to controls following treatment with BL and Ra at the indicated concentrations were validated by reverse transcriptase-polymerase chain reaction (RT-PCR). Transcript levels of seven genes, NADPH-dependent thioredoxin reductase A (NTRA), catalase2 (CAT2), peroxidase, calmodulin- binding transcription factor 3 (CAMTA3), SNF-1 related protein kinase (CIPK1), disease resistance protein (DRP), and ACC oxidase. The bottom panels show the elongation factor α (ef1α) as loading control. Primers for all genes are specific to transcripts from the respective cDNA. The data represents the typical gel image of one of three replicates. BL: blue light; Ra: raphanusanin.