RNA expression profiles and data mining of sugarcane response to low temperature

Abstract

Tropical and subtropical plants are generally sensitive to cold and can show appreciable variation in their response to cold stress when exposed to low positive temperatures. Using nylon filter arrays, we analyzed the expression profile of 1,536 expressed sequence tags (ESTs) of sugarcane (Saccharum sp. cv SP80-3280) exposed to cold for 3 to 48 h. Thirty-four cold-inducible ESTs were identified, of which 20 were novel cold-responsive genes that had not previously been reported as being cold inducible, including cellulose synthase, ABI3-interacting protein 2, a negative transcription regulator, phosphate transporter, and others, as well as several unknown genes. In addition, 25 ESTs were identified as being down-regulated during cold exposure. Using a database of cold-regulated proteins reported for other plants, we searched for homologs in the sugarcane EST project database (SUCEST), which contains 263,000 ESTs. Thirty-three homologous putative cold-regulated proteins were identified in the SUCEST database. On the basis of the expression profiles of the cold-inducible genes and the data-mining results, we propose a molecular model for the sugarcane response to low temperature.

Figure 1.

Examples of macroarray filters used to analyze gene expression under cold acclimation. Filters containing 768 random sugarcane cDNAs in duplicate were probed with [³³P]cDNA reverse-transcribed from total RNA of control (A) and cold-treated (48 h at 4°C; B) sugarcane plantlets. Nor-specific hybridization was monitored using 12 spots representing the empty pSPORT1 vector (dashed rectangles). The signals were detected in a phosphorimager analyzer. The arrows indicate examples of cold-inducible sugarcane ESTs.

Figure 2.

Scatter plots of the expression ratios of all ESTs analyzed. The average of the normalized relative intensities of each EST was used to calculate the expression ratios. The ratios that were higher with cold treatment (3, 6, 12, 24, and 48 h at 4°C) are plotted upward, and those that were higher in the controls are plotted downward. The lateral arrows represent ratios of approximately 1. Letters on the scatter plots indicate examples of cold-inducible sugarcane ESTs. A, EST encoding to no hit protein; B, OsNAC6 gene; C, EST encoding to unknown protein; D, OsNAC4 gene and E, cellulose synthase gene. Asterisks in the 12, 24, and 48 h panels indicate expression ratios that surpassed the ratio scale. Only one replicate macroarray experiment is represented in this figure.

Figure 3.

Examples of the expression profile of cold-inducible sugarcane ESTs. The average expression was calculated from normalized relative intensities of each time point of each EST from two independent experiments (white and black circles). CeSA4, Cellulose synthase 4; Pht1-2, phosphate transporter; and OsNAC4, rice NAC gene 4.

Figure 4.

Relative contribution of the SUCEST libraries used in the macroarray experiments to identify cold-inducible genes. The values represent the percentage of normalized cold-inducible genes identified from each cDNA library (see “Results”) in relation to the total number of ESTs identified as cold inducible in our arrays. CL6, Heat- and cold-treated and untreated callus; AD1, sugarcane plantlets infected with Acetobacter diazotroficans; HR1, plantlets infected with Herbasperillum rubrisubalbicans; and LR1, leaf row tissue.

Figure 5.

Comparison between EST macroarray and RNA-blot analysis for cold-inducible sugarcane ESTs. In RNA gel blots, each lane was loaded with 10 μg of total RNA isolated from plantlets grown at 26°C (0) and plantlets grown at 4°C for 3, 6, 12, 24, and 48 h. The graphs show the induction kinetics observed in the macroarrays (white circles) and RNA blots (black circles).

Figure 6.

Hypothetical model of the sugarcane responses to low temperature. Colored rectangles contain pathways and protein names. The proteins identified by data mining are shown in black, whereas those in red were identified by macroarray expression profiling. Thin solid and dashed arrows represent gene induction (confirmed and putative, respectively) and large solid arrows represent protein interactions. OsCDPK7, rice Ca²⁺-dependent protein kinase 7; NpCaM-1, calmodulin protein; 14-3-3, 14-3-3 protein; ADA2b, transcriptional adaptor; GCN5, HAT-like protein; CBF1, transcription factor containing AP2 DNA-binding motif; GBF-1, ABI3, LIP15, and OCSBF-1, bZIP transcription factors; AIP2,ABI3, interacting protein 2; COR TMC-AP3, chloroplastic amino acid selective channel protein; WCOR413, cold acclimation protein homolog F18B3.110; TLP, thaumatin-like protein; CLP, chitinase-like protein; GLP, glucanase-like protein; CHS, chalcone synthase; GolS, galactinol synthase; L13, ribosomal protein; Pht1-2, phosphate transporter; V-ATPase, vacuolar ATPase; HSC70, heat shock protein; PUMP, plant uncoupling mitochondrial protein; ADH, alcohol dehydrogenase; CeSA4, cellulose synthase 4; se-wap41, Golgi-associated protein; DHN2 and WCOR410b, dehydrin-like proteins; HVA22, ABA-inducible protein; P5CS, Δ (1) pyrroline-5-carboxylase synthetase; PLC1, phosphatidyl-specific phospholipase C protein; NAM-like, OsNAC4, and OsNAC6, NAC proteins. The cis-elements present in cold-inducible gene promoters are indicated by the rectangles: DRE/CRT and ABRE.