Transcriptome profiling of grapefruit flavedo following exposure to low temperature and conditioning treatments uncovers principal molecular components involved in chilling tolerance and susceptibility.

A pre-storage conditioning (CD) treatment of 16 degrees C for 7 d enhanced chilling tolerance of grapefruit and reduced the development of chilling injuries during storage at 5 degrees C. To gain a better understanding of the molecular mechanisms involved in the responses of citrus fruit to low temperatures, we performed genome-wide transcriptional profiling analysis of RNA isolated from grapefruit flavedo using the newly developed Affymetrix Citrus GeneChip microarray. Utilizing very restrictive cut-off criteria, including pair-wise anova comparisons significantly different at P < or = 0.05 and induction or repression of transcript levels by at least fourfold, we found that out of 30 171 probe sets on the microarray, 1345 probe sets were significantly affected by chilling in both control and CD-treated fruits, 509 probe sets were affected by chilling specifically in the CD-treated fruits, and 417 probe sets were specifically expressed in chilling-sensitive control fruits. Overall, exposure to chilling led to expression arrest of general cellular metabolic activity, including concretive down-regulation of cell wall, pathogen defence, photosynthesis, respiration, and protein, nucleic acid and secondary metabolism. On the other hand, chilling enhanced adaptation processes that involve changes in the expression of transcripts related to membranes, lipid, sterol and carbohydrate metabolism, stress stimuli, hormone biosynthesis, and modifications in DNA binding and transcription factors.

Postharvest heat and conditioning treatments activate different molecular responses and reduce chilling injuries in grapefruit.

A combination of hot water (a rinse at 62 degrees C for 20 s) and conditioning (pre-storage at 16 degrees C for 7 d) treatments synergistically reduced chilling injury development in grapefruit (Citrus paradisi, cv. "Star Ruby") during cold storage at 2 degrees C, suggesting that the treatments may activate different chilling tolerance responses. To study the molecular mechanisms involved, chilling- and conditioning-responsive genes were isolated by polymerase chain reaction (PCR) cDNA subtraction, cDNA libraries were constructed from hot water- and conditioning-treated fruit, and cDNA sequencing was used to identify putative stress-responsive and chilling tolerance genes. PCR cDNA subtraction revealed the identification of 17 chilling-responsive and heat- and conditioning-induced genes, and the expression patterns of 11 additional stress-related genes, antioxidant defensive genes, and genes encoding enzymes involved in membrane lipid modifications were characterized. It was found that hot water and conditioning treatments had little effect on gene expression by themselves, but rather had a priming effect, and enabled the fruit to activate their defence responses after subsequent exposure to chilling. RNA gel blot hybridizations revealed that the expression patterns of eight genes, including HSP19-I, HSP19-II, dehydrin, universal stress protein (USP), EIN2, 1,3;4-beta-D-glucanase, and superoxide dismutase (SOD), were specifically regulated by the heat treatment, and four genes, including fatty acid desaturase2 (FAD2) and lipid transfer protein (LTP), were specifically regulated by the conditioning treatment. Furthermore, four more genes were identified, including a translation initiation factor (SUI1), a chaperonin, and alcohol dehydrogenase (ADH), that were commonly regulated by both heat and conditioning treatments. According to these data, it is suggested that pre-storage heat and conditioning treatments may enhance fruit chilling tolerance by activating different molecular mechanisms. The hot water treatment activates mainly the expression of various stress-related genes, whereas the conditioning treatment activates mainly the expression of lipid membrane modification enzymes.