title: Studies of a cold-induced gene encoding a hybrid proline-rich protein in Brassica napus
Creators: name: Goodwin, William H.
Publisher: name: University of Glasgow
Year: 1995
Subjects: subject: Plant sciences.

description: Studies were undertaken to examine molecular events that occur during cold acclimation. This is a process whereby a plant increases its tolerance to low temperatures. The LF5B1 cDNA from Brassica napus had previously been isolated (Pallas, 1992). The cDNA was sequenced, which revealed that it was a composite cDNA, apparently composed of at least three separate cDNAs. The cDNA had been shown to hybridise to cold-induced transcripts, but it was not known which part of the cDNA was hybridising. The restriction enzyme Msp I cut the cDNA into five fragments. Using each of these fragments to probe northern blots allowed identification of the portion of the cDNA that was hybridising to the cold-induced transcripts. One of the Msp I fragments that hybridised to cold-induced transcripts was used to screen both a "cold-induced" leaf cDNA library and a Brassica napus genomic library. Ten cDNAs were isolated, none of which represented a full length cDNA. The cold-induced transcripts had been estimated to be approximately 1.5 kb (Pallas, 1992) whereas the longest cDNA isolated was less that 1 kb. The largest cDNA was sequenced and found to be similar to LF5B1, though not identical. The sequence revealed that the encoded protein was a hybrid proline-rich protein; the N-terminal part was very rich in the amino acid proline while the C-terminal part contained regions that are typical of membrane spanning domains. This cDNA was named BnPRP, this stood for Brassica napus proline-rich protein. Seven genomic clones were also isolated and partially characterised and one of these clones was subcloned and sequenced. The sequence in the corresponding region was identical to the cDNA BnPRP and the clone contained all of the putative coding sequence of BnPRP. This allowed the entire putative protein, BNPRP to be analysed. In addition to the information derived from the cDNA it revealed a putative signal peptide at the N-terminus and three distinct domains within the proline-rich region. The lack of identity of the LF5B1 and BnPRP cDNAs indicates the presence of a gene family. This had been previously suggested based on data from Southern blots of Brassica napus DNA probed with the LF5B1 cDNA (Pallas, 1992). The possible function of the BNPRP protein in relation to cold tolerance is discussed. Preliminary expression studies showed that LF5B1 hybridised to cold-induced transcripts and no expression in response to heat-shock was detected (Pallas, 1992). Further investigations into the expression of BnPRP were undertaken. Transcripts were detectable within eight hours of transfer to 4°C and increased up to 24 hours of cold treatment. Upon removal of the cold stimulus the transcript level rapidly fell; after two hours of deacclimation only very low levels were detectable and no transcripts were detected after eight hours deacclimation. The transcripts were found to be highly abundant in leaf tissue, lower levels were detected in stem tissue and no expression was detected in the root tissue; in all tissues the transcript was only detected after cold treatment. Dehydration stress caused no detectable accumulation of the transcript and no increase in transcript abundance was detected in response to abscisic acid (ABA), heat-shock or wounding. A control probe from a dehydration- and ABA-inducible gene was used to show that dehydration had occurred and that ABA had entered the tissue. The genomic clone contained several kb of the putative coding region and 1.35 kb of this DNA was isolated from the genomic clone by PCR. This was subcloned upstream of the p-glucuronidase gene (GUS) in the PBI101.1 vector to make a promoter-GUS construct. This vector was used to transform Arabidopsis thaliana plants. Three lines of transformants were analysed for GUS activity but no activity was detected in plants maintained at control temperatures (22°C) or cold treated (4°C).