Copyright © Physiologia Plantarum 2000 PHYSIOLOGIA PLANTARUM 108: 382 – 389. 2000 Printed in Ireland —all rights resered ISSN 0031-9317 Involvement of phenylalanine ammonia-lyase in the response of Fortune mandarin fruits to cold temperature Maria T. Sanchez-Ballesta a , Maria T. Lafuente a , Lorenzo Zacarias a and Antonio Granell b, * a Instituto de Agroquı ´mica y Tecnologı ´a de Alimentos, CSIC, Apartado de Correos 73, 46100 Burjassot, Valencia, Spain b Instituto de Biologı ´a Molecular y Celular de Plantas, CSIC-UPV, Camino de Vera s /n, 46022 Valencia, Spain *Corresponding author, e -mail: agranell@ibmcp.up.es Received 30 June 1999; revised 18 November 1999; in final form 5 January 2000 L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) is gener- non-chilling (12°C) temperatures. Northern blot analyses, us- ally recognised as a marker of environmental stress in differ- ing both FPAL1 and FPAL2 cDNAs as probes, recognised a single mRNA that accumulated in fruits exposed to 2°C prior ent plant tissues. To investigate the involvement of PAL in the to the appearance of physical chilling symptoms and the response of citrus fruits to cold temperature, changes in the abundance of PAL mRNA and PAL activity were examined accompanying increase in PAL activity. Once symptoms were obvious, accumulation of PAL transcript and PAL activity in flavedo tissue of the chilling-sensitive Fortune cultivar (Citrus clementina Hort. ex Tanaka ×Citrus reticulata, were restricted to the tissue in and around the necrotic regions. However, exposure to a low non-chilling temperature Blanco). A cDNA library was constructed from flavedo tissue of chilling-stressed fruits and screened with a 660 bp PAL produced an early, moderate and transient increase in PAL probe, obtained by polymerase chain reaction using oligonu- mRNA levels and PAL activity that declined after 1 day. This cleotides derived from conserved sequence regions. Two full- transient induction of both PAL gene expression and activity length cDNA clones (FPAL1 and FPAL2 ) were isolated, and could be part of a rapid adaptive response of the tissue to low the deduced amino acid sequences showed a 75 – 85% similar- temperatures. Interestingly, a rapid and sustained accumula- ity with PAL genes from other plant species. A comparative tion of PAL transcript occurred in the leaves and roots of analysis of the changes in PAL activity and PAL mRNA citrus plants exposed to a low temperature in the absence of levels was conducted in fruits stored at chilling (2°C) and any detectable chilling-induced damage. phenol metabolism, the symptoms of which are necrosis and darkening (Hyodo et al. 1978). In most plants, PAL is encoded by a small multigene family of 2–6 members (Cramer et al. 1989, Growri et al. 1991, Minami and Tanaka 1993, Zhu et al. 1995). Individual members of the PAL gene family are differentially expressed in plant tissues as well as in the response to different stress conditions (Liang et al. 1989, Lois et al. 1989). PAL and other anthocyanin biosynthesis genes have been considered to be cor (cold regulated) genes, and have been proposed as excellent subjects for characterising plant molecular re- sponses to low temperature (Christie et al. 1994). In addi- tion to transcriptional regulation, chilling stress may also affect PAL activity levels (Rhodes and Wooltorton 1977, Introduction Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) is the en- zyme at the entry-point of the phenylpropanoid pathway. PAL catalyses the deamination of L-phenylalanine to form trans -cinnamic acid, a substrate common to the biosynthesis of different classes of phenylpropanoid products: an- thocyanins, flavonoid pigments, ultraviolet (UV) protec- tants, antimicrobial furanocoumarins, isoflavonoid phytoalexins, lignin and wound phenolic esters (Hahlbrock and Scheel 1989, Dixon and Paiva 1995). Due to the nature and function of these products, PAL activity and the activa- tion of PAL under stress conditions have been considered part of a defence mechanism operating in stress-afflicted cells (Dixon and Paiva 1995). PAL may also participate in the development of plant tissue damage related to altered Abbreiations – ACC, 1-aminocyclopropane-1-carboxylic acid; CI, chilling injury; PAL, phenylalanine ammonia-lyase; PCR, polymerase chain reaction. Physiol. Plant. 108, 2000 382