Cloning of a harvest-induced beta-galactosidase from tips of harvested asparagus spears.

Abstract

We are investigating the early changes in physiology, biochemistry, and gene expression that follow harvest of asparagus (Asparagus officinalis L.) spears to identify factors regulating the postharvest senescence of horticultural crops harvested while physiologically immature. The tips of spears comprise immature developing tissues that appear particularly susceptible to harvest stress, and they are usually the first parts of the spear to show symptoms of postharvest senescence, including feathering and browning of bracts, tissue flaccidity, and cellular breakdown (King et al., 1990). These visual symptoms of senescence are preceded by rapid changes in carbon and nitrogen metabolism and gene expression. Within 48 h of harvest, the respiration rate of tips declines markedly, protein is lost, and both free amino acids (especially Asn) and ammonia accumulate (King et al., 1990). Major changes in gene expression occur within 6 h of harvest, including the de novo induction of specific genes (King and Davies, 1992). We previously constructed cDNA libraries from mRNA extracted from tips of spears at harvest and from tips of spears held in the dark at 20°C for 12 h. Differential hybridization screening of these libraries isolated nine cDNA clones whose transcripts had altered expression in tips of harvested asparagus spears (King and Davies, 1992), including a cDNA clone for a harvest-induced Asn synthetase (King and Davies, 1992; Davies and King, 1993). We report here the cloning and characteristics of a full-length cDNA (pTIP31) encoding P-galactosidase using one of our original harvest-induced partial cDNA clones (pTIPl1) as a hybridization probe (Table I). The derived protein sequence of pTIP31 shares 71% amino acid identity with ABGl, a cDNA clone encoding a member of the apple P-galactosidase gene family (Ross et al., 1994), and 63% amino acid identity with DINCARSR12, an ethylene-regulated P-galactosidase cDNA clone isolated from a senescing carnation peta1 library (Ragothama et al., 1991; W.R. Woodson, personal communication). Transcripts homologous to pTIP31 increase in abundance in tips of harvested asparagus spears. Induction of P-galactosidase may explain the observation of Waldron and Selvendran (1990), who noted a 50% reduction in the levels of