Minisymposium 2: Plant Biology in the Tropics

Abs # 12004: Characterization and proteolytic processing of a novel cystatin upregulated in pineapple fruit.

Presenter:	Neupane, Kabi R, kabi@hawaii.edu
Authors	Neupane, Kabi R (A)   Neuteboom, Leon W. (B)   Okazaki, Kristie  (B)   Christopher, David A. (B)
Affiliations:	(A): Division of Math and Natural Sciences, Leeward Community Collge, 96-045 Ala Ike, Pearl City, HI 96782 (B): Department of Molecular Biosciences & Bioengineering, University of Hawaii, 1955 East-West Rd. Honolulu, Hawaii 96822

Cultivated pineapple (Ananas comosus) is the leading agricultural commodity in Hawaii and is the most widely traded tropical fruit worldwide. It utilizes CAM photosynthesis and is capable of adapting to drought and heat stress, making it highly productive under limited water availability. Besides its edible fruit, the pineapple plant is also a source of potent cysteine proteases, ananain and bromelain, which have applications in medicine and the food industry. Breeding and genetic engineering programs seek to improve nematode and virus resistance, flowering, fruit ripening, and sugar content. However, these programs are hampered by limited information on genes, their structure, codon usage, promoters and expression during growth. For example, cDNAs can be used as molecular markers in breeding programs, while tissue-specific promoters can be used to bioengineer pest and pathogen resistance. Therefore, as a first step to increase the permanent genetic resources for pineapple improvement, we have identified and characterized genes involved in root and fruit development. A root-specific cDNA library was differentially screened using tissue-specific probes from root, fruit and aerial tissues. Clones were categorized into 14 classes based on their expression characteristics. 40% of the clones were classified as constitutive, being expressed about equally in all three tissues. Approximately 25% of the clones were expressed higher in roots than the other tissues. A few clones were expressed higher in fruit than any other tissue. One of the fruit-enhanced clones is predicted to encode a cystatin. The expression of the cystatin gene was analyzed in detail in 14 different tissues of a mature plant, as well as in roots and aerial tissue from 3, 6, 13, 20 month-old developing plants and during 5 stages of fruit development. Regardless of tissue and developmental stage analyzed, expression was highest in fruit, particularly the shell. A novel feature of the predicted polypeptide sequence is an unusually long amino-terminus (75 amino acids) not found in any other cystatin isolated to date. Using in vitro transcription-translation of the cystatin cDNA followed by microsomal processing, we provide evidence that the cystatin is processed by microsomal membranes from 20.7 kDa to 17 kDa, and retains ~40 amino acids of its amino terminus. The role of these additional amino acids in cysteine protease inhibition is being determined.