Characterization of a citrus vascular-specific zinc-binding cysteine proteinase inhibitor

Abstract

A 712 bp partial cDNA clone (czbp- 1) of the citrus vascular zinc binding protein (CVZBP) was isolated using reverse transcriptase polymerase chain reaction (RT-PCR). The deduced amino acid sequence of czbp-1 was identical to the N-terminal amino acid sequence for the CVZBP. Czbp- 1 had a 549 bp open reading frame and two putative polyadenylation sites, +20 bp and +103 bp relative to the poly-A tail. The deduced amino acid sequence had identity with members of the Kunitz soybean proteinase inhibitor (KSPI) family. Many members of this family are present in high concentrations in storage organs such as seeds and tubers, increase in response to abiotic stress, and are considered defense or stress response proteins. The CVZBP did not appear to fit in this category. Unlike many members of the KSPI family CVZBP was not detected in citrus seeds and protein levels decreased in response to wounding. Transcript also decreased in response to osmotic stress; a similar result previously was reported for CVZBP protein levels. Accumulation of CVZBP and its transcript increased in Zn deficient citrus seedlings compared to those receiving sufficient levels of Zn, indicating that Zn nutrition can modulate CVZBP expression. Recombinant CVZBP was produced and used to determine the capacity of this protein to inhibit several types of proteinases. The CVZBP inhibited the cysteine proteinase, papain, but not the serine proteinases, trypsin and chymotrypsin. CVZBP protein was immunolocalized primarily to the xylem parenchyma in vascular tissue of citrus midribs. Based on these results it is possible that the CVZBP has a function in vascular differentiation. Cysteine proteinases were identified in developing tracheary elements in Zinnia cell cultures. Addition of inhibitors of cysteine proteinase to these cultures prior to secondary cell wall deposition prevents differentiation of the cells into tracheary elements. Perhaps cysteine proteinase inhibitors, such as the CVZBP, in the xylem, contribute to timing of tracheary element differentiation and determination.