Summary

Clone O141 Encodes a Cysteine Proteinase

Sequence analysis of clone O141 reveals that the 1347 bp cDNA encodes a putative protein of approximately 40 kD and predicted pI of 6.36. The predicted polypeptide is most similar to endopeptidases from Vigna mungo, Phaseolus vulgaris, Vicia sativa and Glycine max, with which it shares 67, 66, 64 and 60% overall identity, respectively The predicted polypeptide also shares significant identity with the well characterized fruit endopeptidases papain (43%) and actinidin (45%), as well as the aleurone specific endopeptidase aleurain (39%). It is likely that O141 encodes an authentic endopeptidase activity, since it contains the consensus sequences surrounding the three key amino acids critical to catalysis (Cys¹⁵⁵, His^290, Asn³¹¹ of O141) and contains 9 of 10 type II glycine residues that are important to the overall conformation of the protein.

Analysis of Gene Expression

Because of its reduced nature, the orchid embryo undergoes very little development prior to seed maturity, and there is little growth and differentiation of other seed structures following fertilization. One obvious event though following fertilization is the degeneration of cells of the outer integument during the formation of the seed coat. Because the time at which cells of the outer integument die corresponds with the window of O141 gene expression, we propose that O141 plays a role in carrying out developmentally the programmed cell death associated with seed coat formation in fertilized ovules. In situ hybridization of immature ovules to clone O141 RNA probes shows no hybridization signal (not shown), indicating that expression of this gene is limited to the latest stage of ovule development (See Time Line). Hybridization to mature or nearly mature ovules occurs in both cell layers of the outer integument of the mature ovule . No other tissues of the ovule were ever observed to show hybridization to O141 above background levels even in very long exposures of several months. O141 hybridizes to only a subset of ovules in each section observed (Panels A and B) indicating that O141 is expressed only during a narrow window of time, such that the slight differences in developmental stage of neighboring ovules is reflected in O141 gene expression. O141 gene expression most likely begins after fertilization because all the ovules in which the gene is expressed show the elongated outer integument characteristic of post-fertilization ovules as seen in Panels C and D. Furthermore, most ovules that express O141 mRNA have integuments that appear slightly degraded in appearance.