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38: 1988-1995). In the present studies, one of the major endopeptidase forms, designated EP-C1, was purified as a 34-kD polypeptide from pods of maturing French bean fruits. EP-C1 was found to be immunologically distinguished from other forms in extracts from pods, but homologous to SH-EP, the major cysteine endopeptidase expressed in cotyledons of germinating Vigna mungo seeds (W. Mitsuhashi, T. Minamikawa 119891 Plant Physiol89: 274-279). The leve1 of endopeptidase that reacted with the antiserum to EP-C1 increased in pods as the fruit maturation proceeded. EP-C1 was also immunologically detected in stems of French bean plants bearing fruits of later maturation stages. Protein immunoblotting showed that a 34-kD polypeptide corresponding to EP-C1 in molecular mass occurred in extracts from 7-to 9-d cotyledons of germinating French bean seeds. In addition, two other polypeptides with slightly higher molecular masses were observed in extracts from 3-to 5-d cotyledons. We suggest that these two polypeptides are intermediates involved in posttranslational processing of EP-C1. RNA blot hybridization with EP-C1 cDNA as a probe showed that EP-C1 mRNA occurred in pods of fruits at later maturing stages and also in cotyledons of 3-to 7-d germinating seeds.

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When French bean (Pbaseolus vulgaris) plants were depodded in the early stages of fruit development, relative levels of a specific protein with a relative molecular weight of 28,000 were enhanced in the young pods that formed later. The protein, designated pod storage protein (PSP), was purified from extracts of newly formed pods from plants that had been previously depodded four times at intervals of 2 weeks. Two-dimensional polyacrylamide gel electrophoresis showed the presence of three forms (designated A, B, and C) of PSP with identical electrophoretic mobilities but different charges. The molecular mass of native PSP was estimated by gel filtration t o be 67 kD; therefore, the protein was most likely present as a dimer. The antisera raised against forms A and C were cross- mobilization in pods during fruit maturation (Tanaka et al., 1993).In the course of studies of endopeptidases in pods of French bean plants, we found that when a large number of pods in the early stages of fruit development were removed from a plant, relative levels of a specific protein with an M, of 28,000 were enhanced in the newly formed pods. The protein accumulated to high levels in the pods of plants that had been previously depodded, whereas a smaller amount was detected in the pods of nontreated plants. It was postulated that enhanced levels of the M, 28,000 protein resulted from a reduction of plant sink size caused by removing pods in the early stages of fruit development.In this report, we characterize this protein from French bean pods and designate it PSP. PSP cDNA clones were isolated, and the deduced amino acid sequence showed 65 to 71% identity with the sequences of VSPs from soybean (Glycine man) leaves (Staswick, 1988) and 40% identity with the sequences of acid phosphatase-1 from tomato (Erion et al., 1991). (Pkaseolus vul-garis) plants, the accumulation of protein in developing pods precedes that in se& (Endo et al., 1987). As reported for other legumes such as field pea (Flinn and Pate, 1968), bean (Oliker, 1978), and cowpea (Peoples et al., 1985a(Peoples et al., , 1985b(Peoples et al., , 1985c, protein accumulates in French bean seeds during fruit development concurrently with active mobilization of protein and free amino acids in the pods. Thus, a proportion of seed nitrogen is temporarily stored in pods Dry seeds of French bean (Phaseolus vulgaris L. cv Goldchased from Sakata Seed (Yokohamat Japan). If stated with 13 h of light (190 p E s-l m-') and 11 h of darkness at 25°C and, at desired developmental stages, maturing fruits were harvested and stored at -20°C until use. For the purification of PSP, pods were collected as follows: just star) and "ybean (Glycine max cv Mikawajima) were purOtherwise, MATERIALS AND METHODS During fruit development in French beanFrench bean plants were grown in a phytotron as proteins, amino acids, and probably small peptides, which are then mobilized to support the growth of seeds.activity throughout the development and subsequent senescence of French bean pods (Endo et al., 1987)....