Takao Minamikawa

Degradation of ribulose-bisphosphate carboxylase by vacuolar enzymes of senescing French bean leaves: immunocytochemical and ultrastructural observations.

SummaryThe possible involvement of vacuolar cysteine proteinases in degradation of ribulose-bisphosphate carboxylase (Rubisco) in senescing French bean leaves was studied by ultrastructural and immunocytochemical analyses with antibodies raised against the large subunit (LSU) of Rubisco and SH-EP, a cysteine proteinase fromVigna mungo that is immunologically identical to one of the major proteinases of French bean plants. Primary leaves of 10-day-old plants were detached and placed at 25 °C in darkness for 0, 4, and 8 days to allow their senescence to proceed. The leaves at each senescence stage were subjected to the conventional electron microscopic and immunocytochemical studies. The results indicated that the chloroplasts of senescing French bean leaves were separated from the cytoplasm of the cell periphery and taken into the central vacuole and that the Rubisco LSU in the chloroplasts was degraded by vacuolar enzymes such as an SH-EP-related cysteine proteinase that developed in senescing leaves. The present results together with the results of previous biochemical studies using vacuolar lysates support the view that Rubisco is degraded through the association of chloroplasts with the central vacuole during the senescence of leaves that were detached and placed in darkness.

Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components

α-Amylase is expressed in cotyledons of germinated Vigna mungo seeds and is responsible for the degradation of starch that is stored in the starch granule (SG). Immunocytochemical analysis of the cotyledon cells with anti–α-amylase antibody showed that α-amylase is transported to protein storage vacuole (PSV) and lytic vacuole (LV), which is converted from PSV by hydrolysis of storage proteins. To observe the insertion/degradation processes of SG into/in the inside of vacuoles, ultrastructural analyses of the cotyledon cells were conducted. The results revealed that SG is inserted into LV through autophagic function of LV and subsequently degraded by vacuolar α-amylase. The autophagy for SG was structurally similar to micropexophagy detected in yeast cells. In addition to the autophagic process for SG, autophagosome-mediated autophagy for cytoplasm and mitochondria was detected in the cotyledon cells. When the embryo axes were removed from seeds and the detached cotyledons were incubated, the autophagosome-mediated autophagy was observed, but the autophagic process for the degradation of SG was not detected, suggesting that these two autophagic processes were mediated by different cellular mechanisms. The two distinct autophagic processes were thought to be involved in the breakdown of SG and cell components in the cells of germinated cotyledon.

C-terminal KDEL sequence of a KDEL-tailed cysteine proteinase (sulfhydryl-endopeptidase) is involved in formation of KDEL vesicle and in efficient vacuolar transport of sulfhydryl-endopeptidase

Sulfhydryl-endopeptidase (SH-EP) is a papain-type vacuolar proteinase expressed in cotyledons of germinated Vigna mungo seeds, and the enzyme possesses a C-terminal propeptide containing KDEL tail, an endoplasmic reticulum retention signal for soluble proteins. SH-EP is transported to vacuoles via a KDEL vesicle (KV) through a Golgi complex-independent route. To see the function of the KDEL sequence of SH-EP, wild-type SH-EP and its KDEL deletion mutant (SH-EPΔKDEL) were heterologously expressed in Arabidopsis and in cultured tobacco Bright Yellow 2 cells, and their intracellular transport pathways and localizations were analyzed. A combination of the results from analyses for transformed Arabidopsis and tobacco (Nicotiana tabacum) cells indicated that wild-type SH-EP is packed into KV-like vesicles through the KDEL sequence and is transported to vacuoles in the cells of transformants. In contrast, KV was not formed/induced in the cells expressing SH-EPΔKDEL, and the mutant protein was mainly secreted. Therefore, the C-terminal KDEL sequence of the KDEL-tailed cysteine proteinase is thought to be involved in the formation of KV, and in the efficient vacuolar transport of the proteins through KV.

Transformation and regeneration of French bean plants by the particle bombardment process

Abstract Stable transformation of French bean (Phaseolus vulgaris L. cv. Goldstar) plants has been achieved using gold particles coated with plasmid pSOG-1016 carrying a chimeric gene encoding β-glucuronidase (GUS) under the control of concanavalin A (Con A) promoter. The bombardment conditions were optimized by the observation of transient expression of the GUS reporter gene bombarded to the shoot apexes of embryonic axes. After bombardment of embryonic axes, many particles were detectable at 20–80 μm of depth from the top of the shoot apex by anatomic observation. The bombarded embryonic axes were cultured for 3 weeks on MS or N6 medium without plant hormones. Regenerated plantlets were transferred to soil and kept in a growth chamber where they subsequently flowered and produced seeds. PCR and Southern blot analysis showed the integration of the Con A::GUS gene. The expression of the GUS activity was observed in the cotyledons and seed coats of the transgenic plants by histochemical staining.

Stored mRNA in cotyledons of Vigna unguiculata seeds: nucleotide sequence of cloned cDNA for a stored mRNA and induction of its synthesis by precocious germination

By differential hybridization screening, we previously selected a class of cDNA clones from a λgt10 cDNA library that was constructed from the total poly(A)+ RNA of mature cowpea cotyledons (Plant Cell Physiol 31: 39–44, 1990). pSAS10, a clone of this class, hybridized with a cDNA probe complementary to poly(A)+ RNA from cotyledons collected 1 day after the onset of imbibition (DAI), but not with the cDNA probe from cotyledons at development stage II (13 to 15 days after flowering, DAF). pSAS10 mRNA was detectable only in cotyledons at development stage III (17 to 19 DAF) or later, and its level began to decline when seeds germinated. We have suggested that pSAS10 mRNA is likely to belong to the class of ‘stored’ mRNA or the mRNA that is formed at the late stage of seed maturation, is conserved in quiescent seeds and becomes functional at the early stage of germination. We determined the nucleotide sequence of pSAS10 cDNA consisting of 459 bp and an approximately 36 bp poly(A) tract, and deduced the amino acid sequence of its product, a 10-kDa cysteine-rich polypeptide. Synthesis of pSAS10 mRNA was induced just before germination began, not only in mature seeds but also in immature seeds even at stages I (9 to 11 DAF) and II (13 to 15 DAF) if they were placed under conditions suitable for germination.

Occurrence of shikimic and quinic acids in angiosperms

Abstract Leaves of 83 angiosperms have been made surveyed for quinic and shikimic acids. The quinic acid content was higher in woody dicotyledons than in herbaceous dicotyledons or in the monocotyledons, substantiating the view that its presence may be correlated with the lignification in plants.

Expression of an Endopeptidase (EP-C1) in Phaseolus vulgaris Plants

Endopeptidase activity increases continually in pods of maturing fruits of French bean (Phaseolus vulgaris L. cv Goldstar) plants and is thought to participate in the protein mobilization in pods during the development of seeds (M. Endo, T. Minamikawa, D. Yamauchi, W. Mitsuhashi [1987] J Exp Bot 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 [1989] Plant Physiol 89: 274–)279). The level 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.

Nucleotide sequence of cDNA for a putative cysteine protease from rice seeds

A Cys endopeptidase (SH-EP) is involved in the degradation of storage globulin in cotyledons of germinating seeds of Vigna mungo (Mitsuhashi and Minamikawa, 1989). The structure and expression of the gene for SH-EP have been reported (Akasofu et al., 1990; Yamauchi et al., 1992). Here we isolated a full-length cDNA for a putative Cys protease from a rice seed cDNA expression library using antiserum against SH-EP, and determined its nucleotide sequence (Table I). A Agtll cDNA library was constructed from total poly(A)+ RNA that was prepared from germinating rice seeds grown in the dark for 9 d. The library was screened with a rabbit polyclonal antiserum raised against SH-EP. The initial screening resulted in the isolation of a partial cDNA clone (designated pRP8). Although this pRP8 clone lacked the initiation Met codon, the deduced amino acid sequence showed high homology to the other known Cys proteases. To obtain a full-length clone, the same library was rescreened with the pRP8 cDNA probe, and a cDNA clone (designated pRP80) was isolated. This cDNA is 1440 bp long and contains 1134 bp of an open reading frame, and 93 and 213 bp of 5’ and 3’ untranslated regions, respectively. There is a polyadenylation signal, AATAAA, in the 3‘ untranslated region. Cys proteases are synthesized as prepropeptides and cotranslationally processed to mature enzymes. The pRP80 clone shows a similar feature. The first 24 amino acids deduced from this clone have the feature of typical eukaryotic signal sequence (Von Heijne, 1983). This putative signal peptide is followed by 117 amino acids of prosequence and 237 amino acids of the mature enzyme. The predicted mo1 wt of the mature protein encoded by pRP80 is 25,477. Comparisons of the deduced amino acid sequence of pRP80 revealed high homology to the other reported Cys proteases. The putative mature protein of pRP80 shows similarity to the mature proteins of SH-EP (67%) (Akasofu et al., 1990), EP-B from barley (64%) (Koehler and Ho, 1990), oryzain a from rice (60%) (Watanabe et al., 1991), and aleurain from barley (42%) (Rogers et al., 1985).

Hydrolytic enzyme activities and degradation of storage components in cotyledons of germinatingPhaseolus mungo seeds

Phaseolus mungo seeds were allowed to germinate in the dark, and time-course changes in contents of protein fractions, starch, soluble α-amino nitrogen and reducing sugars and in hydrolytic enzyme activities in cotyledons were investigated. In cotyledons of germinated seeds, marked increases in proteolytic (caseolytic, globulytic and gelatin-hydrolyzing) activities and amylolytic activity occurred with concurrent mobilization of storage proteins and starch. Removal of axis organs from seeds at very early stages of germination caused the deteriorated breakdown of storage components and decreased development of proteolytic enzymes in the cotyledons, but this treatment did not significantly affect the appearance of amylolytic activity. The experimental results are discussed in comparison with the hydrolytic enzyme activities of germinating seeds of other leguminous species.

A vacuolar cysteine endopeptidase (SH-EP) that digests seed storage globulin: Characterization, regulation of gene expression, and posttranslational processing

Summary A cysteine endopeptidase named SH-EP was isolated as one of the major hydrolytic enzymes involved in the mobilization of storage globulin in the cotyledon of germinated seeds of Vigna mungo . The enzyme, by itself or in combination with a serine endopeptidase, digests the globulin subunits to smaller peptides in vitro . Protein immunoblot analysis with antiserum raised against SH-EP showed that this enzyme is synthesized in the cotyledon after the onset of imbibition and increases until day 4 and decreases thereafter. Application of plant hormones and growth regulators, as well as amino acids as proteolytic end-products, appears to affect the development of SH-EP to a limited extent in the cotyledon of the germinated seeds. SH-EP of 33 kDa is synthesized on membrane-bound polysomes as a large, inactive 45-kDa precursor, which is cotranslationally processed to a 43-kDa intermediate through cleavage of a signal peptide. The amino-terminal propeptide region of the intermediate is cleaved further to form the 33-kDa mature enzyme by a multistep processing. The 43-kDa intermediate is also subjected to the cleavage of the carboxy-terminal decapeptide containing a Lys-Asp-Glu-Leu tail, which is known as a retention signal for the endoplasmic reticulum lumen. An asparaginyl endopeptidase, designated VmPE-1, was isolated as one of the processing enzymes responsible for the cleavage of the amino-terminal propeptide region of the precursor to SH-EP.