The N-terminal propeptide of the precursor to sporamin acts as a vacuole-targeting signal even at the C terminus of the mature part in tobacco cells.

An asparagine-proline-isoleucine-arginine-leucine (NPIRL) and its related sequences in the N-terminal propeptides (NTPP) of several plant vacuolar proteins, including that of sporamin from sweet potato (SPO) function as vacuole-targeting determinants in a manner that is distinct from the vacuole-targeting determinant in the CTPPs of other plant vacuolar proteins. When the mutant precursor to sporamin, SPO-NTPP (in which NTPP was moved to the C terminus of the mature part), was expressed in tobacco (Nicotiana tabacum) cells, the pro-form was efficiently targeted to the vacuole and the NTPP was cleaved off. Unlike the results obtained with the wild-type precursor, substitution of the NPIRL sequence in the C-terminally located NTPP to asparagine-proline-glycine-arginine-leucine in the SPO-isoleucine-28-to-glycine mutant resulted in missorting of less than 20% of the pro-form to the medium. Unlike the vacuolar transport of SPO-NTPP, the vacuolar transport of SPO-isoleucine-28-to-glycine was strongly inhibited by 33 microM wortmannin, which is similar to the C-terminal propeptide-mediated vacuolar transport. These results suggest that the vacuole-targeting function of the NPIRL sequence is not strictly dependent on its location at the N terminus of a protein and that the C-terminally located mutant NTPP acquired some physicochemical properties of the C-terminal vacuole-targeting sequence.     

Colocalization of barley lectin and sporamin in vacuoles of transgenic tobacco plants.

Various targeting motifs have been identified for plant proteins delivered to the vacuole. For barley (Hordeum vulgare) lectin, a typical Gramineae lectin and defense-related protein, the vacuolar information is contained in a carboxyl-terminal propeptide. In contrast, the vacuolar targeting information of sporamin, a storage protein from the tuberous roots of the sweet potato (Ipomoea batatas), is encoded in an amino-terminal propeptide. Both proteins were expressed simultaneously in transgenic tobacco plants to enable analysis of their posttranslational processing and subcellular localization by pulse-chase labeling and electron-microscopic immunocytochemical methods. The pulse-chase experiments demonstrated that processing and delivery to the vacuole are not impaired by the simultaneous expression of barley lectin and sporamin. Both proteins were targeted quantitatively to the vacuole, indicating that the carboxyl-terminal and amino-terminal propeptides are equally recognized by the vacuolar protein-sorting machinery. Double-labeling experiments showed that barley lectin and sporamin accumulate in the same vacuole of transgenic tobacco (Nicotiana tabacum) leaf and root cells.     

Transport of a Sweet Potato Storage Protein, Sporamin, to the Vacuole in Yeast Cells

The propeptide of a precursor to sporamin, a storage proteinof sweet potato, is required for targeting of sporamin to thevacuole in transformed tobacco cells (Matsuoka and Nakamura1991). A fusion gene consisting of an inducible GAL 10 promoterand sporamin cDNA was introduced into Saccharomyces cerevisiaeby use either of a multiple-copy plasmid (YEpSAD16) or of asingle-copy plasmid (YCpSAD16) to control the level of expressionof the precursor. Although we could not detect any sporamin-relatedpolypeptides in cells that harbored YCpSAD16, extracts fromcells that harbored YEpSAD16 contained multiple forms of sporaminrelatedpolypeptides: preprosporamin, prosporamin and several polypeptidesthat were smaller than prosporamin. However, YCpSAD16 directedthe accumulation of prosporamin in pep4 mutant yeast cells thatlack vacuolar proteases, andpep4 mutant cells that harboredYEpSAD16 did not contain any sporamin-related polypeptides smallerthan prosporamin. The vacuole fractions isolated from the wild-typeand pep4 mutant cells contained sporamin-related polypeptidessmaller than prosporamin and prosporamin, respectively. Theseand other results suggest that, at a low level of expressionof the precursor, prosporamin is transported to the vacuoleand degraded by vacuolar proteases. A mutant precursor to sporamin,in which the propeptide and the N-terminal region of maturesporamin were replaced by an unrelated sequence of four aminoacid residues, directed the secretion of sporamin to the culturemedium in transformed tobacco cells. However, this mutationdid not affect the transport of sporamin to the vacuole in yeastcells and none of the sporamin-related polypeptides were secretedto the extracellular space. (Received July 16, 1991; Accepted March 25, 1992)     

The N-terminal propeptide and the C terminus of the precursor to 20-kilo-dalton potato tuber protein can function as different types of vacuolar sorting signals

Two types of vacuolar sorting signals (VSSs), an asparagine-proline-isoleucine-arginine-leucine (NPIRL)-related VSS in the N-terminal propeptides (NTPPs) and a C-terminal VSS in the C-terminal propeptides (CTPPs), function differently in plant cells. A precursor to a 20-kDa protein of potato tuber (PT20) contains two NPIRL-related sequences, NPINL in a short NTPP and NPLDV close to the C terminus of the precursor. We made mutant forms of sweet potato sporamin (SPO), nPT20-SPO, in which the N-terminal pre-pro part was exchanged with that of the precursor to PT20, and SPO-PT20c, in which the C-terminal 13 amino acids of the precursor to PT20 was attached to the C terminus of delta pro-SPO which lacked NTPP. Both nPT20-SPO and SPO-PT20c were efficiently transported to the vacuoles in tobacco cells. Unlike nPT20-SPO, the vacuolar transport of SPO-PT20c was inhibited by wortmannin and by the C-terminal addition of Gly or Gly-Gly suggesting its similarity to the vacuolar transport of sporamin mediated by CTPP of barley lectin. Further analysis of the C-terminal sequence of PT20 indicated that the most C-terminal SFKQVQ sequence functions as the C-terminal VSS. These results suggest that the precursor to PT20 contains both NPIRL-like VSS in its NTPP and C-terminal VSS at the C terminus.     

甘薯贮藏蛋白(sporam in)A基因编码区克隆及序列同源性分析

采用 PCR技术 ,从我国广泛栽培甘薯品种南薯 88基因组中扩增和克隆到甘薯贮藏蛋白 A基因编码区段 ,并测定了其全部核苷酸序列 .该编码区长 65 7bp,编码一个长 2 1 9个氨基酸残基的蛋白质 ,其中信号肽长 37个氨基酸残基 ,成熟蛋白质长 1 82个氨基酸残基 ,其分子量为 2 0 k D.将该片段的核苷酸序列与已登录在 Gen Bank中的另外 6个甘薯贮藏蛋白 A基因编码区序列进行比较 ,发现其同源性高达 90 % ,说明甘薯贮藏蛋白 A基因编码区序列具有高度保守性 .虽然 7个基因编码区的核苷酸总变异为 1 0 % ,但在每两个基因之间的比较则表明其核苷酸的变异范围小于 7% .     

O-glycosylation of a precursor to a sweet potato vacuolar protein, sporamin, expressed in tobacco cells

Sporamin, a vacuolar protein of the sweet potato, is synthesized as a precursor that contains signal peptide and an N-terminal propeptide that functions as a vacuolar targeting determinant. Sporamin, when expressed in tobacco cells, migrated as smeared bands on an SDS-polyacrylamide gel. The smearing was due to O-glycosylation of the precursor to sporamin. The smeared bands were stained by a glycan-specific stain but no N-glycosylation site was found in the amino acid sequence of the precursor to sporamin. The glycan attached to sporamin contained galactose and arabinose as major sugar components. Mutations that altered the Pro³⁶ or Ser³⁹ residue of the precursor to sporamin prevented glycosylation of the protein, and analysis by semiquantitative Edman degradation suggested that a glycan moiety was attached to Pro³⁶ and, possibly, to Ser³⁹. Pulse-labeling and cell-fractionation experiments revealed that the O-glycosylation of the precursor to sporamin occurred in the Golgi apparatus. Thus, this modification serves as a good marker of the transport from the endoplasmic reticulum (ER) to the Golgi apparatus of the precursor to sporamin. Treatment of transformed tobacco cells with brefeldin A (BFA) caused the intracellular accumulation of prosporamin that did not migrate as smeared bands. Thus, it appeared that BFA inhibited the transport of the precursor to sporamin to the Golgi apparatus. This result provides the first biochemical evidence that BFA inhibits transport from the ER to the Golgi apparatus in plant cells.     

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

Post-translational maturation of natural and drug-induced missorted phytohemagglutinin

The bean lectin phytohemagglutinin (PHA) was expressed in transgenic suspension-cultured BY-2 tobacco cells simultaneously with another recombinant vacuolar protein, the sweet potato sporamin. In contrast to previous observations in different transgenic plant systems when expressed in BY-2 tobacco cells, phytohemagglutinin is mostly but not exclusively targeted to the vacuole. Indeed, a small amount of recombinant phytohemagglutinin is secreted into the culture medium of tobacco cells. Furthermore part of this extracellular phytohemagglutinin has no lectin activity and presents an abnormal glycosylation consistent with higher accessibility of glycans N-linked to these extracellular phytohemagglutinin forms. Phytohemagglutinin secretion occurs regardless of recombinant protein expression level. Consequently, missorting in this case is due to an abnormal phytohemagglutinin conformation or oligomerization rather than to receptor saturation. The treatment of BY-2 cells with drugs, such as monensin and wortmannin, increases even more the transport of phytohemagglutinin to the cell surface through a general inhibition of the sorting mechanisms of vacuolar proteins. The sensitivity to wortmannin is similar for the sorting of phytohemagglutinin and endogenous tobacco chitinase and β-1,3-glucanase, suggesting that phytohemagglutinin and COOH-terminal propeptide mediated vacuolar sorting share similar mechanisms. A characterization of glycans N-linked to extracellular phytohemagglutinin secreted by monensin- or wortmannin-treated transgenic tobacco cells illustrates that in contrast with monensin, wortmannin completely inhibits the sorting of vacuolar proteins without having any effect on the efficiency of Golgi processing enzymes.     

Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.     

A Major Jasmonate-Inducible Protein of Sweet Potato, Ipomoelin, is an ABA-Independent Wound-Inducible Protein

Treatment of sweet potato plants cultured in vitro with a vaporof methyl jasmonate (MeJA) induced an accumulation in leavesof a large amount of protein with an apparent molecular massof 18 kDa. This protein, designated ipomoelin, was purified,and the amino acid sequences of proteolytic fragments were determined.Screening a cDNA library of MeJA-treated leaves by oligonucleotideprobes designed from the peptide sequences identified a clonethat could code for a polypeptide with 154 amino acids. Thededuced amino acid sequence of ipomoelin showed an overall aminoacid identity of 25% with the salt-inducible SalT protein ofrice. In addition, the C-terminal 70 amino acid sequence ofipomoelin showed about 50% identity with the C-terminal aminoacid sequences of seed lectins from Moraceae. The gene for ipomoelinwas present in a few copies in the genome of sweet potato. ThemRNA for ipomoelin was detected in leaves and petioles, butnot in stems and tuberous roots, of sweet potato plants grownin the field. Mechanical wounding of leaves induced ipomoelinmRNA both locally and systemically, while treatment of leaveswith ABA, salt, or a high level of sucrose did not induce ipomoelinmRNA. By contrast, ABA-inducible mRNA for sporamin was not inducedby MeJA. These results suggest that ipomoelin is involved indefensive reactions of leaves in response to wounding and thatJA-mediated wound-induction of ipomoelin occurs independentlyof ABA. (Received January 6, 1997; Accepted March 13, 1997)     

Molecular cloning and nucleotide sequence of cDNA for sporamin,the major soluble protein of sweet potato tuberous roots

Summary Sporamin accounts for more than 80% of the total soluble proteins of tuberous roots of sweet potato, but very little, if any, in other tissues of the same plant. In vitro translation of RNA fractions from the tuberous roots in wheat germ extract and subsequent immunoprecipitation with the antibody to sporamin indicated that this protein is synthesized by membrane-bound polysomes as a precursor 4 000 daltons larger than the mature protein. A cDNA expression library was constructed from the total poly(A)⁺ RNA from the tuberous roots by a vector-primer method, and an essentially full-length cDNA clone for the sporamin mRNA was selected by direct immunological screening of the colonies. Northern blot analysis showed that sporamin mRNA is approximately 950 nucleotides in length and is specifically present in tuberous roots and very little, if any, in leaves, petioles and non-tuberous roots. Nucleotide sequence of the cDNA predicts a 37 amino acid extension in the precursor at the amino-terminus of the mature protein.     

Involvement of Ca2+ signalling in the sugar-inducible expression of genes coding for sporamin and β-amylase of sweet potato

Genes coding for sporamin and β-amylase of sweet potato are inducible not only by high levels of metabolizable sugars, such as sucrose, but also by a low concentration of polygalacturonic acid (PGA). Calmodulin inhibitors and EGTA inhibited both the PGA-inducible and the sucrose-inducible accumulation of mRNAs for sporamin and β-amylase in sweet potato. Calmodulin inhibitors, EGTA and La³⁺, also inhibited the sucrose-inducible expression, in leaves of transgenic tobacco, of a fusion gene, β-Amy:GUS, which consists of the promoter of the β-amylase gene and the coding sequence for β-glucuronidase. The sucrose-inducible expression of the β-Amy:GUS fusion gene was also inhibited by two inhibitors of Ca²⁺ channels, diltiazem and nicardipine. These results suggest that the sugar-inducible expression of genes for sporamin and β-amylase involves, at least in part, Ca²⁺-mediated signalling, and that the cytosolic free Ca²⁺ may mediate cross-talk between signals related to carbohydrate metabolism and other stimuli. Treatment of coelenterazine-loaded leaf discs of tobacco expressing a Ca²⁺-binding photoprotein, aequorin, with 0.2 M sucrose for 24 h significantly reduced the level of luminescence that could be induced by cold shock, as compared to cold shock-induced luminescence in coelenterazine-loaded leaf discs treated with water. Repression of cold shock-induced luminescence was due to the conversion of holoaequorin to apoaequorin during the treatment with sucrose. Treatment of coelenterazine-loaded leaf discs with a 0.2 M solution of glucose or fructose, but not of mannitol or sorbitol, also reduced the cold shock-induced luminescence. It is suggested that non-synchronous increases in cytosolic level of free Ca²⁺ occur in leaf discs during treatment with high levels of metabolizable sugars.     

Characterization of major proteins in sweet potato tuberous roots

The tuberous roots, but not other organs, of sweet potato contained large quantities of two proteins which accounted for more than 80% of the total proteins. The two proteins, tentatively named sporamins A and B, were monomeric forms with similar M,s (25 000). They were separated from each other by electrophoresis on polyacrylamide gels in a non-denaturing buffer or a buffer containing sodium dodecyl sulphate without being reduced by dithiothreitol. They were very similar to each other with respect to amino acid composition, peptide map and immunological properties. These proteins decreased in preference to other proteins during sprouting. The amino acid sequencing of the amino terminal part of sporamin A suggested that it consists of at least two molecular species with different combinations of a few amino acids.