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.     

Induction of Expression of Genes Coding for Sporamin and beta-Amylase by Polygalacturonic Acid in Leaf-Petiole Cuttings of Sweet Potato

Sporamin and β-amylase are two major proteins of tuberous storage root of sweet potato (Ipomoea batatas) and their accumulation can be induced concomitantly with the accumulation of starch in leaves and petioles by sucrose (K Nakamura, M Ohto, N Yoshida, K Nakamura [1991] Plant Physiol 96: 902-909). Although mechanical wounding of leaves of sweet potato only occasionally induced the expression of sporamin and β-amylase genes, their expression could be reproducibly induced in leaf-petiole cuttings when these explants were dipped in a solution of polygalacturonic acid or chitosan at their cut edges. Polygalacturonic acid seemed to induce expression of the same genes coding for sporamin and β-amylase that are induced by sucrose. Because polygalacturonic acid and chitosan are known to mediate the induction of wound-inducible defense reactions, these results raise an interesting possibility that β-amylase, in addition to sporamin, may have some role in the defense reaction. Expression of sporamin and β-amylase genes could also be induced by abscisic acid, and this induction by abscisic acid, as well as induction by polygalacturonic acid or sucrose, was repressed by gibberellic acid. By contrast, methyl jasmonate did not cause the significant induction of either sporamin or β-amylase mRNAs. Induction of expression of sporamin and β-amylase genes by polygalacturonic acid or sucrose was inhibited by cycloheximide, suggesting that de novo synthesis of proteins is required for both of the induction processes.     

Sugar-Induced Increase of Calcium-Dependent Protein Kinases Associated with the Plasma Membrane in Leaf Tissues of Tobacco

The sugar-inducible expression of genes for sporamin and [beta]-amylase in leaf explants of sweet potato (Ipomoea batatas) and that of a [beta]-glucuronidase-fusion gene, with the promoter of the gene for [beta]-amylase in leaves of tobacco (Nicotiana tabacum), requires Ca2+ signaling (M. Ohto, K. Hayashi, M. Isobe, K. Nakamura [1995] Plant J 7: 297-307), and it was inhibited by staurosporin and K252a, inhibitors of protein kinases. Autophosphorylation activities of several potential protein kinases in leaves of tobacco were significantly higher in younger leaves than in mature leaves. However, the autophosphorylation activities of these proteins in mature leaves, especially those of the major autophosphorylatable proteins with apparent molecular masses of 56 and 54 kD, increased upon treatment of leaf discs with a 0.3 M solution of sucrose, glucose, or fructose, did not increase with sorbitol or mannitol treatments, and the increase by sucrose was inhibited by cycloheximide. Autophosphorylation of the 56- and 54-kD protein in vitro was dependent on Ca2+ and inhibited by staurosporine, K-252a, and by W-7. These results suggest that they belong to the family of calcium-dependent protein kinases. They were concentrated in the plasma membrane fraction and were released from membrane vesicles by high salt or with sodium carbonate. The possible functions of these sugar-inducible calcium-dependent protein kinases associated with the plasma membrane are discussed.     

Sucrose-Induced Accumulation of beta-Amylase Occurs Concomitant with the Accumulation of Starch and Sporamin in Leaf-Petiole Cuttings of Sweet Potato

β-Amylase of sweet potato (Ipomoea batatas L.), which constitutes about 5% of the total soluble protein of the tuberous root, is absent or is present in only small amounts in organs other than the tuberous roots of the normal, field-grown plants. However, when leaf-petiole cuttings from such plants were supplied with a solution that contained sucrose, the accumulation of β-amylase was induced in both leaf and petiole portions of the explants. The sucrose-induced accumulation of β-amylase in leaf-petiole cuttings occurred concomitant with the accumulation of starch and of sporamin, the most abundant storage protein of the tuberous root. The accumulation of β-amylase, of sporamin and of starch in the petioles showed similar dependence on the concentration of sucrose, and a 6% solution of sucrose gave the highest levels of induction when assayed after 7 days of treatment. The induction of mRNAs for β-amylase and sporamin in the petiole could be detected after 6 hours of treatment with sucrose, and the accumulation of β-amylase and sporamin polypeptides, as well as that of starch, continued for a further 3 weeks. In addition to sucrose, glucose or fructose, but not mannitol or sorbitol, also induced the accumulation of β-amylase and sporamin, suggesting that metabolic effects of sucrose are important in the mechanism of this induction. Treatment of leaf-petiole cuttings with water under continuous light, but not in darkness, also caused the accumulation of small amounts of these components in the petioles, probably as a result of the endogenous supply of sucrose by photosynthesis. These results suggest that the expression of the gene for β-amylase is under metabolic control which is coupled with the expression of sink function of cells in the sweet potato.     

Spatial Patterns of Sucrose-Inducible and Polygalacturonic Acid-Inducible Expression of Genes That Encode Sporamin and r{beta}-Amylase in Sweet Potato

Two major proteins of tuberous roots of sweet potato, sporaminand rß-amylase, were detected in storage parenchymacells, which contain a large amount of starch. In both the leavesand petioles of sweet potato, the sucrose-induced accumulationof mRNAs for sporamin and rß-amylase, and of starchoccurred in a wide variety of cells, first in cells within andaround the vascular tissue and then in various cells distalto them, with the exception of epidermal cells. In the mesophyllcells of leaves treated with sucrose, the accumulation of largenumbers of well-developed starch granules occurred in the preexistingchloroplasts. These results, together with the previous observationthat the sucrose-induced accumulation of sporamin, of rß-amylaseand of starch occurs with similar dependency on the concentrationof sucrose, suggest that an excess supply of sugars to varioustypes of cell triggers a cellular transition that induces thesimultaneous accumulation of these reserve materials that arenormally present in tuberous roots. Accumulation of mRNAs forsporamin and rß-amylase, but not the accumulationof starch, in leaves and petioles can be also induced when leaf-petiolecuttings are supplied with low concentrations of polygalacturonicacid (PGA) at their cut edges. The spatial patterns of accumulationof mRNAs for sporamin and rß-amylase in leaves andpetioles after treatment with PGA were found to be similar tothose observed upon treatment with sucrose. These results suggestthat most of the cells in leaves and petioles have the capacityto respond to both a carbohydrate metabolic signal and a PGA-derivedsignal that is transmitted by diffusion from the vascular system. ⁴Present address: Department of Molecular Biology, NationalInsustitute of Agrobiological Resources, Tsukuba City, Ibaraki,305 Japan.     

Sucrose-Induced Expression of Genes Coding for the Tuberous Root Storage Protein, Sporamin, of Sweet Potato in Leaves and Petioles

Sporamin, a major tuberous root protein of sweet potato, wasfound to accumulate in large quantities in excised leaves andpetioles when such explants were supplied with high concentrationsof sucrose. Although a small amount of sporamin could be detectedin leaves and petioles treated with 1% or lower concentrationsof sucrose, the maximum level of induction required sucroseat a concentration of 3% or higher. The appearance of sporaminpolypeptides in leaves and petioles treated with 3% sucrosefollowed a lag period of about one day, while a significantamount of sporamin mRNAs was already detectable in petiolesafter one day of treatment with sucrose. Addition of silvernitrate to the medium did not affect the accumulation of sporamin,suggesting that this induction is not due to the effect of ethyleneinduced by wounding of the tissue. The accumulation of sporamincould also be induced by glucose and by fructose, but not byman-nitol, suggesting that changes in carbohydrate and/or energymetabolism in the cell may be involved in the induction. Callustissues obtained by treatment of leaf segments with 1-naphthaleneaceticacid did not accumulate sporamin even though these cells werecultured on agar medium that contained 3% sucrose. However,when callus tissues were allowed to grow after transfer to amedium that contained 6-benzylaminopurine and sucrose, accumulationof large amounts of sporamin was induced. These results suggestthat, while expression of genes coding for sporamin can be inducedin organs other than the tuberous root by a process that doesnot accompany the differentiation of tissue, the induction ofexpression of sporamin genes by sucrose requires that cellsbe competent in some specific, but as yet unidentified, way. (Received August 27, 1990; Accepted November 5, 1990)     

Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5′ upstream regions of genes coding for sporamin and β-amylase from sweet potato

We isolated a cDNA encoding a DNA-binding protein, SPF1, of sweet potato that binds to the SP8a (ACTGTGTA) and SP8b (TACTATT) sequences present in the 5 upstream regions of three different genes coding for sporamin and -amylase of tuberous roots. SPF1 comprises 549 amino acids and is enriched in both basic and acidic residues. The amino acid sequence of SPF1 shows no significant homology to any known protein sequences, suggesting that it may represent a new class of DNA-binding protein. Binding studies with ³⁵S-labeled SPF1, synthesized in vitro, and synthetic DNA fragments indicated that, although SPF1 binds to both the SP8a and SP8b sequences, it binds much more strongly to SP8a than to SP8b. SPF1 bound to the SP8a sequence as a monomer. The DNA-binding domain of SPF1 was localized within the C-terminal half of this protein, and a 162-amino acid fragment of SPF1 (Met³¹⁰-Arg⁴⁷²) showed DNA-binding activity with no change in target sequence specificity. This fragment contains a region enriched in basic amino acids adjacent to a highly acidic stretch. A sequence which is highly homologous to a 40-amino acid sequence in the basic region of the DNA-binding domain is duplicated in the N-terminal part of SPF1. The gene coding for SPF1 is present in one or a few copies per haploid genome and the SPF1 mRNA was detected in leaves, stems and tuberous roots of the sweet potato, in addition to petioles. The level of SPF1 mRNA in the petioles decreased when leaf-petiole cuttings were treated with sucrose to induce accumulation of sporamin and -amylase mRNAs.     

Developmental and Hormonal Regulation of Rice [alpha]-Amylase(RAmy1A)-gusA Fusion Genes in Transgenic Rice Seeds

Transgenic seeds of rice (Oryza sativa L.) were used to investigate temporal, spatial, and hormonal regulation of a rice [alpha]-amylase gene, RAmy1A. Two overlapping segments of the RAmy1A promoter were fused to the coding region of the bacterial reporter gene, gusA. The resulting promoter-gusA fusions, pE4/GUS (-232 to +31) and pH4/GUS (-748 to +31), were used separately to transform rice protoplasts. [beta]-Glucuronidase (GUS) activity was detected in germinated transgenic seeds, although the two constructs showed no significant difference in timing or location of GUS expression. Both constructs first expressed GUS in the scutellar epithelium and then in the aleurone layer. Aleurone expression of GUS activity was strongly induced when embryoless half-seeds were treated with gibberellic acid. GUS expression in the aleurone layer was also suppressed by abscisic acid. These results indicate that the 5[prime] regulatory region from -232 to +31 is sufficient for temporal, spatial, and hormonal regulation of RAmy1A gene expression.     

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)     

Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants

Granule-bound starch synthase is the key enzyme in amylose synthesis. The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5 upstream sequence of the granule-bound starch synthase gene from potato and the -glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system. The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low. However, leaves from in vitro grown plantlets exhibited an elevated GUS expression. The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants. The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves. The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene. Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities. Stolons and tubers show high expression.     

StCDPK1 is expressed in potato stolon tips and is induced by high sucrose concentration

StCDPK1 encodes a calcium-dependent protein kinase (CDPK) from Solanum tuberosum, which is transiently induced upon tuberization in swelling stolons. In situ hybridization determined that StCDPK1 mRNA is localized in the apical dome of tuberizing stolon tips, close to the region where sucrose was reported to accumulate. The expression of StCDPK1, and other tuber-specific genes was enhanced when in vitro-cultured potato plants were transferred to high sucrose or high sorbitol containing media. Glucose, fructose or a mixture of both showed no effect on CDPK expression. Okadaic acid blocked sucrose-inducible gene expression, suggesting that phosphatases from the PP1/PP2A family could also participate in the regulation of StCDPK1 and other tuberization-related genes.     

Sugar-Dependent Expression of the CHS-A Gene for Chalcone Synthase from Petunia in Transgenic Arabidopsis

Transgenic Arabidopsis thaliana plants were constructed by introduction of a fusion of the gene for β-glucuronidase (GUS) to the CHS-A gene, which is one of the two genes for chalcone synthase that are actively expressed in the floral organs of petunia. The expression of the fusion gene CHS-A::GUS was low in transgenic Arabidopsis plantlets, but it was enhanced when plantlets or detached leaves were transferred to a medium that contained 0.3 molar sucrose, glucose, or fructose. No enhancement was observed when plantlets were transferred to a medium that contained 0.3 molar mannitol. Measurements of cellular levels of sugars revealed a tight linkage between the level of expression of the CHS-A::GUS gene and the level of accumulation of exogenously supplied sugars, in particular sucrose. The parallelism between the organ-specific accumulation of sugar and the organ-specific expression of the CHS-A::GUS gene was also observed in petunia and A. thaliana plants grown under normal conditions in soil. The consensus sequences for sugar responses, such as boxes II and III in members of the family of sporamin genes from the sweet potato, were found in the promoter region of the CHS-A gene that was used for fusion to the GUS gene. It is suggested that the expression of the CHS-A gene is regulated by sugars, as is the expression of other sugar-responsive genes, such as the genes for sporamin. A putative common mechanism for the control of expression of “sugar-related” genes, including the CHS-A gene, is discussed.     

Vacuolar targeting and posttranslational processing of the precursor to the sweet potato tuberous root storage protein in heterologous plant cells

Sporamin, the tuberous root storage protein of the sweet potato, which is localized in vacuoles, is synthesized as a prepro-precursor with an N-terminal sequence of amino acids that includes a signal peptide and an additional pro-segment of 16 amino acids. A full-length cDNA for sporamin was placed downstream of the 35 S promoter of cauliflower mosaic virus and introduced into tobacco and sunflower genomes by Ti plasmid-mediated transformation. A polypeptide of nearly the same size as mature sporamin from the sweet potato was detected in transformed calli of tobacco and sunflower, as well as in the leaves, stems, and roots of regenerated, transgenic tobacco plants. Amino acid sequence analysis of the nearly mature-sized form of sporamin from the transformed tobacco cells revealed that it is actually longer by three amino acids at its N terminus than authentic sporamin purified from the sweet potato. By pulse labeling of suspension-cultured tobacco cells with [35S]methionine, the pro-form of the precursor to sporamin, but not the prepro-precursor, was detected. The 35S-labeled proform was chased to the nearly mature-sized form via an intermediate form which is slightly larger than the nearly mature-sized form. Analysis by Edman degradation of the intermediate form that was labeled in vivo with [3H]histidine suggested that it is longer by two amino acids at its N terminus than the nearly mature-sized form of sporamin. These results suggest that at least two steps of posttranslational processing of the pro-form occurs sequentially in tobacco cells. The posttranslational processing of the pro-form of the precursor to sporamin was inhibited by monensin, suggesting that this step takes place in the acidic compartment, probably in the vacuole. All of the sporamin polypeptides synthesized in transformed tobacco cells were retained inside the cell and sporamin was localized in the vacuole, as judged from results of subcellular fractionation. These results indicate that sporamin is appropriately targeted to the vacuole in tobacco cells.     

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% .