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.     

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.     

Starch-accumulating Sweet Potato Callus Tissue Devoid of β-Amylase but with Two Starch Phosphorylase Isozymes

By controlling the concentrations of kinetin, auxin, and sucrose in the Murashige–Skoog medium, starch contents in callus culture induced from sweet potato tissues could be manipulated. Activity staining and Western analysis on PAGE plates and activity assays made on starch phosphorylase in the presence and absence of mercuric ions showed that β-amylase is absent in callus cultures regardless of whether their starch content is high or low. This would imply that β-amylase induction in sweet potato calli is not linked to the metabolic control through which the expression of storage function is associated, as proposed by Nakamura et al. [Plant Physiol., 96, 902 (1991)] for sweet potato leaf-petiole cuttings. Analyses of starch phosphorylase in crude extracts suggested the presence of a new starch phosphorylase in tuberous root and callus tissue. This phosphorylase is immunologically different from the tuberous root and leaf enzymes that we studied previously.     

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.     

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)     

Starch Phosphorylase Inhibitor Is beta-Amylase

The proteinaceous noncompetitive inhibitor of starch phosphorylase isolated from the root of sweet potato (Ipomoea batatas [L.] Lam.) (TC Chang, JC Su 1986 Plant Physiol 80: 534-538) has been identified as a β-amylase. The starch phosphorylase inhibitor and β-amylase activities copurified to give a protein indistinguishable from commercial β-amylase by electrophoretic and immunological methods, and the two activities showed parallel responses in pH, temperature, and inhibitor sensitivity tests. The amylolytic pattern of the inhibitor corresponded to that of β-amylase and its inhibitory effect toward starch phosphorylase was due to neither deprivation of starch, the primer for the phosphorylase assay, nor the inhibitory effect of amylolytic products.     

Sweet potato: A novel substrate for pullulan production by Aureobasidium pullulans

A strain Aureobasidium pullulans AP329, was used for the production of pullulan by employing hydrolysed sweet potato as cultivation media. Hydrolysis with α-amylase alone resulted in the lowest yields of pullulan. In contrast continuous hydrolysis with pullulanase and the β-amylase in sweet potato itself gave higher yields, but prolonged hydrolysis with amyloglucosidase decreased the yield. The maximum pullulan yield (29.43 g/l) was achieved at the dextrose equivalent value of 45 and pH of 5.5 for 96 h. As a substitute of sucrose, hydrolysed sweet potato was found to be hopeful and the yield of pullulan was higher than that of glucose and sucrose. The molecular weight of pullulan obtained from hydrolysed sweet potato media was much higher than that of sucrose and glucose media. Results of this work indicated that sweet potato was a promising substrate for the economical production of pullulan.     

Beta-Amylases from Alfalfa (Medicago sativa L.) Roots

Amylase was found in high activity (193 international units per milligram protein) in the tap root of alfalfa (Medicago sativa L. cv. Sonora). The activity was separated by gel filtration chromatography into two fractions with molecular weights of 65,700 (heavy amylase) and 41,700 (light amylase). Activity staining of electrophoretic gels indicated the presence of one isozyme in the heavy amylase fraction and two in the light amylase fraction. Three amylase isozymes with electrophoretic mobilities identical to those in the heavy and the light amylase fractions were the only amylases identified in crude root preparations. Both heavy and light amylases hydrolyzed amylopectin, soluble starch, and amylose but did not hydrolyze pullulan or β-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato β-amylase, while that of bacterial α-amylase was considerably higher. The identification of maltose and β-limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are all β-amylases.     

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.     

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.     

The ram1 Mutant of Arabidopsis Exhibits Severely Decreased β-Amylase Activity

Despite extensive biochemical analyses, the biological function(s) of plant β-amylases remains unclear. The fact that β-amylases degrade starch in vitro suggests that they may play a role in starch metabolism in vivo. β-Amylases have also been suggested to prevent the accumulation of highly polymerized polysaccharides that might otherwise impede flux through phloem sieve pores. The identification and characterization of a mutant of Arabidopsis var. Columbia with greatly reduced levels of β-amylase activity is reported here. The reduced β-amylase 1 (ram1) mutation lies in the gene encoding the major form of β-amylase in Arabidopsis. Although the Arabidopsis genome contains nine known or putative β-amylase genes, the fact that the ram1 mutation results in almost complete loss of β-amylase activity in rosette leaves and inflorescences (stems) indicates that the gene affected by the ram1 mutation is responsible for most of the β-amylase activity present in these tissues. The leaves of ram1 plants accumulate wild-type levels of starch, soluble sugars, anthocyanin, and chlorophyll. Plants carrying the ram1 mutation also exhibit wild-type rates of phloem exudation and of overall growth. These results suggest that little to no β-amylase activity is required to maintain normal starch levels, rates of phloem exudation, and overall plant growth.     

Effects of Sucrose on Petiolar Carbohydrate Accumulation and Photosynthesis in Excised Sinapis Cotyledons

Petioles of Sinapis cotyledons cultured in 6x10^–2 M sucrosein the light increased 28-fold in total carbohydrate contentover a 7-d period compared with an 8-fold increase in petiolesof cotyledons cultured on water. The starch and reducing sugarfractions were the major components of this accumulation. Labelledsucrose applied to the petiole base moved quickly up the petioleand into the main veins of the lamina. Some basipetal redistributionoccurred subsequently and after 24 h radioactivity accumulatedstrongly at the petiole base. Culture in sucrose reduced basalaccumulation and increased acropetal movement of the label. Fixation of ¹⁴CO₂ by petioles remained constant when cotyledonswere cultured in water, whereas in sucrose, fixation fell by50 per cent during the first 2 d. The pattern of inhibitionof fixation matched the pattern of sucrose distribution in thepetiole. Petiolar chlorophyll content remained constant in controlsbut fell rapidly after 4 d culture in sucrose. The results are discussed in relation to the role of petiolarcarbohydrate accumulation in the regulation of CO₂ fixation,primordium development, and senescence in this system.     

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)     

Production and Characteristics of Raw-Potato-Starch-Digesting α-Amylase from Bacillus subtilis 65

A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting α-amylase. The electrophoretically homogeneous preparation of enzyme (molecular weight, 68,000) digested and solubilized raw corn starch to glucose and maltose with small amounts of maltooligosaccharides ranging from maltotriose to maltoheptaose. This enzyme was different from other amylases and could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH. The mixed preparation with Endomycopsis glucoamylase synergistically digested raw potato starch to glucose at 30°C. The raw-potato-starch-digesting α-amylase showed strong digestibility to small substrates, which hydrolyzed maltotriose to maltose and glucose, and hydrolyzed p-nitrophenyl maltoside to p-nitrophenol and maltose, which is different from the capability of bacterial liquefying α-amylase.     

Occurrence of alpha-amylase in the axis of germinating peas

α-Amylase was found in the axis portion of ungerminated pea seeds (Pisum sativum var. Alaska). The occurrence of this enzyme was demonstrated with crude homogenates (also containing β-amylase) using three different methods: the hydrolysis of β-limit dextrin, the change in absorption spectra for the iodine-starch complex, and the increase in reducing materials relative to the decrease in starch. The first method was used to quantitate the changes in α-amylase activity during germination. The increase in total amylase activity (primarily β-amylase) paralleled germination; the accumulation of α-amylase activity was not initiated for an additional day. The increased α-amylase activity was related to epicotyl growth. Approximately half of this activity was found in the etiolated stem, the distribution being higher in growing than in nongrowing portions.