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.     

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.     

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)     

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 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.     

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.     

Detection and Characterization of a Vacuolar Protein (VP24) in Anthocyanin-Producing Cells of Sweet Potato in Suspension Culture

Vacuoles containing large amounts of protein and anthocyaninwere isolated and purified from anthocyanin-producing culturedcells of sweet potato (Ipomoea batatas). A 24 kDa protein (VP24),identified as a major protein in the isolated vacuoles, wasdetected by SDS-polyacrylamide gel electrophoresis. NeitherVP24 nor anthocyanin was detectable in dark-cultured cells,but VP24 appeared three days after the start of irradiationwith light and the level of VP24 increased for up to a week.High concentrations of 2,4-D markedly inhibited the accumulationof both VP24 and anthocyanin. These results indicated that theexpression of VP24, which was induced by exposure to light,was closely accompanied by the accumulation of anthocyanin inthe vacuoles. VP24 was recovered as an insoluble reddish precipitateafter ultracentrifugation of a lysate of anthocyanin-containingvacuoles. Immunoblot analysis indicated that this vacuolar proteinwas distinct from sporamin, a major storage protein of sweetpotato tuberous root. These results suggest that VP24 is notan integral protein of the vacuolar membrane but tends to formaggregates via interactions with anthocyanin during extraction.VP24 may be involved in the formation of intravacuolar pigmentedstructures that develop in vivo in the anthocyanin-containingvacuoles of sweet potato cells in culture. ³Present address: Department of Applied Biology, Faculty ofTextile Science and Technology, Shinshu University, Tokida 3-15-1,Ueda, 386 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.     

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.     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 2. The Incorporation of 13C into Carbohydrates, Organic Acids, Amino Acids and some Storage Compounds

Nodulated soya bean (Glycine max L.) plants at the early floweringstage were allowed to assimilate ¹³CO₂ under steady-state conditions,with a constant ¹³C abundance, for 8 h in the light. The plantswere either harvested immediately or 2 d after the end of the¹³CO₂ feeding, divided into young leaves (including flower buds),mature leaves, stems+petioles, roots and nodules; the ¹³C abundancein soluble carbohydrates, organic acids, amino acids, starchand poly-ß-hydroxybutyric acid was determined witha gas chromatography-mass spectrometry. The rapid turnover of ¹³C in the sucrose pools observed in allorgans of the plants showed that sucrose was the principal materialin the translocation stream of primary products of photosynthesis.At the end of the ¹³CO₂ exposure, sucrose in the mature leavesas the major source organs and in the stems+petioles was labelledwith currently assimilated carbon to about 75 per cent, whereasa much higher labelling of sucrose was found in the roots andin the nodules. This suggests the existence of two or more compartmentedpools of sucrose in mature leaves and also in stems+petioles. The relative labelling patterns of individual organic acidsand amino acids were similar in various plant organs. However,the rapid turnover of succinate and glycine was characteristicof nodules. Treatment with a high concentration of nitrate inthe nutrient media increased the turnover rate of amino acidcarbon in shoot organs and roots, while it markedly decreasedthe labelling of amino acids in nodules. The cyclitols, exceptfor D-pinitol, were significantly labelled with assimilated¹³C in mature leaves, but in nodules, the labelling was verymuch less. In the nodules, which were actively fixing atmospheric nitrogen,a large proportion (80–90 per cent) of currently assimilatedcarbon was found as sucrose and starch at the end of the ¹³CO₂feeding. This was also true of the roots. On the other hand,in young growing leaves, the distribution of currently assimilatedcarbon into sucrose, starch and other soluble compounds wasmuch less. This suggests that a large amount of carbon assimilatedby and translocated to young leaves was used to make up structuralmaterials, mainly protein and cell wall polymers synthesis,during the light period. Glycine max L., soya bean, ¹³CO₂ assimilation, carbon metabolism in nodules     

Source--sink Relationships and Carbon Metabolism in Tomato Leaves 1. 14C Assimilate Compartmentation

Effects of the interaction between assimilate availability andsink demand on the metabolism of ¹⁴C assimilates in tomato leaveshave been examined in plants where the source—sink relationshipof assimilates was simplified to one leaf and one fruit truss. During experimentation the source leaf was exposed to either80 or 20 W m^–2 (PAR), while the truss was either retainedor removed. Under these four source-sink conditions, a timecourse study was made on ¹⁴C assimilate distribution in thesource leaf over a period of 23 h after pulse feeding with ¹⁴CO₂. While truss removal caused a temporary increase of ¹⁴C sucrosein leaves under both irradiances, the principal assimilatesaccumulated were starch and hexoses. Decreased ¹⁴C export followingtruss removal was observed within a day in well-illuminatedleaves but after 3 days in leaves under low light. The accumulationof ¹⁴C sucrose at the end of the light period was affected bytruss removal in high light leaves only 3 days later. These observations suggest that while the compartmentation ofnewly fixed assimilate was affected rapidly by the change ofsource—sink relationship, carbon export, as measured by¹⁴C loss, was affected only gradually. The possible effect of sucrose accumulation on photosynthesisis discussed.     

Mobilization of Starch and Essential Oil Biogenesis during Leaf Ontogeny of Lemongrass (Cymbopogon flexuosus Stapf.)

The levels of starch, soluble sugars, starch mobilizing enzymes(amylases and phosphorylase) and sodium [2-^I4C] acetate incorporationinto essential oil have been examined during leaf ontogeny oflemongrass (Cymbopogon flexuosus Stapf., cv. OD-19). The degradationof starch was predominantly amylolytic and ß-amylasewas the major enzyme involved. Its activity was quite high duringthe period of active leaf growth accompanying active accumulationof essential oil. The activities of a-amylase and phosphorylasewere relatively lower. The change in starch to soluble sugarsratio was inversely related to ß-amylase activity.The time-course (12 h light followed by 12 h dark) monitoringof the [^I4C]-radioactivity in starch and essential oil, afterexposure of the immature (15 days after emergence) leaf to ¹⁴CO₂,revealed a progressive loss of label from starch and a parallelincrease in radioactivity in essential oil. The results havebeen discussed in relation to degradation of transitory starchserving as the source of carbon precursor for essential oil(monoterpene) biogenesis in the tissue. The amount of exogenouslysupplied acetate incorporated into essential oil increased tremendouslywith 5-10 fold decrease in specific activity of the labelledacetate (2,110 GBq mole^–1). The effect was largely manifestedin ‘citral’, the chief (ca. 80%) constituent oflemongrass essential oil. Ontogenetically, the amount of essentialoil synthesized from the exogenously supplied precursor (acetate)was much higher in young (10 days after emergence) than in mature(30 days after emergence) leaf. Thus, the leaf developmentalphase influences the expression of essential oil metabolismand actual synthesis. Only young lemongrass leaves are substantiallyactive to synthesize essential oil. The oil biosynthetic phaseappears to be coordinated/integrated with the development ofelevated levels of certain primary metabolic activities likestarch mobilization. ¹CIMAP Publication No. 706 ²Present address: CSIR Complex, Palampur-176 061, Kangra Distt.Himachal Pradesh, India ^J Present address (until October 10, 1991): Department of Biology,Queen's University, Kingston, Ontario, K7L 3N6, Canada (Received November 30, 1990; Accepted May 31, 1991)