Transport of Stachyose and Sucrose by Vacuoles of Japanese Artichoke (Stachys sieboldii) Tubers

Vacuoles are the stores for large amounts of stachyose [αgal (1,6) αgal (1,6) αglc (1,2) βfru] in tubers of Japanese artichoke (Stachys sieboldii). The uptake of stachyose by these vacuoles was examined and compared with that of sucrose. The uptake mechanisms of both sugars were quite similar. The kinetics showed a single saturable response to increasing external concentrations of ¹⁴C-sugars with similar apparent K_m values of about 50 and 30 millimolar for stachyose and sucrose, respectively. The uptake rates, however, were always higher for stachyose than for sucrose. Stachyose and sucrose uptake was inhibited by fructose and raffinose, and, reciprocally, by sucrose and stachyose, but not by glucose or galactose. The main structural feature common to all sugars recognized by the uptake systems seems to be a terminal fructosyl residue. The uptake of both sugars was stimulated by Mg-ATP and inorganic pyrophosphate, suggesting a proton-sugar antiport system. The possibility that stachyose and sucrose might be transported by the same carrier is discussed.     

Further Evidence for Stachyose and Sucrose/H+ Antiporters on the Tonoplast of Japanese Artichoke (Stachys sieboldii) Tubers

Vacuoles of Japanese artichoke (Stachys sieboldii) tubers accumulate up to 180 mM stachyose ([alpha]-galactose-[1->6]-[alpha]-galactose-[1->6]-[alpha]-glucose-[1 <->2]-[beta]-fructose) against a concentration gradient, probably by means of an active stachyose/H+ antiporter situated on the tonoplast. The goal of this study was to use isolated tonoplast vesicles to provide further evidence for the existence of such a transport mechanism. Therefore, vesicles were prepared from purified vacuoles of dormant tubers. ATP- and pyrophosphate (PPi)-dependent fluorescence quenching of the [delta]pH probe 9-amino-6-chloro-2-methoxyacridine (ACMA) indicated that these vesicles were capable of building up a pH gradient ([delta]pH, inside acid). The potent V-type H+-ATPase inhibitor bafilomycin prevented the formation of a [delta]pH in the vesicles. Bafilomycin (as well as nitrate, but not vanadate) also inhibited ATP hydrolysis, confirming the tonoplast origin of the isolated vesicles. Addition of stachyose (or sucrose, but not of mannitol) to energized vesicles caused a recovery of ACMA fluorescence, indicating a sugar-dependent dissipation of [delta]pH. The rate of fluorescence recovery was dependent on the external sugar concentration used. It displayed a single saturable response to increasing sugar concentrations. Apparent Km values of 52 and 25 mM were computed for stachyose and sucrose antiporter activities, respectively. It was also demonstrated that energized vesicles showed a much higher rate of [14C]stachyose (3 mM) and [14C]sucrose (1 mM) uptake than deenergized vesicles. The results obtained with isolated tonoplast vesicles were very similar to those obtained earlier with intact vacuoles and, therefore, confirm the existence of active stachyose and sucrose/H+ antiporters on the tonoplast of Stachys tuber vacuoles.     

Sucrose Synthase, a Cytosolic Enzyme in Protoplasts of Jerusalem Artichoke Tubers (Helianthus tuberosus L.)

The exact subcellular location of sucrose synthase (UDP-d-glucose: d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13) in Helianthus tuberosus tubers was studied by comparison of its activity in protoplasts with that of vacuoles isolated from them. Assuming 100% of the β-N-acetylglucosaminidase activity to be of vacuolar origin, less than 5% of both the sucrose synthase activity and the extravacuolar marker NAD-malate dehydrogenase was detected in the vacuole preparations. Sucrose synthase is therefore an extravacuolar enzyme. Its role in the inulin metabolism of H. tuberosus is discussed.     

Tuber Formation in Excised Stem Tips of Stachys sieboldii

The excised stem-tips of the germinating tubers of Stachys sieboldii Miq. were cultivated under the laboratory condition. As the room temperature decreased, the formation of tubercles on the tips of the explanted stems was observed. The excised tips of the rhizome at its different stages of development, when cultured in vitro, demonstrated variations in tuber formation. When the excised tips were taken from the rhizomes which had just emerged from the base of the aerial shoots, they usually failed to grow. However, existence of normal growth was observed in the cultured excised tips of the rhizomes that had become elongated at which time flowering occurred on the aerial shoot. The tips became swollen as the environmental temperature decreased. In early September, as the process of underground tuberization initiated, cultures taken from the nontuberized tips again grew poorly and usually failed to form tubercles.     

地灵组培白化苗的超微结构变化研究

应用超薄切片和电镜观察了唇形科水苏属植物地灵组培白化苗和正常苗叶片的超微结构。结果表明：在超微结构上,白化苗与正常苗叶片细胞具有明显差异。多数白化苗细胞中胞质含量稀少,叶绿体数量较少,叶绿体的形态变化较大,极少数为正常形态,多数呈部分解体状态,还有部：分叶绿体呈现膜融合状态。而细胞壁、细胞核以及细胞形态等结构,白化苗与正常苗细胞并无大的差异。     

植物肌醇半乳糖苷合酶的生理功能和调控机制

植物肌醇半乳糖苷合酶（galactinol synthase, GolS）是高等植物棉子糖类寡糖合成途径中的关键酶,为棉子糖系列寡糖提供活化的半乳糖基,调控植物体内棉子糖（raffinose, RFO）系列寡糖的生物合成与积累。编码该酶的基因属于糖基转移酶（glycosyltransferases, GTs）GT8基因家族的亚家族。GolS参与合成的最终产物棉子糖家族低聚糖（raffinose family oligosaccharides,RFOs）是植物中重要的碳水化合物存在形式,在细胞内可溶性强,可作为脱水保护剂;还能发挥稳定膜结构的作用。同时,GolS催化合成的直接产物肌醇半乳糖苷（galactinol）和RFOs都能作为羟基自由基捕获分子参与活性氧的清除。因此,GolS参与的代谢途径在植物碳同化物的贮存与运输、生物和非生物逆境响应、种子的脱水效应等生命过程中均发挥了重要作用。GolS基因结构差异与表达模式不同,导致不同GolS基因参与的生物学功能具有很大的差异。研究植物中不同GolS基因的结构特征,组织特异性表达特性及它们响应不同生长发育阶段、环境变化的表达特性,对了解GolS参与的生物学功能具有重要意义。同时,在分子生物学水平上,深入了解调控植物GolS基因的分子调控机制,为通过遗传工程或分子辅助育种等手段,利用GolS改良农林作物的经济性状提供理论支持。本文针对近年来植物中GolS基因的生理功能和调控机制的研究进行了综述。     

UDP-glucose Pyrophosphorylase from Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

UDP-glucose pyrophosphorylase of Jerusalem artichoke tubers was purified 90-fold over the crude extract. The purified enzyme preparation absolutely required magnesium ions for activity. Cobalt ions were 60% as effective as magnesium ions; other divalent cations including manganese showed little or no effect. This enzyme had a pH optimum of 8.5 and a temperature optimum of 40°C. ATP and UDP inhibited the activity of this enzyme in both forward and backward directions. Km values for UDP-glucose, inorganic pyrophosphate, glucose-1-phosphate and UTP were determined to be 4.45 × 10^?4 M, 2.33 × 10^?4 M, 9.38 × 10^?4 M and 2.98 × 10^?4 M, respectively. These results are discussed in comparison with those of UDP-glucose pyrophosphorylases isolated from other plants.     

植物肌醇半乳糖苷合酶的生理功能和调控机制

植物肌醇半乳糖苷合酶（galactinol synthase, GolS）是高等植物棉子糖类寡糖合成途径中的关键酶,为棉子糖系列寡糖提供活化的半乳糖基,调控植物体内棉子糖（raffinose, RFO）系列寡糖的生物合成与积累。编码该酶的基因属于糖基转移酶（glycosyltransferases, GTs）GT8基因家族的亚家族。GolS参与合成的最终产物棉子糖家族低聚糖（raffinose family oligosaccharides,RFOs）是植物中重要的碳水化合物存在形式,在细胞内可溶性强,可作为脱水保护剂;还能发挥稳定膜结构的作用。同时,GolS催化合成的直接产物肌醇半乳糖苷（galactinol）和RFOs都能作为羟基自由基捕获分子参与活性氧的清除。因此,GolS参与的代谢途径在植物碳同化物的贮存与运输、生物和非生物逆境响应、种子的脱水效应等生命过程中均发挥了重要作用。GolS基因结构差异与表达模式不同,导致不同GolS基因参与的生物学功能具有很大的差异。研究植物中不同GolS基因的结构特征,组织特异性表达特性及它们响应不同生长发育阶段、环境变化的表达特性,对了解GolS参与的生物学功能具有重要意义。同时,在分子生物学水平上,深入了解调控植物GolS基因的分子调控机制,为通过遗传工程或分子辅助育种等手段,利用GolS改良农林作物的经济性状提供理论支持。本文针对近年来植物中GolS基因的生理功能和调控机制的研究进行了综述。     

Sugar Transport into Storage Tubers of Stachys sieboldii Miq.: Evidence for Symplastic Unloading and Stachyose Uptake into Storage Vacuoles by an H+-Antiport Mechanism*

Following assimilation of ¹⁴CO₂ by leaves of Stachys sieboldii, ¹⁴C-stachyose is translocated into the tubers. Stachyose is accumulated and stored in the vacuoles of the pith parenchyma. Protoplasts and vacuoles were isolated and the uptake of sugars was examined. Uptake of sucrose and sucrosyl oligosaccharides of the raffinose family by protoplasts was very low compared to glucose. Transport parameters for glucose indicated a carrier mediated transport in the lower concentration range which was superimposed by diffusion at higher concentrations (> 10 mM). The very low sugar uptake by protoplasts and the sparse enzyme activities of stachyose synthase in the storage parenchyma as well as acid invertase and α-galactosidase in the cell walls indicated symplastic unloading of stachyose in the tubers. Experiments on ¹⁴C-stachyose uptake by isolated vacuoles confirmed previous observations by Keller (1992). Isolated vacuoles exhibited ATP and PP hydrolysis and were capable of generating a proton gradient across the tonoplast by a V-type H⁺-ATPase and H⁺-PPase. This was demonstrated by fluorescence quenching of quinacrine. Fluorescence could be restored by the addition of gramicidin and partly recovered by the addition of stachyose; mannitol, sorbitol and glucose had no effect. Fluorescence recovery depended on the concentration of stachyose and revealed saturation kinetics (K_m = 28 mM). Comparable results have been obtained with tonoplast vesicles by Greutert and Keller (1993). Experimental data presented here provide circumstantial evidence for symplastic unloading of stachyose in the tubers of Stachys sieboldii and demonstrate that the stachyose concentration in the cytoplasm of storage parenchyma cells is kept low by active stachyose transport into the vacuoles. The results suggest a stachyose/H⁺-antiport system.     

Arrangement and Structure of Plastids in Dormant and Cultured Tissue from Artichoke Tubers

The plastids in the tubers of the Jerusalem artichoke are foundboth scattered in the peripheral cytoplasm of the cells andalso in compact clusters usually near the nucleus. The clustersmay be a stage in the formation of plastids. The membrane-boundstructures within the plastids themselves can be assigned toone or other of two systems: a peripheral system of irregulartubules and cisternae some of which connect with the inner membraneof the plastid envelope; and a central system of highly variablemorphology which appears not to connect with the peripheralsystem. It is, apparently, from the central system that thethylakoids of stroma lamellae and grana develop during the greeningof illuminated cultures.     

VR-ACS6 is an Auxin-Inducible l-Aminocyclopropane-l-carboxylate Synthase Gene in Mungbean (Vigna radiata)

We have isolated four cDNA clones of ACC synthase from etiolatedmungbean seedlings treated with auxin. PVR-ACS2, pVR-ACS3 andpVR-ACS6 contained the same sequences as the previously reportedDNA fragments, pMAC2, pMAC3 (Botella et al. 1992b) and pMBAl(Kim et al. 1992), respectively. pVR-ACSl was identical withpAIM-1 (Botella et al. 1992a). VR-ACS6 was specifically induced in response to the auxin signal.The IAA-induction of VR-ACS6 was very rapid (within 30 min)and insensitive to cycloheximide treatment at concentrationsup to 100 µM. Significant accumulation of VR-ACS6 mRNAwas detected at 1 µM.IAA.The IAA-induced expression ofVR-ACS6 was suppressed by ABA and ethylene, but enhanced byBA. These characteristics of VR-ACS6 expression were well correlatedwith the physiological data of auxin-induced ethylene productionin mungbean hypocotyls. VR-ACS1 was strongly induced by cycloheximide, but was foundto be not auxin-specific. Inhibitors of either ethylene biosynthesis(AOA) or action (NBD) increased the basal level of VR-ACS1 mRNA. (Received May 7, 1996; Accepted November 25, 1996)     

Molecular Cloning and Characterization of GhGolS1, a Novel Gene Encoding Galactinol Synthase from Cotton (Gossypium hirsutum)

Galactinol synthase (GolS) is a key initial regulatory enzyme in the synthesis of raffinose family oligosaccharides (RFOs), sugars that play essential roles in plant growth and development. We have cloned the GhGolS1 gene (GenBank accession number: JF813792), which encodes the cotton GolS1 protein by rapid amplification of cDNA ends?Cpolymerase chain reaction (RACE?CPCR). The full-length cDNA is 1,729-bp long and encoded an open reading frame (ORF) of 343 amino acids with a deduced molecular weight of 39.2?kDa. The GhGolS1 protein shared 74?C78% identity at the amino acid level with GolS isolated from Ricinus communis, Populus trichocarpa, Solanum lycopersicum, Arabidopsis thaliana and Capsicum annuum. The corresponding genomic DNA, which contained three exons and two introns, was isolated and analyzed. The 5?? flanking region was also analyzed to identify a group of putative cis-acting elements. DNA gel blot analysis showed that in the cotton genome, the GhGolS gene family contains at least three members. Real-time PCR (RT-PCR) analysis revealed that GhGolS1 is expressed in cotton leaves, anthers and 35 DPA fibers, and its expression level in anthers is much higher than that in leaves and 35 DPA fibers. It was minimally expressed in other tissues. Additionally, the GhGolS1 protein was localized to the cell membrane. On the basis of these results, we propose that GhGolS1 has critical roles in cotton male fertility, fiber quality and seed development.     

Correlation between Changes in Mitochondrial Membranes of Artichoke Tubers and Their Hardening and Dormancy

Spin labeling studies using mitochondrial membranes of Jerusalem artichoke (Helianthus tuberosus L.) showed that the decrease during winter in the temperatures of the upper and lower lipid transitions correlated with the development of freezing hardiness of the tubers. The killing temperature for tuber tissue reached a minimum of −12 C, about 5 C degrees lower than the lower transition. Freeze-hardiness decreased when the lower transition increased at the time of sprouting.     

Biochemical Properties of the Plasmalemma ATPase of Jerusalem Artichoke (Helianthus tuberosus L.) Tubers in Relation to Dormancy

Enzymological properties of the ATPase of plasmalemma-enrichedfractions obtained from the parenchyma of Jerusalem artichoke(Helianthus tuberosus L.) tubers were studied in relation tothe morphogenetic properties of the tubers. Preparations fromdormant tubers showed higher V_max and K_m values than those fromnon-dormant tubers. These differences persisted after solubilizationof the enzyme with octylglucoside, but were slightly modifiedafter addition of K$ to the reaction medium. The implicationsof these results are discussed in relation to the morphogeneticproperties of the tubers (Received August 24, 1987; Accepted March 19, 1988)     

Presence of putative Cycas necrotic stunt virus RNAs in Japanese primrose (Primula sieboldii E. Morren)

The study screened the putative viral RNA sequences in the cDNA library of Japanese primrose, and conducted a molecular approach in determining its presence in selected Primula sieboldii accessions showing characteristic viral symptoms. Three putatively viral non-homologous sequence groups of RNA were identified; however, coding for different proteins representing a complete virus structure, it was determined to be singly originating from Cycas necrotic stunt virus (CNSV). Subsequently, sequence-specific primers were customised based from the non-homologous-sequence groups; however, amplification data showed no association between the presence of the putative viral RNA sequences and the identified characteristic virus symptoms. Despite this, amplification of the three non-homologous sequences is fully correlated. Thus, Japanese primrose was potentially identified as an alternate host of CNSV.     

Galactinol synthase gene of Coptis japonica is involved in berberine tolerance

Many plant secondary metabolites show strong biological activities and are potentially also toxic to plants, while plants producing such active compounds are usually insensitive to their own metabolites, suggesting that they have species-specific detoxification mechanisms. In order to clarify the detoxification mechanism of alkaloids, we used cultured cells of Coptis japonica, which are capable of producing a yellow benzylisoquinoline alkaloid, berberine, and accumulate it in the vacuole. Unlike other plant cells that do not produce berberine, C. japonica shows strong tolerance to this alkaloid. We established a fission yeast strain that was sensitive to berberine and performed functional screening using a C. japonica cDNA library. One cDNA clone, which conferred clear berberine tolerance, encoded galactinol synthase (CjGolS). The possible role of CjGolS in berberine tolerance is discussed.     

Change in the spectra of peroxidases in regenerants of Stachys sieboldii (Miq.) as a result of hormonal and mutagenic effects

Nodular segments of the stem and stolons of aseptic Stachys sieboldii (Miq.) plants were cultured in Gamborg's B5 medium at a low (1-2 mg/l) or high (10 mg/l) concentration of cytokinins (6-BAP, kinetin) and produced many adventitious buds and shoots without callus formation. Several plants were regenerated from nodular segments incubated at various concentrations of 5-bromodeoxiuridine (5-BdU) for three weeks. All regenerants were morphologically normal. Peroxidase electrophoretic patterns were analyzed to determine whether somaclonal variation is associated with S. sieboldii micropropagation. The frequency of changed forms was higher with the high 6-BAP concentration and with mutagenic treatment. Changed forms were not detected in plants grown at a low cytokinin concentration, which can be used in S. sieboldii micropropagation.     

The Influence of Seasonal Variation on Oxidative Phosphorylation in Sub-mitochondrial Particles Prepared from Jerusalem Artichoke Tubers

A method for the preparation of sub-mitochondrial particlesfrom Jerusalem artichoke tubers by sonication is described.The particles carried out a rapid oxidation of NADH, succinate,and ascorbate saturated with N',N',N,N-tetramethylphenylenediamine(TMPD). The difference spectrum (dithionite reduced minus oxidized)of sub-mitochondrial particles was similar to that of wholemitochondria. In the autumn phosphorylation accompanied electrontransport only when supernatant from the preparation procedurewas added to the sub-mitochondrial particles in the presenceof magnesium. In the winter and spring there was a decreasein the effect of supernatant on phosphorylation, and the sub-mitochondrialparticles alone synthesized ATP during substrate oxidation.It is suggested that the reconstitution of phosphorylation inJerusalem artichoke sub-mitochondrial particles is essentiallysimilar to that observed in beef heart electron transport particles,but that phosphorylation in the former is subject to seasonalvariations.     

The Isolation and Characterization of the Acid-Soluble Nucleotides from the Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

The acid-soluble nucleotides were extracted from the tubers of Jerusalem artichoke with percbloric acid, and separated and purified by means of adsorption on and elution from active charcoal, repeated chromatography on columns of Dowex I (Cl^-), followed by paper chromatography. The following nucleotides have been characterized and/or identified: 5′-AMP, 3′-AMP, ADP, ATP, 5′-GMP, 2′-GMP, 3′-GMP, 2′,3′-cyclic GMP, GDP, GTP, 5′-UMP, UDP, UTP, NADP, UDP-glucose, UDP-galactose, UDP-fructose, UDP-N-acetylhexosamine and GDP-mannose.^** Neither cytosine ribonucleotides nor deoxyribonucleotides have been detected. The significance of these observations is discussed.