Isolation and characterization ofo-diphenol-O-methyltransferase cDNA clone in hot pepper (Capsicum annuum L.)

A cDNA clone,CaOMTl encoding ano-diphenol-O-methyltransferase (OMT), which is involved in capsaicin biosynthesis, was isolated by screening of a cDNA library prepared from the mRNA of pepper (Capsicum annuum L.) pericarp. Nucleotide sequence analysis ofCaOMTl revealed that it had an open reading frame of 1080 bp which encodes a polypeptide with a predicted molecular weight of 39,430 D, corresponding well with the size of the known OMT’s of tobacco, poplar, aspen, alfalfa, and cabbage. It also had five conserved boxes which appear in all known OMT’s. The nucleotide sequence ofCaOMTl had 89–74% identity with the OMT cDNA’s of tobacco, aspen, alfalfa, and poplar, but a relatively lower identity of 59% with the OMT cDNA of maize. Amino acid sequence analysis also revealed that CaOMT1 has high identity with the known OMT’s which have a substrate ofo-diphenolic compounds, especially 5-hydroxyferulic acid and caffeic acid. It supportsCaOMTl which encodes an OMT. Southern blot analysis suggested thatCaOMTl might exist in the form of multiple copies in the pepper genome.CaOMTl is expressed preferentially in pepper fruit and its expression levels increased during pepper fruit development, but decreased during fruit ripening, suggesting that theCaOMTl gene is fruit development-related.CaOMTl is the first reported cDNA clone for enzymes related to the phenlypropanoid pathway in pepper.     

Glycerolipid-fatty-acid desaturase deficiencies in chloroplasts from fruits of Capsicum annuum L.

Chloroplasts from fruits and leaves of Capsicum annuum cv. Bell Tower were purified on sucrose gradients, and the lipids were separated by column and thin-layer chromatography. The glycerolipids mono- and digalactosyldiacylglycerol (MGDG, DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG) were quantified, and the fatty-acid composition at the 1 and 2 positions of the glycerol moiety (sn-1 and sn-2) was determined after hydrolysis with position-specific lipases. In fruit chloroplasts, ³-trans hexadecenoate (trans-3-161) was absent and replaced by palmitate (160) at sn-2 of PG, and ^7,10,13-hexadecatrienoate (163) at sn-2 of MGDG was greatly reduced and largely replaced by linoleate (182). The ratio of 182 to linolenate (183) was consistently greater in glycerolipids from fruit compared with leaf chloroplasts. The lower percentage of C-16 fatty acids at sn-2 indicated that prokaryotic molecular species were reduced by 15% in DGDG, 40% in SQDG, and 90% in MGDG, in fruit compared with leaf chloroplasts. The MGDGDGDG ratios in fruit and leaf chloroplasts were 1.21 and 2.21, respectively. Taken together, the data indicate that chloroplasts in Capsicum fruit are deficient in three desaturases: those that convert 1) 160 to ³-trans-161 at sn-2 of PG, 2) 160 to ⁷–cis-161 at sn-2 of MGDG, and 3) 182 to 183 at both sn-1 and sn-2 of various chloroplast glycerolipids.Abbreviations Chl chlorophyll - DGDG digalactosyldiacylglycerol - FS free sterol - GL galactolipid - MGDG monogalactosyldiacylglycerol - PE phosphatidyl ethanolamine - PG phosphatidylglycerol - PL phospholipid - SQDG sulfoquinovosyldiacylglycerol We are grateful to Dr. Roger Calza for providing us with the tobacco gt11 cDNA expression library and to Dr. Eric Huttner for his advice throughout the screening procedure. We also wish to thank M. Gosse for his assistance in growing and maintaining our plants. T.W.B. was supported by a BAP research grant from the Commission of the European Communities.     

Cytomorphology of induced octoploid Chili pepper (Capsicum annuum L.)

Summary Octoploidy was induced in Chili pepper (Capsicum annuum cultivar cerasiformis) through the application of colchicine and the cytomorphological features of two octoploid plants were described. In general, the octoploids did not exhibit gigas characters when compared to the tetraploids; on the contrary they were less vigorous, suggesting that the optimum and desirable ploidy level for Capsicum is probably tetraploid. Chromosome associations such as octovalents and hexavalents, in addition to IVs, IIIs, IIs and Is, were recorded at diakinesis and metaphase I. Meiosis was highly irregular and the pollen and seed fertility was very low. Cytological features of octoploid Chili peppers are compared with octoploids of Physalis and Petunia.     

Influence of BA and sucrose on the competence and determination of pepper (Capsicum annuum L. var. Sweet Banana) hypocotyl cultures during shoot formation

Summary The simultaneous presence of 6-benzyladenine (BA) and sucrose in a Murashige and Skoog medium (SIM) during the initial stages of shoot initiation have been found to be obligatory for high-frequency shoot formation in the Capsicum annuum L. var. Sweet Banana upper hypocotyl explants. The explants are determined for shoot formation following a minimum of 8 days of culture on SIM. Deprivation of exogenous sucrose from day 6 to day 20 of culture had no effect on the shoot forming response of the explants. BA and sucrose appear to act independently on different aspects of the competence of explants to respond to SIM during shoot initiation.Abbreviations BA N⁶-benzyladenine - MS Murashige and Skoog medium - SIM shoot induction medium - HFM hormone free medium - SUC sucrose minus medium     

Biological control of Phytophthora blight in red pepper (Capsicum annuum L.) using Bacillus subtilis

Phytophthora blight is one of the most important devastating diseases of red pepper plants. Forty-one bacterial isolates were obtained from rhizosphere soil and subsequently tested for antagonistic activity under in vitro and in vivo conditions. Among the 41 isolates tested, 12 exhibited a maximum antagonistic activity in dual culture assay. These 12 isolates were further screened for disease suppression on red pepper plants in both natural and greenhouse conditions. All the antagonists showed varying levels of antagonism, whereas the isolates R33 and R13 exhibited the maximum (86.8 and 71%) ability to reduce the disease severity in in vivo conditions. Based on the 16S rDNA sequencing, the most effective isolates were identified as Bacillus subtilis. In addition, the isolates were also screened for siderophores, hydrogen cyanide and hydrolytic enzymes. Further, the isolates increased the root and shoot length of the red pepper, which is an added advantage of the isolates while performing the desired function.     

Cytokinins in the perennial herb Urtica dioica L. as influenced by its nitrogen status

The effect of nitrogen on the cytokinin relations of Urtica dioica, the stinging nettle, has been investigated. The plants were grown in quartz sand and nutrient solutions providing levels of nitrate ranging from 1 to 22 mM. Nitrogen supply did not affect biomass production within the range of 3–15 mM NO ₃ ^- . However, the shoot: root ratio of biomass was significantly higher at 15 mM (standard plants) than at 3 mM (low-nitrogen plants) nitrate supply. The cytokinin patterns of the roots, stems and adult, as well as meristematic leaves of plants grown at these two levels of nitrate supply, were determined by means of high-performance liquid chromatography (HPLC) and immunoassays. Enzyme-linked immunosorbent assays (ELISAs) for zeatin riboside, dihydrozeatin riboside, isopentenyladenosine, benzyladenosine and o-hydroxybenzyladenosine enabled the quantification of 17 cytokinins, 13 of which were found in the various tissues of Urtica. trans-Zeatin and its conjugates were the predominant cytokinins in all examined samples. While the free base trans-zeatin and its O-glucoside were the major cytokinins in adult leaves, trans-zeatin riboside was prominent in the other tissues of at least the standard plants. Glucosides of the trans-zeatin type cytokinins were present only in lower amounts. However, considerable amounts of a compound, tentatively identified as cis-zeatin riboside-O-glucoside, were found, particularly in roots and meristematic leaves. Comparatively high amounts of trans-zeatin nucleotide as well as isopentenyladenosine phosphate were also demonstrated in these tissues. Analysis of the root-pressure exudates similarly showed trans-zeatin riboside and, at a lower concentration, trans-zeatin to be the only substantial components. In the low-nitrogen plants, shortage of nitrogen was manifest only in the roots; the nitrogen contents of the shoots did not respond to the nitrogen supply. Likewise, the total content of cytokinins in the shoots of the low-nitrogen plants equaled that of the standard-plant shoots, while it was lower by about 25% in the roots of the low-nitrogen plants. In the latter, the amounts of cytokinins exuded via the root-pressure fluid were also approximately 25% lower. Since the levels of only the trans-zeatin cytokinins in the roots showed a linear correlation with the shoot-to-root ratios, these cytokinins may play an important role in biomass partitioning in Urtica dioica.Abbreviations DHZ dihydrozeatin - ELISA enzyme-linked immunosorbent assay - -G glucoside - HPLC high-performance liquid chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - -N nucleotide (ribotide) - -OG O-glucoside - -R riboside - S/R shoot-to-root (ratio) - Z zeatin This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the SFB 137. The authors wish to thank Mrs. A. Fischbach for skilful technical assistence and Dr. Paul Ziegler (Lehrstuhl für Pflanzenphysiologie, University of Bayreuth, FRG) for linguistic suggestions.     

An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method

In this work we report a new method forin vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 ± 2°C in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 ± 2°C under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 mol m-² s-^-1) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation ( 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10–14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L^–1 for 3 h and then subculturing on MS medium without growth regulators.     

Carbohydrate metabolism during fruit development in sweet pepper (Capsicum annuum) plants

Growth, accumulation of sugars and starch, and the activity of enzymes involved in sucrose mobilization were determined throughout the development of sweet pepper fruits. Fruit development was roughly divided into three phases: (1) an initial phase with high relative growth rate and hexose accumulation, (2) a phase with declining growth rate and accumulation of sucrose and starch, and (3) a ripening phase with no further fresh weight increase and with accumulation of hexoses, while sucrose and starch were degraded. Acid and neutral invertase (EC 3.2.1.26) were closely correlated to relative growth rate until ripening and inversly correlated to the accumulation of sucrose. Acid invertase specifically increased during ripening, concurrently with the accumulation of hexoses. Sucrose synthase (EC 2.4.1.13) showed little correlation to fruit development, and in periods of rapid growth the activity of sucrose synthase was low compared to the invertases. However, during late fruit growth sucose synthase was more active than the invertases. We conclude that invertase activities determine the accumulation of assimilates in the very young fruits, and a reactivation of acid invertase is responsible for the accumulation of hexoses during ripening. During late fruit growth, before ripening, sucrose synthase is transiently responsible for the sucrose breakdown in the fruit tissue. Results also indicate that pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) and its activator fructose-2,6-bisphosphate (Fru2,6bisP) are involved in the regulation of the sink metabolism of the fruit tissue.     

An integrated genetic linkage map of pepper (Capsicum spp.)

An integrated genetic map of pepper including 6 distinct progenies and consisting of 2262 markers covering 1832 cM was constructed using pooled data from six individual maps by the Keygene proprietary software package INTMAP. The map included: 1528 AFLP, 440 RFLP, 288 RAPD and several known gene sequences, isozymes and morphological markers. In total, 320 anchor markers (common markers in at least two individual maps) were used for map integration. Most anchor markers (265) were common to two maps, while 27, 26 and 5 markers were common to three, four and five maps, respectively. Map integration improved the average marker density in the genome to 1 marker per 0.8 cM compared to 1 marker per 2.1 cM in the most dense individual map. In addition, the number of gaps of at least 10 cM between adjacent markers was reduced in the integrated map. Although marker density and genome coverage were improved in the integrated map, several small linkage groups remained, indicating that further marker saturation will be needed in order to obtain a full coverage of the pepper genome. The integrated map can be used as a reference for future mapping studies in Capsicum and to improve the utilization of molecular markers for pepper breeding.These authors contributed equally to the work described in this paper(e-mail:     

The use of seed protein electrophoresis in the study of phylogenetic relationships in Chili pepper (Capsicum L.)

Summary The seed protein profile of eight taxa of Chili peppers obtained by disc electrophoresis was found to be a diagnostic character in the study of phylogenetic relationships. The distinctness of each species and the wild and cultivated nature of concerned taxa has been confirmed. While the clustering of wildC. annuum var. glabriusculum withC. baccatum types indicated that the former is the progenitor of the latter group, the marked differences discernible in the seed protein profile of all other taxa suggest a polyphyletic origin for the genusCapsicum.     

Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis

Summary Chile pepper (Capsicum annuum L.) plants were regenerated from cotyledon explantsin vitro in four major stages: bud induction, bud enlargement, shoot elongation, and root development. Bud induction medium contained 0.5 mg/L (2.9μM) indole-3-acetic acid and 2 mg/L (8.9 μM) N⁶-benzyladenine. Bud enlargement occurred, and an occasional shoot appeared when medium with 2 mg/L (6μM) gibberellic acid, 2 mg/L (8.9 μM) N⁶-benzyladenine, and 5 mg/L (29.4 μM) silver nitrate was used. Most shoots elongated after placement on a third medium without plant growth regulators or on fresh plates of bud enlargement medium. Incubations were for 2, 2, and 4 weeks, respectively, at 28.5°C and continuous light. Treatment with silver nitrate was necessary for multiple shoot production and elongation to occur in the third culture stage and was most effective when present in the second-stage medium but not in the bud induction medium. Sixteen to 26% of the shoots rooted in medium with 1 mg/L (5.4 μM) 1-naphthaleneacetic acid after 1 month. Additional shoots transferred to a second rooting medium with 0.1 or 1.0 mg/L (0.54 or 5.4 μM) 1-naphthaleneacetic acid developed roots, increasing the overall rooting efficiency to 70–72%. Most rooted shoots grew well and produced viable seeds when grown in the greenhouse. Other cytokinins tested for plant regeneration were zeatin and thidiazuron. Zeatin induced few shoots and fewer well-developed plants. Thidiazuron induced multiple shoots 4 months after culture began, but many were small and did not elongate further. Phytagar tissue culture grade proved superior to other agars tested, increasing bud induction frequency from 0-33% to 80–93% and eliminating explant hyperhydricity.     

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves

Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1 -dimethylurea - OC osmotic concentration - RL red light - TX tentoxin - WL white light We thank Dr. G.E. Templeton, University of Arkansas, Fayetteville, USA, for initially supplying us with TX, and also Dr. Stephen O. Duke, Southern Weend Science Laboratory, Stoneville, Miss., USA, for suggesting this compound for our experiments. We are grateful to Professor E. Ballio for his generous gift of fusicoccin.     

Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.)

Various systems of anther and microspore cultures were studied to establish an efficient doubled haploid production method for Indonesian hot pepper (Capsicum annuum L.). A shed-microspore culture protocol was developed which outperformed all the previously reported methods of haploid production in pepper. The critical factors of the protocol are: selection of flower buds with more than 50% late unicellular microspores, a 1 day 4°C pretreatment of the buds, followed by culture of the anthers in double-layer medium system for 1 week at 9°C and thereafter at 28°C in continuous darkness. The medium contained Nitsch components and 2% maltose, with 1% activated charcoal in the solid under layer and 2.5 μM zeatin and 5 μM indole-3-acetic acid in the liquid upper layer. All the ten genotypes of hot pepper tested, responded to this protocol. The best genotypes produced four to seven plants per original flower bud. This protocol can be used as a potential tool for producing doubled haploid plants for hot pepper breeding.     

The effect of leaf exudates on the spore germination of phylloplane mycoflora of chilli (Capsicum annuum L.) cultivars

Summary Ninetten aminoacids, twelve sugars, eleven organic acids and ten phenols were detected in the leaf exudates of three cultivars of chilli. The number of aminoacids, sugars, organic acids and phenols increased as the plants grew older. More aminoacids and sugars were detected in the exudate from the susceptible cultivar (Malwa). More organic acids and phenols were detected from the resistant cultivar (Simla). The leaf exudate of the resistant cultivar (Simla) inhibited spore germination of the pathogen (Alternaria solani) while that of susceptible (Malwa) stimulated spore germination. The cultivar ‘Patna’ which is moderately resistant, occupied an intermediate position. Spore germination of the isolated fungi was enhanced in leaf exudate of susceptible cultivar (Malwa), while leaf exudates of the moderately resistant (Patna) and resistant (Simla) inhibited spore germination of the majority of fungi isolated. Most of the antagonistic fungi were not isolated from the susceptible cultivar and the percentage spore germination of these fungi was less in leaf exudate of the susceptible cultivar, while leaf exudates of resistant cultivars enhanced the percentage spore germination of antagonistic fungi,viz Aspergillus flavus, A. fumigatus, A. versicolor, Penicillium citrinum, P. restrictum andTrichoderma viride.     

The synthetic potential of immobilised cells of Capsicum frutescens Mill cv. annuum

Cells of Capsicum frutescens Mill. cv. annuum, immobilised in reticulate polyurethane foam, produced higher yields of capsaicin, the pungent principle of Chilli pepper fruits, than did freely-suspended cells, when batch-cultured in a medium conducive to culture growth. In the absence of specific precursors to capsaicin, immobilised cells produced between two and three orders of magnitude higher yields than did suspended cells over 5-d or 10-d culture periods (typically up to 4 or 5 mg capsaicin g^-1 dry weight l^-1 medium compared with up to 30 g g^-1l^-1, respectively). These results were reflected by an increased rate and extent of incorporation of L-[U-¹⁴C]phenylalanine into capsaicin in immobilised as compared with freely-suspended cells, and evidence is presented for an inverse relationship between incorporation of [¹⁴C]phenylalanine into protein and capsaicin. The accumulation of capsaicin can be experimentally manipulated and increased by supplementing the medium with precursors of capsaicin such as phenylalanine and isocapric acid and by reducing the growth rate of immobilised cells by omitting growth regulators from the medium. The importance of these observations is discussed.Abbreviations HPLC high-performance liquid chromatography - Phe phenylalanine - TLC thin-layer chromatography     

Microspore-derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development

Background information. In vitro-cultured microspores, after an appropriate stress treatment, can switch towards an embryogenic pathway. This process, known as microspore embryogenesis, is an important tool in plant breeding. Basic studies on this process in economically interesting crops, especially in recalcitrant plants, are very limited and the sequence of events is poorly understood. In situ studies are very convenient for an appropriate dissection of microspore embryogenesis, a process in which a mixture of different cell populations (induced and non-induced) develop asynchronically.Results. In the present study, the occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, an horticultural crop of agronomic interest, in relation to proliferation and differentiation events. Haploid plants of Capsicum annuum L. (var. Yolo Wonder B) have been regenerated from in vitro anther cultures by a heat treatment at 35 degrees C for 8 days. Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels, using low-temperature-processed, well-preserved specimens. The comparison with the normal gametophytic development revealed changes in cell organization after embryogenesis induction, and permitted the characterization of the time sequence of a set of structural events, not previously defined in pepper, related to the activation of proliferative activity and differentiation. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.Conclusions. The reported changes can be considered as markers of the microspore embryogenesis. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies, especially in agronomically interesting crops.     

Daily changes in nitrate uptake and metabolism in Capsicum annuum

The diurnal pattern of nitrate uptake by Capsicum annuum L. cv. California Wonder in a constant environment is described by a Fourier harmonic, with the maximum uptake in the middle of the photoperiod and the minimum in the middle of the dark period. Comparison of the uptake pattern with that of nitrate reductase (EC 1.6.6.1.) activity suggests against a direct control of one process by the other. This was confirmed by the observation that the pattern of nitrate reductase activity was not altered by restricting nitrate uptake to one hour per day. Translocation of ¹⁵N from the roots is much greater in the lightperiod than in the dark period. Reduction of ¹⁵N in the leaves occurs in the lightperiod but very little is reduced in the dark period. Amino acid levels showed marked daily fluctuations but in the roots neither amino acids, sucrose, fructose, glucose nor malate showed fluctuations. The amino acid composition of roots and leaves differed: glutamine+glutamate were relatively more important in leaves than in roots whereas alanine was a more important constituent of roots than of leaves.Abbreviation NR nitrate reductase     

Spatial distribution of growth rates and of epidermal cell lengths in the elongation zone during leaf development in Lolium perenne L.

Relative elemental growth rates (REGR) and lengths of epidermal cells along the elongation zone of Lolium perenne L. leaves were determined at four developmental stages ranging from shortly after emergence of the leaf tip to shortly before cessation of leaf growth. Plants were grown at constant light and temperature. At all developmental stages the length of epidermal cells in the elongation zone of both the blade and sheath increased from 12 m at the leaf base to about 550 m at the distal end of the elongation zone, whereas the length of epidermal cells within the joint region only increased from 12 to 40 m. Throughout the developmental stages elongation was confined to the basal 20 to 30 mm of the leaf with maximum REGR occurring near the center of the elongation zone. Leaf elongation rate (LER) and the spatial distributions of REGR and epidermal cell lengths were steady to a first approximation between emergence of the leaf tip and transition from blade to sheath growth. Elongation of epidermal cells in the sheath started immediately after the onset of elongation of the most proximal blade epidermal cells. During transition from blade to sheath growth the length of the blade and sheath portion of the elongation zone decreased and increased, respectively, with the total length of the elongation zone and the spatial distribution of REGR staying near constant, with exception of the joint region which elongated little during displacement through the elongation zone. Leaf elongation rate decreased rapidly during the phase when only the sheath was growing. This was associated with decreasing REGR and only a small decrease in the length of the elongation zone. Data on the spatial distributions of growth rates and of epidermal cell lengths during blade elongation were used to derive the temporal pattern of epidermal cell elongation. These data demonstrate that the elongation rate of an epidermal cell increased for days and that cessation of epidermal cell elongation was an abrupt event with cell elongation rate declining from maximum to zero within less than 10 h.Abbreviations LER leaf elongation rate - REGR relative elemental growth rates     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.