Expression analysis of the auxin efflux carrier family in tomato fruit development

Auxin transport network, which is important in the integration of plant developmental signals, depends on differential expression of the auxin efflux carrier PIN gene family. We cloned three tomato PIN (referred as SlPIN) cDNAs and examined their expression patterns in fruit and other organs. The expression of SlPIN1 and SlPIN2 was highest in very young fruit immediately after anthesis, whereas the expression of SlPIN3 was low at this same stage of fruit development. SlPIN2::GUS was expressed in ovules at anthesis and in young developing seeds at 4 days after anthesis, while SlPIN1::GUS was expressed in whole fruit. The DR5::GUS auxin-responsive reporter gene was expressed in the fruit and peduncle at anthesis and was higher in the peduncle 4 days after anthesis. These studies suggest that auxin is likely transported from young seeds by SlPIN1 and SlPIN2 and accumulated in peduncles where SlPIN gene expression is low in tomato. The possible role of SlPINs in fruit set was discussed.     

Correlation between Lack of Receptacle Growth in Response to Auxin and Accumulation of a Specific Polypeptide in a Strawberry (Fragaria ananassa Duch.) Variant Genotype

Receptacle growth in strawberry (Fragaria ananassa Duch. cv.Ozark Beauty) occurred after either pollination or auxin treatment.In a strawberry variant genotype (Washington State UniversitySelection No. 12/13), pollination did not lead to receptaclegrowth but application of -naphthaleneacetic acid (NAA) at anthesisresulted in normal receptacle growth. The receptacles of OzarkBeauty retained their ability to respond to auxin at least upto 36 days after anthesis. However, delay of auxin applicationto the receptacles of the variant genotype resulted in decreasedauxin-responsive growth and auxin application after the 10thday of anthesis led to very little growth. The loss of auxin-responsivegrowth of the receptacle of the variant genotype was not associatedwith any loss of auxin binding activity of receptacle membranes.If auxin was not supplied to the receptacles of the variantgenotype at anthesis, the receptacles did not grow and a polypeptideof 52,000 M_r accumulated. Application of NAA to the receptaclesof the variant genotype at anthesis or on the fifth day afteranthesis resulted in the growth of the receptacle and the 52,000M_r polypeptide did not accumulate. Application of NAA to thereceptacles of the variant genotype on the 10th or the 15thday after anthesis led to very little growth of the receptacleand the 52,000 M_r polypeptide accumulated to high levels. Theseresults suggested a correlation between the lack of receptaclegrowth in response to auxin and accumulation of the 52,000 M_rpolypeptide. ¹ Current adress: The Institute of Applied Research, Ben GurionUniversity of the Negev, Beer-Sheva, Israel. (Received August 6, 1984; Accepted December 11, 1984)     

Influence of Sink-Source Interaction on Dry Matter Production in Tomato

Sink-source ratio in tomato was manipulated, in six glasshouseexperiments, by fruit pruning (trusses pruned to two to sevenfruits immediately after fruit set of each truss), truss pruning(removal of every other truss at anthesis) and truss pruningin plants with two shoots. Periodic destructive harvest wereconducted for about 100 d after flowering of the first truss.Dry matter production was not influenced by sink-source ratio,whereas dry matter distribution between fruits and vegetativeparts was greatly affected. The fraction of dry matter distributedto the fruits at the end of the fruit pruning experiments (F_fruits)could be described accurately as a saturation-type functionof number of fruits retained per truss (N_f): F_fruits = 0.660(l-e^-0.341N_f). Specific leaf area and internode length decreasedand plant leaf area increased when sink-source ratio was reduced.Removal of every other truss at anthesis did reduce dry matterpartitioning into the fruits, but it did not influence internodelength. Plant development (number of visible leaves at the endof the experiments) was not influenced by sink-source ratio.In four experiments some plants were pruned to one fruit pertruss. Final dry matter production was 8-24% lower for theseplants, compared with plants with more than one fruit per truss.This was, at least party, the result of less light interceptionby these plants, which had strongly curled leaves pointing downwards. Results indicate that effects of sink demand on dry matter productionper unit of intercepted radiation and probably on leaf photosyntheticrate in commercial tomato production can be ignored.Copyright1995, 1999 Academic Press Dry matter production, feedback control, glasshouse, growth analysis, Lycopersicon esculentum, pruning, sink demand, sink-source ratio, tomato     

Endogenous Cytokinins in Developing Fruits of Seeded and Seedless Citrus Cultivars

This report describes the isolation and identification of endogenouscytokinins from Citrus ovaries. Cytokinin active fractions wereobtained by extraction with 80% (v/v) ethanol, followed by purificationwith hexane, n-butanol and a polyvinylpyrrolidone and SephadexLH-20 column chromatography. Five fractions with cytokinin activity were found in the organicphase, using the tobacco callus assay. The main active compoundsin these fractions were separated by HPLC, bioassayed and identifiedby GC—MS as ribosyl zeatin, zeatin and isopentenyl adenosine.Hydrolysis of the first fraction with B-glucosidase gave cytokininactive compounds that in paper chromatography had R_F's similarto those of zeatin and ribosyl zeatin. Treating the aqueousphase with alkaline phosphatase produced a cytokinin activecompound that in paper chromatography had the same R_F as isopentenyladenosine indicating that their ribotide was probably the majorphosphorylated cytokinin present in Citrus ovaries. Key words: Citrus, cytokinin fruit set and development     

l-Ascorbic acid metabolism in parthenocarpic and seeded cherry tomatoes

The auxin treatment in tomato plants during anthesis has been extensively used for setting fruits in adverse climatic conditions (e.g., low temperatures and inadequate light), which is well known that reduces pollen availability and fertility. Since auxin application may affect fruit composition and quality, we examined l-ascorbic acid metabolism in seeded fruit (set by natural pollination) and parthenocarpic fruit (set by auxin) in cherry tomato cv. Conchita. Specifically, we studied the oxidized and total ascorbic acid contents, the expression of all characterized genes of l-ascorbic acid metabolism, the activity of ascorbate peroxidase and dehydroascorbate reductase and the immunolocalization of ascorbate peroxidase. Differences were detected between seeded and parthenocarpic fruits, in the expression of some of the genes of ascorbic acid metabolism. However, strong presence of l-ascorbic acid peroxidase protein was detected on the developing seeds. Our data indicate that induced parthenocarpy in auxin treated plants has a significant influence in ascorbic acid metabolism comparing to seeded tomato fruits.     

Changes in Endogenous Plant Hormones in Cherry Tomato Fruits during Development and Maturation

Hormonal extracts of cherry tomato fruits (Lycopersicon esculentum Mill.) cv. Small Fry at different stages of fruit development and maturation were bioassayed for their auxin, gibberellin, cytokinin and growth inhibitor activities. In general, the levels of endogenous growth promoters were much higher in the young developing fruits than in the more mature fruits. Free cytokinin levels were highest in the first two weeks of development then declined rapidly. However, cytokinin activity in the ribotide fraction, after treatment with alkaline phosphatase, decreased during thefirst three weeks of development then increased rapidly over the following four weeks. Auxin levels increased during early development to reach a maximum in three-week-old fruits after anthesis. Gibberellin levels during the first two weeks of development were much lower than those of auxins and cytokinins, but then increased to reach a peak in the fourth week after anthesis. A growth inhibiting substance with Rf similar to that of abscisic acid was found in the acidic fraction of the fruit extracts. This inhibitor increased gradually during fruit growth and development and reached a peak at the age of five weeks which coincides with the green mature stage.     

Inactivation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase,which catalyses the final step in the biosynthesis of ethylene,showed a non-linear time-course in vitro and activity decayedwith a half-life of around 14 min. This loss of activity wasstudied using tomato ACC oxidase purified from Escherichia coiltransformed with the cDNA clone pTOM13. Inactivation was notdue to end-product inhibition by dehydroascorbic acid or cyanide.Preincubatlon of enzyme in the combined presence of Fe²⁺ ascorbateand ACC, which together allowed catalytic turnover, resultedin almost total loss of ACC oxidase activity. Enzyme Inactivatedby catalysis could not be reactivated by passage through SephadexG-25 or by treating with combina tions of DTT and CO₂ A non-lineartime-course and inactivation in the presence of all substratesand cofactors was also shown for the enzyme assayed in vivowith melon fruit discs. Using the purified tomato enzyme a distinctascorbate-dependent inactivation was also observed, which occurredIn the absence of catalysis and was prevented, although notreversed, by catalase. This ascorbate-dependent inactivationmay thus be due to H₂O₂ attack on ACC oxidase. Key words: 1-aminocyclopropane-1-carboxylate (ACC) oxidase, catalase, catalytic inactivation, ethylene     

Potential Dry Matter and Water Import Rates in the Tomato Fruit in Relationship to Fruit Size

The tomato fruit was compared to a sphere with a radius R. Radialgrowth rates in the fruit (F_IW and F_ID) due to water importor to dry matter import, respectively, which are also the waterimport rate or dry matter import rate per unit surface areaof fruit, were calculated from two sets of published results.This data referred to fruits which swelled in such a way thatthe availability of assimilates had little effect on growth.Two varieties differentiated the two series of results and inone series, three trials were differentiated by the salinityof the nutrient solution. In all trials, it was found that F_IW and F_ID decreased whenR increased. Two phases were observed for F_IW: after a firstphase, F_IW decreased more quickly and almost linearly when Rincreased. F_ID was constant or decreased with respect to R.Except at the beginning of growth at the greatest salinity,there were clearly linear regressions between F_IW and F_ID suchas F_ID = aF_IW-b; where b was lower with higher salinity. Thechanges of the concentration of imported dry matter (F_ID/F_IWwere examined in terms of R and F_IW/R. The mechanisms controllingthe changes in F_IW and F_ID were discussed. The results suggestedfruit radius was an important parameter of these mechanisms.Thus, water import rate and dry matter import rate could eachbe considered to be the product of two factors: fruit surfacearea, which is directly dependent on fruit radius, and waterimport rate or dry matter import rate per unit of fruit surfacearea.Copyright 1993, 1999 Academic Press Dry matter, fruit growth, logistic model, sink size, tomato, water transfer     

Water Import Rate in Tomato Fruit: A Resistance Model

A model of the water import rate in tomato fruit is proposed.It compares the fruit to a hollow sphere (P) with external radiusR and internal radius R_G, corresponding to pericarp, and containingan internal spherical part (G). The pathway limiting water inputrate at any point I at a distance r from the fruit centre wasassumed to be proportional: (a) in P, to the length of the arcwhich has a radius r and which goes from I to the pedicel extension;(b) in G, to r. The water input rate at I was modelled basedon a law similar to Darcy's law which takes into account thedifference between the water potential at entry of fruit andthe water potential at point I. This latter potential was thesum of fruit osmotic potential and pressure potential due toresistance of tissue to deformation. This potential was proportionalto R-r or R_G -r. The model was expressed at fruit level by alaw such that water mass imported per unit time per unit surfacearea of fruit (f_rw) was a linear function of R. The model wascompared to linear regressions of this rate in terms of R whichhad been found during fruit swelling from published results,and which were obtained at different values of nutrient solutionsalinity. The results suggested that water input in tomato fruitis conditioned by passive forces depending on fruit size.Copyright1994, 1999 Academic Press Fruit, growth, model, resistance, salinity, size, tomato, transfer, water     

Increased activity of ornithine decarboxylase in tomato ovaries induced by auxin

Auxin, which mimics natural pollination in tomato flowers, induces fruit setting as well as a 3-fold increase in ornithine decarboxylase activity. In pollinated ovaries ornithine decarboxylase activity increased 4.7 fold. The possible connection between pollination, auxin treatment, fruit set, cell division and ornithine decarboxylase activity are discussed.     

Relationship between set, development and activities of growth regulators in tomato fruits

The actions of plant regulators in set and development of fruitsare well known. However, the presence and function of endogenoushormones in parthenocarpic fruits have still not been sufficientlyinvestigated. A comparison between seeded and seedless fruitsmakes it possible to obtain a more accurate understanding ofsome relationships between growth regulators and stages of fruitdevelopment. Endogenous auxin and gibberellin activity levelsand some growth parameters (fresh and dry weight, cell numberand cell volume, DNA content) have been determined in tomatofruits (Lycopersicon esculentum Mill.) of the cultivar Venturaand of its isogenic parthenocarpic mutant. In both genotypes,auxin and gibberellin are present in the first week after anthesis,though at different concentrations and with different patterns.These two activities are involved in fruit setting. The simultaneousoccurrence of maximum auxin concentration and of the beginningof cell enlargement, in both genotypes, shows that the activitypresent at this time starts fruit development and possibly determinesthe size of the fruits. High auxin activity is observed only in seeded fruits 20–40days after anthesis, and it is probably synthesized by seeds.Gibberellin activity is present, corresponding to the changein fruit development from the mature green to pink stages. (Received February 16, 1978; )     

Effects of Gibberellin on Auxin-Induced Hyperpolarization of Cell Membranes in Hypocotyls of Vigna unguiculata

Auxin activates pumping of protons from the symplast to theapoplast and causes hyperpolarization of the symplast membranein the elongation zone of Vigna stems prior to the accelerationof growth. This auxin-induced hyperpolarization has been studiedin most cases in hypocotyl segments excised from the elongationzone. In the present study, mature-zone segments were perfusedwith IAA by the xylem perfusion technique in an effort to determinewhether or not IAA has any effects in the mature zone. Althoughno hyperpolarization of the symplast membrane was observed uponthe perfusion with auxin alone, auxin-induced hyperpolarizationwas observed when mature-zone segments had been pretreated withGA₃, in the absence of an increase in the growth rate. Theseresults suggest that cells in the mature zone have lost theability to activate the proton-pumping machinery in responseto auxin but that this ability can be restored by treatmentwith GA₃. This effect of GA₃ suggests the possibility that theconcentration of gibberellin in a tissue controls one of thecell's responses to auxin, namely, activation of the protonpump. (Received January 10, 1994; Accepted June 11, 1994)     

Under-expression of the Auxin Response Factor Sl-ARF4 improves post-harvest behavior of tomato fruits

Auxin is one of the most prominent phytohormones regulating many aspects of fleshy fruit development including fruit set, fruit size through the control of cell division and cell expansion, and fruit ripening. To shed light on the role of auxin fruit ripening, we have previously shown that Sl-ARF4 is a major player in mediating the auxin control of sugar metabolism in tomato fruit (cv MicroTom). Further extending this study, we show here that down-regulation of Sl-ARF4 in tomato alters some ripening-related fruit quality traits including enhanced fruit density at mature stage, increased firmness, prolonged shelf-life and reduced water (weight) loss at red ripe stage. These findings suggest that Sl-ARF4 plays a role in determining fruit cell wall architecture and thus providing a potential genetic marker for improving post-harvest handling and shelf life of tomato fruits.     

Effect of indole-3-acetic acid on cell wall loosening: Changes in mechanical properties and noncellulosic glucose content of Avena coleoptile cell wall

The effect of indole-3-acetic acid on cell wall loosening andchemical modifications of noncellulosic components of the cellwall in Avena coleoptile segments was studied and the followingresults were obtained. (1) Auxin decreased both the minimum stress-relaxation time(T_o) and the noncellulosic glucose content of the cell wall. (2) Decreases were observed in the absence or presence of mannitolsolution at concentrations lower than 0.20 M which osmoticallysuppressed auxin-induced extension, while at concentrationshigher than 0.25 M, there was little auxin effect, indicatingthat it is turgor-dependent. (3) The decrease in T_o of the cell wall and that in the noncellulosicglucose content caused by auxin in the presence of mannitolsolutions of various concentrations paralleled each other (thecorrelation coefficient was 0.897). (4) Both decreases in T_o and glucose content caused by auxinwere inhibited by nojirimycin (5-amino-5-deoxy-D-glucopyranose)in the presence of mannitol. The results suggest that auxin-induced cell wall loosening iscaused by the degradation of noncellulosic rß-glucanin the cell wall. (Received December 24, 1976; )     

寄主对桔小实蝇耐寒性的影响

为了研究寄主营养对桔小实蝇耐寒性的作用,测定了以15种果蔬饲养的桔小实蝇1日龄蛹的过冷却点(supercooling points,SCP); 再选取南瓜、西红柿、柑桔、番石榴和杨桃等5种果蔬,测定了桔小实蝇3龄老熟幼虫、1日龄蛹、3日龄蛹、5日龄蛹、7日龄蛹和雌雄成虫的过冷却点,并观察了1日龄蛹的低温存活力。结果表明：（1）15种果蔬饲养所得的桔小实蝇1日龄蛹SCP均值在-11.03℃～-13.17℃,不同寄主发育的桔小实蝇SCP值存在显著性差异,其中以取食蒲桃的最高,为-11.03℃,取食苦瓜的最低,为-13.17℃。（2）5种果蔬饲养所得的桔小实蝇各虫态的SCP均值存在极显著差异(F_(4,863)＝35.6,P<0.01); 同一寄主上的桔小实蝇不同虫态之间SCP均值也达到极显著性差异(F_(6,863)＝392.9,P<0.01); 且寄主和发育龄期之间存在着极显著的交互作用(F_(24,863)＝9.4,P<0.01)。（3）桔小实蝇各发育阶段,SCP值表现一定变化： 老熟幼虫发育至1日龄蛹,SCP值变化不大; 蛹发育至3、5和7天过冷却能力明显增强,降至-20℃左右,但他们之间没有明显区别; 羽化后3～5天的成虫SCP值又升高至-10℃左右。老熟幼虫、1日龄蛹和2～3日龄成虫与3日龄、5日龄和7日龄蛹的SCP值之间有显著性差异。（4）将5种果蔬饲养所得的桔小实蝇1日龄蛹置于6℃和-3℃下进行较长时间(1～8天)和较短时间(1～8 h)的低温处理,发现番石榴、杨桃和南瓜发育的蛹经低温处理后的校正羽化率较西红柿和柑桔发育的蛹高; 同样在0℃、3℃、6℃和9℃处理(2天)的实验中,得出相似的结果。因此,本实验结果表明桔小实蝇幼虫由于生活寄主的不同使得其下一代蛹的耐寒性产生了差异,引起其差异的原因值得进一步研究。     

Agrobacterium rhizogenes-transformed roots of coffee (Coffea arabica): conditions for long-term proliferation, and morphological and molecular characterization

Background and Aims: The aims of this study were to set up proliferation conditionsfor hairy roots of Coffea arabica regenerated after transformationby Agrobacterium rhizogenes strain A4-RS, and to carry out themorphological and molecular characterization of hairy root clonesmaintained over the long term. Methods: Auxin supply, light conditions and sucrose concentration weremodified with the aim of establishing efficient root proliferationconditions. The morphological variability among 62 establishedhairy root clones was phenotyped by scanning the roots and analysingthe images using ‘whinRHIZO’ software procedures.PCR analysis of integration in transformed root cells of roland aux oncogenes from the T-DNA of the Ri plasmid was usedto study the molecular variability among clones. Key Results: Auxin supply was necessary to obtain and stimulate growth andbranching, and IBA applied at 0·5 µM was the mostefficient auxin. Significant differences were shown among the62 clones for total root length and for the percentage of fineroots. These variables were stable across subcultures and couldhence be used for efficient characterization of hairy root clones.The majority of hairy root clones (86 %) exhibited non-significantphenotype differences with non-transformed roots. Eight cloneswere significantly different from the non-transformed controlsin that they possessed a low proportion of fine roots. Two otherhairy root clones grew significantly faster than the other clones.The PCR analysis revealed a low variability in the integrationof rol and aux oncogenes in transformed root cells. The T_R-DNAwas never integrated as aux1 and aux2 genes were not found,although rolB and rolC genes from the T_L-DNA were always present. Conclusions: The discovery of low morphological variability among coffeehairy roots together with the identification of morphologicalvariables allowing easy identification of phenotypically alteredclones represent two important results. They make hairy rootsa possible, and efficient, tool for functional-genomic studiesof coffee root genes.     

Studies on cutin-esterase I. Preparation of cutin and its fatty acid component from tomato fruit peel

Examination was made of the fatty acid component of tomato cutinvia gas-liquid chromatography and thin layer chromatography.Dihydroxyeicosanoic acid was identified as a major componentof tomato cutinic acid in contrast with the results of BAKERand MARTIN (1) who recognized 10,16-dihydroxyhexadecanoic acidas the dominant acid of cutin in all plants tested. On the thinlayer chromatograms we found more than nine kinds of fatty acidsin the cutin hydrolysate which was saponified with ethanol-potashsolution. The gas-liquid chromatogram for trimethylsilyl etherderivatives of methyl cutinate showed somewhat different results,i.e., unsaturated decanoic, t_R 1.4, unsaturated stearic, t_R4.2 and unsaturated octadecanedioic acid, t_R 16.0 as unsaturatedfatty acids. Two more than C₂₂-hydroxyfatty acids were recognizedas minor components. Beside these components, octanoic, t_R 0.9,hydroxydecanoic, t_R 7.0 and cis-epoxy-hydroxyoctadecanoic acid,t_R 18.7 were identified. The biosynthesis of cutin is positednot to be fulfilled or to be delayed due to less lipoxidaseactivity in tomato fruit. ¹Biological Laboratory, Research Department, Nihon Noyaku Co.Ltd., Kawachinagano, Osaka, Japan (Received December 8, 1969; )     

Fruit Set and Growth in Strawberry, Fragaria x ananassa Duch.

The application of GA₃in aqueous solution to leaves or flowersof hermaphrodite cultivars of strawberry, Redgauntlet and Rabunda,prevented growth of the receptacle despite hand pollination.This inhibitory effect occurred only when GA₃ was applied priorto anthesis. Although viable pollen was produced and germinatedto grow down the styles of treated plants, no seeds were formed.Receptacle growth failed underneath the unfertilized carpels,but the basal region devoid of carpels enlarged and ripened.The effect of GA₃ was the same in vivo and for flowers grownin vitro. ABA and BAP also inhibited growth of pollinated flowersin vitro, but neither substance stimulated growth of the baseof the receptacle. 2-NOA stimulated receptacle growth of pollinatedflowers but did not overcome the inhibitory effect of GA₃. Removal of fertile carpels 9 days after pollination preventedfurther receptacle growth. GA₃ treatment of the bare receptaclere-started growth but was less effective than 2-NOA. No growth substance treatment induced parthenocarpic developmentin these cultivars when unopened buds were emasculated and culturedwithout pollination, although GA₃ induced some swelling of thereceptacle base. Fragaria x ananassa Duch., strawberry fruit set, fruit growth, growth regulators     

The Role of Growth Substances in the Regulation of Onion Bulb Dormancy

The correlation between sprouting and changes in endogenousgrowth substances was investigated in stored onion bulbs (Alliumcepa c.v. Rijnsburger and Lancastrian). In the main experimentsbulbs were removed from store at approximately fortnightly intervals,samples were assessed for percentage sprouting and non-sproutingbulbs were either extracted for hormone assay or treated withgrowth substances in an attempt to induce sprouting. In otherexperiments the hormone content of bulbs at different stagesof sprouting was assessed. Growth-inhibitor and gibberellinactivity decreased before sprouting, but there was an increasein gibberellin and auxin activity as sprouting commenced. Gibberellinactivity was highest in bulbs with well-developed sprouts whereasauxin activity occurred mainly in bulbs in which early sproutdevelopment was visible only on their being cut open. Therewas no conclusive evidence that bulb dormancy could be brokenby application of the gibberellins GA₃ and GA_4/7, or the auxin1-naphthylacetic acid (NAA). Maleic hydrazide (MH) completelyinhibited root and sprout development but the growth retardant(2-chloroethyl)trimethylammonium chloride (CCC) was mainly effectivein reducing root development and sprouting was only slightlyinhibited.