Germination and Dormancy of Abscisic Acid- and Gibberellin-Deficient Mutant Tomato (Lycopersicon esculentum) Seeds (Sensitivity of Germination to Abscisic Acid,Gibberellin, and Water Potential)

Germination responses of wild-type (MM), abscisic acid (ABA)-deficient (sitw), and gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds to ABA, GA4+7, reduced water potential ([psi]), and their combinations were analyzed using a population-based threshold model (B.R. Ni and K.J. Bradford [1992] Plant Physiol 98: 1057-1068). Among the three genotypes, sitw seeds germinated rapidly and completely in water, MM seeds germinated more slowly and were partially dormant, and gib-1 seeds did not germinate without exogenous GA4+7. Times to germination were inversely proportional to the differences between the external osmoticum, ABA, or GA4+7 concentrations and the corresponding threshold levels that would either prevent ([psi]b, log[ABAb]) or promote (log[GAb]) germination. The sensitivity of germination to ABA, GA4+7, and [psi] varied widely among individual seeds in the population, resulting in a distribution of germination times. The rapid germination rate of sitw seeds was attributable to their low mean [psi]b (-1.17 MPa). Postharvest dormancy in MM seeds was due to a high mean [psi]b (-0.35 MPa) and a distribution of [psi]b among seeds such that some seeds were unable to germinate even on water. GA4+7 (100 [mu]M) stimulated germination of MM and gib-1 seeds by lowering the mean [psi]b to -0.75 MPa, whereas ABA inhibited germination of MM and sitw seeds by increasing the mean [psi]b. The changes in [psi]b were not due to changes in embryo osmotic potential. Rather, hormonal effects on endosperm weakening opposite the radicle tip apparently determine the threshold [psi] for germination. The analysis demonstrates that ABA- and GA-dependent changes in seed dormancy and germination rates, whether due to endogenous or exogenous growth regulators, are based primarily upon corresponding shifts in the [psi] thresholds for radicle emergence. The [psi] thresholds, in turn, determine both the rate and final extent of germination within the seed population.     

Studies on the Changes in the Major Nucleic Acid Components of Tomato Seeds (Lycopersicon esculentum Mill.) Resulting from Osmotic Presowing Treatment

Extensive accumulation of nucleic acid may be one of the factorsinvolved in the acceleration of tomato seed germination by osmoticpresowing treatment. The experiments reported here were designedto characterize the changes in the major nucleic acids duringosmotic pretreatment and subsequent germination. Polyacrylamidegel electrophoresis showed that ribosomal RNA comprised themajor part of this net synthesis, but it was more difficultto ascertain whether or not there was a proportional increasein sRNA, as part of the latter peak may comprise comigratingfragments of degraded rRNA. The mass ratio of 25S: 18S rRNAsextracted from dry seeds was near unity, while during normalgermination and during pretreatment this ratio increased toabout 1?9:I, close to the theoretical ratio assuming 25S and18S rRNAs occur in equimolar proportions. This leads to thespeculation that replacement of old ribosomes may occur duringearly germination and pretreatment, although this would notaccount for all the synthetic activity observed during the latterprocess. Use of the diphenylamine colour reaction showed that no increasein DNA results from pretreatment and that the onset of the firstS-phase during subsequent germination of pretreated seeds wascommensurate with the advancement of the time to 50% germination.     

Amounts of Nuclear DNA and Internal Morphology of Gibberellin- and Abscisic Acid-deficient Tomato (Lycopersicon esculentum Mill.) Seeds During Maturation, Imbibition and Germination

Using X-ray photography and flow cytometry, the internal morphologyand DNA replication activity of wild type (wt), GA- (gib-1 )and ABA-deficient (sit^w ) tomato (Lycopersicon esculentum Mill.cv. Moneymaker) mutant seeds were studied. During seed formation,from 30 to 45 d after pollination (DAP) the endosperm becomessolid and the seed starts to gain desiccation tolerance. Atthis time significant changes occur in the amounts of DNA inradicle tip cells. At 30 DAP, radicle tip cells of the threegenotypes manifest about 60% of 2C, 30% of 4C and 10% of 8Camounts of DNA. Upon maturation (45 DAP onwards), most cellsin the seeds of the three genotypes arrest in the G₁phase ofthe cell-cycle with 2C amounts of DNA. However, a relativelyhigh proportion of cells with 4C amounts of DNA was detectedin the radicle tip cells ofsit^w compared with wild type andgib-1. At the well-matured stage (60 DAP), there were about 2% ofseeds with free space in wild type andgib-1 , and about 13%insit^w . At the over-matured stage (75 DAP), even more seedswith free space were found insit^w , whereas no increase in theproportion of the seeds with free space was detected in theother two genotypes. In -1.0 MPa PEG-6000 with or without 10µM GA₄₊₇, no germination occurred in well-matured wildtype andgib-1 seeds, whether or not they were dried after harvest.However,sit^w seeds were able to germinate both in over-maturefruit and in -1.0 MPa PEG-6000. Priming of dried seeds in -1.0MPa PEG induced a large amount of free space in almost all seedsof the three genotypes, and nuclear DNA synthesis in the radicletip cells of wild type andsit^w seeds. However, PEG priming offresh (non-dried) seeds had no effect on the amount of freespace and 2C/4C DNA ratios in wild type orgib-1 seeds, but didinduce free space in about 20–25% ofsit^w seeds and provoked4C signals insit^w seeds. Removal of the endosperm and testaopposite the radicle tip of seeds resulted in root protrusion,the induction of free space and an increase of 4C DNA signalsin the three genotypes. It is concluded that ABA is crucialfor the efficient arrest of tomato embryo radicle tip cellsin G₁phase upon maturation, whereas GAs play an important rolein re-initiating 4C DNA levels upon germination. Dormancy; flow cytometry; free space; Lycopersicon esculentum ; maturation; priming; seed; tomato     

Properties and Activity Changes of Chlorogenic Acid:Glucaric Acid Caffeoyltransferase From Tomato (Lycopersicon esculentum)

A novel acyltransferase from cotyledons of tomato (Lycopersicon esculentum Mill.), which catalyzes the transfer of caffeic acid from chlorogenic acid (5-O-caffeoylquinic acid) to glucaric and galactaric acids, was purified with a 2400-fold enrichment and a 4% recovery. The enzyme showed specific activities (theoretical V_max per milligram of protein) of 625 nanokatals (caffeoylglucaric acid formation) and 310 nanokatals (caffeoylgalactaric acid formation). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it gave an apparent M_r of 40,000, identical to the value obtained by gel filtration column chromatography. Highest activity was found at pH 5.7, which was constant over a range of 20 to 120 millimolar K-phosphate. The isoelectric point of the enzyme was at pH 5.75. The reaction temperature optimum was at 38°C and the apparent energy of activation was calculated to be 57 kilojoules per mole. The apparent K_m values were 0.4 millimolar for glucaric acid, 1.7 millimolar for galactaric acid, and with both acceptors as second substrates 20 millimolar for chlorogenic acid. The relative ratio of the V_max/K_m values for glucaric acid and galactaric acid was found to be 100:12. Substrate-competition experiments support the conclusion that one single enzyme is responsible for both the glucaric and galactaric acid ester formation with marked preference for glucaric acid. It is proposed that the enzyme be called chlorogenic acid:glucaric acid O-caffeoyltransferase (EC 2.3.1.-). The three caffeic acid-dependent enzyme activities involved in the formation of the glucaric and galactaric acid esters, the chlorogenic acid:glucaric acid caffeoyltransferase as the key activity as well as the caffeic acid:CoA ligase and the caffeoyl-CoA:quinic acid caffeoyltransferase as the preceding activities, were determined. The time course of changes in these activities were followed during development of the seedling in the cotyledons and growth of the young plant in the first and second leaf. The results from tomato seedlings suggest a sequential appearance of these enzymes.     

Descriptive Physiology and Biochemistry of the Abnormally Ripening Tomato Fruit (Lycopersicon esculentum) cv. Snowball

Aspergillus tamarii Kita grew and sporulated best at 30°C. The best pH for growth and sporulation were 5.5 and 6.5, respectively. Among the carbon sources employed, glucose supported the highest growth and sporulation. Best growth was obtained with sodium nitrate as nitrogen source and best sporulation with ammonium tar-tarate. When glucose was used as carbon source, the carbohydrates found in the mycelium included myoinositol. dulcitol, fructose, arabinose and ribose.     

Utilization of Caffeic Acid Phenethyl Ester to Control Alternaria alternata Rot in Tomato (Lycopersicon esculentum Mill.) Fruit

Chemical fungicides that are related with resistant strains develop negative effects on human health and environment. Propolis is a resinous substance collected by bees with positive effects on human health and inhibitory activity against Alternaria alternata. Caffeic acid phenethyl ester (CAPE) is a component of the propolis. The objective of this experiment was to test the effect of CAPE on fungi infecting tomato fruit using as a model the pathosystem A. alternata‐tomato. CAPE was chemically synthesized in our laboratory and analysed with nuclear magnetic resonance spectroscopy. Different concentrations (0, 16, 32, 48, 64, 80, 90 and 100 μm ) of CAPE were tested on A. alternata growing in vitro. For the in vivo experiment, red ripe tomato fruit was inoculated with A. alternata and untreated or treated with 1, 50 and 100 μm of CAPE. After that, the fruit was stored at 25°C for up to 20 days. Colony size (CS) was recorded in vitro. In tomato fruits, the severity of infection (SI), respiration rate (RR), ethylene production (EP), pH, total soluble solids (TSS), weight loss (WL) and titratable acidity (TA) were evaluated during the storage time. CAPE melting point and spectral data probed to be the right molecule. In vitro, 64 and 100 μm of CAPE reduced CS by 30%. In vivo, 50 and 100 μm of CAPE reduced SI higher than the fungicide Captan^® with no effects on RR, EP, WL, pH, TSS and TA. It was concluded that CAPE controls A. alternata infection better than a commercial fungicide without negative effects on tomato fruit ripening and fruit quality.     

Ripening Physiology of Fruit from Transgenic Tomato (Lycopersicon esculentum) Plants with Reduced Ethylene Synthesis

The physiological effects of reduced ethylene synthesis in a transgenic tomato (Lycopersicon esculentum) line expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase enzyme have been examined. Fruit from the transgenic line 5673 ripen significantly slower than control fruit when removed from the vine early in ripening. In contrast, fruit that remain attached to the plants ripen much more rapidly, exhibiting little delay relative to the control. Ethylene determinations on attached fruit revealed that there was significantly more internal ethylene in attached than detached fruit. The higher ethylene content can fully account for the observed faster on-the-vine ripening. All of the data are consistent with a catalytic role for ethylene in promoting many, although not all, aspects of fruit ripening. Biochemical analyses of transgenic fruit indicated no significant differences from controls in the levels of ACC oxidase or polygalacturonase. Because transgenic fruit are significantly firmer than controls, this last result indicates that other enzymes may have a significant role in fruit softening.     

Dormancy and Germination of Abscisic Acid-Deficient Tomato Seeds : Studies with the sitiens Mutant

The role of abscisic acid (ABA) in the dormancy induction of tomato (Lycopersicon esculentum) seeds was studied by comparison of the germination behavior of the ABA-deficient sitiens mutant with that of the isogenic wild-type genotype. Freshly harvested mutant seeds, in contrast to wild-type seeds, always readily germinate and even exhibit viviparous germination in overripe fruits. Crosses between mutant and wild-type and self-pollination of heterozygous plants show that in particular the ABA fraction of embryo and endosperm is decisive for the induction of dormancy. After-ripened wild-type seeds fully germinate in water but are more sensitive toward osmotic inhibition than mutant seeds. Germination of both wild-type and mutant seeds is equally sensitive toward inhibition by exogenous ABA. ABA content of mature wild-type seeds is about 10-fold the level found in mutant seeds. Nevertheless, it is argued that the differences in dormancy between the seeds of both genotypes are not a result of actual ABA levels in the mature seeds or fruits but a result of differences in ABA levels during seed development. It is hypothesized that the high levels of ABA that occur during seed development in wild-type seeds induce an inhibition of cell elongation of the radicle that can still be observed after long periods of dry storage.     

Pectin Methylesterase Isoforms in Tomato (Lycopersicon esculentum) Tissues (Effects of Expression of a Pectin Methylesterase Antisense Gene)

We have identified two major groups of pectin methylesterase (PME, EC 3.1.1.11) isoforms in various tissues of tomatoes (Lycopersicon esculentum). These two groups exhibited differential immuno-cross-reactivity with polyclonal antibodies raised against tomato fruit PME or flax callus PME and differences in their accumulation patterns in tissues of wild-type and transgenic tomato plants expressing a PME antisense gene. The group I isoforms with isoelectric points (pls) of 8.2, 8.4, and 8.5 are specific to fruit tissue, where they are the major forms of PME activity. The group II PME isoforms, with pl values of 9 and above, are observed in both vegetative and fruit tissues. The group I isoforms cross-react with polyclonal antibodies raised to a PME isoform purified from fruit, whereas the group II isoforms cross-react with antibodies to a PME purified from flax callus. Expression of a fruit-specific PME anti-sense gene impairs accumulation of the group I PME isoforms, with no apparent effect on the accumulation of the group II PME isoforms. The absence of any noticeable effects on growth and development of transgenic plants suggests that the group I PME isoforms are not involved in plant growth and development and may play a role under special circumstances such as cell separation during fruit ripening.     

Characterization of the Stimulation of Ethylene Production by Galactose in Tomato (Lycopersicon esculentum Mill.) Fruit

We have characterized the stimulation of ethylene production by galactose in tomatoes (Lycopersicon esculentum Mill.). The effect of concentration was studied by infiltrating 0, 4, 40, 100, 200, 400, or 800 micrograms galactose for each gram of fresh fruit weight into mature green `Rutgers' fruit. Both 400 and 800 micrograms per gram fresh weight consistently stimulated a transient increase in ethylene approximately 25 hours after infiltration; the lower concentrations did not. Carbon dioxide evolution of fruit infiltrated with 400 to 800 micrograms per gram fresh weight was greater than that of lower concentrations. The ripening mutants, rin and nor, also showed the transient increase in ethylene and elevated CO₂ evolution by 400 micrograms per gram fresh weight galactose. 1-Aminocyclopropane-1-carboxylic acid (ACC) content and ACC-synthase activity increased concurrently with ethylene production. However, galactose did not stimulate ACC-synthase activity in vitro. The infiltrated galactose in pericarp tissue was rapidly metabolized, decreasing to endogenous levels within 50 hours. Infiltrated galacturonic acid, dulcitol, and mannose stimulated transient increases in ethylene production similar to that of galactose. The following sugars produced no response: sucrose, fructose, glucose, rhamnose, arabinose, xylose, raffinose, lactose, and sorbitol.     

Electrical Stimulation and its Effects on Indoleacetic Acid and Peroxidase Levels in Tomato Plants (Lycopersicon esculentum)

The effect of applied direct electrical currents (3–7µA) on the levels of indoleacetic acid (IAA) and peroxidasein tomato plants was determined. Six-week-old treated and controltomato plants were divided into four parts (petiole, leaf, root,and internode). The levels of IAA and peroxidase were higherin the treated tissues (leaf and petiole) compared to the controls.There was not a significant difference in peroxidase levelsin internodal tissue between treated and control plants; however,the peroxidase level in treated root tissue was lower than incontrol plants. It is suggested that small currents may alterthe levels of IAA and peroxidase in different plant tissues.     

Ethylene and Glycosidase Promotion in GA3- and IAA-treated Tomato Fruit (Lycopersicon esculentum Mill.)

apd: 15 September 2000     

番茄"白化"突变体果实的几个生理生化特性检测

在番茄“白化”突变体果实发育过程中,总叶绿素、叶绿素a、叶绿素b的含量均小于正常植株的果实;果肉细胞内含有白色体;果实在贮藏期间的硬度较大。而果实品质,如糖酸比、维生素C和可溶性固形物的含量与正常果实差异不大。     

Phosphate-Regulated Induction of Intracellular Ribonucleases in Cultured Tomato (Lycopersicon esculentum) Cells

Four intracellular RNases were found to be induced in cultured tomato (Lycopersicon esculentum) cells upon phosphate starvation. Localization studies revealed three (RNases LV 1-3) in the vacuoles and one (RNase LX) outside these organelles. All of these RNases were purified to homogeneity and were shown to be type I RNases on the basis of type of splitting, substrate, and base specificity at the cleavage site, molecular weight, isoelectric point, and pH optimum. Moreover, RNase LV 3 was shown by fingerprinting of tryptic digests on reversed-phase high-performance liquid chromatography and sequencing the N terminus and two tryptic peptides to be structurally very similar to a recently characterized extracellular RNase LE which is also phosphate regulated (Nürnberger et al. [1990] Plant Physiol 92: 970-976; Jost et al. [1991] Eur J Biochem 198: 1-6). Expression of the four intracellular RNases is induced by depleting the cells of phosphate and repressed by adding phosphate. Our studies indicate that higher plants, in addition to secreting enzymes for scavanging phosphate under starvation conditions, also induce intracellularly emergency rescue systems.     

Re-evaluation of Conditions for Plant Regeneration and Agrobacterium-Mediated Transformation from Tomato (Lycopersicon esculentum)

Conditions for plant regeneration from explants of tomato (Lycopersiconesculentum) cv. UC82B were studied for optimizing transformationprocedure. The best regeneration rate was obtained from cotyledonexplants from 8–10-d-old seedlings on a modified Murashigeand Skoog medium (1962) with 0·5 mg dm^–3 zeatinand 0·5 mg dm^–3 indolylacetic acid. Tomato cultivars(UC82B, Castone, Fl Ferline, Monalbo) and a Lycopersicon peruvkmum‘CMV sel. INRA’ were studied. The cultivarUC82Band the wild Lycopersicon species showed an efficient shootregeneration potential. Early events in the transformation of tomato cotyledons wereanalysed using an Agrobacterium tumefaciens strain carryinga binary vector with an nptII (pnos) gene and a reporter GUS-intron(p35S) chimeric gene. Two days after infection, GUS activityappeared specifically at the cut surface. Subepidermal cellswere more susceptible to transformation than epidermal cells.When selection for kanamycin resistance was applied 2 d afterinoculation, transformed cells were efficiently recovered. Preculturewith feeder cells stimulated cell transformation, but reducedregenerationcapacity from transformed cells. The optimal transformationrate was observed witha time of preculture of 1 and 2 d. Transformationevents for two tomato cultivars (UC82B and Monalbo) occurredat the same rate as 55% of the inoculated explants developedkanamycin resistant calli. However, transformed plants wereobtained at different rates of 8% and 14% for cv. Monalbo andcv. UC82B. Key words: Agrobacterium tumefaciens, ß-glucuronid, Lycopersicon esculentum, plant regeneration, transformation     

乙酰水杨酸在番茄果实发育期间对蔗糖代谢相关酶活性的影响

研究不同浓度乙酰水杨酸(ASA)对番茄品种‘辽园多丽’果实发育期间蔗糖代谢相关酶影响的结果表明:ASA可抑制果实的维管束和胶质胎座中酸性转化酶(AI)和中性转化酶(NI)活性,而提高蔗糖合成酶(SS)与蔗糖磷酸合成酶(SPS)活性;心室隔壁和中果肉中ASA的作用与此相反。ASA促进果实维管束中可溶性糖积累主要通过调控AI和NI活性实现,而在胶质胎座中主要通过调控SS活性实现;在中果肉和心室隔壁中主要通过调控SS和AI活性实现。     

1-甲基环丙烯采后处理对樱桃番茄果实成熟过程的影响

研究了不同浓度(0、0.035、0.07和0.11μL/L)的乙烯受体竞争性抑制剂1-甲基环丙烯(1-MCP)采后处理对绿熟期樱桃番茄的乙烯合成、果实软化、果实色素(叶绿素、茄红素、β-胡萝卜素)含量消长的影响.0.07 μL/L及其以上浓度的1-MCP降低了前期乙烯合成,同时推迟了乙烯释放高峰,但0.035 μL/L浓度的1-MCP处理并不能抑制内源乙烯合成.1-MCP显著延迟了果实软化和叶绿素降解,但并不影响这两个过程的启动.茄红素合成的启动和积累均受到了1-MCP抑制,而1-MCP并不推迟β-胡萝卜素合成的启动,只抑制其积累.这些结果提示了乙烯调节成熟生理过程的不同机制.对于绿熟期的樱桃番茄,0.07～0.11μL/L的1-MCP是实用的有效处理浓度.1-MCP有效浓度可能用于了解果实的乙烯受体水平和乙烯敏感性.