Phytochrome A does not mediate reduced stem extension from cool day‐temperature treatments

Stem elongation can be suppressed by a temperature drop at the onset of the photoperiod (DROP) or with a cooler day than night temperature (DT and NT, respectively), commonly described as DIF (DT ‐ NT). To test our hypothesis that phytochrome A (phyA) mediated the reduction of stem elongation caused by −DIF and DROP, we conducted experiments with photomorphogenic mutants of tomato ( Solarium lycopersicon L.) and transgenic potato ( Solarium tuberosum L.). The plants studied were tomato mutants fri ¹ (deficient in phyA) and tri ³ (deficient in phytochrome B1 [phyBl]) and their isogenic wild‐type (WT) cv. Moneymaker, nontransformed potato, and two lines each of antisense phyA (15‐9 and 15‐11) and overexpressed phyA (PS‐2 and PS‐4). Plants were placed in three temperature regimens with a daily mean of 20°C: a constant 20°C (0 DIF), an 8°C DROP for 3 h, and a ‐ 8°C DIF. For all tomato genotypes, −DIF and DROP reduced intemode length by ≥ 21% and stem elongation by 30% compared to that of plants at 0 DIF. Interactions between temperature treatment and genotype were nonsignificant. For potato, −DIF, but not DROP, significantly reduced intemode length of WT (by 39%) and both antisense lines (by 36 or 48%) but only one of the two lines of overexpressed phyA plants (by 18%). The −DIF significantly reduced stem length for only antisense phyA (by 36 or 48%) and WT (by 35%) plants. Thus, at least for tomato and potato, it appears that phyA does not control stem extension in relation to cool‐temperature treatments.     

Structure and expression of an ethylene-related mRNA from tomato.

Messenger RNAs homologous to a cDNA clone (pTOM 13) derived from a ripe-tomato-specific cDNA library are expressed during tomato fruit ripening and after the wounding of leaf and green fruit material. Both responses involve the synthesis of the hormone ethylene. Accumulation of the pTOM 13--homologous RNA during ripening is rapid and sustained, and reaches its maximum level in orange fruit. Following mechanical wounding of tomato leaves a pTOM 13--homologous RNA shows rapid induction within 30 minutes, which occurs before maximal ethylene evolution (2-3 h). This RNA also accumulates following the wounding of green tomato fruit. Northern blot analysis of poly(A)+ RNA indicates that the length of the mRNA is about 1400 nucleotides. Nucleotide sequence analysis showed the cDNA insert to contain the complete coding region of the pTOM 13 protein (33.5 kD) and an unusual 5' structure of ten dT-nucleotides. Hybridisation of the pTOM 13 cDNA insert to Southern blots of tomato DNA indicates the presence of only a small number of homologous sequences in the tomato genome.     

Elevated pyruvate,orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene

Changes in carbon metabolism and δ¹³C value of transgenic potato plants with a maize pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) gene are reported. PPDK catalyzes the formation of phospho enol pyruvate (PEP), the initial acceptor of CO₂ in the C₄ photosynthetic pathway. PPDK activities in the leases of transgenic potatoes were up to 5.4‐fold higher than those of control potato plants (wild‐type and treated control plants). In the transgenic potato plants, PPDK activity in leaves was negatively correlated with pyruvate content (r²= 0.81), and was positively correlated with malate content (r²= 0.88). A significant increase in the δ¹³C value was observed in the transgenic potato plants, suggesting a certain contribution of PEP carboxylase as the initial acceptor of atmospheric CO₂. These data suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C₄‐type carbon metabolism. However, since parameters associated with CO₂ gas exchange were not affected, the altered carbon metabolism had only a small effect on the total photosynthetic characteristics of the transgenic plants.     

Soil compaction. A role for ethylene in regulating leaf expansion and shoot growth in tomato?

The role of ethylene in regulating growth in tomato (Lycopersicon esculentum Mill.) during compaction stress was examined using wild-type (cv Ailsa Craig) and transgenic (ACO1_AS) genotypes; the latter has a reduced capacity to produce ethylene. Ethephon or silver ions were applied to increase ethylene production or block its action. Shoot growth in both genotypes was comparable in uncompacted (1.1 g cm⁻³) and uniformly compacted soil (1.5 g cm⁻³). However, a 1.1/1.5-g cm⁻³ split-pot treatment invoked marked genotypic differences: growth was reduced in cv Ailsa Craig but was comparable to uncompacted control plants in ACO1_AS. As xylem sap abscisic acid levels were similar, abscisic acid was not responsible for inhibiting growth in cv Ailsa Craig. These genotypic differences in growth were accompanied by increased ethylene evolution in cv Ailsa Craig, suggesting that the ability of ACO1_AS to maintain growth in the split-pot treatment reflected its lower ethylene levels, a view supported by the observation that excising the roots in the compacted compartment reduced ethylene evolution and restored shoot growth in cv Ailsa Craig. Treatment with silver restored shoot growth in cv Ailsa Craig, whereas treatment with ethephon reduced growth in ACO1_AS. Thus, ethylene apparently has a key role in determining growth when tomato plants encounter differential soil compaction.     

Does an antagonistic relationship between ABA and ethylene mediate shoot growth when tomato (Lycopersicon esculentum Mill.) plants encounter compacted soil?

This study tested the hypothesis that antagonistic interactions between abscisic acid (ABA) and ethylene mediate the effects of soil compaction on shoot growth. Isogenic wild‐type (Ailsa Craig), ABA‐deficient (notabilis) and a transgenic (ACO1_AS) tomato genotype with a reduced capacity to synthesize ethylene were examined. Exogenous ABA was also applied. Leaf area was comparable when Ailsa Craig and ACO1_AS were grown in uncompacted (1·1 g cm^?3) or compacted (1·5 g cm^?3) soil, but was lower in notabilis. However, a 1·1/1·5 g cm^?3 split‐pot treatment invoked marked genotypic differences, whereby leaf area was comparable to 1·1 g cm^?3 control plants in ACO1_AS but was intermediate between the 1·1 and 1·5 g cm^?3 treatments in Ailsa Craig and notabilis. ABA may be discounted as the root‐sourced signal responsible for reducing leaf area when the roots encountered compacted soil as Ailsa Craig and ACO1_AS showed differing responses despite similar increases in xylem sap ABA concentration; leaf area was invariably lower in notabilis. These genotypic differences were correlated with ethylene evolution; thus the greater leaf area in ACO1_AS was associated with its reduced ability to synthesize ethylene, whereas the reductions in leaf expansion observed when Ailsa Craig and notabilis encountered compacted soil were accompanied by increased ethylene production. Application of ABA had little effect on ACO1_AS, but promoted a recovery of leaf expansion in notabilis, and more surprisingly in Ailsa Craig. These results suggest that antagonistic interactions between ABA and ethylene may regulate leaf expansion when the root system simultaneously encounters uncompacted and compacted soil.     

Suitability of tomato leaves for larval development of Potato Tuber Moth,Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

The potato tuber moth (PTM) is an important pest of the potato plant and its tuber. With the expansion of its geographic range, the PTM could be potential threat to the tomato (Solanum lycopersicum L.), a congeneric species of the potato. To assess that risk, we tested larval feeding and development of PTM on leaves of five cultivated tomato varieties namely Moneymaker, Campari, Ailsa Craig, LA3475, E6203 and one wild tomato variety S. pimpinellifolium. PTM larvae accepted all tested plant leaves and developed into adults. Larval development was fastest on the Ailsa Craig variety. Pupae developed fastest on the Moneymaker variety and slowest on LA3475. Host acceptance and survival were highest on Ailsa Craig and lowest on LA3475. The significantly highest male proportion occurred on LA3475 variety. The study showed that PTM could be a potential threat to tomato cultivation which is rapidly increasing in temperate regions owing to climate change.     

Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild-type plants indicated that ACC oxidase mRNA accumulation normally begins in pre-senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild-type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild-type plants. Northern analysis of the photosynthesis-associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild-type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening-related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild-type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild-type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild-type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay-green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.     

Influence of long photoperiods on plant development and expression of Crassulacean acid metabolism in Mesembryanthemum crystallinum

Abstract. In the natural habitat plants of Mesembryanthemum crystallinum are induced to perform Crassulacean acid metabolism (CAM) after 3 months, and reproductive growth begins after 5 months (Winter, Liittge & Winter, 1978, Oecologia (Berlin), 34, 225-237). The life cycle of M. crystallinum and the extent of growth required prior to induction of enzymes of Crassulacean acid metabolism (CAM) are dramatically shortened by growing seedlings with a long photoperiod (3=16h/8h light/dark). Reproductive growth begins as soon as five weeks after germination when plants are grown in continuous light (under 600μmol quanta m⁻² s⁻¹, 30°C). In plants grown under well-watered conditions, the activities of PEP carboxylase and NADP-malic enzyme begin increasing markedly 2 weeks after germination, with plants grown under longer photoperiods having higher enzyme activities. After 3 weeks of growth, leaves accumulated a large amount of malate, but the microequivalents of malate present were up to nine times greater than the total titratable acidities. Interestingly, plants from a 24h/0h or a 20h/4h photo-period showed no diurnal fluctuation of malate, but did produce malate in the light as a major photosynthetic end product. That is, under these environmental conditions, principal enzymes of CAM can be induced without the plants performing CAM. However, plants grown in a 16h/8h photoperiod did exhibit nocturnal accumulation of malate after 3 weeks of growth. In plants of all three growth conditions, the activities of NADP-malic enzyme and PEP carboxylase were further increased two- to live-fold by irrigating 3-week-old-plants with 350mol m⁻³ NaCl. Such early enhancement of these enzymes by salt and the shortened life cycle may be due to an accelerated development under the long photoperiods.     

Wild-type levels of abscisic Acid are not required for heat shock protein accumulation in tomato

Levels of endogenous abscisic acid (ABA) in wild type were not required for the synthesis of heat shock proteins in detached leaves of tomato (Lycopersicon esculentum Mill., cv Ailsa Craig). Heat-induced alterations in gene expression were the same in the ABA-deficient mutant of tomato, flacca, and the wild type. Heat tolerance of the mutant was marginally less that the wild type, and in contrast, ABA applications significantly reduced the heat tolerance of wild-type leaves. It was concluded that elevated levels of endogenous ABA are not involved in the tomato heat shock response.     

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus × domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.     

Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes.

The last step in biosynthesis of the plant hormone ethylene, oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC), is catalysed by the elusive ethylene-forming enzyme (EFE). EFE is induced by fungal elicitors in suspension-cultured tomato cells. We demonstrate that Xenopus laevis oocytes injected with RNA from elicitor-treated tomato cells gain the ability to convert ACC to ethylene. The enzyme expressed in the oocytes under the direction of plant RNA is indistinguishable from genuine plant EFE with regard to its saturation kinetics, its iron dependency and its stereospecificity to the diastereomeric ethyl derivatives of ACC, allocoronamic acid and coronamic acid. In tomato cells stimulated for different times with elicitor, the level of EFE correlates with the level of RNA directing EFE expression in oocytes. Hybridization and co-injection experiments demonstrate that the tomato RNA species directing EFE expression in oocytes are homologous to clone pTOM13 which has been shown to inhibit ethylene production in plants when expressed in antisense. Using a cDNA library from elicitor-stimulated tomato cells, we have isolated several homologues of pTOM13 and identified one of them, pHTOM5, as a clone of EFE on the basis of its functional expression in the Xenopus oocytes.     

Inhibition of tomato shoot growth by over‐irrigation is linked to nitrogen deficiency and ethylene

Although physiological effects of acute flooding have been well studied, chronic effects of suboptimal soil aeration caused by over‐irrigation of containerized plants have not, despite its likely commercial significance. By automatically scheduling irrigation according to soil moisture thresholds, effects of over‐irrigation on soil properties (oxygen concentration, temperature and moisture), leaf growth, gas exchange, phytohormone [abscisic acid (ABA) and ethylene] relations and nutrient status of tomato (Solanum lycopersicum Mill. cv. Ailsa Craig) were studied. Over‐irrigation slowly increased soil moisture and decreased soil oxygen concentration by 4%. Soil temperature was approximately 1°C lower in the over‐irrigated substrate. Over‐irrigating tomato plants for 2 weeks significantly reduced shoot height (by 25%) and fresh weight and total leaf area (by 60–70%) compared with well‐drained plants. Over‐irrigation did not alter stomatal conductance, leaf water potential or foliar ABA concentrations, suggesting that growth inhibition was not hydraulically regulated or dependent on stomatal closure or changes in ABA. However, over‐irrigation significantly increased foliar ethylene emission. Ethylene seemed to inhibit growth, as the partially ethylene‐insensitive genotype Never ripe (Nr) was much less sensitive to over‐irrigation than the wild type. Over‐irrigation induced significant foliar nitrogen deficiency and daily supplementation of small volumes of 10 mM Ca(NO₃)₂ to over‐irrigated soil restored foliar nitrogen concentrations, ethylene emission and shoot fresh weight of over‐irrigated plants to control levels. Thus reduced nitrogen uptake plays an important role in inhibiting growth of over‐irrigated plants, in part by stimulating foliar ethylene emission.     

Involvement of abscisic acid and ethylene in the responses of citrus seedlings to salt shock

The responses of salt‐sensitive citrus rootstocks to 200 m M NaCl were periodically determined on seedlings of citrange Carrizo ( Citrus sinensis [L.] Osbeck × Poncirus trifoliata [L.] Raf) during 30 days. The stressed seedlings adjusted osmotically, reduced stomatal conductance, increased proline content and ethylene production, and showed massive leaf abscission (92%). The salt shock also increased abscisic acid (ABA) and aminocyclopropane‐1‐carboxylic acid (ACC) in roots, xylem fluid and leaves, and in addition promoted Cl⁻ accumulation. The pattern of change of ABA, ACC and proline followed a two‐phase response: an initial transient increase (10‐12 days) overlapping with a gradual and continuous accumulation. This biphasic response appears to be compatible with the proposal that the transitory hormonal rises are induced by the osmotic component of salinity, whereas the Cl⁻ increase determines the subsequent accumulations. During the second phase, Cl⁻ levels correlated with abscission in leaves. Production of leaf ethylene was also concomitant with the increase in the abscission rate. Salt‐induced abscission was either reduced with CoCl₂ (52%) or inhibited with silver thiosulphate (14%). The results suggest that in salt‐stressed citrus, leaf abscission is induced by the chloride build‐up through a mechanism that stimulates leaf ACC synthesis and further conversion to ethylene.     

Apoplastic and membrane‐associated Ca2+ in leaves and roots as affected by boron deficiency

A combination of fluorescein‐isothiocyanate (FITC), coumarin‐benzothiazol (BTC), and chlorotetracycline (CTC) fluorescence was used to simultaneously monitor apoplastic pH, apoplastic free Ca²⁺, and plasma membrane‐bound Ca²⁺. As early boron deficiency reactions supposedly include alterations of plasma membrane‐bound transport processes besides rapid effects on cell wall physical properties, the corresponding changes were followed in leaves and roots of Vicia faba L. cv. Troy.
Boron deficiency did not alter the apoplastic pH, but it reduced plasma membrane‐bound Ca²⁺ in roots at 4 h and leaves at 3 days after starting the deficiency treatment. The decrease in plasma membrane‐bound Ca²⁺ coincided with an increase in apoplastic free Ca²⁺ and K⁺, and occurred before the first visible symptoms were noticed.
It is proposed that less Ca²⁺ is bound to the plasma membrane due to a reduction of specific Ca²⁺‐binding sites (borate esters with vic ‐diols or polyhydroxy‐carboxylates) before plasma membrane integrity deteriorates.     

5'-Methylthioadenosine nucleosidase and 5-methylthioribose kinase activities in relation to stress-induced ethylene biosynthesis

The activities of 5'-methylthioadenosine (MTA) nucleosidase (EC 2.2.2.28) and 5-methylthioribose (MTR) kinase (EC 2.7.1.100) were related to changes in ethylene biosynthesis in tomato ( Lycopersicon esculentum Mill. cv. Rutgers) and cucumber ( Cucumis sativus Mill. cv. Poinsett 76) fruit following wounding and chemically induced stresses. Stress ethylene formation in wounded tomato and cucumber tissue continued to increase after wounding, reached its peak by 3h, and then declined. The activities of MTA nucleosidase and MTR kinase increased parallel to stress ethylene in both tissues. At peak ethylene formation, MTA and MTR kinase activities were 2- to 4-fold higher in wounded than in intact tissue. Wounded, mature-green tomato tissue treated with specific inhibitors of MTA nucleosidase and MTR kinase showed a significant reduction in the activities of these enzymes, which was concomitant with a decline in stress ethylene biosynthesis. When mature-green tomato discs were infiltrated with [¹⁴CH₃] MTA and wounded, radioactive MTR and methionine were formed. Incubation of mature-green tomato discs with Cu²⁺ and Li⁺ in the presence of kinetin increased ethylene biosynthesis. MTA nucleosidase activity was higher than that of the control in the presence of Cu²⁺ but not in the presence of Li⁺, while MTR kinase activity was lower than that of the control in both Cu²⁺ and Li⁺ treatments. Data indicate that MTA nucleosidase and MTR kinase are required for wound-induced ethylene biosynthesis but not for chemical stress-induced ethylene by Cu²⁺ or Li⁺ treatments.     

Tolerance to atmospheric ozone in transgenic tobacco over‐expressing glutathione synthetase in plastids

A cross between transgenic tobacco ( Nicotiana tabacum L.) plants which over‐expressed either γ‐glutamylcysteine synthetase (cpGSHI) or glutathione synthetase (cpGSHII) in their chloroplasts was used to compare the consequences of over‐expression of components of the glutathione synthetic pathway on tolerance to atmospheric O₃ or paraquat. A high proportion (50%) of those progeny which carried the cpGSHII transgene alone showed tolerance to atmospheric O₃ but not to paraquat. Progeny of an additional two, independent, self‐pollinated primary transgenic lines, which segregated in a Mendelian fashion for the presence of the cpGSHII transgene and therefore included both transformed and non‐transformed (recessive, wild‐type) plants, were also challenged by fumigation with O₃. Again, in both cases, about 50% of the plants expressing the epGSHII transgene were found to be O₃‐tolerant on the basis of reduced ethylene emissions and increased or unchanged total pigment concentrations. However, this tolerance was not due to specific changes in stomatal densities.     

Regulation of the loss of frost hardiness in Pinus radiata by photoperiod and temperature

Abstract. In controlled environments, the interactive effects of warm (16: 8°C, day: night) and cool (12: 4°C, day: night) temperatures and long (13.5 h) and short (10 h) photoperiods on the dehardening of seedlings of Pinus radiata D. Don were investigated. In another experiment, the effect of four photoperiods from 9 to 14 h was examined. In a third, dehardening at constant temperatures from 5 to 17°C was followed. There was no evidence for an interaction between photoperiod and temperature. Dehardening was temporarily delayed by photoperiods below about 10 h, but there was no other quantitative effect of photoperiod. At constant temperatures, the rate of dehardening was initially constant but declined as the minimum summer frost hardiness was reached. In the initial phase the rate of dehardening was a linear function of temperature, increasing from 0.05°C day⁻¹ at 8°C to 0.30 °C day⁻¹ at 17°C. Temperature controlled the loss of frost hardiness by regulating the rate of dehardening.     

Continuous light may induce photosynthetic downregulation in onion – consequences for growth and biomass partitioning

Onions were grown in environmentally controlled growth chambers for 85 days to investigate the effect of relatively low light intensity (350 µmol m⁻² s⁻¹) at two different total irradiance periods (12-h and 24-h photoperiods) on growth and photosynthetic performance. To test whether photosynthetic downregulation occurred due to carbohydrate feedback, we used onions that differed in bulb-forming capacity. Allium fistulosum (L. cv. 'Kinka') is a non-bulbing onion, with potentially limited carbohydrate storage capacity, while Allium cepa (L. cv. 'Cal 296') is a bulb-forming onion with possibly greater carbohydrate storage capacity. In A . fistulosum , photosynthetic downregulation was observed in 24-h plants as indicated by reductions in the light- and CO₂-saturated photosynthetic capacity ( A _sat and A _max, respectively) by 26%, reduced maximum rate of carboxylation ( V _cmax) by ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) by 33%, reduced maximum rate of electron transport ( J _max) by 27% and 3-fold higher foliar sugar concentration. In contrast, the photosynthetic and biochemical capacity of A . cepa was not affected by exposure to 24-h photoperiod, presumably because substantial amounts of foliar carbohydrates were re-allocated to bulbs. In 24-h A . cepa , up to 84% of total plant mass was allocated to bulbs, while in 12-h plants, more mass was allocated to leaves. Production of greater leaf area in 12-h plants compared with 24-h plants compensated for lower total daily irradiance such that 12-h and 24-h plants of both species exhibited similar daily total leaf net CO₂ exchange and plant mass at the end of the experiment.