The effect of reduced activity of phytoene synthase on isoprenoid levels in tomato pericarp during fruit development and ripening

Carotenoids, gibberellins (GAs), sterols, abscisic acid and -amyrins were analysed in tomato (Lycopersicon esculentum Mill.) pericarp during fruit development and ripening. The contents of these isoprenoids in wild-type (cv. Ailsa Craig) fruit were compared with those in fruit of the carotenoid-deficient R-mutant and a transgenic plant containing antisense RNA to a phytoene synthase gene. In both carotenoid-deficient genotypes, a 14-fold reduction in carotene and twofold decrease in xanthophyll content, compared to the wild type, was found in ripe fruit. Immature green fruit from wild type and R-mutant plants contained similar amounts of the C₁₉-GAs, GA₁, and GA₂₀, and their C₂₀ precursor, GA₁₉. Immature fruit from the transgenic plants contained three- to fivefold higher contents of these GAs. In wild-type fruit at the mature green stage the contents of these GAs had decreased to < 10% of the levels in immature fruit. A similar decrease in GA₁₉ content occurred in the other genotypes. However, the contents of GA₁ and GA₂₀ in fruit from phytoene synthase antisense plants decreased only to 30% between the immature and mature green stages and did not decrease at all in R-mutant fruit. At the breaker and ripe stages, the contents of each GA were much reduced for all genotypes. The amount of abscisic acid was the same in immature fruit from all three genotypes, but, on ripening, the levels of this hormone in antisense and R-mutant fruit were ca. 50% of those in the wild type. Quantitative differences in the amounts of the triterpenoid -amyrins, total sterols, as well as individual sterols, such as campesterol, stigmasterol and sitosterol, were apparent between all three genotypes during development. Amounts of free sterols of wild type and antisense fruit were greatest during development and decreased during ripening, whereas the opposite was found in the R-mutant. This genotype also possessed less free sterol and more bound sterol in comparison to the other varieties. These data provide experimental evidence to support the concept of an integrated metabolic relationship amongst the isoprenoids.Abbreviations ABA abscisic acid - dpb days post breaker - FDP farnesyl diphosphate - GA gibberellin - GGDP geranyl-geranyl diphosphate We thank Mr. Paul Gaskin (Long Ashton Research Station) for the qualitative GC-MS of triterpenoids and Dr. R. Horgan (University of Wales, Aberystwyth) for a gift of [6-³H₂]ABA. The work was supported by a research grant (No. PG111/617) to P.M.B. from the Agricultural and Food Research Council to whom we express our thanks.     

YFT1基因突变影响番茄果色和硬度

番茄（Solanum lycopersicum）是全球第一大经济作物,其品质倍受科学家和消费者关注,果色和硬度是决定番茄品质的重要经济性状。为了解析YFT1（YELLOW FRUIT TOMATO1）调控番茄果色发育和硬度形成机制,本文对野生型cv.M82和突变体yft1的果色、硬度和果皮显微结构进行了比较分析。结果显示,在35 dpa（days post anthesis）,yft1和cv.M82番茄果色、硬度、果皮细胞大小和形状无明显差异。随着果实发育cv.M82番茄由绿色转为橙色/浅红（47 dpa）和红色（54 dpa）;并由硬变软,54 dpa的果实硬度（13.68±1.78N）仅相当于35 dpa的（35.51±1.09N）1/3。同时,从转色期（47 dpa）果皮细胞由内向外逐渐变大;内果皮薄壁细胞由圆形（35 dpa）依次变成卵形（47 dpa）和不规则形（54 dpa）,并观察到了细胞内陷和胞间空隙消失（54 dpa）。与cv.M82不同,yft1番茄的果色、硬度和果皮细胞显微结构随果实发育（35~54 dpa）变化不明显;而其果实硬度（47~54 dpa）显著高于cv.M82。这些结果均暗示YFT1突变使番茄果实发育延迟,并影响了果色发育和硬度的形成。该研究将为番茄果色发育和果色形成机制的揭示提供重要的表型证据。     

Pectin Modification in Cell Walls of Ripening Tomatoes Occurs in Distinct Domains

The class of cell wall polysaccharides that undergoes the most extensive modification during tomato (Lycopersicon esculentum) fruit ripening is pectin. De-esterification of the polygalacturonic acid backbone by pectin methylesterase facilitates the depolymerization of pectins by polygalacturonase II (PGII). To investigate the spatial aspects of the de-esterification of cell wall pectins and the subsequent deposition of PGII, we have used antibodies to relatively methylesterified and nonesterified pectic epitopes and to the PGII protein on thin sections of pericarp tissue at different developmental stages. De-esterification of pectins and deposition of PGII protein occur in block-like domains within the cell wall. The boundaries of these domains are distinct and persistent, implying strict, spatial regulation of enzymic activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins strongly associated with cell walls of pericarp tissue at each stage of fruit development show ripening-related changes in this protein population. Western blots of these gels with anti-PGII antiserum demonstrate that PGII expression is ripening-related. The PGII co-extracts with specific pectic fractions extracted with imidazole or with Na2CO3 at 0[deg]C from the walls of red-ripe pericarp tissue, indicating that the strong association between PGII and the cell wall involves binding to particular pectic polysaccharides.     

Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits

Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the β-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype—Pusa Rohini. We found that expression of phytoene synthase and β-carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.     

Fruit-specific overexpression of wound-induced tap1 under E8 promoter in tomato confers resistance to fungal pathogens at ripening stage

Based on high economic importance and nutritious value of tomato fruits and as previous studies employed E8 promoter in fruit ripening-specific gene expression, we have developed transgenic tomato plants overexpressing tomato anionic peroxidase cDNA (tap1) under E8 promoter. Stable transgene integration was confirmed by polymerase chain reaction (PCR) and Southern analysis for nptII. Northern blotting confirmed elevated tap1 levels in the breaker- and red-ripe stages of T(1) transgenic fruits, whereas wild-type (WT) plants did not show tap1 expression in these developmental stages. Further, tap1 expression levels were significantly enhanced in response to wounding in breaker- and red-ripe stages of transgenic fruits, whereas wound-induced expression of tap1 was not detected in WT fruits. Confocal microscopy revealed high accumulation of phenolic compounds at the wound site in transgenic fruits suggesting a role of tap1 in wound-induced phenolic polymerization. Total peroxidase activity has increased remarkably in transgenic pericarp tissues in response to wounding, while very less or minimal levels were recorded in WT pericarp tissues. Transgenic fruits also displayed reduced post-harvest decay and increased resistance toward Alternaria alternata and Fusarium solani infection with noticeable inhibition in lesion formation. Conidiospore germination and mycelial growth of F. solani were severely inhibited when treated with E8-tap1 fruit extracts compared to WT fruits. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed reduced spore viability when incubated in E8-tap1 fruit extracts. Thus, fruit-specific expression of tap1 using E8 promoter is associated with enhanced total peroxidase activity and high phenolic accumulation in fruits with minimized post-harvest deterioration caused by wounding and fungal attack in tomato fruits.     

Ripening-related changes in raspberry cell wall composition and structure

Cell walls were prepared from the fruit of two cultivars of raspberry at three stages of ripening; green, white and red (ripe). The cultivars. Glen Clova and Glen Prosen, are subjectively classified, at harvest by growers, as soft and firm fruit, respectively. The cell walls were analysed for neutral sugar composition, uronic acid content, degree of methyl esterification, lignin and ferulic acid-derived dehydrodimers. Solid-state 31C NMR and diffuse reflectance infrared (DRIFT) spectra were acquired for the cell wall residues. For both cultivars the progression from green to white produced minimal changes, save for a reduction in pectin. NMR analyses indicated that the solubilized pectin was acetylated. Progression to the red (ripe) stage, in both cultivars, was accompanied by a reduction in the ordered cellulose and a dramatic reduction in pectin content and the degree of methyl-esterification. Significantly, the softer fruit (Glen Clova) exhibited greater reductions in both parameters, implicating increased pectin hydrolysis, as one of the main factors contributing to the difference in firmness between the cultivars. A relative increase in cell wall-associated protein was seen at the red stage. The nature and function of the protein(s) are, as yet unknown.     

不同成熟度菠萝贮藏特性的差异

随着菠萝果皮由绿转黄,果肉硬度及可溶性固形物含量降低,可溶性糖醛酸含量增加,多聚半乳糖醛酸酶(PG)活性持续升高,而果胶脂酶(PE)活性在过熟期达到高峰并开始下降。研究表明,同一菠萝不同部位果肉硬度、PG活性及可溶性糖醛酸含量差异较大。     

Silencing of SlPL,which encodes a pectate lyase in tomato,confers enhanced fruit firmness,prolonged shelf‐life and reduced susceptibility to grey mould

Pectate lyase genes have been documented as excellent candidates for improvement of fruit firmness. However, implementation of pectate lyase in regulating fruit postharvest deterioration has not been fully explored. In this report, 22 individual pectate lyase genes in tomato were identified, and one pectate lyase gene SlPL (Solyc03g111690) showed dominant expression during fruit maturation. RNA interference of SlPL resulted in enhanced fruit firmness and changes in pericarp cells. More importantly, the SlPL‐RNAi fruit demonstrated greater antirotting and pathogen‐resisting ability. Compared to wild‐type, SlPL‐RNAi fruit had higher levels of cellulose and hemicellulose, whereas the level of water‐soluble pectin was lower. Consistent with this, the activities of peroxidase, superoxide dismutase and catalase were higher in SlPL‐RNAi fruit, and the malondialdehyde concentration was lower. RNA‐Seq results showed large amounts of differentially expressed genes involved in hormone signalling, cell wall modification, oxidative stress and pathogen resistance. Collectively, these data demonstrate that pectate lyase plays an important role in both fruit softening and pathogen resistance. This may advance knowledge of postharvest fruit preservation in tomato and other fleshy fruit.     

Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit

Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO₂ and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit.     

Antisense suppression of a beta-galactosidase gene (TB G6) in tomato increases fruit cracking

Antisense suppression of a tomato beta-galactosidase gene (TBG6) was used to study its role in fruit development, cell wall-modification, and fruit firmness. TBG6 mRNA is highly abundant during the early stages of fruit development, but the levels decline sharply after the breaker stage with the start of the respiratory climacteric and a concomitant increase in ethylene production. Two antisense lines were obtained with significantly reduced levels of TBG6 mRNA at all stages of fruit development. At 30 d after pollination (dap), TBG6 mRNA levels were reduced by up to 98% and 88% in lines 6-2 and 6-10, respectively. Morphological phenotypes observed in the antisense lines included increased fruit cracking, reduced locular space, and a doubling in the thickness of the fruit cuticle. Two biochemical changes in antisense lines, compared with wild-type lines, were a reduction of exo-galactanase activity at the breaker +3 d stage and a reduction in the cell wall galactosyl content at the 20 dap stage. In addition, transgenic lines exhibited a 35-39% reduction in fruit firmness at the 20 dap stage, but their texture was equivalent to the wild type at 30 dap and beyond. Although the exact function of the TBG6 product is still unknown, these results implicate an important role for this enzyme in early fruit growth and development in tomato.     

Physiology and firmness determination of ripening tomato fruit

Tomato ( Lycopersicon esculentum Mill.) genotypes varying in intrinsic firmness were examined to determine the quantitative relationships between polygalacturonase (EC 3.2.1.15) activity, firmness and other ripening parameters including rate (days from mature-green to full red) and intensity (rate of ethylene production at climacteric peak) of ripening. Texture, respiration and ethylene production were monitored in the immature-green through the red (ripe) stages of development. Polygalacturonase activity was measured by direct assay of salt-extractable wall protein or by monitoring the release of pectins from isolated, enzymically active wall. In all fruit, polygalacturonase activity was highly correlated with pericarp softening, but only moderately correlated with softening of whole fruit (r = 0.920 and 0.757, respectively). Polygalacturonase activity was positively correlated with cell-wall autolytic activity in pink (r = 0.969) and red (r = 0.900) fruit. Firmer genotypes exhibited lower rates of respiration and ethylene production during ripening. Polygalacturonase activity in isolates prepared from fruit at the climacteric peak was positively correlated with ethylene production and respiration, and negatively correlated with days to ripening (r = 0.929, 0.805, and -0.791, respectively). The data demonstrate the importance of selecting the appropriate method of firmness determination and are consistent with the hypothesis that pectin fragments released by polygalacturonase contribute to the production of autocatalytic (system II) ethylene.     

Regulatory Role of Cystathionine-γ-Synthase and de novo Synthesis of Methionine in Ethylene Production during tomato Fruit Ripening

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the “turning” stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.     

Partial demethylation of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence responses in wild strawberry (Fragaria vesca)

In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGA), are essential components for elicitation of defence responses. To investigate how modifications of pectin alter defence responses, we expressed the fruit-specific Fragaria  ×  ananassa pectin methyl esterase FaPE1 in the wild strawberry Fragaria vesca . Pectin from transgenic ripe fruits differed from the wild-type with regard to the degree and pattern of methyl esterification, as well as the average size of pectin polymers. Purified oligogalacturonides from the transgenic fruits showed a reduced degree of esterification compared to oligogalacturonides from wild-type fruits. This reduced esterification is necessary to elicit defence responses in strawberry. The transgenic F. vesca lines had constitutively activated pathogen defence responses, resulting in higher resistance to the necrotropic fungus Botrytis cinerea . Further studies in F. vesca and Nicotiana benthamiana leaves showed that the elicitation capacity of the oligogalacturonides is more specific than previously envisaged.     

Altered cell wall disassembly during ripening of Cnr tomato fruit: implications for cell adhesion and fruit softening

The Cnr ( C olourless n on- r ipening) tomato ( Lycopersicon esculentum Mill.) mutant has an aberrant fruit-ripening phenotype in which fruit do not soften and have reduced cell adhesion between pericarp cells. Cell walls from Cnr fruit were analysed in order to assess the possible contribution of pectic polysaccharides to the non-softening and altered cell adhesion phenotype. Cell wall material (CWM) and solubilised fractions of mature green and red ripe fruit were analysed by chemical, enzymatic and immunochemical techniques. No major differences in CWM sugar composition were detected although differences were found in the solubility and composition of the pectic polysaccharides extracted from the CWM at both stages of development. In comparison with the wild type, the ripening-associated solubilisation of homogalacturonan-rich pectic polysaccharides was reduced in Cnr. The proportion of carbohydrate that was chelator-soluble was 50% less in Cnr cell walls at both the mature green and red ripe stages. Chelator-soluble material from ripe-stage Cnr was more susceptible to endo-polygalacturonase degradation than the corresponding material from wild-type fruit. In addition, cell walls from Cnr fruit contained larger amounts of galactosyl- and arabinosyl-containing polysaccharides that were tightly bound in the cell wall and could only be extracted with 4 M KOH, or remained in the insoluble residue. The complexity of the cell wall alterations that occur during fruit ripening and the significance of different extractable polymer pools from cell walls are discussed in relation to the Cnr phenotype.     

Changes in the cell-wall polysaccharides of outer pericarp tissues of kiwifruit during development.

Changes in pectin, hemicelluloses and cellulose in the cell walls of outer pericarp tissues of kiwifruit (Actinidia deliciosa cv. Hayward) were determined during development. An extensive amylase digestion was employed to remove possible contaminating starch before and after fractionation of wall polysaccharides. An initial treatment of crude cell walls with alpha-amylase and iso-amylase or DMSO, was found to be insufficient removing the contaminating starch from wall polysaccharides. After EDTA and alkaline extraction, the pectic and hemicellulose fractions were again treated with the combination of alpha-amylase and iso-amylase. The amounts of predominant pectic sugars Gal, Rha and Ara, unaffected by the first and second amylase digestion, decreased markedly during the early fruit enlargement (8-12 weeks after anthesis, WAA), then increased during 16-20 WAA, and finally declined during fruit maturity (20-25 WAA). The molecular-mass of pectic polysaccharides decreased during fruit enlargement (8-16 WAA), and then changed little during fruit maturity. The higher molecular-mass components of hemicelluloses in HC-I and HC-II fractions detected at the early stage of fruit enlargement (8-12 WAA) were degraded at the late stage of fruit enlargement (16 WAA), but then remained stable at the much lower molecular-mass till fruit maturity. The amount of Xyl in the HC-II fraction decreased during the early fruit enlargement and fruit maturity, an observation that was consistent with xyloglucan (XG) content. The gel permeation profiles of XG showed a slight increase in higher molecular-mass components during 8-12 WAA, but thereafter there was no significant down-shift of molecular-mass until harvest time. The cellulose fraction increased steadily during fruit enlargement through maturity, but the XG contents in HC-I and HC-II fractions remained at a low level during these stages. Methylation analysis of HC-I and HC-II fractions confirmed the low level of XG in the hemicellulosic fractions. It was suggested that pectin in the outer pericarp of kiwifruit was degraded at the early stage of fruit enlargement, but XG remains constant during fruit enlargement and maturation.     

Pectin methylesterases induce an abrupt increase of acidic pectin during strawberry fruit ripening

The decrease of strawberry (Fragariaxananassa Duch.) fruit firmness observed during ripening is partly attributed to pectolytic enzymes: polygalacturonases, pectate lyases and pectin methylesterases (PMEs). In this study, PME activity and pectin content and esterification degree were measured in cell walls from ripening fruits. Small green, large green, white, turning, red and over-ripe fruits from the Elsanta cultivar were analyzed. Using the 2F4 antibody directed against the calcium-induced egg box conformation of pectin, we show that calcium-bound acidic pectin was nearly absent from green and white fruits, but increased abruptly at the turning stage, while the total pectin content decreased only slightly as maturation proceeded. Isoelectrofocalisation performed on wall protein extracts revealed the expression of at least six different basic PME isoforms. Maximum PME activity was detected in green fruits and steadily decreased to reach a minimum in senescent fruits. The preliminary role of PMEs and subsequent pectin degradation by pectolytic enzymes is discussed.     

Molecular cloning of a ripening-specific lipoxygenase and its expression during wild-type and mutant tomato fruit development.

A 94-kD protein that accumulates predominately in tomato (Ly-copersicon esculentum) fruit during ripening was purified, and antibodies specific for the purified protein were used to isolate cDNA clones from a red-ripe fruit cDNA library. A sequence analysis of these cDNAs and cross-reactivity of the 94-kD-specific antibodies to the soybean lipoxygenase (LOX) L-1, L-2, and L-3 proteins and soybean LOX L-1-specific antibodies to the 94-kD protein identified it as a member of the LOX gene family. Maximum levels of the 94-kD LOX mRNA and protein are present in breaker to ripe and red-ripe stages, respectively. Expression of 94-kD LOX in different tissues from mature green and red-ripe tomato fruits was found to be greatest in the radial walls of ripe fruit, but immunocytolocalization using tissue printing suggests that the highest accumulation of its protein occurs in locular jelly. None of 94-kD LOX is expressed in nonripening mutant fruits of any age. Never-ripe mutant fruit accumulate the 94-kD LOX mRNA to levels similar to those obtained in wild-type fruit, but fail to accumulate the 94-kD LOX protein. Collectively, the results show that expression of 94-kD LOX is regulated by the ripening process, and ethylene may play a role in its protein accumulation.     

Chlorophyll a fluorescence, enzyme and antioxidant analyses provide evidence for the operation of alternative electron sinks during leaf senescence in a stay-green mutant of Festuca pratensis

Mutation of the sid gene in Festuca pratensis prevents chlorophyll degradation. The senescing leaves retain their chlorophyll complement and stay green. Nevertheless, CO2 assimilation and ribulose-bisphosphate carboxylase/oxygenase content decline in both mutant and wild-type plants. Photosynthesis and chlorophyll a fluorescence measurements were performed in air and at low oxygen to prevent photorespiration. The maximum extractable activity of ribulose 1,5 bisphosphate carboxylase was higher in the senescent mutant leaves than in those of the wild-type control hut Mas much lower than that observed in the mature leaves of either genotype. The activation state of this enzyme was similar in mutant and wild-type lines at equivalent stages of development. Analysis of chlorophyll a fluorescence quenching with varying irradianco showed similar characteristics for mature leaves of the two genotypes. Genotypic variations in photosystem II (I'SII) efficiency were observed only in the senescent leaves. Photochemical quenching and the quantum efficiency of PSII were greater in the senescent mutant leaves than in (he wild type at a given irradiance. The calculated electron flux through PSII was substantially higher in the mutant with a greater proportion of electrons directed to photorespiration. Maximum catalytic activities of ascorbate peroxidase decreased in senescent compared to mature leaves of both genotypes, while glutathione reductase and monodehydroascorbate reductase were unchanged in both cases. Superoxide dismutase activity was approximately doubled and dehydroascorbate reductase activity was three times higher in senescent leaves compared with the mature leaves of both genotypes. In no case was there a difference in enzyme activities between mutant and wild type at equivalent growth stages. The pool of reduced ascorbate was similar in the mature leaves of the two genotypes, whereas it was significantly higher in the senescent leaves of the mutant compared with the wild type. Conversely, the hydrogen peroxide content was significantly higher in the mature leaves of the wild type than in those of the mutant, but in senescent leaves similar values were obtained. In leaves subjected to chilling stress the reduced ascorbate pool was higher in both mature and senescent leaves of the mutant than in their wild-type counterparts. Similarly, the hydrogen peroxide pool was significantly lower in both mature and senescent leaves of the mutant than in the wild type. We conclude that, in spite of deceased CO₂ assimilation, the mutant is capable of high rates of electron Slow. The high ascorbate/hydrogen peroxide ratio observed in the mutant, particularly at low temperatures, suggests that the senescent leaves are not subject to enhanced oxidative stress.