Dual role for tomato heat shock protein 21: protecting photosystem II from oxidative stress and promoting color changes during fruit maturation

The tomato (Lycopersicon esculentum) chloroplast small heat shock protein (sHSP), HSP21, is induced by heat treatment in leaves, but also under normal growth conditions in developing fruits during the transition of chloroplasts to chromoplasts. We used transgenic tomato plants constitutively expressing HSP21 to study the role of the protein under stress conditions and during fruit maturation. Although we did not find any effect for the transgene on photosystem II (PSII) thermotolerance, our results show that the protein protects PSII from temperature-dependent oxidative stress. In addition, we found direct evidence of the protein's role in fruit reddening and the conversion of chloroplasts to chromoplasts. When plants were grown under normal growth temperature, transgenic fruits accumulated carotenoids earlier than controls. Furthermore, when detached mature green fruits were stored for 2 weeks at 2 degrees C and then transferred to room temperature, the natural accumulation of carotenoids was blocked. In a previous study, we showed that preheat treatment, which induces HSP21, allowed fruit color change at room temperature, after a cold treatment. Here, we show that mature green transgenic fruits constitutively expressing HSP21 do not require the heat treatment to maintain the ability to accumulate carotenoids after cold storage. This study demonstrates that a sHSP plays a role in plant development under normal growth conditions, in addition to its protective effect under stress conditions.     

Changes in peel pigmentation during ripening of mango fruit (Mangifera indica var. Tommy Atkins)

The pigments in the peel of Tommy Atkins mango were analysed at six stages during ripening at 22 ^oC. The loss of green colour and the development of yellow colouration was associated with an almost complete loss of chlorophyll and an increase in carotenoids. Anthocyanin content showed a slight decrease during ripening. An ultrastructural study showed plastids in green fruit with a well developed grana network system. On ripening the chloroplasts underwent extensive disorganisation which was associated with the development of large osmiophilic globules.     

Possible Mechanism of the Detached Unripe Green Tomato Fruit Turning Red

In general, a mature green tomato will ripen and turn red on the vine or off the vine. Interestingly, an unripe green (UG) tomato also will turn red after being detached from the plant, but the mechanism behind this is unclear. Our study showed that detached UG fruits were able to become red-ripened at the room temperature (25?°C), although fruit quality was lower than that of the vine-ripened fruit. In addition, detached UG fruits exposed to light accumulated more lycopene and total soluble sugars than those incubated in darkness. When the detached UG fruits were stored at a low temperature (10?°C), the fruit-ripening process was nearly blocked, which displayed a non-ripening phenotype. At a high temperature (35?°C), the detached UG fruit showed a yellow-color on ripening, and fruit qualities such as lycopene and soluble sugars were obviously lower than those stored at 25?°C. Moreover, detaching the UG fruit from the plant evoked a rapid increase in total respiration as well as alternative pathway respiration, but ethylene production was not stimulated during the first 24 h of storage. Importantly, the application of nPG, an inhibitor of alternative pathway respiration, markedly suppressed the wound-induced rise in total respiration and delayed the ripening of detached UG fruit. These findings imply that wound-induced respiration might be a signature for launching the onset of the ripening of detached UG fruit, and the optimal conditions of light and temperature are beneficial for the color change and the ripening processes.     

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.     

Anthocyanin, Carotenoid, and Chlorophyll Changes in the Peel of Cox's Orange Pippin Apples during Ripening on and off the Tree

The concentrations of various peel pigments of Cox’s OrangePippin apples have been measured during ripening on the treeand during storage at 12 °C. Total chlorophyll decreased and total carotenoid increased atthe time of the respiration climacteric. These changes weremore pronounced in fruit maturing on the tree where a significantincrease of anthocyanin occurred; it did not occur in storedfruit. There was no consistent or marked difference in the ratesof destruction of chlorophylls a and b. The carotenoids found in the unripe fruit were those characteristicof photosynthetic tissue, ß-carotene, lutein, violaxanthin,and neoxanthin. These decreased to a greater or lesser extent,and at different rates, on and off the tree. Other carotenoidswhich increased greatly during ripening were identified as esters,mainly of violaxanthin. During the climacteric there is a transition from an assemblageof pigments associated with the chloroplast to that typicalof a chromoplast.     

Changes in chlorophyll,polyamines and chloroplast ultrastructure of Puccinia striiformis induced ‘green islands’ on detached leaves of Triticum aestivum

Biochemical and ultrastructure features of ‘green islands’ were investigated using detached wheat leaves infected with the yellow rust Puccinia striiformis. Chlorophylls appear to culminate 10 d after inoculation at which point ‘green islands’ were fully developed. These changes were paralleled by an increase in spermidine and spermine content which play an important role in formation of ‘green islands’. Retention of chlorophyll was demonstrated in leaf tissues of wheat plants supplied with exogenous putrescine, spermidine and spermine. Putrescine was least and spermidine and spermine most effective in retarding loss of chlorophylls. Ultrastructural observation revealed that chloroplasts were regenerated in ‘green islands’ where many proplastids were detected. The regeneration of chloroplasts coincided with the high concentration of chlorophylls and polyamines particularly spermidine and spermine. The ultrastructural changes of chloroplasts in leaf cell containing infection structures were parallel to physiological changes.     

Patterns of pigment changes in apple fruits during adaptation to high sunlight and sunscald development

Reflectance spectra of four apple (Malus domestica Borkh.) cultivars were studied and chlorophyll, carotenoid, anthocyanin and flavonoid content in sunlit and shaded peel was determined. In all cases sunlit peel accumulated high amounts of phenolics (flavonoid glycosides). Adaptation to strong sunlight of an apple cultivar with limited potential for anthocyanin biosynthesis (Antonovka) was accompanied by a decrease in chlorophyll and a significant increase in total carotenoid content. The increase in carotenoids also took place in sunlit sides of the Zhigulevskoye fruits, accumulating high amounts of anthocyanins, but chlorophyll content in sunlit peel was higher than that in shaded peel. Significant increases in carotenoids and anthocyanins were detected during fruit ripening when chlorophyll content fell below 1.5–1.8 nmol cm^–2. Chlorophyll in sunlit fruit surfaces of both cultivars was considerably more resistant to photobleaching than in shaded (especially of Zhigulevskoye) sides. Induced by sun irradiation, the photoadaptive responses were cultivar-dependent and expressed at different stages of fruit ripening even after storage in darkness. The development of sunscald symptoms in susceptible apple cultivars (Granny Smith and Renet Simirenko) led to a dramatic loss of chlorophylls and carotenoids, which was similar to that observed during artificial photobleaching. The results suggest that apple fruits exhibit a genetically determined strategy of adaptation of their photoprotective pigments to cope with mediated by reactive oxygen species photodynamic activity of chlorophyll under strong solar irradiation. This includes induction of synthesis and accumulation of flavonoids, anthocyanins and carotenoids that could be expressed, if necessary, at different stages of fruit development     

Galactolipids and phospholipids of orange peel and juice chromoplasts

Chromoplasts from yellow orange (Citrus sinensis) fruit peel contain monogalactosyl diglycerides (MGDG), digalactosyl diglycerides (DGDG) and phosphatidyl glycerol (PG) in amounts similar to those found in chloroplasts from green fruit peel. Juice chromoplasts contain relatively little MGDG and no DGDG with high levels of phosphatidyl choline and phosphatidyl ethanolamine but no PG.     

Partial Restoration of the High Rate of Plastid Pigment Development and the Ultrastructure of Plastids in Detached Water-stressed Wheat Leaves

Detached etiolated wheat (Triticum aestivum L. cv. Chris) leaves accumulated plastid pigments at a high rate, developed chloroplasts with stacked thylakoids, and stored plastid starch when wetted on filter paper in light. A moderate water deficit of — 10 bars markedly reduced the accumulation of chlorophyll and carotenoids in the 8-day-old detached leaves during greening. δ-Aminolevulinic acid treatment of stressed leaf segments resulted in slightly increased pigment accumulations but benzyladenine application restored plastid pigment formation in stressed tissue to within 15% of the pigment content of the nonstressed detached leaves. The addition of δ-aminolevulinic acid to benzyladenine-treated stressed leaf segments improved both chlorophyll and carotenoid formation to nearly the amounts found in nonstressed leaf tissue. Stressed leaf sections developed plastids that were small, lacked starch, contained few thylakoids per granum, and possessed dilated thylakoids. Benzyladenine application to the stressed leaf segments did not restore normal plastid stacking but benzyladenine induced the formation of extended intergranal lamellae and stimulated pigment accumulations in both stressed and nonstressed detached leaves. Starch was absent in plastids of benzyladeninetreated leaf sections.     

Chloroplast to chromoplast transition in tomato fruit: spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated plastids and time-lapse recording on intact live tissue

Background and Aims

There are several studies suggesting that tomato (Solanum lycopersicum) chromoplasts arise from chloroplasts, but there is still no report showing the fluorescence of both chlorophylls and carotenoids in an intermediate plastid, and no video showing this transition phase.

Methods

Pigment fluorescence within individual plastids, isolated from tomato fruit using sucrose gradients, was observed at different ripening stages, and an in situ real-time recording of pigment fluorescence was performed on live tomato fruit slices.

Key results

At the mature green and red stages, homogenous fractions of chloroplasts and chromoplasts were obtained, respectively. At the breaker stage, spectral confocal microscopy showed that intermediate plastids contained both chlorophylls and carotenoids. Furthermore, an in situ real-time recording (a) showed that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell; and (b) confirmed that all chromoplasts derived from pre-existing chloroplasts.

Conclusions

These results give details of the early steps of tomato chromoplast biogenesis from chloroplasts, with the formation of intermediate plastids containing both carotenoids and chlorophylls. They provide information at the sub-cellular level on the synchronism of plastid transition and pigment changes.     

Sucrose deficiency delays lycopene accumulation in tomato fruit pericarp discs

Tomato (Solanum lycopersicum) fruit ripening is characterized by a massive accumulation of carotenoids (mainly lycopene) as chloroplasts change to chromoplasts. To address the question of the role of sugars in controlling carotenoid accumulation, fruit pericarp discs (mature green fruits) were cultured in vitro in the presence of various sucrose concentrations. A significant difference in soluble sugar content was achieved depending on external sucrose availability. Sucrose limitation delayed and reduced lycopene and phytoene accumulation, with no significant effect on other carotenoids. Chlorophyll degradation and starch catabolism were not affected by variations of sucrose availability. The reduction of lycopene synthesis observed in sucrose-limited conditions was mediated through metabolic changes illustrated by reduced hexose accumulation levels. In addition, variations of sucrose availability modulated PSY1 gene expression. Taken together our results suggest that the modulation of carotenoid accumulation by sucrose availability occurs at the metabolic level and involves the differential regulation of genes involved in carotenoid biosynthesis.     

Effect of Temperature on Photosynthetic Activities of Senescing Detached Wheat Leaves

Changes in various components of photosynthetic activity duringthe dark induced senescence of detached wheat leaves, maintainedat 25°C (control) and 35°C (mildly elevated temperaturetreatment), were examined. Senescence-associated decline measuredup to 96 h, in photosynthetic activity was appreciably hastenedat 35°C, than at 25°C as evident by the relative higherlosses of chlorophyll, photosystem (PS) II and PS I catalyzedphotochemical activities and ribulose-1,5-bisphosphate (RuBP)carboxylase activity. In addition, a comparatively higher risein light scattering profile of isolated chloroplasts was notedat 35°C than at 25°C. Senescence-induced degradationof chlorophyll was faster at 35°C than at 25°C; on theother hand, the degradation of carotenoids was faster at 25°Cthan at 35°C. Furthermore, the ratio of carotenoids to chlorophyllincreased with senescence up to 96 hours, higher ratio beingobtained at 35°C than at 25°C. Both PS II and PS I activitiesshowed a transient rise in the beginning phase of dark incubation,whereas loss in chlorophyll was continuous throughout the periodof senescence. The initial rise observed in photochemical activitieswas attributable to the uncoupling of electron transport fromphotophosphorylation. Elevated temperature treatment resultedin greater inactivation of RuBP carboxylase than control. Itappears that during senescence the loss in chlorophyll and RuBPcarboxylase activity are triggered simultaneously. (Received June 7, 1985; Accepted October 30, 1985)     

An ATP synthase harboring an atypical γ–subunit is involved in ATP synthesis in tomato fruit chromoplasts

Chromoplasts are non‐photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes in non‐photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report the involvement of a plastidial ATP synthase harboring an atypical γ–subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast γ–subunits. Silencing of this atypical γ–subunit during fruit ripening impairs the capacity of isolated chromoplast to synthesize ATP de novo. We propose that the replacement of the γ–subunit present in tomato leaf and green fruit chloroplasts by the atypical γ–subunit lacking the dithiol domain during fruit ripening reflects evolutionary changes, which allow the operation of chromoplast ATP synthase under the particular physiological conditions found in this organelle.     

Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing

Virus-induced gene silencing (VIGS) is a technology that has rapidly emerged for gene function studies in plants. Many advances have been made in applying this technique in an increasing number of crops. Recently, VIGS has been successfully used to silence genes in tomato fruit through agroinfiltration of fruit attached to the plant. The phytoene desaturase (Pds) gene has been widely used as a reporter gene in VIGS experiments, although little is known about the changes that occur due to its silencing in plants. In this paper, we describe the efficient silencing of the Pds gene through the VIGS approach in detached tomato fruits, which makes the VIGS procedure even more versatile and applicable. After 16 days of agroinfiltration, approximately 75% of the tomatoes showed Pds silencing symptoms, although the distribution of silenced areas was variable among fruits. To study the potential effects caused by Pds silencing in detached tomatoes, carotenoids and other semi-polar secondary metabolites were analyzed using Liquid Chromatography-Mass Spectrometry. In addition, potential differences in primary metabolites were analyzed using Gas Chromatography-Mass Spectrometry. The results indicated that the yellow phenotype observed in Pds-silenced fruit was mainly due to the lack of the red-colored lycopene and therefore to a more pronounced contribution of the yellow-orange carotenoids (lutein, violaxanthin, and zeaxanthin) to the final color of the fruits. Furthermore, the biochemical changes observed in Pds-silenced detached tomatoes suggested that carotenoid and other pathways, e.g. leading to alkaloids and flavonoids, might be affected by the silencing of this reporter gene, and this should be taken into consideration for future experimental designs.     

A Comparative Study of Chlorophyll Production in Cotyledons

Chlorophyll production was studied in four species with cotyledonsdiffering widely in photosynthetic potential. When cotyledons were detached from dark-grown seedlings at intervalsduring germination and exposed to light for 24–48 h, maximumchlorophyll production was found after 8 days of dark-germinationin french bean, runner bean, and lupin, and after 5 days inmustard. Chlorophyll formation was reduced when the cotyledonswere detached earlier or later than this time, and during thefirst 1–2 days of germination the cotyledons producedvery little chlorophyll. With later times of excision the abilityto accumulate chlorophyll increased, rapidly in lupin and mustardand more slowly in the beans. A comparison of chlorophyll accumulation by attached cotyledonsand those excised during the first 24 h of germination showeda fourfold enhancement of chlorophyll content in detached french-beancotyledons and a fourfold reduction in chlorophyll content indetached mustard cotyledons, whilst chlorophyll accumulationwas similar in both the attached and detached cotyledons oflupin. The results are discussed in relation to the possible promotiveand inhibitory influence of the seedling axis on greening ofcotyledons differing in their potential for expansion and inthe amount of reserve material stored.     

Physiological changes associated with senescence and abscission in mature citrus fruit induced by 5-chloro-3-methyl-4-nitro-1H-pyrazole and ethephon application

This research compares effects of the compound 5-chloro-3-methyl-4-nitro-1 H -pyrazole (CMNP), a plant growth regulator that selectively promotes abscission in mature citrus fruit ( Citrus sinensis ), and the ethylene-releasing agent ethephon (2-chloroethylphosphonic acid). Application of CMNP and ethephon to mature citrus fruit reduced fruit detachment force and changed peel color from green to orange. More total chlorophyll was extracted from flavedo in early season (November) than late season (January), and both compounds caused a similar reduction in chlorophyll. In contrast, total carotenoid content was similar in November and January. Both abscission compounds increased total carotenoids, but induction was greater in January, and CMNP was more effective in both months. Phospholipase A₂ (PLA₂) activity increased after CMNP but not ethephon application. Electrolyte leakage increased 2 h after CMNP treatment, and total protein content was reduced by 50% after 72 h. Ethephon caused only minor changes in electrolyte leakage and total protein content. Inhibition of PLA₂ activity with aristolochic acid did not reduce leakage but inhibited total protein loss and reduced visual peel damage associated with CMNP. Ultrastructural observations indicated decreased number, and length of starch grains 3 h after CMNP treatment. A transient increase in soluble sugars was measured 3 h after CMNP application. Ethephon had little effect on soluble sugar content and changes in starch grains. Collectively, the results indicate that CMNP and ethephon induced color change in peel and advanced mature fruit abscission. However, CMNP but not ethephon promoted other physiological changes associated with senescence.     

Carotenoid Accumulation and Carotenogenic Genes Expression During Two Types of Persimmon Fruit (<Emphasis Type="Italic">Diospyros kaki</Emphasis> L.) Development

Persimmon (Diospyros kaki L.), belonging to the Ebenaceae family, has been used not only as a fresh fruit, but also for many medicinal uses. Carotenoids are the main pigment in persimmon fruit, which contribute significantly to fruit color and nutritional quality due to their composition and content. In this study, fruit quality indices, carotenoid contents and expression of carotenogenic genes were analyzed in two types of persimmon fruit. The results demonstrated that there was a positive correlation between fruit color and the contents of main composition and total carotenoids. Carotenoid accumulation in persimmon fruit resulted from the interaction of carotenogenic genes, but the molecular mechanisms responsible for accumulation of carotenoids in two types of persimmon fruit had a few differences. As a complete unit, the relatively low expression level of phytoene synthase gene (DkPSY) in “Niuxinshi” resulted in low carotenoid contents or even under the detection limit at the early fruit developmental stages; but low carotenoid contents in “Nishimurawase” were due to the relatively low expression level of carotenogenic genes other than DkPSY. At the late fruit developmental stages, increased expression levels of DkPSY, phytoene desaturase gene and beta-carotene hydroxylase gene (DkBCH) induced elevated carotenoid contents; because all carotenogenic genes strongly expressed in “Nishimurawase”, a large amount of carotenoids were accumulated. In addition, β-cryptoxanthin was the main composition whose content increased with the fruit maturity changes, which was mainly because of DkBCH which might lead more conversion of β-carotene to β-cryptoxanthin.     

Polyamine content and chilling susceptibility are affected by seasonal changes in temperature and by conditioning temperature in cold-stored 'Fortune' mandarin fruit

‘Fortune’ mandarins are prone to develop pitting and necrosis upon exposure to low temperatures. We have examined the effect of field temperature during fruit maturation and the effect of conditioning temperatures (from 2 to 37°C) prior to cold storage on the content of polyamines (PAs) and on chilling susceptibility in order to understand the role of PAs in maturation and chilling tolerance of this citrus cultivar. Chilling susceptibility and the content of PAs were more affected by seasonal changes in field temperature than by the stage of fruit maturity. The highest putrescine (Put) and spermidine (Spd) content was found in fruits exposed to the lowest field temperatures. These fruits were in turn more susceptible to develop chilling injury (CI) after storage at 2°C. Spermine (Spm), however, decreased in attached fruit with time of exposure to temperatures below 12°C. Temperature pretreatments for 3 days above 20°C of fruits detached from the tree reduced CI, the more so the higher the conditioning temperature. Put and Spd increased with temperature conditioning in detached fruits, differing from the response of fruits attached to the tree. No direct relationship between induced levels of these PAs and the tolerance to CI was found. Levels of Put and Spd increased at temperatures (22, 30 and 37°C) which increased the tolerance and also at temperatures (6 and 12°C) which accelerated the appearance of chilling symptoms. In contrast, a significant increase in Spm levels was only found after conditioning at 30 or 37°C. After cold storage a general decline in PA levels occurred in all temperature‐conditioned mandarins. In most cases no significant differences among fruit exposed to effective and non‐effective pretreatments were observed. PA content increased again after transferring cold‐stored fruits to 20°C, whereas the CI index was barely affected. In conclusion, PA changes in the flavedo of ‘Fortune’ mandarins appear to be related to variations in temperature rather than to stage of maturity or tolerance to chilling.