Chlorophyllase (PsCLH1) and light-harvesting chlorophyll a/b binding protein 1 (PsLhcb1) and PsLhcb5 maintain petal greenness in Paeonia suffruticosa 'Lv Mu Yin Yu'.

INTRODUCTION: Green flowers are not an adaptive trait in natural plants due to the challenge for pollinators to discriminate from leaves, but they are valuable in horticulture. The molecular mechanisms of green petals remain unclear. Tree peony (Paeonia suffruticosa) is a globally cultivated ornamental plant and considered the 'King of Flowers' in China. The P. suffruticosa 'Lv Mu Yin Yu (LMYY)' cultivar with green petals could be utilized as a representative model for understanding petal-specific chlorophyll (Chl) accumulation and color formation. OBJECTIVES: Identify the key genes related to Chl metabolism and understand the molecular mechanism of petal color changes. METHODS: The petal color parameter was analyzed at five developmental stages using a Chroma Spectrophotometer, and Chl and anthocyanin accumulation patterns were examined. Based on comparative transcriptomes, differentially expressed genes (DEGs) were identified, among which three were functionally characterized through overexpression in tobacco plants or silencing in 'LMYY' petals. RESULTS: During flower development and blooming, flower color changed from green to pale pink, consistent with the Chl and anthocyanin levels. The level of Chl demonstrated a similar pattern with petal epidermal cell striation density. The DEGs responsible for Chl and anthocyanin metabolism were characterized through a comparative transcriptome analysis of flower petals over three critical developmental stages. The key chlorophyllase (PsCLH1) and light-harvesting chlorophyll a/b binding protein 1 (PsLhcb1) and PsLhcb5 influenced the Chl accumulation and the greenness of 'LMYY' petals. CONCLUSION: PsCLH1, PsLhcb1, and PsLhcb5 were critical in accumulating the Chl and maintaining the petal greenness. Flower color changes from green to pale pink were regulated by the homeostasis of Chl degradation and anthocyanin biosynthesis. This study offers insights into underlying molecular mechanisms in the green petal and a strategy for germplasm innovation.

5-Azacytidine accelerates mandarin fruit post-ripening and enhances lignin-based pathogen defense through remarkable gene expression activation.

5-Azacytidine (AZ) is a DNA methylation inhibitor that has recently demonstrated potential in regulating fruit quality through exogenous application. In this study, we treated mandarin fruits for 4-day storage. Noteworthy were the induced degreening and the enhanced citrus aroma of fruits under AZ treatment, involving the promotion of chlorophyll degradation, carotenoid biosynthesis, and limonene biosynthesis. Key genes associated with these processes exhibited expression level increases of up to 123.8 times. Additionally, AZ treatment activated defense-related enzymes and altered phenylpropanoid carbon allocation towards lignin biosynthesis instead of flavonoid biosynthesis. The expression levels of lignin biosynthesis-related genes increased by nearly 100 times, leading to fortified lignin that is crucial for citrus defense against Penicillium italicum. Currently, the underlying mechanisms of such intense AZ-induced changes in gene expressions remain unclear and further research could help establish AZ treatment as a viable strategy for citrus preservation.

The response of Midknight Valencia oranges to ethephon degreening varies in the turning and regreening stages.

BACKGROUND: Late-ripening citrus plays an important role in the stability of the global citrus industry. However, the regreening phenomenon in Valencia oranges impacts the peel color and commercial value. Ethylene degreening is an effective technique to improve the color of citrus fruits, but this effect may be delayed in regreened oranges. To better clarify this phenomenon, plastid morphology, pigment and phytohormone content in ethephon-degreened Midknight Valencia oranges harvested in different stages were evaluated. RESULTS: Results showed that in fruits harvested at the turning stage, ethephon degreening treatment induced a chloroplast-to-chromoplast transition, and chlorophyll degradation and carotenoid accumulation were accelerated. Conversely, in fruits harvested at the regreening stage, the changes in plastid morphology were minimal, with delayed changes in chlorophyll and carotenoids. Genes related to ethylene biosynthesis and signaling pathways supported these responses. Variations in endogenous auxin, jasmonic acid, abscisic acid and gibberellins could partially explain this phenomenon. CONCLUSION: The response of Midknight Valencia oranges to ethephon degreening was delayed in the regreening stage, possibly due to the dynamic variations in endogenous phytohormones. © 2024 Society of Chemical Industry.

Graph-Based Pangenome of Actinidia chinensis Reveals Structural Variations Mediating Fruit Degreening.

Fruit ripening is associated with the degreening process (loss of chlorophyll) that occurs in most fruit species. Kiwifruit is one of the special species whose fruits may maintain green flesh by accumulating a large amount of chlorophyll even after ripening. However, little is known about the genetic variations related to the fruit degreening process. Here, a graph-based kiwifruit pangenome by analyzing 14 chromosome-scale haplotype-resolved genome assemblies from seven representative cultivars or lines in Actinidia chinensis is built. A total of 49,770 non-redundant gene families are identified, with core genes constituting 46.6%, and dispensable genes constituting 53.4%. A total of 84,591 non-redundant structural variations (SVs) are identified. The pangenome graph integrating both reference genome sequences and variant information facilitates the identification of SVs related to fruit color. The SV in the promoter of the AcBCM gene determines its high expression in the late developmental stage of fruits, which causes chlorophyll accumulation in the green-flesh fruits by post-translationally regulating AcSGR2, a key enzyme of chlorophyll catabolism. Taken together, a high-quality pangenome is constructed, unraveled numerous genetic variations, and identified a novel SV mediating fruit coloration and fruit quality, providing valuable information for further investigating genome evolution and domestication, QTL genes function, and genomics-assisted breeding.

ABSCISIC ACID INSENSITIVE 4 coordinates eoplast formation to ensure acquisition of seed longevity during maturation in Medicago truncatula.

Seed longevity, the capacity to remain alive during dry storage, is pivotal to germination performance and is essential for preserving genetic diversity. It is acquired during late maturation concomitantly with seed degreening and the de-differentiation of chloroplasts into colorless, non-photosynthetic plastids, called eoplasts. As chlorophyll retention leads to poor seed performance upon sowing, these processes are important for seed vigor. However, how these processes are regulated and connected to the acquisition of seed longevity remains poorly understood. Here, we show that such a role is at least provided by ABSCISIC ACID INSENSITIVE 4 (ABI4) in the legume Medicago truncatula. Mature seeds of Mtabi4 mutants contained more chlorophyll than wild-type seeds and exhibited a 75% reduction in longevity and reduced dormancy. MtABI4 was necessary to stimulate eoplast formation, as evidenced by the significant delay in the dismantlement of photosystem II during the maturation of mutant seeds. Mtabi4 seeds also exhibited transcriptional deregulation of genes associated with retrograde signaling and transcriptional control of plastid-encoded genes. Longevity was restored when Mtabi4 seeds developed in darkness, suggesting that the shutdown of photosynthesis during maturation, rather than chlorophyll degradation per se, is a requisite for the acquisition of longevity. Indeed, the shelf life of stay green mutant seeds that retained chlorophyll was not affected. Thus, ABI4 plays a role in coordinating the dismantlement of chloroplasts during seed development to avoid damage that compromises the acquisition of seed longevity. Analysis of Mtabi4 Mtabi5 double mutants showed synergistic effects on chlorophyll retention and longevity, suggesting that they act via parallel pathways.

Examining the Role of Low Temperature in Satsuma Mandarin Fruit Peel Degreening via Comparative Physiological and Transcriptomic Analysis.

Peel degreening is the most conspicuous aspect of fruit ripening in many citrus fruits because of its importance for marketability. In this study, peel degreening in response to propylene (an ethylene analog) and at varying storage temperatures was characterized in Satsuma mandarin (Citrus unshiu Marc.) fruit. Propylene treatment triggered rapid peel degreening (within 4-6 days), indicated by an increase in the citrus color index (CCI) and chlorophyll loss. Peel degreening was also observed in fruit at 10°C and 15°C after 28-42 days, with gradual CCI increase and chlorophyll reduction. However, fruit at 5°C, 20°C, and 25°C remained green, and no substantial changes in peel CCI and chlorophyll content were recorded during the 42-day storage duration. The transcriptomes of peels of fruit treated with propylene for 4 days and those stored at varying temperatures for 28 days were then analyzed by RNA-Seq. We identified three categories of differentially expressed genes that were regulated by (i) propylene (and by analogy, ethylene) alone, (ii) low temperature (5°C, 10°C, or 15°C vs. 25°C) alone, and (iii) either propylene or low temperature. Gene-encoding proteins associated with chlorophyll degradation (such as CuSGR1, CuNOL, CuACD2, CuCAB2, and CuLHCB2) and a transcription factor (CuERF114) were differentially expressed by propylene or low temperature. To further examine temperature-induced pathways, we also monitored gene expression during on-tree fruit maturation vs. postharvest. The onset of on-tree peel degreening coincided with autumnal drops in field temperatures, and it was accompanied by differential expression of low temperature-regulated genes. On the contrary, genes that were exclusively regulated by propylene (such as CuCOPT1 and CuPOX-A2) displayed insignificant expression changes during on-tree peel degreening. These findings indicate that low temperatures could be involved in the fruit ripening-related peel degreening independently of ethylene.

Low temperature delays degreening of apple fruit by inhibiting pheophorbide a oxygenase (PAO) pathway and chlorophyll oxidation during ripening.

The effects of low temperature (LT) on chlorophyll (Chl) degradation in peel of apple fruit during ripening were investigated. Apples collected at commercial maturity were stored at 4 ± 0.5°C. Our data indicated that LT treatment reduced respiration rate and ethylene production and slowed down softening of apple fruit during ripening. The LT treatment delayed increase in L*, a*, and b* values and decrease in Chl content compared with controls. The LT treatment reduced hydrogen peroxide (H2 O2 ) and malondialdehyde (MDA) contents and decelerated superoxide anion (O2 ·- ) production rate in chloroplast of peel compared with controls during ripening. The LT treatment differentially reduced activities of pheophytin pheophorbide hydrolase (PPH), Mg-dechelatase (MDcase), chlorophyll-degrading peroxidase (Chl-POX), and Chl oxidase, while enhanced SOD activity in chloroplast of peel during ripening. Expression levels of MdHCARa, MdNYC1, MdNYC3, MdNYE1, MdRCCR2, MdPPH1, MdPAO6, MdPAO8, and MdNOL2 in peel were differentially reduced by LT treatment during ripening. Our results indicated that LT treatment might delay Chl degradation through inhibiting PAO pathway and Chl oxidation during ripening of apple fruit. PRACTICAL APPLICATIONS: The LT is a common practice used to extend storage life of apple fruit. Degreening caused by Chl degradation is an integral part of fruit ripening, and elucidating its mechanism is an important subject for fruit quality maintenance. Our data indicated that LT delayed degreening of apple fruit by inhibiting PAO pathway and Chl oxidation during ripening. These results will provide useful information for clarifying molecular mechanisms of LT in regulation of degreening and also for quality maintenance of apple fruit.

The role of hydrogen water in delaying ripening of banana fruit during postharvest storage.

Experiments were conducted to identify the role of hydrogen water (HW) in banana fruit ripening. Banana fruit soaked with 0.8 ppm HW showed longer ripening than control fruit. HW treatment significantly reduced ethylene production and respiratory rate, and inhibited the expressions of ethylene synthesis- and signaling-related genes. Similarly, HW treatment inhibited the down-regulation of chlorophylls binding proteins and delayed the increase of chromaticity a*, b* and L* in banana peel. Furthermore, HW-treated peel exhibited lower expressions of cell wall degradation-related genes and higher levels of fruit firmness, pectin, hemicellulose and lignin. In addition, HW-treated pulp exhibited higher levels of starch, lower level of total soluble solids (TSS) and lower expression of flavor-related genes. Microstructural observation further confirmed that HW treatment delayed the degradations of starch and cell walls. Those results indicated that HW treatment delayed banana ripening via the role of ethylene in relation to degreening, flavor and softening.

Diversity of Plastid Types and Their Interconversions.

Plastids are pivotal subcellular organelles that have evolved to perform specialized functions in plant cells, including photosynthesis and the production and storage of metabolites. They come in a variety of forms with different characteristics, enabling them to function in a diverse array of organ/tissue/cell-specific developmental processes and with a variety of environmental signals. Here, we have comprehensively reviewed the distinctive roles of plastids and their transition statuses, according to their features. Furthermore, the most recent understanding of their regulatory mechanisms is highlighted at both transcriptional and post-translational levels, with a focus on the greening and non-greening phenotypes.

VviNAC33 promotes organ de-greening and represses vegetative growth during the vegetative-to-mature phase transition in grapevine.

Plants undergo several developmental transitions during their life cycle. In grapevine, a perennial woody fruit crop, the transition from vegetative/green-to-mature/woody growth involves transcriptomic reprogramming orchestrated by a small group of genes encoding regulators, but the underlying molecular mechanisms are not fully understood. We investigated the function of the transcriptional regulator VviNAC33 by generating and characterizing transgenic overexpressing grapevine lines and a chimeric repressor, and by exploring its putative targets through a DNA affinity purification sequencing (DAP-seq) approach combined with transcriptomic data. We demonstrated that VviNAC33 induces leaf de-greening, inhibits organ growth and directly activates the expression of STAY-GREEN PROTEIN 1 (SGR1), which is involved in Chl and photosystem degradation, and AUTOPHAGY 8f (ATG8f), which is involved in the maturation of autophagosomes. Furthermore, we show that VviNAC33 directly inhibits AUXIN EFFLUX FACILITATOR PIN1, RopGEF1 and ATP SYNTHASE GAMMA CHAIN 1T (ATPC1), which are involved in photosystem II integrity and activity. Our results show that VviNAC33 plays a major role in terminating photosynthetic activity and organ growth as part of a regulatory network governing the vegetative-to-mature phase transition.

Enhanced Chlorophyll Degradation Triggers the Pod Degreening of "Golden Hook," a Special Ecotype in Common Bean (Phaseolus vulgaris L.).

To reveal genetic factors or pathways involved in the pod degreening, we performed transcriptome and metabolome analyses using a yellow pod cultivar of the common bean "golden hook" ecotype and its green pod mutants yielded via gamma radiation. Transcriptional profiling showed that expression levels of red chlorophyll catabolite reductase (RCCR, Phvul.008G280300) involved in chlorophyll degradation was strongly enhanced at an early stage (2 cm long) in wild type but not in green pod mutants. The expression levels of genes involved in cellulose synthesis was inhibited by the pod degreening. Metabolomic profiling showed that the content of most flavonoid, flavones, and isoflavonoid was decreased during pod development, but the content of afzelechin, taxifolin, dihydrokaempferol, and cyanidin 3-O-rutinoside was remarkably increased in both wild type and green pod mutant. This study revealed that the pod degreening of the golden hook resulting from chlorophyll degradation could trigger changes in cellulose and flavonoids biosynthesis pathway, offering this cultivar a special color appearance and good flavor.

Roles of stay-green (SGR) homologs during chlorophyll degradation in green plants.

Chlorophyll (Chl) degradation is one of the most obvious signs of leaf senescence and fruit ripening. Stay-green (SGR) homologs that can remove magnesium from Chl a are the most important components in Chl degradation pathway in green plants. SGR homologs are not only universally involved in Chl breakdown during the senescence of green organs, but also play crucial roles in other organs during plant growth and development, such as fruit mature and nodule development. In this review, we focus on the diverse functions of SGR homologs in plant growth and development. A better understanding of SGR would be helpful for providing a theoretical basis for further illustrating the regulatory mechanism of SGR homologs.

Low temperature modulates natural peel degreening in lemon fruit independently of endogenous ethylene.

Peel degreening is an important aspect of fruit ripening in many citrus fruit, and previous studies have shown that it can be advanced by ethylene treatment or by low-temperature storage. However, the important regulators and pathways involved in natural peel degreening remain largely unknown. To determine how natural peel degreening is regulated in lemon fruit (Citrus limon), we studied transcriptome and physiochemical changes in the flavedo in response to ethylene treatment and low temperatures. Treatment with ethylene induced rapid peel degreening, which was strongly inhibited by the ethylene antagonist, 1-methylcyclopropene (1-MCP). Compared with 25 ºC, moderately low storage temperatures of 5-20 °C also triggered peel degreening. Surprisingly, repeated 1-MCP treatments failed to inhibit the peel degreening induced by low temperature. Transcriptome analysis revealed that low temperature and ethylene independently regulated genes associated with chlorophyll degradation, carotenoid metabolism, photosystem proteins, phytohormone biosynthesis and signalling, and transcription factors. Peel degreening of fruit on trees occurred in association with drops in ambient temperature, and it coincided with the differential expression of low temperature-regulated genes. In contrast, genes that were uniquely regulated by ethylene showed no significant expression changes during on-tree peel degreening. Based on these findings, we hypothesize that low temperature plays a prominent role in regulating natural peel degreening independently of ethylene in citrus fruit.

Effects of elevated CO2 on pigment metabolism of postharvest mandarin fruit for degreening.

Degreening is widely used in citrus fruit to extend the market season for economic gains and increase the consumer acceptance. Elevated CO2 was exogenously applied to Satsuma mandarins (Citrus unshiu Marc.) for degreening and its effect on pigment metabolism was investigated. The results revealed 15% CO2 treatment accelerated the citrus fruit peel color change along with the chlorophyll degradation, ß-cryptoxanthin and flavonoids accumulation. The expression of CitSGR, CitNYC, CitChlase, CitPPH, CitPAO and CitRCCR genes involved in chlorophyll metabolism and a set of genes involved in producing ß, ß-xanthophylls were up-regulated by elevated CO2. For flavonoid metabolism, the up-regulated expressions of CitPAL, CitCHS and CitCHI partly explained the increased total flavonoids content. These results showed that 15% CO2 treatment improved the visual appearance of citrus fruits due to its impact on pigment metabolism and also maintained their nutritional value, thus could be employed as a potential commercial technique for citrus degreening.

Pruning and dormancy breaking make two sustainable grape-cropping productions in a protected environment possible without overlap in a single year.

In table grape production, protected cultivation in a vineyard in different regions and climates is currently a commonly used practice. The aims of this study were to provide key approaches to sustainably produce two crops of grape without overlap under protected environment in a single year. Spraying the degreening chemicals 400 mg/L ethephon +0.4% sulfur at 4 weeks of vine nutrient restoration after the harvest of the summer crop resulted in the highest percentage of sprouting inflorescence. The retention of 7-10 buds in the base shoot results in the high percentage of sprouting inflorescence. Bud breaking chemicals with 2.5% hydrogen cyanamide+2.0% Baoguoliang +0.02% Shenzhonggen significantly led to sprout inflorescence more efficiently. Cluster and fruit weights of the winter crop weighed significantly less than those of the summer crop. However, the contents of total soluble sugar and titratable acidity were higher than those of the summer crop. The anthocyanin content in the peel of the winter fruit was significantly higher than that in the summer fruit. The yield of the winter crop is controlled by the yield of the summer fruit. To maintain the stability of the two crops for one year, the ratio of yield in the winter to the summer should be controlled from 2:5 to 3:5 to ensure the sustainable production of two crops without overlap for 'Summer Black' grape. These results may help grape growers to overcome the impacts of rainy and hot climates with the help of protected facilities, and it could enable the use of solar radiation and heat resources in subtropical and tropical areas.

Auto- and mutual-regulation between two CitERFs contribute to ethylene-induced citrus fruit degreening.

Citrus fruit postharvest degreening is a critical stage in marketing, carried out by exposure to ethylene or ethephon. Genome-wide screening of the AP2/ERF superfamily indicated that a novel ERF-II (CitERF6) was shown to trans-activate the CitPPH promoter. Expression of CitERF6 is associated with both developmental and postharvest degreening in citrus fruit. Transient and stable over-expression of CitERF6 in Nicotiana tabacum leaves and 'Ponkan' fruit also results in rapid chlorophyll degradation. Auto- and mutual-regulation was also found between CitERF6 and the previously characterized CitERF13 using the dual-luciferase and yeast one-hybrid assays. Moreover, substitution of the 35S promoter for endogenous promoters showed that both pCitERF6::CitERF6 and pCitERF13::CitERF13 were effective in trans-activating their promoters or triggering chlorophyll degradation. It is proposed that ethylene is one of the triggers activating promoters of CitERF6 and CitERF13, and subsequent auto- and mutual-regulation between CitERF6 and CitERF13 might facilitate the effect of ethylene, leading to fruit degreening.

Acidity Drop and Coloration in Clementine: Implications for Fruit Quality and Harvesting Practices.

The commercial quality of fruit is the result of a combination of internal (acidity, sugars, juice, etc.) and external characteristics (shape, size, color, visual defects, etc.). On citrus, the internal maturity of fruit is often reached prior and independently to their external maturity, inducing the use of degreening practices to artificially color fruit. However, for some sectors where degreening is not authorized, such as organic farming or up-market, it is important to understand the co-occurrence between fruit coloration and internal ripening, and its impact on fruit quality and harvesting management. Our study was based on a monitoring of the color and acidity of Protected Geographical Indication "Clémentine de Corse" orchards of producers in 2013 and 2014. Our results show that: (i) the dynamics of acidity drop during maturation are similar from one plot to another but staggered in time; (ii) fruit coloring occurs at different times during acidity drop; (iii) the synchronization between the coloring process and acidity drop determines both the quality of harvested fruit and the period during which orchards are harvestable, which we called the "harvestability window." This study sheds new light on the quality of citrus harvested without fruit degreening and leads to propose actions to anticipate internal maturity evolution according to the coloring and spreading of the harvest period. The fruit acidity model obtained in this study will be extended to a practical application tool to predict fruit acidity and quality for a better-controlled harvest management.

Photoinhibition and photoprotection during flower opening in lilies.

Although most studies to extend vase life in cut flowers have focused on flower senescence thus far, flower opening is a complex process of major biological significance in the determination of flower commercialization. In order to better understand flower opening, this study evaluated to what extent photoinhibition and photo-oxidative stress are associated with tepal de-greening during flower opening in lilies (Lilium "Litouwen"). We estimated the degree of photoinhibition, the capacity for photo- and antioxidant protection, and the extent of lipid peroxidation at four flower opening stages, from closed flowers to anthesis. Additionally, we evaluated to what extent and by which mechanisms related to photo- and antioxidant protection, Promalin® (a combination of gibberellins and cytokinins) delays flower opening. Results showed that chlorophyll content decreased progressively during flower opening, while a sharp decrease of the maximum PSII efficiency (Fv/Fm ratio) was observed just before anthesis. Moreover, content of secondary lipid peroxidation products (malondialdehyde and jasmonic acid) increased just before anthesis, which was preceded by an enhanced production of primary lipid peroxidation products (lipid hydroperoxides). While both tocopherols and tocotrienols (vitamin E) increased during flower opening, ß-carotene and xanthophyll content decreased sharply, which may be associated with the sharp decline in the Fv/Fm ratio before anthesis. Flowers treated with Promalin®, which showed delayed opening, experienced transient increases of lipid hydroperoxide and jasmonic acid contents at early stages of flower opening, together with reduced vitamin E and malondialdehyde contents just prior to anthesis. We conclude that the extent of photoinhibition, the capacity of photo- and antioxidant protection and the production of primary and secondary products of lipid peroxidation are finely controlled in a time-dependent manner to allow a correct development of lily flowers.

Sensitivity to Ethephon Degreening Treatment Is Altered by Blue LED Light Irradiation in Mandarin Fruit.

Although citrus fruits are not climacteric, exogenous ethylene is widely used in the degreening treatment of citrus fruits. Irradiation with blue light-emitting diode (LED) light (450 nm) for 10 h can promote the formation of good coloration of ethephon-degreened fruit. This study evaluated the effect of blue LED light irradiation on the pigments contents of ethephon-degreened fruit and evaluated whether the blue LED light irradiation could influence the sensitivity of mandarin fruit to ethylene. The results indicated that blue light can accelerate the color change of ethephon-degreened fruit, accompanied by changes in plastid ultrastructure and chlorophyll and carotenoid contents. Ethephon-induced expressions of CitACS1, CitACO, CitETR1, CitEIN2, CitEIL1, and CitERF2 were enhanced by blue LED light irradiation, which increased the sensitivity to ethylene in ethephon-degreened fruits. These results indicate that blue LED light-induced changes in sensitivity to ethylene in mandarin fruit may be responsible for the improved coloration of ethephon-degreened mandarin fruits.