Beyond Photoprotection: The Multifarious Roles of Flavonoids in Plant Terrestrialization.

Plants evolved an impressive arsenal of multifunctional specialized metabolites to cope with the novel environmental pressures imposed by the terrestrial habitat when moving from water. Here we examine the multifarious roles of flavonoids in plant terrestrialization. We reason on the environmental drivers, other than the increase in UV-B radiation, that were mostly responsible for the rise of flavonoid metabolism and how flavonoids helped plants in land conquest. We are reasonably based on a nutrient-deficiency hypothesis for the replacement of mycosporine-like amino acids, typical of streptophytic algae, with the flavonoid metabolism during the water-to-land transition. We suggest that flavonoids modulated auxin transport and signaling and promoted the symbiosis between plants and fungi (e.g., arbuscular mycorrhizal, AM), a central event for the conquest of land by plants. AM improved the ability of early plants to take up nutrients and water from highly impoverished soils. We offer evidence that flavonoids equipped early land plants with highly versatile "defense compounds", essential for the new set of abiotic and biotic stressors imposed by the terrestrial environment. We conclude that flavonoids have been multifunctional since the appearance of plants on land, not only acting as UV filters but especially improving both nutrient acquisition and biotic stress defense.

Photoprotective Role of Photosynthetic and Non-Photosynthetic Pigments in Phillyrea latifolia: Is Their "Antioxidant" Function Prominent in Leaves Exposed to Severe Summer Drought?

Carotenoids and phenylpropanoids play a dual role of limiting and countering photooxidative stress. We hypothesize that their "antioxidant" function is prominent in plants exposed to summer drought, when climatic conditions exacerbate the light stress. To test this, we conducted a field study on Phillyrea latifolia, a Mediterranean evergreen shrub, carrying out daily physiological and biochemical analyses in spring and summer. We also investigated the functional role of the major phenylpropanoids in different leaf tissues. Summer leaves underwent the most severe drought stress concomitantly with a reduction in radiation use efficiency upon being exposed to intense photooxidative stress, particularly during the central hours of the day. In parallel, a significant daily variation in both carotenoids and phenylpropanoids was observed. Our data suggest that the morning-to-midday increase in zeaxanthin derived from the hydroxylation of ß-carotene to sustain non-photochemical quenching and limit lipid peroxidation in thylakoid membranes. We observed substantial spring-to-summer and morning-to-midday increases in quercetin and luteolin derivatives, mostly in the leaf mesophyll. These findings highlight their importance as antioxidants, countering the drought-induced photooxidative stress. We concluded that seasonal and daily changes in photosynthetic and non-photosynthetic pigments may allow P. latifolia leaves to avoid irreversible photodamage and to cope successfully with the Mediterranean harsh climate.

Antioxidant Defenses in Plants: A Dated Topic of Current Interest.

Plants have been challenged against oxidative stress since their appearance on land [...].

Unveiling the shade nature of cyanic leaves: A view from the "blue absorbing side" of anthocyanins.

Anthocyanins have long been suggested as having great potential in offering photoprotection to plants facing high light irradiance. Nonetheless, their effective ability in protecting the photosynthetic apparatus from supernumerary photons has been questioned by some authors, based upon the inexact belief that anthocyanins almost exclusively absorb green photons, which are poorly absorbed by chlorophylls. Here we focus on the blue light absorbing features of anthocyanins, a neglected issue in anthocyanin research. Anthocyanins effectively absorb blue photons: the absorbance of blue relative to green photons increases from tri- to mono-hydroxy B-ring substituted structures, reaching up to 50% of green photons absorption. We offer a comprehensive picture of the molecular events activated by low blue-light availability, extending our previous analysis in purple and green basil, which we suggest to be responsible for the "shade syndrome" displayed by cyanic leaves. While purple leaves display overexpression of genes promoting chlorophyll biosynthesis and light harvesting, in green leaves it is the genes involved in the stability/repair of photosystems that are largely overexpressed. As a corollary, this adds further support to the view of an effective photoprotective role of anthocyanins. We discuss the profound morpho-anatomical adjustments imposed by the epidermal anthocyanin shield, which reflect adjustments in light harvesting capacity under imposed shade and make complex the analysis of the photosynthetic performance of cyanic versus acyanic leaves.

Are Flavonoids Effective Antioxidants in Plants? Twenty Years of Our Investigation.

Whether flavonoids play significant antioxidant roles in plants challenged by photooxidative stress of different origin has been largely debated over the last few decades. A critical review of the pertinent literature and our experimentation as well, based on a free-of-scale approach, support an important antioxidant function served by flavonoids in plants exposed to a wide range of environmental stressors, the significance of which increases with the severity of stress. On the other side, some questions need conclusive answers when the putative antioxidant functions of plant flavonoids are examined at the level of both the whole-cell and cellular organelles. This partly depends upon a conclusive, robust, and unbiased definition of "a plant antioxidant", which is still missing, and the need of considering the subcellular re-organization that occurs in plant cells in response to severe stress conditions. This likely makes our deterministic-based approach unsuitable to unveil the relevance of flavonoids as antioxidants in extremely complex biological systems, such as a plant cell exposed to an ever-changing stressful environment. This still poses open questions about how to measure the occurred antioxidant action of flavonoids. Our reasoning also evidences the need of contemporarily evaluating the changes in key primary and secondary components of the antioxidant defense network imposed by stress events of increasing severity to properly estimate the relevance of the antioxidant functions of flavonoids in an in planta situation. In turn, this calls for an in-depth analysis of the sub-cellular distribution of primary and secondary antioxidants to solve this still intricate matter.

Functional and Structural Leaf Plasticity Determine Photosynthetic Performances during Drought Stress and Recovery in Two Platanus orientalis Populations from Contrasting Habitats.

In the context of climatic change, more severe and long-lasting droughts will modify the fitness of plants, with potentially worse consequences on the relict trees. We have investigated the leaf phenotypic (anatomical, physiological and biochemical) plasticity in well-watered, drought-stressed and re-watered plants of two populations of Platanus orientalis, an endangered species in the west of the Mediterranean area. The two populations originated in contrasting climate (drier and warmer, Italy (IT) population; more humid and colder, Bulgaria (BG) population). The IT control plants had thicker leaves, enabling them to maintain higher leaf water content in the dry environment, and more spongy parenchyma, which could improve water conductivity of these plants and may result in easier CO2 diffusion than in BG plants. Control BG plants were also characterized by higher photorespiration and leaf antioxidants compared to IT plants. BG plants responded to drought with greater leaf thickness shrinkage. Drought also caused substantial reduction in photosynthetic parameters of both IT and BG plants. After re-watering, photosynthesis did not fully recover in either of the two populations. However, IT leaves became thicker, while photorespiration in BG plants further increased, perhaps indicating sustained activation of defensive mechanisms. Overall, our hypothesis, that plants with a fragmented habitat (i.e., the IT population) lose phenotypic plasticity but acquire traits allowing better resistance to the climate where they became adapted, remains confirmed.

Phellem Cell-Wall Components Are Discriminants of Cork Quality in Quercus suber.

Cork is a renewable, non-wood high valued forest product, with relevant ecological and economic impact in the Mediterranean-type ecosystems. Currently, cork is ranked according to its commercial quality. The most valuable planks are chosen for cork stoppers production. Cork planks with adequate thickness and porosity are classified as stoppable quality cork (SQC). The chemical composition of cork is known, but the regulation of metabolic pathways responsible of cork production and composition, hence of cork quality, is largely unknown. Here, we tested the hypothesis that post-genomic events may be responsible for the development of SQC and N-SQC (non-stoppable quality cork). Here, we show that combined proteomics and targeted metabolomics (namely soluble and cell wall bound phenolics) analyzed on recently formed phellem allows discriminate cork planks of different quality. Phellem cells of SQC and N-SQC displayed different reducing capacity, with consequential impact on both enzymatic pathways (e.g., glycolysis) and other cellular functions, including cell wall assembly and suberization. Glycolysis and respiration related proteins were abundant in both cork quality groups, whereas the level of several proteins associated to mitochondrial metabolism was higher in N-SQC. The soluble and cell wall-bound phenolics in recently formed phellem clearly discriminated SQC from N-SCQ. In our study, SQC was characterized by a high incorporation of aromatic components of the phenylpropanoid pathway in the cell wall, together with a lower content of hydrolysable tannins. Here, we propose that the level of hydrolysable tannins may represent a valuable diagnostic tool for screening recently formed phellem, and used as a proxy for the quality grade of cork plank produced by each tree.

Dissecting Adaptation Mechanisms to Contrasting Solar Irradiance in the Mediterranean Shrub Cistus incanus.

Molecular mechanisms that are the base of the strategies adopted by Mediterranean plants to cope with the challenges imposed by limited or excessive solar radiation during the summer season have received limited attention. In our study, conducted on C. incanus plants growing in the shade or in full sunlight, we performed measurements of relevant physiological traits, such as leaf water potential, gas exchange and PSII photochemistry, RNA-Seq with de-novo assembly, and the analysis of differentially expressed genes. We also identified and quantified photosynthetic pigments, abscisic acid, and flavonoids. Here, we show major mechanisms regulating light perception and signaling which, in turn, sustain the shade avoidance syndrome displayed by the 'sun loving' C. incanus. We offer clear evidence of the detrimental effects of excessive light on both the assembly and the stability of PSII, and the activation of a suite of both repair and effective antioxidant mechanisms in sun-adapted leaves. For instance, our study supports the view of major antioxidant functions of zeaxanthin in sunny plants concomitantly challenged by severe drought stress. Finally, our study confirms the multiple functions served by flavonoids, both flavonols and flavanols, in the adaptive mechanisms of plants to the environmental pressures associated to Mediterranean climate.

Review: ABA, flavonols, and the evolvability of land plants.

There is evidence that the ABA signaling pathway has greatly contributed to increase the complexity of land plants, thereby sustaining their ability to adapt in an ever-changing environment. The regulatory functions of the ABA signaling pathway go well beyond the movements of stomata and the dormancy of seeds. For instance, the ABA signaling regulates the flavonoid biosynthesis, consistent with the high integration of ABA and light signaling pathways, which occurs at the level of key signaling components, such as the bZIP transcription factors HY5 and ABI5. Here we focus on the regulation of 'colorless' (UV-absorbing) flavonol biosynthesis by the ABA signaling and, about how flavonols may regulate, in turn, the ABA signaling network. We discuss very recent findings that quercetin regulates the ABA signaling pathway, and hypothesize this might occur at the level of second messenger and perhaps of primary signaling components as well. We critically review old and recent suggestions of the primary roles played by flavonols, the ancient class of flavonoids already present in bryophytes, in the evolution of terrestrial plants. Our reasoning strongly supports the view that the ABA-flavonol relationship may represent a robust trait of land plants, and might have contributed to their adaptation on land.

Seasonal and daily variations in primary and secondary metabolism of three maquis shrubs unveil different adaptive responses to Mediterranean climate.

Maquis species play a central role in the maintenance of coastal ecosystems thanks to anatomical, physiological and biochemical features evolved to cope with severe stress conditions. Because the seasonal and daily dynamics of physiological and biochemical traits of maquis species are not fully addressed, we performed a field study on three coexisting Mediterranean shrubs (Pistacia lentiscus L. and Phillyrea latifolia L., evergreen schlerophylls, and Cistus incanus L., semi-deciduous) aiming at detecting the main adaptive differences, on a seasonal and daily basis, in primary and secondary metabolism along with the principal climatic determinants. These species differed in their physiological and biochemical responses especially on a seasonal level. In P. latifolia, a great investment in antioxidant phenylpropanoids contributed to maintain high photosynthetic rates throughout the whole growing season. In C. incanus, high carotenoid content associated with chlorophyll (Chl) regulation alleviated oxidative damage during the hot and dry summers and help recover photosynthesis in autumn. In P. lentiscus, high abscisic acid levels allowed a strict control of stomata, while fine Chla/Chlb regulation concurred to avoid photoinhibition in summer. Temperature resulted the most important climatic factor controlling the physiological and biochemical status of these coexisting shrubs and, thus, in determining plant performances in this Mediterranean coastal habitat.

Physiological and structural adjustments of two ecotypes of Platanus orientalis L. from different habitats in response to drought and re-watering.

Platanus orientalis covers a very fragmented area in Europe and, at the edge of its natural distribution, is considered a relic endangered species near extinction. In our study, it was hypothesized that individuals from the edge of the habitat, with stronger climate constrains (drier and warmer environment, Italy, IT ecotype), developed different mechanisms of adaptation than those growing under optimal conditions at the center of the habitat (more humid and colder environment, Bulgaria, BG ecotype). Indeed, the two P. orientalis ecotypes displayed physiological, structural and functional differences already under control (unstressed) conditions. Adaptation to a dry environment stimulated constitutive isoprene emission, determined active stomatal behavior, and modified chloroplast ultrastructure, ultimately allowing more effective use of absorbed light energy for photochemistry. When exposed to short-term acute drought stress, IT plants showed active stomatal control that enhanced instantaneous water use efficiency, and stimulation of isoprene emission that sustained photochemistry and reduced oxidative damages to membranes, as compared to BG plants. None of the P. orientalis ecotypes recovered completely from drought stress after re-watering, confirming the sensitivity of this mesophyte to drought. Nevertheless, the IT ecotype showed less damage and better stability at the level of chloroplast membrane parameters when compared to the BG ecotype, which we interpret as possible adaptation to hostile environments and improved capacity to cope with future, likely more recurrent, drought stress.

Metabolic plasticity in the hygrophyte Moringa oleifera exposed to water stress.

Over the past decades, introduction of many fast-growing hygrophilic, and economically valuable plants into xeric environments has occurred. However, production and even survival of these species may be threatened by harsh climatic conditions unless an effective physiological and metabolic plasticity is available. Moringa oleifera Lam., a multipurpose tree originating from humid sub-tropical regions of India, is widely cultivated in many arid countries because of its multiple uses. We tested whether M. oleifera can adjust primary and secondary metabolism to efficiently cope with increasing water stress. It is shown that M. oleifera possesses an effective isohydric behavior. Water stress induced a quick and strong stomatal closure, driven by abscisic acid (ABA) accumulation, and leading to photosynthesis inhibition with consequent negative effects on biomass production. However, photochemistry was not impaired and maximal fluorescence and saturating photosynthesis remained unaffected in stressed leaves. We report for the first time that M. oleifera produces isoprene, and show that isoprene emission increased three-fold during stress progression. It is proposed that higher isoprene biosynthesis helps leaves cope with water stress through its antioxidant or membrane stabilizing action, and also indicates a general MEP (methylerythritol 4-phosphate) pathway activation that further helps protect photosynthesis under water stress. Increased concentrations of antioxidant flavonoids were also observed in water stressed leaves, and probably cooperate in limiting irreversible effects of the stress in M. oleifera leaves. The observed metabolic and phenotypic plasticity may facilitate the establishment of M. oleifera in xeric environments, sustaining the economic and environmental value of this plant.

Modulation of Phytohormone Signaling: A Primary Function of Flavonoids in Plant-Environment Interactions.

The old observation that plants preferentially synthesize flavonoids with respect to the wide range of phenylpropanoid structures when exposed to high doses of UV-B radiation has supported the view that flavonoids are primarily involved in absorbing the shortest solar wavelengths in photoprotection. However, there is compelling evidence that the biosynthesis of flavonoids is similarly upregulated in response to high photosynthetically active radiation in the presence or in the absence of UV-radiation, as well as in response to excess metal ions and photosynthetic redox unbalance. This supports the hypothesis that flavonoids may play prominent roles as scavengers of reactive oxygen species (ROS) generated by light excess. These 'antioxidant' functions of flavonoids appears robust, as maintained between different life kingdoms, e.g., plants and animals. The ability of flavonoids to buffer stress-induced large alterations in ROS homeostasis and, hence, to modulate the ROS-signaling cascade, is at the base of well-known functions of flavonoids as developmental regulators in both plants and animals. There is both long and very recent evidence indeed that, in plants, flavonoids may strongly affect phytohormone signaling, e.g., auxin and abscisic acid signaling. This function is served by flavonoids in a very low (nM) concentration range and involves the ability of flavonoids to inhibit the activity of a wide range of protein kinases, including but not limited to mitogen-activated protein kinases, that operate downstream of ROS in the regulation of cell growth and differentiation. For example, flavonoids inhibit the transport of auxin acting on serine-threonine PINOID (PID) kinases that regulate the localization of auxin efflux facilitators PIN-formed (PIN) proteins. Flavonoids may also determine auxin gradients at cellular and tissue levels, and the consequential developmental processes, by reducing auxin catabolism. Recent observations lead to the hypothesis that regulation/modulation of auxin transport/signaling is likely an ancestral function of flavonoids. The antagonistic functions of flavonoids on ABA-induced stomatal closure also offer novel hypotheses on the functional role of flavonoids in plant-environment interactions, in early as well as in modern terrestrial plants. Here, we surmise that the regulation of phytohormone signaling might have represented a primary function served by flavonols for the conquest of land by plants and it is still of major significance for the successful acclimation of modern terrestrial plants to a severe excess of radiant energy.

Dissecting the role of isoprene and stress-related hormones (ABA and ethylene) in Populus nigra exposed to unequal root zone water stress.

Isoprene is synthesized through the 2-C-methylerythritol-5-phosphate (MEP) pathway that also produces abscisic acid (ABA). Increases in foliar free ABA concentration during drought induce stomatal closure and may also alter ethylene biosynthesis. We hypothesized a role of isoprene biosynthesis in protecting plants challenged by increasing water deficit, by influencing ABA production and ethylene evolution. We performed a split-root experiment on Populus nigra L. subjected to three water treatments: well-watered (WW) plants with both root sectors kept at pot capacity, plants with both root compartments allowed to dry for 5 days (DD) and plants with one-half of the roots irrigated to pot capacity, while the other half did not receive water (WD). WD and WW plants were similar in photosynthesis, water relations, foliar ABA concentration and isoprene emission, whereas these parameters were significantly affected in DD plants: leaf isoprene emission increased despite the fact that photosynthesis declined by 85% and the ABA-glucoside/free ABA ratio decreased significantly. Enhanced isoprene biosynthesis in water-stressed poplars may have contributed to sustaining leaf ABA biosynthesis by keeping the MEP pathway active. However, this enhancement in ABA was accompanied by no change in ethylene biosynthesis, likely confirming the antagonistic role between ABA and ethylene. These results may indicate a potential cross-talk among isoprene, ABA and ethylene under drought.

Isoprene Responses and Functions in Plants Challenged by Environmental Pressures Associated to Climate Change.

The functional reasons for isoprene emission are still a matter of hot debate. It was hypothesized that isoprene biosynthesis evolved as an ancestral mechanism in plants adapted to high water availability, to cope with transient and recurrent oxidative stresses during their water-to-land transition. There is a tight association between isoprene emission and species hygrophily, suggesting that isoprene emission may be a favorable trait to cope with occasional exposure to stresses in mesic environments. The suite of morpho-anatomical traits does not allow a conservative water use in hygrophilic mesophytes challenged by the environmental pressures imposed or exacerbated by drought and heat stress. There is evidence that in stressed plants the biosynthesis of isoprene is uncoupled from photosynthesis. Because the biosynthesis of isoprene is costly, the great investment of carbon and energy into isoprene must have relevant functional reasons. Isoprene is effective in preserving the integrity of thylakoid membranes, not only through direct interaction with their lipid acyl chains, but also by up-regulating proteins associated with photosynthetic complexes and enhancing the biosynthesis of relevant membrane components, such as mono- and di-galactosyl-diacyl glycerols and unsaturated fatty acids. Isoprene may additionally protect photosynthetic membranes by scavenging reactive oxygen species. Here we explore the mode of actions and the potential significance of isoprene in the response of hygrophilic plants when challenged by severe stress conditions associated to rapid climate change in temperate climates, with special emphasis to the concomitant effect of drought and heat. We suggest that isoprene emission may be not a good estimate for its biosynthesis and concentration in severely droughted leaves, being the internal concentration of isoprene the important trait for stress protection.

Phenotypic differences determine drought stress responses in ecotypes of Arundo donax adapted to different environments.

Arundo donax has been identified as an important biomass and biofuel crop. Yet, there has been little research on photosynthetic and metabolic traits, which sustain the high productivity of A. donax under drought conditions. This study determined phenotypic differences between two A. donax ecotypes coming from stands with contrasting adaptation to dry climate. We hypothesized that the Bulgarian (BG) ecotype, adapted to drier conditions, exhibits greater drought tolerance than the Italian (IT) ecotype, adapted to a more mesic environment. Under well-watered conditions the BG ecotype was characterized by higher photosynthesis, mesophyll conductance, intrinsic water use efficiency, PSII efficiency, isoprene emission rate and carotenoids, whereas the IT ecotype showed higher levels of hydroxycinnamates. Photosynthesis of water-stressed plants was mainly limited by diffusional resistance to CO2 in BG, and by biochemistry in IT. Recovery of photosynthesis was more rapid and complete in BG than in IT, which may indicate better stability of the photosynthetic apparatus associated to enhanced induction of volatile and non-volatile isoprenoids and phenylpropanoid biosynthesis. This study shows that a large phenotypic plasticity among A. donax ecotypes exists, and may be exploited to compensate for the low genetic variability of this species when selecting plant productivity in constrained environments.

Dissecting molecular and physiological response mechanisms to high solar radiation in cyanic and acyanic leaves: a case study on red and green basil.

Photosynthetic performance and the expression of genes involved in light signaling and the biosynthesis of isoprenoids and phenylpropanoids were analysed in green ('Tigullio', TIG) and red ('Red Rubin', RR) basil. The aim was to detect the physiological and molecular response mechanisms to high sunlight. The attenuation of blue-green light by epidermal anthocyanins was shown to evoke shade-avoidance responses with consequential effects on leaf morpho-anatomical traits and gas exchange performance. Red basil had a lower mesophyll conductance, partially compensated by the less effective control of stomatal movements, in comparison with TIG. Photosynthesis decreased more in TIG than in RR in high sunlight, because of larger stomatal limitations and the transient impairment of PSII photochemistry. The methylerythritol 4-phosphate pathway promoted above all the synthesis and de-epoxidation of violaxanthin-cycle pigments in TIG and of neoxanthin and lutein in RR. This enabled the green leaves to process the excess radiant energy effectively, and the red leaves to optimize light harvesting and photoprotection. The greater stomatal closure observed in TIG than in RR was due to enhanced abscisic acid (ABA) glucose ester deglucosylation and reduced ABA oxidation, rather than to superior de novo ABA synthesis. This study shows a strong competition between anthocyanin and flavonol biosynthesis, which occurs at the level of genes regulating the oxidation of the C2-C3 bond in the dihydro-flavonoid skeleton.

Grape Ripening Is Regulated by Deficit Irrigation/Elevated Temperatures According to Cluster Position in the Canopy.

The impact of water deficit on berry quality has been extensively investigated during the last decades. Nonetheless, there is a scarcity of knowledge on the performance of varieties exposed to a combination of high temperatures/water stress during the growing season and under vineyard conditions. The objective of this research was to investigate the effects of two irrigation regimes, sustained deficit irrigation (SDI, 30% ETc) and regulated deficit irrigation (RDI, 15% ETc) and of two cluster positions within the canopy (east- and west-exposed sides) on berry ripening in red Aragonez (Tempranillo) grapevines. The study was undertaken for two successive years in a commercial vineyard in South Portugal, monitoring the following parameters: pre-dawn leaf water potential, berry temperature, sugars, polyphenols, abscisic acid (ABA) and related metabolites. Additionally, expression patterns for different transcripts encoding for enzymes responsible for anthocyanin and ABA biosynthesis (VviUFGT, VvNCED1, VvßG1, VviHyd1, VviHyd2) were analyzed. In both years anthocyanin concentration was lower in RDI at the west side (RDIW- the hottest one) from véraison onwards, suggesting that the most severe water stress conditions exacerbated the negative impact of high temperature on anthocyanin. The down-regulation of VviUFGT expression revealed a repression of the anthocyanin synthesis in berries of RDIW, at early stages of berry ripening. At full-maturation, anthocyanin degradation products were detected, being highest at RDIW. This suggests that the negative impact of water stress and high temperature on anthocyanins results from the repression of biosynthesis at the onset of ripening and from degradation at later stages. On the other hand, berries grown under SDI displayed a higher content in phenolics than those under RDI, pointing out for the attenuation of the negative temperature effects under SDI. Irrigation regime and berry position had small effect on free-ABA concentration. However, ABA catabolism/conjugation process and ABA biosynthetic pathway were affected by water and heat stresses. This indicates the role of ABA-GE and catabolites in berry ABA homeostasis under abiotic stresses. Principal component analysis (PCA) showed that the strongest influence in berry ripening is the deficit irrigation regime, while temperature is an important variable determining the improvement or impairment of berry quality by the deficit irrigation regime. In summary, this work shows the interaction between irrigation regime and high temperature on the control of berry ripening.