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ARS Home » Pacific West Area » Wenatchee, Washington » Physiology and Pathology of Tree Fruits Research » Research » Research Project #427870

Research Project: Developmental Genomics and Metabolomics Influencing Temperate Tree Fruit Quality

Location: Physiology and Pathology of Tree Fruits Research

2019 Annual Report

The long-term goal of this project is to develop various tools to assist in quality management of deciduous tree fruits. Specifically, during the next five years we will focus on the following objectives. Objective 1: Integrate pre- and postharvest environment and commercial horticultural management practices with genomic and metabolomic regulation of apple and pear fruit quality.[C1; PS 1.A] Sub-objective 1A: Determine how fruit position within the tree impacts pear metabolic profile, superficial scald, postharvest quality, and ripening. Sub-objective 1B: Determine if inconsistent post-storage ripening of 1- methylcyclopropene (1-MCP) treated d’Anjou pears is relatable to differential absorbance (DA) value at harvest. Sub-objective 1C: Determine if pre-storage light exposure impacts apple peel metabolic responses to postharvest chilling. Objective 2: Enable new apple, pear and sweet cherry fruit biomarker-based quality management strategies. [C1; PS 1.A] Sub-objective 2A: Determine if apple aroma volatile production changes when fruit are stored in environments conducive to development of physiological disorders. Sub-objective 2B: Develop biomarker-based risk monitoring protocols using existing validated gene expression and metabolic biomarkers for early detection of apple and pear peel and cortex storage disorders. Sub-objective 2C: Determine if sweet cherry fruit pitting, cracking and browning is relatable to fruit epidermis and wax composition. The two objectives both rely on metabolomic and genomic techniques to investigate field and postharvest factors that impact fruit quality. The link from sub-objective 2B to 1A reflects biomarkers identified in the previous project period to be validated for apple (2B) as well as applied initially for pear (1A, 2B). Objective 1 is focused on enhancing knowledge of how field horticulture impacts postharvest fruit quality with emphasis on fruit physiological disorders and ripening. The sub-objectives (1A, 1B) are designed to generate new information regarding the impact of pear field horticulture on fruit quality and ripening metabolism, particularly disorder-related metabolomics and genomics. Sub-objective 1C also is focused on generating disorder-related metabolomic information for apple fruit sun damage originating prior to harvest. Application of metabolomic and genomic techniques to disorders arising in the postharvest environment is the basis for Objective 2. Can assessment of apple fruit volatiles accumulating during storage in environments known to cause disorders provide a means to avoid disorder development. (2A) Biomarkers identified for apple disorders in the previous project plan will be validated with multiple fruit lots and cultivars (2B). Disorder metabolism of sweet cherries (2C) will be explored using a metabolomic approach.

Fruit from commercial orchards will be harvested then stored at ARS-Wenatchee and in commercial CA rooms. Fruit quality, metabolites, and mRNA will be characterized at harvest and after storage using standard methods. Hypothesis 1A: Fruit position on the tree directly impacts maturation, superficial scald susceptibility, ripening and storability, and associated metabolism. Pears from two extreme light environments within the tree canopy will be grouped based on differential absorbance (DA). Fruit quality and disorders will be assessed at harvest and after storage. Metabolites and mRNA in peel collected from each canopy location/DA group will be analyzed. Results will be mined for metabolites and mRNA associated with physiological disorders. Hypothesis 1B: Inconsistent post-storage ripening of 1-MCP treated d’Anjou pears is relatable to differential absorbance (DA) value at harvest. Pears will be exposed at harvest to 1-MCP for 16 hours, then stored at 1 degree C. After storage fruit will be evaluated for disorders and fruit quality characterized. Hypothesis 1C: Apple peel metabolism following cold storage imposition is altered by pre-harvest light exposure. ‘Granny Smith’ apples exposed to direct sunlight will be harvested and sorted by sun damage. Apples will be stored at 1 degree C and after storage, untargeted metabolic profiling of ~800 metabolites will be performed on peel and cortex tissue collected at each sampling date. Multivariate and univariate statistical approaches will be employed to link changes in specific areas of metabolism with sunscald and superficial scald development. Hypothesis 2A: Apple aroma volatile production changes when fruit are stored in environments conducive to development of physiological disorders. ‘Honeycrisp’ apples stored in controlled atmosphere chambers will be subjected to atmospheres known to cause physiological disorders. Volatile compound samples will be collected after various intervals and after 180 days, fruit will be removed from storage and evaluated for incidence and severity of external disorders. Hypothesis 2B: Metabolic and gene expression superficial scald based risk assessment can be used to indicate when scald risk in a storage room is elevated.‘Granny Smith’ and ‘Delicious’ apples, and Anjou pears will be stored in CA at 1 degree C. Superficial scald will be evaluated following various lengths of storage. Storage atmospheres will be evaluated for volatile compounds determined to be useful for scald risk assessment. Hypothesis 2C: Pitting and cracking incidence of sweet cherries is associated with altered epidermal metabolic profile compared with undamaged fruit. ‘Rainier’ sweet cherries will be subjected to uniform bruising using a steel ball dropped onto the fruit. Micro-cracking and stomata/lenticel number will be estimated by staining fresh whole fruit with acridine orange and counting micro-cracks at five random positions on each fruit using florescence microscopy. Waxes extracted from fruit will be analyzed using HPLC-QTOF-MS. Metabolic differences linked with cracking and epidermal defects will be identified using untargeted metabolic profiling methods developed for apple.

Progress Report
Research continued on Sub-objective 1A, in determining how fruit position within the tree impacts pear metabolic profile, superficial scald, postharvest quality, and ripening. We determined pears attached to the outside of the tree canopy mature and ripen differently compared with fruit on the inside of the tree canopy. These differences are reflected in peel chemistry including natural chemicals which manifest fruit quality. Other chemicals potentially link fruit on-tree position with future peel condition during the cold chain. Likewise, these differences in fruit quality are also reflected in global gene expression patterns. The capacity to detect elevated levels of natural chemicals responsible for protection from sunlight may provide additional opportunities for accurate fruit sorting which could make stored pear more consistent going into the cold chain, the worldwide temperature controlled supply chain. Similarly, biomarker genes could be monitored and used to guide sorting strategies. Ultimately, our results indicate that fruit on-tree position impacts ripeness and quality all of the way to the consumer and would impact every cold chain and retail management decision beginning immediately at harvest. Research in support of Sub-objective 1B, continued in determining if inconsistent post-storage ripening of 1-methylcyclopropene (1-MCP) treated d’Anjou pears is relatable to differential absorbance (DA) value at harvest. An initial experiment with ‘Bosc’ pears did not support the hypothesis that DA value at harvest could be useful as a predictor of post-storage ripening of 1-MCP treated pears. ‘Bosc’ pears were sorted at harvest using a DA meter into four classes representing less to more peel chlorophyll content. Half the fruit was treated with the ripening inhibitor 1-MCP, then all fruit was stored at 31 degrees Fahrenheit for six or nine months. While a differential in pear firmness, as an indicator of ripeness, was observed after cold storage due to 1-MCP treatment at harvest, no relationship was observed due to DA class at harvest. This result supports some of the previous work with ‘d’Anjou’ where in two of three experiments, DA value at harvest was not a reliable predictor of ripening for 1-MCP treated fruit after cold storage. Research in support of Sub-objective 1C, continued in determining if pre-storage light exposure impacts how apple peel metabolism responds to chilling. Comparison of apple fruit peel from the sun facing side compared with the less exposed side of for varieties of apples revealed vastly different metabolism indicating different ripening and quality alongside a sometimes-different appearance. Each variety manifested sun-related damage in different ways including some consistent differences in metabolism. Natural compounds in the peel associated with fruit quality and ripening were different even on the same apple depending upon relative sun exposure. Levels of natural peel chemicals in the outer coating and those responsible for photoprotection were elevated with sun exposure. Hot air treatment at different timepoints prior to harvest caused symptoms similar to sunscald to develop during storage indicating that solar radiance within the infrared region contributes to disorder development. Also, depending upon timing, heat treatment could “acclimate” peel forming regions where peel was resistant to sunscald. This indicates that peel previously acclimated may not develop the disorder. Microscopic analysis of the cuticle of sun facing, shaded, and sunscalded peel shows that the natural peel coating, cutin, is deposited around the outer epidermal cells of sun-facing peel. This indicates apple peel can create extra protective layers as a defense against high solar irradiation and water loss. We are also determining gene expression differences associated with sun exposure. We have identified natural peel with unique reflectance properties that could potentially be used to sort and remove sun stress-compromised apples before they enter storage. Research in support of Sub-objective 2B, focused on the development of biomarker-based risk monitoring protocols using existing validated gene expression and metabolic biomarkers for early detection of apple and pear peel and flesh storage disorders. Superficial scald (scald) risk assessment biomarker levels indicated high risk fruit lots or lots stored under sub-optimal conditions for scald control during distribution, delivery, and retail. The chilling injury that causes scald accumulates within the first two months of cold storage only to develop following 3-6 months storage. Controlled atmosphere storage conditions were identified that may allow for post-storage treatment using crop protect for effective scald control. The postharvest period where chilling causes injury that leads to scald symptoms later is reduced or halted by ultra-low oxygen (ULO) storage regimes, and post-storage application of scald control compounds and heat treatments are nearly as effective as pre-storage treatments assuming appropriate ULO atmospheres are used on fruit within less than one week following harvest. These new findings open up a number of cold chain management strategies for reducing or eliminating superficial scald from both crop protectant restricted and conventional apple cold chains. We have identified peel chemicals where abundance may be monitored to predict the effectiveness of warming treatments or predict superficial scald risk. These potential biomarkers might be useful to make management decisions during the very early storage period by indicating superficial scald risk including estimating how much chilling injury has accumulated and, consequently, whether post-ULO storage scald mitigation treatments would confer additional control. We have also identified gene activity that may be useful to estimate sufficient warming time that allows the fruit to recover from the chilling injury and avoid superficial scald. A critical step in identifying gene activity signatures that could be developed into biomarkers for predicting future fruit quality is accurate gene activity measurements. Research continued on Sub-objective 2C, in determining if sweet cherry postharvest disorders are relatable to fruit epidermis and wax composition. Tip Top sweet cherries were harvested on multiple dates allowing the risk of peel defects to increase with later harvests. Fruit peel was microscopically examined and surface defect incidence increased with a later harvest date. Physical and chemical differences in fruit surface and peel properties consistent with injury susceptibility were identified. The work to date links sweet cherry peel physical and chemical properties with injury (browning) susceptibility and may provide a means to segregate fruit lots according to browning risk in advance of symptom development.

1. Improving pear quality by identifying metabolites associated with ripening consistency. Pear fruit quality inconsistency can contribute to significant annual losses due to spoilage. This variability can be detected in the natural pear chemistry affecting flavor, appearance, and texture. Changes in chemistry revealed sun exposure may be a prominent source of inconsistency based on fruit on-tree position. Differences in natural fruit chemistry and final product following storage indicates this variability would likely impact every cold chain management decision. ARS researchers in Wenatchee, Washington, determined that sorting pears on the basis of levels of specific natural peel chemicals, prior to entering the cold chain, will enhance fruit quality consistency. Knowledge provided under this accomplishment will benefit industry farmers, processors, and retailers by providing the basis for further tools that would reduce losses due to spoilage and food waste and provide a more consistently ripened product.

2. Reducing apple cold chain losses caused by excess sun exposure. Exposure to excessive sunlight can contribute to 10-25 percent annual losses due to multiple disorders in many of the world’s most productive apple growing regions including the Western U.S. ARS researchers in Wenatchee, Washington, demonstrated that heat events in the orchard contribute to sunscald development, a common source of postharvest losses of susceptible apple varieties. Results reveal physical changes to apple peel surface that may confer resistance to solar stress that leads to sun related postharvest disorders. They identified novel peel chemicals associated with elevated sun exposure and stress as well as fruit quality and ripening. Natural fruit chemicals were identified that may be used to non-destructively sort apples according to sunscald risk before they enter storage.

3. New management strategies for apple superficial scald control. Superficial scald (scald), a postharvest browning disorder of apple peel, continues to contribute to annual fruit quality loss, especially in markets where residues of scald control compounds are restricted. ARS scientists in Wenatchee, Washington, have identified gene activity that, after the chilling injury that causes scald occurs, may be useful to define storage management practices to prevent superficial scald. ARS scientists have also found that chilling injury is cumulative and preventable with optimal controlled atmosphere (low oxygen, high carbon dioxide relative to air) storage conditions started within the first week following harvest, and also demonstrated that post-storage crop-protectant or hot water treatment effectively controls scald if the chilling injury that induces scald has been controlled. Monitoring levels of natural compounds in fruit associated with superficial scald risk may indicate if this criterion is met and post-storage treatments will be effective. These findings indicate novel strategies for reducing or eliminating scald from both crop protectant restricted and organic cold chains are possible using existing commercial technologies. These findings will reduce commodity and economic losses for apple producers, distributers, and retailers for markets where no consistent superficial scald mitigation strategy previously existed.

Review Publications
Vieria, M., Argenta, L., Mattheis, J.P. 2018. Relationship between dry matter content at harvest and maturity index and post-harvest quality of "Fuji" apples. Revista Brasileira de Fruticultura. 40:e-596.
Honaas, L.A., Hargarten, H.L., Ficklin, S., Hadish, J., Wafula, E., dePamphilis, C., Mattheis, J.P., Rudell Jr, D.R. 2019. Co-expression networks provide insights into molecular mechanisms of postharvest temperature modulation of apple fruit to reduce superficial scald. Postharvest Biology and Technology. 149:27-41.
Lee, J., Mattheis, J.P., Rudell Jr, D.R. 2019. High storage humidity affects fruit quality attributes and incidence of fruit cracking in cold-stored ‘Royal Gala’ apples. HortScience. 54(1):149-154.
Serra, S., Sullivan, N., Mattheis, J.P., Musacchi, S., Rudell Jr, D.R. 2018. Canopy attachment position influences metabolism and peel constituency of European pear fruit. Biomed Central (BMC) Plant Biology. 18:364.
Lee, J., Cheng, L., Rudell Jr, D.R., Nock, J.F., Watkins, C.B. 2019. Antioxidant metabolism in stem and calyx end tissues in relation to flesh browning development during storage of 1-methylcyclopropene treated ‘Empire’ apples. Postharvest Biology and Technology. 149:66-73.
Hargarten, H.L., Waliullah, S., Kalcsits, L., Honaas, L.A. 2018. Leveraging transcriptome data for enhanced gene expression analysis in apple. Journal of the American Society for Horticultural Science. 143(5):333-346.