In addition to ABA, other hormone biosynthesis genes exhibited a similar expression such as auxin and cytokinin, both of which have been implicated in non-climacteric fruit maturation and ripening . Thus, pistachio should be considered a non-climacteric fruit.While ethylene was not important for ripening in pistachio, ethylene may be more critical prior to kernel development at the end of Stage II where a rise in biosynthesis occurs . The exact function of the hormone at this stage of development is unknown but has previously been suggested to be involved in bud abscission in alternate bearing years and needs further investigation . JA-related genes were also elevated during Stage II and were among the highest expressed hormone-related genes. JA is best known as a stress hormone involved in many responses to abiotic and biotic stress but can also function in fruit development . JA biosynthesis genes were previously shown to be differentially expressed in pistachio vegetative tissues that underwent a salinity treatment, compared to the control . However, there were no known stresses occurring during our samplings to explain elevated expression. This suggests JA is a critical hormone in pistachio fruit Stage II development prior to kernel initiation, or other events occurring concurrently. As pistachio is known to be tolerant to environmental stress, JA and ethylene levels may play a role evolutionarily to adapt to these extreme environments.The hull functions as a protective tissue encapsulating the shell and kernel. The breakdown of the hull caused by senescence can lead to a lower quality commodity; for example, plastic grow bag the shell becomes more vulnerable to staining from the hull and the kernel becomes more accessible to pests. The hull is rich in volatile compounds mainly composed of terpenes . We examined the dynamic changes of volatile compounds in the hull during Stage III and IV to gain insight into the events leading to hull ripening and senescence.
We saw a rise of volatile compounds at the onset of ripening that may offer a signal of developmental changes occurring We observed limonene and alpha-terpinolene to be the highest produced monoterpenes consistently across years. Alpha-terpinolene had also been found in high proportions of Tunisian pistachio variety but did not have as high of concentrations of limonene, which could be due to varietal differences . Limonene has been shown to accumulatein orange peel with ripening in order to attract insects and pathogens . This relationship between limonene and other organisms was proposed to have evolved to facilitate seed dispersal, opening up the fruit to expose the seeds. This could also be the case for pistachio, in which the volatile production at the start of ripening signals that the kernel is mature and attracts seed dispersing organisms. Volatile signals further define the events leading to ripening in pistachio and have additional implications for management practices to time treatments against insects.Like canonical fleshy fruit, pistachio fruit quality is determined during fruit ripening. Ripening changes in the hull coincide with important quality traits and can be used to anticipate the best harvest time. Therefore, understanding the timing and relationship between the hull and kernel during Stage IV allows for increased quality. Our study integrates multiple approaches, including physiology, biochemistry, and genomics, to provide the most thorough understanding of pistachio fruit development to date. Fruit ripening in pistachio to our knowledge has not been previously explored. The hull undergoes changes in composition preceding harvest consistent with fruit ripening including, softening and color change which we define as an additional stage, Stage IV . These changes are important attributes that help determine harvest time and maximize fruit quality. For example, hull softening allows fruits to be detached from the tree, however, if overripe the hull senesces and the degradation can cause shell stain and make kernels vulnerable to pests and disease decreasing its nutritional and market value.
We integrated gene expression data with the observed physical changes to inform the events occurring leading to fruit ripening. Color change is a characteristic of ripening and provides a visual indication of when fruit are ready for harvest. Previous studies have identified anthocyanin, carotenoids, and chlorophyll compounds in pistachio hulls . However, the proportion of these compounds present depended on the stage sampled and variety, with measure-ments limited to pistachio green hulls prior to ripening. Thus, it is unclear which specific compounds lead to the pink colorations in the hull during ripening. Our gene expression analysis indicated that the flavonoid pathways were active in the hull, however there was not a strong expression of anthocyanin reductase genes, the critical final step for anthocyanin production, giving the compounds their pigmentation. It is clear that the fruit produce some anthocyanins because they have been identified in the purple colored seed coat surrounding the kernel . We found stronger gene expression of carotenoid biosynthesis. Among the pigments identified in Grace et al., lutelin was the highest measured in pistachio hulls . We observed high expression of ctrZ which is annotated to act in the step leading to lutein biosynthesis. The change in green coloration to yellow at the end of Stage III also indicates chlorophyll degradation may be occurring. We identified several chlorophyll degradation genes expressed in modules with ripening patterns, such as STAY GREEN , a chlorophyllide reductase that regulates chlorophyll protein degradation . Thus, from our analysis we hypothesize that hull color becomes yellow from chlorophyll degradation and shades of pink from carotenoids. It is well known that fruit softening is mediated by cell wall degrading enzymes in both climacteric and non-climacteric fruits, such as tomato and strawberry . Cell wall degrading enzymes acting on the backbone of pectin molecules, such as polygalacturonase and pectin lyase, are highly expressed in other fruit and exhibit a ripening-specific gene expression pattern . Further, α-LAFase is highly expressed and acts as a catalyst with other coexpressed cell wall degrading enzymes . Thus, we were interested if these enzymes were expressed in a ripening-specific pattern. Pectate lyase was among the highest expressed CWDEs annotated from the CAZy database in pistachio and began to rise in expression at Stage IV .
Consistent with this, pectins have been measured in pistachio hulls and were proposed as a potential source of commercial pectins . The presence of a large proportion of pectins in green hulls and the high expression of pectin-degrading enzymes suggest these enzymes promote pectin degradation and softening of the tissue. The mechanisms involved in this still need to be explored. Overall, knowing how and when hull softening occurs during the growing season can help advise the optimal time for harvest.Kernel growth during Stage III leads to the maturity of the seed and ripening of the hull. Understanding when the kernel is most desirable for consumption and when the fruit is ready for harvest can improve management practices and fruit quality. Maturity can be observed when kernels reach their maximum size and fat content at the start of Stage IV, as ripening progresses . From our gene expression and metabolite data we see that fatty acid biosynthesis occurs early on in kernel development and is primarily composed of unsaturated fatty acids, with much of the production reaching its maximum during ripening . Further, pe grow bag the kernel shows hormonal indications of seed maturity with an increase of GA at the start of Stage IV and ABA increasing throughout Stage IV. Pistachio kernels are consumed for their unique flavor and nutritional benefits. Kernels are made up of primarily unsaturated fats, including both poly- and mono- unsaturated fatty acids . Although PUFAs provide essential fruitrients to the human diet, they make kernels more vulnerable to rancidity, reducing their shelf life . Thus, the ratios of PUFA and MUFA are important for considering nutritional benefits and shelf life. We identified important enzymes in our gene expression data explaining the accumulation of specific unsaturated fats . Interestingly, our study showed fluctuations in the composition of unsaturated fatty acids through kernel development similar to a previous study . The mono-unsaturated fatty acid oleic acid increases through time while the poly-unstaturated linoleic acid decreases. These fluctuations were not completely explainable with our expression data, but are likely caused by other fluxes in the fatty acid metabolism downstream of these compounds.Calcium is an essential plant nutrient required for proper plasma membrane function, in storage organelles to counterbalance anionic charges, in the cytosol for cellular signalling responses, and in the apoplast for cell wall structure . Ca2+ deficiency disorders in fruit have been attributed to lower total tissue Ca2+ content, as well as abnormal regulation of cellular Ca2+ partitioning and distribution . The symptoms of Ca2+ deficiency disorders in fruit start with cell plasmolysis and the water-soaked appearance of blossom-end tissues that eventually becomesdark brown as cells die . Although Ca2+ is believed to move in the plant exclusively through the xylem vascular tissue , the mechanisms regulating Ca2+ partitioning and allocation in tomato plants and fruit remain poorly understood. Consistent with xylem sap flow, the direction and rate of xylemic Ca2+ flow in the plant should be determined by water potential gradients in response to different tissue transpiration and growth rates . In that case, higher transpiration and growth rates can reduce water potential and increase tissue strength as sinks for xylemic Ca2+. Therefore, the partitioning of Ca2+ flowing from the roots toward leaves and fruit will depend on the xylem sap Ca2+ concentration, as well as leaf and fruit transpiration and growth rates. Accordingly, leaves have much higher transpiration rates than fruit, which results in much higher Ca2+ content in the leaves than in the fruit .
Previous studies have shown that specifically reducing leaf transpiration by decreasing atmospheric vapour pressure deficit or treating tomato plants with abscisic acid can potentially decrease xylemic Ca2+ movement into the leaves, and increase its movement into the fruit . However, direct measurements of xylemic Ca2+ concentration and xylem sap flow rates into leaves and fruit in response to reduced leaf transpiration rates have not been reported. Spraying whole plants with ABA increases fruit total tissue and apoplastic Ca2+ concentrations, and reduced fruit cell membrane leakage and the incidence of blossom-end rot . These studies suggest that ABA may affect not only total fruit tissue Ca2+ concentration but also the regulation of cellular Ca2+ distribution, which could affect fruit susceptibility to Ca2+ deficiency disorders such as BER . Since these studies were based on whole-plant ABA sprays, the results cannot be specifically attributed to whole plant or fruit responses to ABA . Fruit-specific ABA studies are still needed to understand if the prevention of BER development is a whole-plant, a fruit specific, or a combination response to ABA. The objectives of this study were to determine Ca2+ partitioning and allocation in tomato plants and fruit in response to whole-plant and fruit-specific ABA treatments, as well as to analyse the effect of changes in Ca2+ partitioning and allocation on fruit susceptibility to BER under water stress conditions. The HRM was developed to measure low net sap flow rates that can take place in either direction in the vascular tissue , but for the current study only the xylem sap flow rate was determined by heat girdling the middle leaf pedicel or fruit peduncle. The heat girdling was accomplished by passing an electrical signal for 20 s across a constant an wire with 0.8mm diameter looped twice around the pedicel or peduncle 1 cm upstream of each heat sensor before starting the sap flow measurements . Heat girdling destroys the phloem cells, obstructing phloem sap movement, while the xylem sap flow remains intact and functional due to its non-living cells. This technique has been used to isolate and quantify phloem and xylem sap flow rates . After heat girdling, sap flow measurements were made over a 24h period. After xylem sap flow measurements, zero sap flow readings were determined by cutting the middle leaf pedicel or fruit peduncle 1 cm downstream of each sensor. The zero xylem sap flow readings were used to determine the baseline accurately for each sap flow sensor after sap flow measurements. After determining the zero sap flow rate, the middle leaf pedicel or fruit peduncle was cut at the heat sensor region to measure the diameter of the xylem vascular tissue, which was used to calculate the volume of xylem sap moving into the leaf and fruit over time. One fully expanded top leaf and one tagged fruit on each plant replication were used for the sap flow analysis.