Here we present analyses of gene expression changes that occurred in the fruit of two blueberry cultivars, ‘Bluecrop’ (known for poor firmness retention) and ‘Legacy’ (known for good firmness retention) during post-harvest storage. Whilst a genome reference sequence for tetraploid blueberry cultivar ‘Draper’ recently became available [22], which provides a reliable genomic resource for transcriptomic analysis, separate de novo transcriptome assemblies were performed here for ‘Bluecrop’ and ‘Legacy’ to avoid multiple- and/or mis-alignments within unigenes produced from a hybrid or reference assembly. Overall, the number of transcripts the ‘Legacy’ and ‘Bluecrop’ unigene sets resolved was 41,896 and 39,335 for respectively, comparable to those of the published genome that contained an average of 32,140 gene predictions per haplotypes, and the majority of the assembled transcripts returned highly similar, or identical matches to predicted genes within the ‘Draper’ reference genome [22].
The data presented here revealed that cell, cell part, intracellular, membrane-bound organelle part, cellular and metabolic processes, and catalytic activity were the most represented GO categories within the assembled unigene set. These categories were strongly represented in other studies of ‘Bluecrop’ [27, 28] as well as in studies of the cultivars ‘Northland’ [24], ‘O’Neal’ [21] and ‘Duke’ [26]. The vast majority of factors affecting post-harvest quality are under strong genetic control [29] and ‘Bluecrop’ and ‘Legacy’ previously displayed significant differences in their response to prolonged periods of low-temperature post-harvest storage [7], resulting in very different post-storage texture profiles of the two cultivars. A recent study of Zhang et al., 2020 [26] used the cultivar ‘Duke’ which previously displayed texture characteristics very similar to ‘Legacy’ [7] at harvest, but which displayed a significant decline in texture during post-harvest storage. The analysis of differentially expressed genes in ‘Duke’ identified 1,167 upregulated, and 685 down-regulated genes following 30 days storage at 0 degrees C, which contrasted to the 2,370 down-regulated and 937 up-regulated, and 1,379 down-regulated and 542 up-regulated genes in ‘Bluecrop’ and ‘Legacy’ respectively following 21 days storage at 4 °C and three days at 18 °C in this study.
Here, a greater number of down and up regulated genes was observed in ‘Bluecrop’ than ‘Legacy’, suggesting ‘Bluecrop’ fruit is less well adapted to the physiological stresses associated with prolonged storage at 4 °C and subsequent shelf life (21 °C) than ‘Legacy’, as reflected in a greater decline in fruit quality and texture attributes observed in ‘Bluecrop’ over ‘Legacy’ [7]. Numerous DEGs associated within diverse cell, molecular, and biological pathways were identified as candidate unigenes with a potential role in firmness changes during storage in this study. Most of the DEGs observed between harvest and post-harvest in ‘Bluecrop’ and in ‘Legacy’ were predominantly categorized into amino acid metabolism, carbohydrate metabolism and cofactor and vitamins metabolism, followed by energy metabolism in ‘Bluecrop’ and by nucleotide metabolism in ‘Legacy’. Furthermore, most of the DEGs identified between harvest and post-harvest between both cultivars were putatively involved in cell wall metabolism, composition of the skin wax layer, adaptation to abiotic stress and solute transport. Zhang et al., 2020 [26] found membrane lipid metabolism, proline, glutathione and flavonoid metabolism, and hormone biosynthesis and signal transduction as the main pathways in which genes were differentially expressed in ‘Duke’, during storage at 0℃. Whilst many of the categories of DEGs observed were similar between the two studies, the differences observed in gene expression profiles between those reported here for ‘Bluecrop’ and ‘Legacy’, and those reported for ‘Duke’ most probably reflect the differences in the temperature storage protocols employed and most importantly differences in the point of sampling between the two studies. The post-harvest sampling conditions of [26] more closely resembled the sampling point in this investigation immediately following post-harvest storage when fruit was still at 4 °C. Zhang et al., 2020 [26], used a temperature of 0ºC, and whilst that has also been used in other blueberry studies to compare the fruit quality including firmness at different temperatures and days post-harvest [10, 11], it is not representative of temperatures in retail or home refrigeration settings.
Scrutiny of the differentially expressed transcripts in ‘Bluecrop’ and ‘Legacy’ was focussed on genes that could have played a putative role in the stress response during post-harvest cold-storage, and genes that could play a role in the retention of texture and fruit quality. Previous studies have shown that genes involved in determining the composition of the wax layer [bloom] in blueberry, as well as those involved in cell wall metabolism, adaptation to biotic or abiotic stress, calcium and solute transport could all play a role in maintaining good fruit quality during post-harvest storage, and from these classes, a set of 21 differentially-expressed candidate genes were characterised.
Epicuticular wax metabolism
The waxy layer representing the bloom of blueberries is a key protective mechanism against a range of abiotic stresses, including moisture loss and temperature fluctuations, and has been shown to reduce deterioration in fruit quality during post-harvest storage [31]. Variations in the composition of the wax compounds in plant cuticles can affect their mechanical properties under storage [35–37] and these variations have been correlated with textural changes in blueberry [38], pepper and tomato [39–41] fruits during storage. Unigene 8556 shown to have high homology to wax ester synthase/diacylglycerol acyltransferase (WSD1), an enzyme which catalyses the synthesis of wax ester compounds in the stem, flowers and leaves of Arabidopsis [42]. Acyltransferase genes have been reported to be strongly upregulated in Arabidopsis in response to abiotic/biotic stress [43] and in Euruca vesicaria seedlings in response to drought stress [44]. ‘Bluecrop’ softens more rapidly during storage than ‘Legacy’ [7], indicating a greater rate of moisture loss in ‘Bluecrop’ fruits during storage [12]. Here, unigene 8556 was highly down-regulated in ‘Legacy’, but up-regulated in ‘Bluecrop’, suggesting fruit of ‘Bluecrop’ may be triggering stress response pathways and increasing wax production in response to moisture loss during extended exposure to low temperatures and reduced % relative humidity experienced in post-harvest storage.
Cell wall metabolism under post-harvest storage
Changes in cell wall structure during development and ripening occur due to degradation of cell wall constituents primarily through the disassembly of the cellulose-hemicellulose network [7, 45–47]. The cellulose and hemicellulose matrix comprise microfibrils linked by hydrogen bonds, that increase the strength of the cell wall. In addition, a pectin matrix interlaces the cellulose-hemicellulose backbone conferring cell wall adhesion [25]. Thus, cell wall modifications and disassembly form part of the regulation of fruit softening, which is a process that has been well characterised in blueberries [17, 32, 48], apple [49], peach [50], strawberries [51, 52] and tomatoes [53, 54].
Glycoside hydrolases have been reported to affect blueberry firmness during ripening and post-harvest stages [25, 26] and their enzymatic activity depends on their specificity to individual carbohydrate components forming the cellulose (homopolymer of glucose) and hemicellulose (xylans, glucans, xyloglucans, callose, mannans and glucomannans) structures [14, 55, 56]. In this investigation, homologues of three glycoside hydrolases; GH17 [Unigene 3134), GH3 (Unigene 6494) and GH28 (Unigene 8703) were shown to be differentially-expressed during post-harvest cold-storage. The GH17 family encodes endoglucanases which degrade cellulose structures (56) and play a role in cell wall degradation in blueberry fruit and banana during ripening stages (25, 57). Down-regulation of unigene 3134 (GH17) in ‘Bluecrop’ and ‘Legacy’ suggests a reduction in activity of this glycoside hydrolase during cold storage, supporting the findings of [25, 46] who reported reduced glycoside hydrolase activity in blueberry fruits during cold storage. Reduction in activity may be purely temperature related or possibly an adaptation mechanism triggered in both cultivars in response to post-harvest cold-storage [25, 46]. GH3 is a xylosidase enzyme [56] with a role in degrading cell walls and contributing to softening in blueberry fruits [14, 48], whilst GH28 showed high homology to a polygalacturonase enzyme which degrades pectin by hydrolysing the homogalacturonan backbone of the cell wall [58]. This family of enzymes has been shown to be upregulated during post-harvest storage and to a play a role in the softening of blueberry fruits and cell wall degradation [32]. Both GH3 (Unigene 6494) and GH28 (unigene 8703) were highly upregulated in ‘Bluecrop’ and down regulated in ‘Legacy’ during cold storage and thus may play a role in faster cell wall degradation in ‘Bluecrop’, leading to a greater degree of softening.
The role of expansin proteins in cell wall loosening has been well characterised in Arabidopsis [59], tomato [60, 61], strawberry [62–64], peach [65] and kiwi fruits [66] where its activity is upregulated during the softening of ripening fruits [67]. Expansins initiate cell wall loosening and extension through the breakage of hydrogen bonds between cellulose and hemicellulose molecules, in particular xyloglucans [68, 69], and their expression is regulated by cross-talk between many plant growth regulators including abscisic acid, indol-3-acetic acid, auxins, brassinosteroids, cytokines, ethylene [67]. Expansin activity has been shown to be enhanced by low pH, and low temperatures in an absence of an ethylene peak during storage [66]. Unigene 21016 was shown to be highly upregulated in ‘Bluecrop’ and down regulated in ‘Legacy’ during post-harvest storage, suggesting that expansins expression in ‘Bluecrop’ may contribute to the increased fruit softening observed by Giongo et a., 2013 [7], as has been reported in kiwi [66] and in tomato [61], where overexpression of expansins produced softer fruit and silencing was correlated to an increase of firmness and extended shelf life.
Abiotic stress and solute transport
Adaptation to abiotic stress in plants include the control of cell turgor, the induction of cell signalling pathways, an increase in respiration rates and deployment of protective mechanisms against Reactive Oxygen Species (ROS). The APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) is one of the mediators to plant external abiotic responses and developmental processes [70–73], and has been reported to regulate cold adaptation responses in the flower buds of ‘Bluecrop’ [74], and in cold-stored blueberry fruit of ‘Duke’ [26]. Unigene 7737 and Unigene 13947 display high homology to AP2/ERF and were found in differentially regulated clusters in this investigation, suggesting that members of this family of transcription factors may be repressed or induced under the same environmental conditions. AP2/ERF homologs in potato (CIP353) conferred low-temperature acclimation to tubers exposed to long-term cold-storage [72], whilst in tomato the overexpression of PTi4 encoding an AP2/ERF induced a family of expansin genes linked to roles in cell wall integrity [75]. AP2/ERF genes have also been reported to regulate the adaptation to osmotic, water stress and drought tolerance in Arabidopsis [76], and osmotic differences between protoplast and the apoplast have been shown to drive changes in turgor pressure in plant cells [77], changes which are linked to post-harvest softening in apple [78] and softening in blueberries [10, 12, 15]. ‘Legacy’ exhibits a greater retention of firmness than ‘Bluecrop’ during post-harvest cold-storage [7] and unigene 13947 was upregulated in ‘Legacy’, and down-regulated in ‘Bluecrop’ suggesting that its expression may play a role in the maintenance of firmness in blueberry fruits during post-harvest cold-storage.
The mitochondria play an important function in buffering cytoplasmic calcium, with mitochondrial calcium uniporters (MCU) acting as calcium sensors and active channel protein for calcium uptake [30, 79, 80]. Mitochondrial uptake plays a role in ATP synthesis and regulating calcium concentration in the cytoplasm [Ca]cyt which is essential for regulating its role as a secondary messenger in response to abiotic stress. Moreover, [Ca]cyt plays a role in regulating fruit quality, specifically by increasing the resilience of fruit to low temperature stress by enhancing the energy status of the cells and maintaining osmotic conditions [81]. Unigene 13589 displayed homology to mitochondrial calcium uniporters [MCU] and was down regulated during post-harvest storage for both cultivars, which may cause loss of mitochondrial function and reduced cellular calcium uptake during post-harvest cold-storage, contributing to fruit quality deterioration during storage.