Savithri U. Nambeesan

Background: Fruit ripening is a coordinated process that leads to an increase in sugars, decrease in acids and accumulation of pigments. Blueberry fruit exhibit an atypical climacteric ripening behavior. These fruit display an increase in respiration and ethylene production during ripening, however ethylene synthesis is developmentally regulated. In this study, the effect of ethylene on blueberry fruit ripening was investigated via preharvest applications of ethylene-releasing plant growth regulators (PGRs), ethephon and 1-aminocyclopropane 1-carboxylic acid (ACC), in one southern highbush cultivar, Miss Lilly in 2019, and two rabbiteye cultivars, Premier and Powderblue in 2019 and 2020. Further, the effects of these two PGRs on fruit metabolism during ripening in the two rabbiteye cultivars, and postharvest fruit quality in all three cultivars were evaluated. Results: Both PGRs increased ethylene evolution within 1–3 days after treatment (DAT). Ethephon and ACC applications increased the rate of ripening within 5 DAT in all cultivars, and increased ripe (blue) fruit by up to 35% and 29%, respectively between 7 to 10 DAT compared to the control. Metabolite analysis revealed that PGR treatments resulted in an immediate, but transient increase in sucrose, glucose and fructose, in ‘Premier’ at 3 DAT. Malate decreased at 3 DAT in response to both PGR treatments in ‘Premier’, and at 5 DAT in ethephon treatment in both cultivars. A rapid increase in the concentration of multiple anthocyanins was noted at 3 DAT in response to both PGRs in ‘Premier’ and ‘Powderblue’. Gene expression analysis revealed an increase in transcript abundance of VACUOLAR INVERTASE (vINV) and multiple anthocyanin biosynthesis genes between 1 and 3 DAT after PGR treatments in both cultivars, supporting the metabolite changes. However, the alteration in fruit metabolite concentrations were not sustained, and similar in PGR-treated fruit compared to the control in ripe fruit harvested at 10 DAT. Postharvest fruit quality attributes, such as firmness, total soluble solids, titratable acidity, and visual quality, were not consistently affected by the PGR applications compared to control treatments across all cultivars. A decrease in fruit weight was noted, although not consistently, in response to PGR treatments. Conclusions: Overall, this study demonstrates that ethylene plays a crucial role in promoting ripening via rapid and transient stimulation of sugar, acid and anthocyanin metabolism. The promotion of fruit ripening by ethylene-releasing PGRs can lead to minimal but inconsistent changes in fruit quality attributes during postharvest storage.

Fruit ripening is a highly coordinated process involving molecular and biochemical changes that collectively determine fruit quality. The underlying metabolic programs and their transitions leading to fruit ripening remain largely under-characterized in blueberry (Vaccinium sp.), which exhibits atypical climacteric behavior. In this study, we focused on sugar, acid and anthocyanin metabolism in two rabbiteye blueberry cultivars, Premier and Powderblue, during fruit development and ripening. Concentrations of the three major sugars, sucrose (Suc), glucose (Glc), and fructose (Fru) increased steadily during fruit development leading up to ripening, and increased dramatically by around 2-fold in ‘Premier’ and 2- to 3-fold in ‘Powderblue’ during the final stage of fruit ripening. Starch concentration was very low throughout fruit development in both cultivars indicating that it does not serve the role of a major transitory carbon (C) storage form in blueberry fruit. Together, these patterns indicate continued import of C, likely in the form of Suc, throughout blueberry fruit development. Concentrations of the predominant acids, malate and quinate, decreased during ripening, and may contribute to increased shikimate biosynthesis which, in-turn, allows for downstream phenylpropanoid metabolism leading to anthocyanin synthesis. Consistently, anthocyanin concentrations were highest in fully ripened blue fruit. Weighted gene co-expression network analysis (WGCNA) was performed using a ‘Powderblue’ fruit ripening transcriptome and targeted fruit metabolite concentration data. A ‘dark turquoise’ module positively correlated with sugars and anthocyanins, and negatively correlated with acids (malate, quinate), was identified. Gene Ontology (GO) enrichment analysis of this module identified transcripts related to sugar, acid, and phenylpropanoid metabolism pathways. Among these, increased transcript abundance of a VACUOLAR INVERTASE during ripening was consistent with sugar storage in the vacuole. In general, transcript abundance of the glycolysis pathway genes was upregulated during ripening. The transcript abundance of PHOSPHOENOLPYRUVATE (PEP) CARBOXYKINASE increased during fruit ripening and was negatively correlated with malate concentration, suggesting increased malate conversion to PEP, which supports anthocyanin production during fruit ripening. This was further supported by the co-upregulation of several anthocyanin biosynthesis-related genes. Together, this study provides insights into important metabolic programs, and their underlying gene expression patterns during fruit development and ripening in blueberry.

Background: Blueberry fruit exhibit atypical climacteric ripening with a non-auto-catalytic increase in ethylene coincident with initiation of ripening. Further, application of ethephon, an ethylene-releasing plant growth regulator, accelerates ripening by increasing the proportion of ripe (blue) fruit as compared to the control treatment. To investigate the mechanistic role of ethylene in regulating blueberry ripening, we performed transcriptome analysis on fruit treated with ethephon, an ethylene-releasing plant growth regulator. Results: RNA-Sequencing was performed on two sets of rabbiteye blueberry (‘Powderblue’) fruit: (1) fruit from divergent developmental stages; and (2) fruit treated with ethephon, an ethylene-releasing compound. Differentially expressed genes (DEGs) from divergent developmental stages clustered into nine groups, among which cluster 1 displayed reduction in expression during ripening initiation and was enriched with photosynthesis related genes, while cluster 7 displayed increased expression during ripening and was enriched with aromatic-amino acid family catabolism genes, suggesting stimulation of anthocyanin biosynthesis. More DEGs were apparent at 1 day after ethephon treatment suggesting its early influence during ripening initiation. Overall, a higher number of genes were downregulated in response to ethylene. Many of these overlapped with cluster 1 genes, indicating that ethylene-mediated downregulation of photosynthesis is an important developmental event during the ripening transition. Analyses of DEGs in response to ethylene also indicated interplay among phytohormones. Ethylene positively regulated abscisic acid (ABA), negatively regulated jasmonates (JAs), and influenced auxin (IAA) metabolism and signaling genes. Phytohormone quantification supported these effects of ethylene, indicating coordination of blueberry fruit ripening by ethylene. Conclusion: This study provides insights into the role of ethylene in blueberry fruit ripening. Ethylene initiates blueberry ripening by downregulating photosynthesis-related genes. Also, ethylene regulates phytohormone-metabolism and signaling related genes, increases ABA, and decreases JA concentrations. Together, these results indicate that interplay among multiple phytohormones regulates the progression of ripening, and that ethylene is an important coordinator of such interactions during blueberry fruit ripening.

Background: Arbuscular mycorrhizal fungi (AMF) are beneficial soil fungi which can effectively help plants with acquisition of mineral nutrients and water and promote their growth and development. The effects of indigenous and commercial isolates of arbuscular mycorrhizal fungi on pear (Pyrus betulaefolia) trees, however, remains unclear.Methods: Trifolium repens was used to propagate indigenous AMF to simulate spore propagation in natural soils in three ways: 1. the collected soil was mixed with fine roots (R), 2. fine roots were removed from the collected soil (S), and 3. the collected soil was sterilized with 50 kGy ⁶⁰Co γ-radiation (CK). To study the effects of indigenous AMF on root growth and metabolism of pear trees, CK (sterilized soil from CK in T. repens mixed with sterilized standard soil), indigenous AMF (R, soil from R in T. repens mixed with sterilized standard soil; S, soil from S in T. repens mixed with sterilized standard soil), and two commercial AMF isolates (Rhizophagus intraradices(Ri) and Funneliformis mosseae (Fm)) inoculated in the media with pear roots. Effects on plant growth, root morphology, mineral nutrient accumulation, metabolite composition and abundance, and gene expression were analyzed.Results: AMF treatment significantly increased growth performance, and altered root morphology and mineral nutrient accumulation in this study, with the S treatment displaying overall better performance. In addition, indigenous AMF and commercial AMF isolates displayed common and divergent responses on metabolite and gene expression in pear roots. Compared with CK, most types of flavones, isoflavones, and carbohydrates decreased in the AMF treatment, whereas most types of fatty acids, amino acids, glycerolipids, and glycerophospholipids increased in response to the AMF treatments. Further, the relative abundance of amino acids, flavonoids and carbohydrates displayed different trends between indigenous and commercial AMF isolates. The Fm and S treatments altered gene expression in relation to root metabolism resulting in enriched fructose and mannose metabolism (ko00051), fatty acid biosynthesis (ko00061) and flavonoid biosynthesis (ko00941).Conclusions: This study demonstrates that indigenous AMF and commercial AMF isolates elicited different effects in pear plants through divergent responses from gene transcription to metabolite accumulation.

Background: Blueberries (Vaccinium sp.) are native to North America and breeding efforts to improve blueberry fruit quality are focused on improving traits such as increased firmness, enhanced flavor and greater shelf-life. Such efforts require additional genomic resources, especially in southern highbush and rabbiteye blueberries. Results: We generated the first full-length fruit transcriptome for the southern highbush and rabbiteye blueberry using the cultivars, Suziblue and Powderblue, respectively. The transcriptome was generated using the Pacific Biosciences single-molecule long-read isoform sequencing platform with cDNA pooled from seven stages during fruit development and postharvest storage. Raw reads were processed through the Isoseq pipeline and full-length transcripts were mapped to the ‘Draper’ genome with unmapped reads collapsed using Cogent. Finally, we identified 16,299 and 15,882 non-redundant transcripts in ‘Suziblue’ and ‘Powderblue’ respectively by combining the reads mapped to Northern Highbush blueberry ‘Draper’ genome and Cogent analysis. In both cultivars, > 80% of sequences were longer than 1,000 nt, with the median transcript length around 1,700 nt. Functionally annotated transcripts using Blast2GO were > 92% in both ‘Suziblue’ and ‘Powderblue’ with overall equal distribution of gene ontology (GO) terms in the two cultivars. Analyses of alternative splicing events indicated that around 40% non-redundant sequences exhibited more than one isoform. Additionally, long non-coding RNAs were predicted to represent 5.6% and 7% of the transcriptomes in ‘Suziblue’ and ‘Powderblue’, respectively. Fruit ripening is regulated by several hormone-related genes and transcription factors. Among transcripts associated with phytohormone metabolism/signaling, the highest number of transcripts were related to abscisic acid (ABA) and auxin metabolism followed by those for brassinosteroid, jasmonic acid and ethylene metabolism. Among transcription factor-associated transcripts, those belonging to ripening-related APETALA2/ethylene-responsive element-binding factor (AP2/ERF), NAC (NAM, ATAF1/2 and CUC2), leucine zipper (HB-zip), basic helix-loop-helix (bHLH), MYB (v-MYB, discovered in avian myeloblastosis virus genome) and MADS-Box gene families, were abundant. Further we measured three fruit ripening quality traits and indicators [ABA, and anthocyanin concentration, and texture] during fruit development and ripening. ABA concentration increased during the initial stages of fruit ripening and then declined at the Ripe stage, whereas anthocyanin content increased during the final stages of fruit ripening in both cultivars. Fruit firmness declined during ripening in ‘Powderblue’. Genes associated with the above parameters were identified using the full-length transcriptome. Transcript abundance patterns of these genes were consistent with changes in the fruit ripening and quality-related characteristics. Conclusions: A full-length, well-annotated fruit transcriptome was generated for two blueberry species commonly cultivated in the southeastern United States. The robustness of the transcriptome was verified by the identification and expression analyses of multiple fruit ripening and quality–regulating genes. The full-length transcriptome is a valuable addition to the blueberry genomic resources and will aid in further improving the annotation. It will also provide a useful resource for the investigation of molecular aspects of ripening and postharvest processes.

Climacteric fruits display an increase in respiration and ethylene production during the onset of ripening, while such changes are minimal in non-climacteric fruits. Ethylene is a primary regulator of ripening in climacteric fruits. The ripening behavior and role of ethylene in blueberry (Vacciniumsp.) ripening is controversial. This work aimed to clarify the fruit ripening behavior and the associated role of ethylene in blueberry. Southern highbush (Vaccinium corymbosumhybrids) and rabbiteye (Vaccinium ashei) blueberry displayed an increase in the rate of respiration and ethylene evolution, both reaching a maxima around the Pink and Ripe stages of fruit development, consistent with climacteric fruit ripening behavior. Increase in ethylene evolution was associated with increases in transcript abundance of its biosynthesis genes,AMINOCYCLOPROPANE CARBOXYLATE(ACC)SYNTHASE1(ACS1) andACC OXIDASE2(ACO2), implicating them in developmental ethylene production during ripening. Blueberry fruit did not display autocatalytic system 2 ethylene during ripening asACStranscript abundance and ACC concentration were not enhanced upon treatment with an ethylene-releasing compound (ethephon). However,ACOtranscript abundance was enhanced in response to ethephon, suggesting thatACOwas not rate-limiting. Transcript abundance of multiple genes associated with ethylene signal transduction was upregulated concomitant with developmental increase in ethylene evolution, and in response to exogenous ethylene. As these changes require ethylene signal transduction, fruit ripening in blueberry appears to involve functional ethylene signaling. Together, these data indicate that blueberry fruit display atypical climacteric ripening, characterized by a respiratory climacteric, developmentally regulated but non-autocatalytic increase in ethylene evolution, and functional ethylene signaling.

Sustaining the fourfold increase in blueberry (Vaccinium sp.) production witnessed during the previous two decades requires better understanding of its mineral nutrient physiology. The primary goals of this review are to evaluate our current understanding of the physiology of nitrogen (N) and calcium (Ca) nutrition in blueberry. Nitrogen concentration in blueberry ranges from 0.4% to >2% across organs. Blueberry uses N in various forms (organic and inorganic), but it appears to display preference for ammonium (NH₄⁺) over nitrate (NO₃⁻). The roles of N acquisition, translocation and assimilation in determining N-source preference in blueberry are evaluated. Calcium plays important roles in determining fruit quality owing to its function in maintaining cell wall and membrane integrity. It is unique in its translocation characteristics being transported primarily via the xylem. Fruit [Ca²⁺] typically declines from around 0.2% during early development to 2+] are discussed. Areas where further research is warranted to improve our understanding of N and Ca physiology in blueberry are identified. Such knowledge is essential for sustainable nutrient management, improving productivity, and enhancing fruit quality in blueberry.

Potassium (K) plays a crucial role in multiple physiological and developmental processes in plants. Its deficiency is a common abiotic stress that inhibits plant growth and reduces crop productivity. A better understanding of the mechanisms involved in plant responses to low K could help to improve the efficiency of K use in plants. However, such responses remain poorly characterized in fruit tree species such as pears (Pyrus sp). We analyzed the physiological and transcriptome responses of a commonly used pear rootstock, Pyrus betulaefolia, to K-deficiency stress (0 mM). Potassium deprivation resulted in apparent changes in root morphology, with short-term low-K stress resulting in rapidly enhanced root growth. Transcriptome analyses indicated that the root transcriptome was coordinately altered within 6 h after K deprivation, a process that continued until 15 d after treatment. Potassium deprivation resulted in the enhanced expression (up to 5-fold) of a putative high-affinity K⁺ transporter, PbHAK5 (Pbr037826.1), suggesting the up-regulation of mechanisms associated with K⁺ acquisition. The enhanced root growth in response to K-deficiency stress was associated with a rapid and sustained decrease in the expression of a transcription factor, PbMYB44 (Pbr015309.1), potentially involved in mediating auxin responses, and the increased expression of multiple genes associated with regulating root growth. The concentrations of several phytohormones including indoleacetic acid (IAA), ABA, ETH, gibberellin (GA₃), and jasmonic acid (JA) were higher in response to K deprivation. Furthermore, genes coding for enzymes associated with carbon metabolism such as SORBITOL DEHYDROGENASE (SDH) and SUCROSE SYNTHASE (SUS) displayed greatly enhanced expression in the roots under K deprivation, presumably indicating enhanced metabolism to meet the increased energy demands for growth and K⁺ acquisition. Together, these data suggest that K deprivation in P. betulaefolia results in the rapid re-programming of the transcriptome to enhance root growth and K⁺ acquisition. These data provide key insights into the molecular basis for understanding low-K-tolerance mechanisms in pears and in other related fruit trees and identifying potential candidates that warrant further analyses.

Blueberry fruit are perishable after harvesting due to fruit softening, water loss and susceptibility to pathogens. Light, especially blue light, increases the accumulation of anthocyanins and reduces postharvest decay in some fruits, but the effect of blue light on postharvest fruit quality attributes in blueberries is unknown. In this study, we evaluated the effect of blue light on fruit quality, anthocyanin accumulation and disease development during postharvest cold storage (2 °C–4 °C) in two experiments with southern highbush blueberry ‘Star’ and rabbiteye blueberry ‘Alapaha’. Overall, diurnal blue light did not affect postharvest fruit quality attributes, such as visual defects, fruit compression, skin puncture, total soluble solid content and titratable acidity, in the two cultivars compared with their respective controls (diurnal white light or continuous darkness). Further, there was no effect of blue light on fruit color and anthocyanin accumulation. Fruit disease incidence in ‘Star’ ranged from 19.0% to 27.3% after 21 days and in ‘Alapaha’ from 44.9% to 56.2% after 24 days in postharvest storage, followed by 4 days at room temperature, but blue light had no consistent effect on postharvest disease incidence for either cultivar. Disease progression following artificial inoculations with Alternaria tenuissima and Colletotrichum acutatum in ‘Star’ was not influenced by light treatment prior to inoculation and during fruit storage. In a separate experiment, we tested the effect of blue light on color development in ‘Farthing’, a southern highbush blueberry cultivar with fruit prone to non-uniform ripening, whereby the stem-end remains green as the rest of the fruit turns blue. Although green stem-end spots turned blue over time, there was no statistically significant effect of the blue light treatment. Overall, these data indicate that blue light does not affect fruit quality attributes or disease development in ripe blueberry fruit during postharvest storage in the conditions investigated here.