Table 2 - uploaded by Lisa Klima Johnson
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Gene names and abbreviations, accession numbers and putative functions of the 12 candidate reference genes along with the accession numbers of the closest Arabidopsis thaliana homologs
Source publication
Application of transcriptomics approaches can greatly enhance our understanding of blueberry physiology. The success of transcriptomics approaches is dependent on the extraction of high-quality RNA which is complicated by the abundance of polyphenolics and polysaccharides in blueberry. Additionally, transcriptomics requires the accurate quantificat...
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Citations
... Each sample was frozen in liquid N 2 and then stored at -80 °C until further processing. Six to eight fruit for each sample were ground into fine powder and total RNA was isolated from the tissue using the cetyltrimethyl ammonium bromide (CTAB)-based method described in [77]. High quality RNA (RIN > 8.0) from each sample was used for RNA-Seq library construction with KAPA Stranded mRNA-Seq Kit (KAPA Biosystems, USA) for the Illumina platform, following the instructions provided in the manufacturer's manual. ...
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.
... For example, in cranberries of the same family and genus, PP2A is the most stable internal reference gene among different cultivars [12]. In rabbiteye blueberry, UBC28/RH8 is the best internal reference gene among the six organs [13]. Actin is the most stably expressed gene in different tissues and fruit development stages of dragon fruit [14]. ...
(1) Background: Vaccinium vitis-idaea is a nutritionally and economically valuable natural wild plant species that produces berries useful for treating various diseases. There is growing interest in lingonberry, but there is limited information regarding lingonberry reference genes suitable for gene expression analyses of different tissues under various abiotic stress conditions. The objective of this study was to identify stable reference genes suitable for different lingonberry tissues in response to abiotic stress. (2) Methods: The delta Ct method and the GeNorm v3.5 and NormFinder v20 programs were used to comprehensively analyze gene expression stability. (3) Results: Actin Unigene23839 was the best reference gene for analyzing different cultivars, whereas Actin CL5740.Contig2 was the most suitable reference gene for analyzing different tissues and alkali stress. In contrast, 18S rRNA CL5051.Contig1 was the most stable reference gene under drought conditions. (4) Conclusions: These suitable reference genes may be used in future qRT-PCR analyses of different lingonberry tissues and the effects of abiotic stresses. Furthermore, the study data may be useful for functional genomics studies and the molecular breeding of lingonberry. In summary, internal reference genes or internal reference gene combinations should be carefully selected according to the experimental conditions to ensure that the generated gene expression data are accurate.
... Samples were processed and prepared for RNA extraction following the methods described by Larson et al. (2023). Total RNA was extracted from frozen tissue according to the CTAB method (Vashisth et al. 2011). After extraction, RNA quantity and concertation were determined using a spectrophotometer (ND-1000; Thermo Fisher Scientific, Wilmington, DE, USA). ...
... After extraction, RNA quantity and concertation were determined using a spectrophotometer (ND-1000; Thermo Fisher Scientific, Wilmington, DE, USA). cDNA was synthesized in a 20-mL reaction volume using 1 mg of DNase-treated RNA (Vashisth et al. 2011) and diluted six-fold for quantitative real-time polymerase chain reaction (PCR). ...
Aminoethoxyvinylglycine (AVG) is widely used in commercial apple ( Malus × domestica Borkh.) production to reduce preharvest fruit drop (PFD) and delay ripening for harvest management. Recently, the maximum allowable concentration of AVG was doubled (up to 264 mg⋅L ⁻¹ ). Reports of the relationship between the AVG concentration and fruit growth, size, and quality have been contradictory. We evaluated the relationship between the AVG concentration and PFD, fruit size, fruit quality, and expression of ethylene signaling-related and cell wall modification-related genes. Experiments were conducted in 2019 and 2020 using mature ‘Red Delicious’ in western North Carolina. The AVG treatments [0 and 132 (AVG-1x) and 264 mg⋅L ⁻¹ (AVG-2x)] were applied 3 weeks before the expected harvest. The AVG treatments reduced fruit drop and internal ethylene concentration relative to the control in both years. There was no difference in drop between AVG-1x and AVG-2x applications. Only in 2020 did AVG treatments delay fruit softening and starch hydrolysis and reduce soluble solids concentration. There were no effects on red fruit color development. Fruit size was unaffected by AVG in 2019, but it was reduced in 2020 with the AVG-2x application. AVG reduced ethylene synthesis and altered signaling, evidenced by decreased relative expression of genes related to ethylene signaling ( ARGOS1, ARGOS2 ). AVG applications also reduced the expression of EXPA8;1 , suggesting that reduced cell wall disassembly was associated with a reduction in fruit softening. These results indicate that preharvest applications of 132 mg⋅L ⁻¹ AVG effectively reduced PFD via altering ethylene evolution and signaling. Use of a higher AVG concentration was of limited benefit.
... The extraction of RNA from grapes was based on the method of Vashisth et al. [20] and slightly improved. The 'Cabernet Sauvignon' grapes were placed in a mortar with liquid nitrogen for protection. ...
The types and contents of organic acids in wine grapes determine wine quality. To explore the effects of different rootstocks on the acid metabolism of ‘Cabernet Sauvignon’ grapes, various perennial rootstock–scion combinations were used as experimental materials. High-performance liquid chromatography (HPLC) was used to determine citric acid, tartaric acid, and malic acid contents during fruit development. Succinic acid and oxalic acid contents and the activity of related enzymes were measured using spectrophotometry. The expression levels of related genes were measured using a real-time fluorescence quantitative method. The results showed that all four rootstock types significantly reduced oxalic acid and citric acid contents in the grapes, while increasing succinic acid content to varying degrees. Employing 110R, SO4, and Kangzhen3 rootstocks increased tartaric acid and malic acid contents. Enzyme activity analysis revealed that 110R, SO4, and Kangzhen3 rootstocks increased the NAD-MDH enzyme activity, which positively correlated with malic acid content. Simultaneously, these rootstocks reduced the NADP-ME enzyme activity level. NAD-MDH and PEPC gene expression levels were higher in ‘Cabernet Sauvignon’ grapes grafted with 110R, SO4, and Kangan3 rootstocks compared to control self-rooted seedlings. Grafting these three rootstocks enhanced malic acid accumulation in ‘Cabernet Sauvignon’ grapes.
... Total RNA was extracted using the CTAB method and treated with DNase I to eliminate residual genomic DNA [71]. The extracted RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo, Waltham, MA, USA), and its integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and by denaturing agarose gel electrophoresis with ethidium bromide staining [72]. For each sample, the RNA concentration and amount exceeded 300 ng/µL and 15 µg, respectively. ...
Amaranth plants contain abundant betalains and flavonoids. Anthocyanins are important flavonoids; however, they cannot coexist in the same plant with betalains. Blue light influences metabolite synthesis and hypocotyl elongation; accordingly, analyses of its effects on betalain and flavonoid biosynthesis in Amaranthus tricolor may provide insight into the distribution of these plant pigments. We analyzed the betalain and flavonoid content and transcriptome profiles in amaranth hypocotyls under blue light and dark conditions. Furthermore, we analyzed the expression patterns of key genes related to betalains and flavonoids. Amaranth hypocotyls were shorter and redder and showed higher betalain and flavonoid content under blue light than in dark conditions. Key genes involved in the synthesis of betalains and flavonoids were upregulated under blue light. The gene encoding DELLA was also upregulated. These results suggest that blue light favors the synthesis of both betalains and flavonoids via the suppression of bioactive gibberellin and the promotion of DELLA protein accumulation, which also suppresses hypocotyl elongation. The metabolite profiles differed between plants under blue light and dark conditions. These findings improve our understanding of the environmental cues and molecular mechanisms underlying pigment variation in Amaranthus.
... The qRT-PCR expression analysis of selected genes was conducted using the primers designed by Primer 3 (Table S4). The POLYUBIQUITIN 3 (UBQ3b) gene was used as an internal control since it was found to be one of the most stably expressed genes across multiple organs in rabbiteye blueberry (Vashisth et al. 2011). The qRT-PCR with SYBR Premix Ex Taq II (TaKaRa) was repeated at least three times, and the data were analyzed using the 2 −(ΔΔCt) method (Livak et al. 2001). ...
Key message
The genomic location and stage-specific expression pattern of GH9 genes reveal their critical roles during fruit abscission zone formation in Vaccinium ashei.
Abstract
Glycosyl hydrolase family 9 (GH9) cellulases play a crucial role in both cellulose synthesis and hydrolysis during plant growth and development. Despite this importance, there is currently no study on the involvement of GH9-encoding genes, specifically VaGH9s, in abscission zone formation of rabbiteye blueberries (Vaccinium ashei). In this study, we identified a total of 61 VaGH9s in the genome, which can be classified into 3 subclasses based on conserved motifs and domains, gene structures, and phylogenetic analyses. Our synteny analysis revealed that VaGH9s are more closely related to the GH9s of Populus L. than to those of Arabidopsis, Vitis vinifera, and Citrus sinensis. In silico structural analysis predicted that most of VaGH9s are hydrophilic, and localized in cell membrane and/or cell wall, and the variable sets of cis-acting regulatory elements and functional diversity with four categories of stress response, hormone regulation, growth and development, and transcription factor-related elements are present in the promoter sequence of VaGH9s genes. Transcriptomic analysis showed that there were 22 differentially expressed VaGH9s in fruit abscission zone tissue at the veraison stage, and the expression of VaGH9B2 and VaGH9C10 was continuously increased during fruit maturation, which were in parallel with the increasing levels of cellulase activity and oxidative stress indicators, suggesting that they are involved in the separation stage of fruit abscission in Vaccinium ashei. Our work identified 22 VaGH9s potentially involved in different stages of fruit abscission and would aid further investigation into the molecular regulation of abscission in rabbiteye blueberries fruit.
... Total RNA was extracted using a cetyltrimethylammonium bromide-based method (Vashisth et al. 2011). RNA was quantified using a nanodrop spectrophotometer (ND2000C, Thermo Fisher Scientific, Waltham, MA, USA). ...
To assess the effects of MYB genes on total flavonoid content and flavonoid component content in Cerasus humilis fruits, members of the MYB gene family were identified and analyzed based on C. humilis genomic data and using bioinformatics. In total, 223 ChMYB genes were identified. According to the Myb DNA-binding domain, the 223 ChMYB genes were divided into four groups, i.e., 1R, R2R3, 3R, and 4R, comprising 112, 106, 4, and 1 ChMYB genes, respectively. The ChMYB genes were distributed unevenly on eight chromosomes, and some large replication fragments were observed. Promoter analysis showed that ChMYB genes play an important role in hormone responses and regulation of flavonoid biosynthesis. According to the transcriptome data and qRT-PCR analysis, several ChMYB genes may play an important role in different tissues such as leaf, seed, and fruit tissue and during fruit development. We tested correlations between expression levels of different genes and the content of total flavonoids and flavonoid components, resulting in three important ChMYB genes which were closely associated with the content of flavonoid components. The relative expression levels of ChMYB1 and ChMYB117 were strongly associated with the content of catechin and quercetin-7-O-β-D-glucopyranoside, respectively, and ChMYB52 was strongly associated with the change in peel color. We identified several potential candidate genes that regulate the synthesis of C. humilis flavonoids. Our results provide comprehensive insights into the functions of ChMYB family genes and may constitute a foundation for gene function verification in the future; after knowing the effect of ChMYB on flavonoid synthesis of C. humilis, we can accelerate the breeding of C. humilis with high flavonoid content by transgenic technology.
... Total RNA was extracted from frozen tissue following the CTAB method presented by Vashisth et al. (2011). Following tissue grinding in liquid nitrogen, $0.8 g of cortex and $0.4 g of pedicel tissue was used for RNA extraction. ...
... After extraction, RNA concentration and 260:280-nm absorbance was quantified with a spectrophotometer (NanoDrop ND-1000; Thermo Fisher Scientific, Inc., Waltham, MA, USA). One microgram of DNase (Promega Corp, Madison, WI, USA) treated RNA was used for cDNA synthesis in a 20-mL reaction volume (Vashisth et al. 2011). cDNA was diluted 6-fold for quantitative real-time polymerase chain reaction (RT-qPCR). ...
Apple ( Malus × domestica ) growers can incur significant economic losses when fruit drop before they can be harvested [preharvest fruit drop (PFD)]. In some years and cultivars, more than 30% of potential yield can be lost. Growers frequently apply plant bioregulators to reduce PFD, either via delay in maturity [aminoethoxyvinylglycine (AVG), 1-methylcycolpropene] or via inhibition in production of cell hydrolysis enzymes in the fruit pedicel [naphthalene acetic acid (NAA)]. Finding a physiological indicator of PFD would allow growers to assess the susceptibility of fruit to PFD. Due to its lignification, xylem is believed to be the last tissue to break down in the fruit pedicel, leading to PFD. To determine whether loss in xylem functionality can be used as an indicator of PFD potential, studies were conducted in 2020 and 2021 with ‘Red Delicious’ treated with AVG (132 µL·L ⁻¹ ), NAA (10 µL·L ⁻¹ ), and an ethylene-producing compound [ethephon (150 µL·L ⁻¹ in 2020, 200 µL·L ⁻¹ in 2021)] to generate a range of PFD potentials. Xylem functionality was assessed in the fruit cortex. Internal ethylene content (IEC), fruit maturity indices, and PFD rates were quantified weekly throughout the harvest period. Expression of genes encoding for cell hydrolysis enzymes ( MdEG1 and MdPG2 ) was quantified to relate xylem functionality to fruit abscission mechanisms. In 2020 and 2021, AVG reduced PFD compared with the untreated control by decreasing IEC. Although ethephon did not result in higher PFD than untreated fruit, NAA reduced PFD in 2020 but not 2021. For all treatments in both years, there was a linear decrease in xylem functionality throughout the measurement period. Cumulative PFD exponentially decreased as xylem functionality neared zero and the climacteric rise in ethylene began. Concurrent with the rise in IEC and PFD was an increase in the expression of MdEG1 and MdPG2 in the fruit pedicel of the control compared with AVG-treated fruit. AVG-treated fruit lost xylem functionality at a similar rate to the untreated control but had lower expression of MdEG1 and MdPG2 . These results indicate that xylem functionality is not a sole direct indicator of PFD. The concurrent increase in PFD and expression of MdEG1 / MdPG2 supports previous research indicating that these two genes may serve as potential markers for PFD.
... RNA extraction was performed following the procedure described by Vashisth et al. (2011). Extractions were performed on tissue samples collected at 0, 6, 12, 19, 26, and 52 DAFB in 2019 ('Cresthaven') and at 10, 17, 40 and 59 DAFB in 2021 ('O'Henry'). ...
Early fruit growth in peach is characterized by cell production. Cytokinins have established roles in regulating cell division and may regulate cell production during early fruit growth. However, the role of active cytokinins and regulation of their metabolism are not well characterized in the peach fruit. In this study, fruit growth parameters, concentrations of active cytokinin bases and a cytokinin riboside, and expression of three key cytokinin metabolism-related gene families were determined during early fruit growth. Early fruit growth was associated with intensive cell production until around 40 days after full bloom. During the early stages of this period, trans-zeatin (tZ), isopentenyladenine (iP), dihydrozeatin (DHZ) and tZ-riboside (tZR), displayed higher abundance which declined rapidly by 3.5- to 16-fold during the later stages. Changes in concentration of active cytokinin bases were consistent with roles for them in regulating cell production. Expression analyses of members of cytokinin biosynthesis-related gene families, ISOPENTENYL TRANSFERASE (IPT) and LONELY GUY (LOG), further indicated that mechanisms of synthesis of cytokinin metabolites and their activation are functional within the fruit pericarp. Changes in expression of multiple members of the LOG family paralleled changes in active cytokinin concentrations. Specifically, transcript abundance of LOG3 and LOG8 were correlated with concentrations of tZ, and iP and DHZ, respectively, suggesting that the direct activation pathway is an important route for active cytokinin base synthesis during early fruit development. Transcript abundance of two CYTOKININ OXIDASE (CKX) genes, CKX1 and CKX2, was consistent with roles in cytokinin catabolism during later stages of early fruit growth. Together, these data support a role for active cytokinins synthesized in the fruit pericarp in regulating early fruit growth in peach.
... The qRT-PCR expression analysis of selected genes was conducted using the primers designed by Primer 3 (Table S4). The POLYUBIQUITIN 3 (UBQ3b) gene was used as an internal control since it was found to be one of the most stably expressed genes across multiple organs in rabbiteye blueberry (Vashisth et al. 2011). The qRT-PCR with SYBR Premix Ex Taq II (TaKaRa) was repeated at least three times, and the data were analyzed using the 2 −(ΔΔCt) method (Livak et al. 2001). ...
Glycosyl hydrolase family 9 (GH9) cellulases play a crucial role in both cellulose synthesis and hydrolysis during plant growth and development. Despite this importance, there is currently no study on the involvement of GH9-encoding genes, specifically VaGH9s, in abscission zone formation of rabbiteye blueberries (Vaccinium ashei). In this study, we identified a total of 61 VaGH9s in the genome, which can be classified into three subclasses based on conserved motifs and domains, gene structures, and phylogenetic analyses. Our synteny analysis revealed that VaGH9s are more closely related to the GH9s of Populus L. than to those of Arabidopsis, Vitis vinifera, and Citrus sinensis. In-silico structural analysis predicted that most of VaGH9s are hydrophilic, and localized in cell membrane and/or cell wall, and the variable sets of cis-acting regulatory elements and functional diversity with four categories of stress response, hormone regulation, growth and development, and transcription factor-related elements are present in the promoter sequence of VaGH9s genes. Transcriptomic analysis showed that there were 22 differentially expressed VaGH9s in fruit abscission zone tissue at the veraison stage, and the expression of VaGH9B2 and VaGH9C10 was continuously increased during fruit maturation, which were in parallel with the increasing levels of cellulase activity and oxidative stress indicators, suggesting that they are involved in the separation stage of fruit abscission in Vaccinium ashei. Our work identified 22 VaGH9s potentially involved in different stages of fruit abscission and would aid further investigation into the molecular regulation of abscission in rabbiteye blueberries fruit.
