Expression Patterns of ACS and ACO Gene Families and Ethylene Production in Rachis and Berry of Grapes

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  • 1 College of Horticulture, Henan Agricultural University, 95 Wenhua Road, 450002 Zhengzhou, People’s Republic of China

Ethylene is important during the berry development and in the last stages of rachis development or rachis senescence. Since grapes develop in a cluster that comprises both the fruit berry and the nonfruit rachis, we measured the release of ethylene from both tissues. Detached berries from Vitis vinifera ‘Ruby Seedless’ and ‘Thompson Seedless’ showed that ethylene release peaks at the beginning of berry development and at veraison. Ethylene production in the rachis was higher than that in the berry and had an obvious peak before harvest in ‘Thompson Seedless’. In both cultivars, ethephon treatment induced ethylene production in the rachis but not in the berry. Expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO) genes showed diverse temporal and spatial patterns in ‘Thompson Seedless’ and ‘Ruby Seedless’. For most gene family members, the low ACS expression levels were observed in berry and rachis. Expression levels of most of the ACS and ACO genes did not correlate with ethylene released in the same organ. The transcriptional level of VvACS1 did correlate with ethylene evolution in rachis of ‘Thompson Seedless’ during berry development and storage, which suggested that VvACS1 may have important roles in rachis senescence. In berries of ‘Thompson Seedless’ and ‘Ruby Seedless’, the transcriptional levels of VvACO1, VvACS2, and VvACS6 coincided with ethylene production, indicating possible roles in berry development. Expression of VvACS2VvACO9 and VvACO1VvACO3 was not consistent with ethylene production during storage or in response to ethephon treatment, which suggests that the expression of ACS and ACO was affected by other stress factors after harvest.

Abstract

Ethylene is important during the berry development and in the last stages of rachis development or rachis senescence. Since grapes develop in a cluster that comprises both the fruit berry and the nonfruit rachis, we measured the release of ethylene from both tissues. Detached berries from Vitis vinifera ‘Ruby Seedless’ and ‘Thompson Seedless’ showed that ethylene release peaks at the beginning of berry development and at veraison. Ethylene production in the rachis was higher than that in the berry and had an obvious peak before harvest in ‘Thompson Seedless’. In both cultivars, ethephon treatment induced ethylene production in the rachis but not in the berry. Expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO) genes showed diverse temporal and spatial patterns in ‘Thompson Seedless’ and ‘Ruby Seedless’. For most gene family members, the low ACS expression levels were observed in berry and rachis. Expression levels of most of the ACS and ACO genes did not correlate with ethylene released in the same organ. The transcriptional level of VvACS1 did correlate with ethylene evolution in rachis of ‘Thompson Seedless’ during berry development and storage, which suggested that VvACS1 may have important roles in rachis senescence. In berries of ‘Thompson Seedless’ and ‘Ruby Seedless’, the transcriptional levels of VvACO1, VvACS2, and VvACS6 coincided with ethylene production, indicating possible roles in berry development. Expression of VvACS2VvACO9 and VvACO1VvACO3 was not consistent with ethylene production during storage or in response to ethephon treatment, which suggests that the expression of ACS and ACO was affected by other stress factors after harvest.

Grapes (V. vinifera L.) are widely cultivated around the world and can be consumed as fresh fruit (table grape) or processed into wine, juice, molasses, or raisins (Mencarelli et al., 2005). Grape berry has a thin skin and a soft, juicy flesh with high sugar content. The berry connects to the main rachis via a short pedicel with a small brush. Table grapes are stored and sold in bunches, and dry, browning rachis and berry drop are unfavorable for storage, transportation, and sale (Balic et al., 2012; Lichter, 2016).

Ethylene plays an important role in preharvest ripening in both climacteric and nonclimacteric fruit and is involved either directly or indirectly in the regulation of grape development and organ senescence (Chervin et al., 2008; Pech et al., 2008). Previous studies reported that rachis browning were induced by exogenous abscisic acid (ABA) and ethephon (Hedberg and Goodwin, 1980; Li et al., 2015), and were restrained by ethylene inhibitors, such as 1-methylcyclopropene (1-MCP) (Li et al., 2015), or other plant hormones, such as cytokinin (Balic et al., 2012) and gibberellin (Raban et al., 2013). Although many studies have linked ethylene to grape berry ripening, there is little research on the role of ethylene in rachis development or senescence before and after harvest. Increased understanding of the synthesis and regulation of ethylene production during development and senescence of grape rachis and berry would lay a foundation for prolonging shelf and storage life.

The key enzymes of ethylene biosynthesis in higher plants are ACS and ACO (Yang and Hoffman, 1984), both of which are encoded by multigene families. For example, eight ACS genes and four ACO genes have been identified in tomato (Rottmann et al., 1991; Yip et al., 1992). Expression of ACS and ACO genes are differentially regulated both temporally and spatially, resulting in specific expression patterns. For instance, EjACS1 and EjACO1 are specifically expressed in loquat fruit (Jiang et al., 2011), and LeACO1 is specifically expressed in tomato fruit (Barry et al., 1996). In grapes, there are at least 10 VvACS and 3 VvACO genes (Munoz-Robredo et al., 2013; Xu and Wang, 2012). The highest expression of VvACO1 gene was concomitant with an ethylene peak around veraison (Chervin et al., 2008; Munoz-Robredo et al., 2013; Pech et al., 2008). Moreover, there is little published data about the temporal and spatial expression of ACS and ACO genes in grapes. To understand how ACS and ACO genes control ethylene synthesis during development and senescence of both the rachis and the berry, the expression levels of nine ACS genes and three ACO genes and the production of ethylene were measured in berry and rachis of two seedless table grape cultivars Thompson Seedless and Ruby Seedless.

Material and Methods

Plant materials.

Grapevines (V. vinifera) of two cultivars, namely Thompson Seedless and Ruby Seedless, were grown in the National Vitis Repository of Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences. ‘Ruby Seedless’ is not susceptible to berry drop, and ‘Thompson Seedless’ berry is more susceptible to berry drop (Cooper et al., 1993). Grape berry and rachis of ‘Thompson Seedless’ were sampled from adult vines at 21, 35, 49, 57, 64, and 71 d after full bloom (DAFB), with the final sample collection at harvest [71 DAFB, 21.3% total soluble solids (TSS) and 0.61% tartaric acid]. Grape berry and rachis of V. vinifera ‘Ruby Seedless’ were sampled on 35, 49, 57, 64, 81, 91, and 102 DAFB (harvest, 16.9% TSS and 0.68% tartaric acid) (Supplemental Fig. 1; Supplemental Table 1). Samples (the third to eighth node) of leaf, petiole, and tendril, young stem (the first to third node), and shoot tip were collected from the two cultivars when the berry was harvested. All samples were frozen in liquid nitrogen and stored at −80 °C until analysis. For each sample, berries and rachis were collected from three or five clusters with the same maturity. The experiment was conducted in the 2014 growing season. For ethylene analysis and physiological characterization, fresh berry and rachis were taken from ‘Thompson Seedless’ and ‘Ruby Seedless’ on the above date. Fresh samples were transported to the laboratory immediately.

Ethephon treatment.

To investigate the expression patterns of ACO and ACS genes in response to exogenous ethephon, grape clusters of ‘Thompson Seedless’ and ‘Ruby Seedless’ from 12 grapevines were sprayed with 400 mg·L−1 ethephon (Sigma, China) or distilled water on the vine before harvest. Then the grape clusters were harvested and kept in a chamber at 15 °C and 85% to 90% relative humidity. Three days after ethephon treatments berries and rachis were collected every 1 or 2 d during storage. Nine clusters from 12 plants were selected for each treatment, and ethylene production of berry and rachis from nine clusters was measured three times.

Measurement of maturity parameters.

To define the stage of berry development, we randomly selected 20 berries on each sampling date, measured the level of TSS with a digital refractometer (PAL-1; ATAGO, Japan). Titratable acidity (TA), diameter, and weight parameters were recorded during berry developing stages of ‘Thompson Seedless’. To guarantee berry and rachis with the similar maturity degree in the individual cultivar, different clusters with the similar TSS were collected together in each sampling date (Munoz-Robredo et al., 2013). Three replicates were conducted for each sampling.

GC analysis of ethylene production.

Ethylene analysis for each sample was performed using gas chromatography (GC; GC-2010 PLUS, Shimadzu, Japan) with a flame ionization detector as described (Li et al., 2015). At each berry development stage, 200–400 g berries and 5–20 g rachis were sampled from clusters and then sealed in either a 65-mL or a 250-mL bottle or box at 20 °C. Three hours later, 1 mL of the gas from the sample vial was injected into a GC Packed Column (GDX-502, Shimadzu, Janpan) for analysis. The temperatures of the injector port, column, and detector were set at 75, 80, and 60 °C, respectively. For each sample, berries and rachis were collected from three or five clusters with the similar maturity. Each experiment was repeated at least three times.

Quantitative real-time RT-PCR analysis and sequencing.

Rachis and berries from nine clusters were homogenized in groups of three clusters and RNA extraction was performed on 100–200 mg samples from each replicate using a modified CTAB method (Gambino et al., 2008). RNA quality was evaluated in a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA), selecting RNA samples with an A260/280 ratio between 1.8 and 2.0 and an A260/230 ratio of more than 2.0 for subsequent analysis. The integrity of RNA samples was assessed by electrophoresis on a 1.5% agarose gel. The primer sequences for ACS and ACO genes were designed in Primer Express version 3.0 (Applied Biosystems, Madrid, CA), and primer efficiency for each primer pair was calculated (Table 1).

Table 1.

Primer description of ACS and ACO genes.

Table 1.

First-stand cDNA synthesis from 1 μg of total RNA was conducted with the FastQuant RT Kit (with gDNase I; Tiangen Biotech, Beijing, China). Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using the SYBR® Select Master Mix (Applied Biosystems) in an ABI PRISM 7500 Fast Sequence Detection System (Applied Biosystems). Reactions contained 1 μL primer mix (0.5 μm forward and reverse primers), 2 μL template cDNA, 10 μL SYBR® Select Master Mix, and 7 μL RNase-free water to a total volume of 20 μL. Reactions were conducted using the following thermal cycling conditions: 50 °C for 2 min (1 cycle), 95 °C for 2 min (1 cycle), 95 °C for 3 s, and 60 °C for 30 s (40 cycles). The PCR products for each primer pair were sequenced (Shanghai Shenggong Company, Shanghai, China) and searched against the NCBI database using BLASTN to ensure data quality and reliability (data not shown). Each experiment was repeated three times with three biological replicates.

Data analysis and statistical analysis.

Efficiency of each primer pair was calculated using standard curve method (Larionov et al., 2005). Actin, GAPDH, and EF-1α genes were used as internal controls (Reid et al., 2006), from which the most stably expressed gene was used as a reference gene, according to geNorm analysis (Vandesompele et al., 2002). For each sample, relative expression was calculated by LinRegPCR method (Ruijter et al., 2009). For gene expression in berry development, the value of the sample at harvest was set as “1” for ACS and ACO. To compare expression in various organs, the value of the shoot tip was set as “1” for ACS and ACO gene expression.

All statistics were analyzed using SPSS 17.0 with three replications of each experiment. The Duncan’s multiple range test was applied at P = 0.05 level to evaluate the significant differences among data.

Results

Ethylene production in berry and rachis during berry development and in storage.

To understand ethylene production patterns in ‘Thompson Seedless’ and ‘Ruby Seedless’ fruit, ethylene release rates of rachis and berry were recorded during berry development and after harvest in storage (Fig. 1). There was a general upward trend in ethylene production in the rachis of ‘Thompson Seedless’ during berry development, and a sharp rise of ethylene production was observed at harvest, 71 DAFB (Fig. 1A). In rachis of ‘Ruby Seedless’, two weak ethylene peaks were observed at 49 and 81 DAFB before harvest (Fig. 1C). In ‘Thompson Seedless’ and ‘Ruby Seedless’ berry, a general downward trend in ethylene production was displayed as the berry developed (Fig. 1E and G), with the highest ethylene production at 35 DAFB. In general, ethylene production in the rachis (1–4.5 µL·kg−1·h−1) was higher than that of the berry (0.02–1 µL·kg−1·h−1) in both ‘Ruby Seedless’ and ‘Thompson Seedless’ (Fig. 1). Comparing the two grapevine cultivars, ethylene production in berry and rachis of ‘Thompson Seedless’ was generally higher than that of ‘Ruby Seedless’. For example, ethylene release rate from berry of ‘Thompson Seedless’ and ‘Ruby Seedless’ at 35 DAFB was 1.0 and 0.4 µL·kg−1·h−1, respectively, and release rate from rachis at 71 DAFB was 4.3 and 2.2 µL·kg−1·h−1, respectively (Fig. 1).

Fig. 1.
Fig. 1.

Ethylene production rate from berry and rachis during berry development and in storage. Ethylene production rates in (A) rachis of Vitis vinifera ‘Thompson Seedless’ during berry development, (B) rachis of V. vinifera ‘Thompson Seedless’ in storage, (C) rachis of V. vinifera ‘Ruby Seedless’ during berry development, (D) rachis of V. vinifera ‘Ruby Seedless’ in storage, (E) berry of ‘Thompson Seedless’ during berry development, (F) berry of ‘Thompson Seedless’ in storage, (G) berry of ‘Ruby Seedless’ during berry development, and (H) berry of ‘Ruby Seedless’ in storage. CK, untreated control; ETH, samples that were treated with ethephon at harvest; H, harvest date. The veraison stage of ‘Thompson Seedless’ was at 49 d after full bloom (DAFB), and ‘Ruby Seedless’ was at 81 DAFB. Different letters indicated significant differences between transgenic lines and the wild type at P < 0.05.

Citation: HortScience horts 52, 3; 10.21273/HORTSCI11050-16

Little changes in ethylene production were observed in berry of either of the two cultivars during storage (Fig. 1F and H). At harvest, the berries produced very little ethylene, and that continued through the 10–15 d of storage. Ethylene production in the rachis of ‘Thompson Seedless’ continued to decrease in storage (Fig. 1B). On the other hand, ethylene production in the rachis of ‘Ruby Seedless’ first increased from 0.75 to 1.5 µL·kg−1·h−1 over 7 d, and then began to decrease at 7 d after harvest (DAH) (Fig. 1D).

In response to ethephon treatment, there was little change in ethylene production in berries of either ‘Thompson Seedless’ or ‘Ruby Seedless’ (Fig. 1F and H), whereas ethylene production of the rachis in both cultivars was increased (Fig. 1B and D).

Expression of ACS and ACO genes in various tissues and organs.

Expression levels of nine ACS gene family members were analyzed in various tissues and organs at harvest. The results indicated that grapevine ACS (VvACS) genes were differentially expressed in ‘Thompson Seedless’ and ‘Ruby Seedless’ (Fig. 2).

Fig. 2.
Fig. 2.

Expression patterns of ACS and ACO genes from different organs in Vitis vinifera ‘Thompson Seedless’ and ‘Ruby Seedless’. Expressions of ACS genes in (A) V. vinifera ‘Thompson Seedless’, (B) V. vinifera ‘Ruby Seedless’, (C) V. vinifera ‘Thompson Seedless’, and (D) V. vinifera ‘Ruby Seedless’. The value of shoot tip was set as “1” for ACS and ACO relative gene expression analysis.

Citation: HortScience horts 52, 3; 10.21273/HORTSCI11050-16

In ‘Thompson Seedless’, expression of VvACS1 was detected at the highest level in grape rachis, at a level 44 times higher than that in shoot tip or leaves. The highest expression of VvACS2 was observed in young stem, and lowest in berry and rachis. Transcriptional levels of VvACS3, VvACS7, VvACS8, and VvACS9 were higher in leaf and lower or undetectable in grape berry and rachis. Expression of VvACS4–VvACS6 was highest in the shoot tip and lowest in rachis (Fig. 2A).

In ‘Ruby Seedless’, VvACS4 and VvACS7VvACS9 expression levels were high in young stem, but low in berries and rachises (Fig. 2B). Transcript levels of VvACS5 were higher in shoot tip, young stem, and berry, but quite lower in rachis. The highest expression of VvACS3 was in rachis at a level 10 times higher than that in berry (Fig. 2B). The highest expression of VvACS1 and VvACS6 was observed in leaves (Fig. 2B).

The expression trends of ACO1 and ACO3 genes were similar in ‘Thompson Seedless’ and ‘Ruby Seedless’ (Fig. 2C and D). The highest expression level of VvACO1 was in the tendril, although the relative levels between the cultivars were quite different. The highest expression level of VvACO3 was in the berry, and the lowest expression level was in the rachis, especially in ‘Thompson Seedless’ (Fig. 2C and D). Transcript level of VvACO2 was highest in the rachis of ‘Thompson Seedless’, and quite lower in the rachis of ‘Ruby Seedless’ (Fig. 2C and D).

Expression of ACS and ACO genes in berry and rachis during grape berry development.

Expression patterns of the VvACS genes in berry and rachis were analyzed during grape berry development. The expression levels of these genes were quite different between ‘Thompson Seedless’ and ‘Ruby Seedless’ (Fig. 3). In the rachis of ‘Thompson Seedless’, expression levels of VvACS2, VvACS5, VvACS6, VvACS8, and VvACS9 were highest at 57 DAFB, then lower at 21 DAFB, and the lowest appeared at harvest (Fig. 3A and B). The expression peaks of VvACS1 and VvACS3 were the most consistent with ethylene production in rachis, with the highest expression level of these two genes at harvest (Fig. 3A).

Fig. 3.
Fig. 3.

Expression patterns of ACS genes in berry and rachis of ‘Thompson Seedless’ and ‘Ruby Seedless’ during berry development. Expressions of ACS genes in (A, B) rachis of ‘Thompson Seedless’, (C, D) berry of ‘Thompson Seedless’, (E, F) rachis of ‘Ruby Seedless’, and (G, H) berry of ‘Ruby Seedless’. The value of sample at harvest was set as “1” for ACS relative gene expression analysis. H = harvest date.

Citation: HortScience horts 52, 3; 10.21273/HORTSCI11050-16

In the ‘Thompson Seedless’ berry, expression levels of VvACS1, VvACS2, VvACS4, VvACS6, and VvACS8 were highest at 21 DAFB, then lower at 35 DAFB, and remained at low level until ripening, similar to ethylene production (Fig. 3C and D). The highest expression levels of VvACS5 and VvACS9 were observed at harvest (Fig. 3B).

In ‘Ruby Seedless’ rachis (Fig. 3E and F), the highest transcript accumulations of VvACS1 and VvACS6 were observed at 35 and 49 DAFB, respectively, and the lowest at 81 DAFB. The highest transcriptional levels of VvACS2 and VvACS5 were observed at 91 DAFB, with relatively lower levels at other berry developmental stages. The expression of VvACS3 gradually decreased from 35 to 57 DAFB, and then slightly increased at harvest. Expression of VvACS4, VvACS7, and VvACS8 peaked at 49 DAFB. Little change in rachis expression of VvACS9 was observed through ‘Ruby Seedless’ berry development.

In ‘Ruby Seedless’ berry (Fig. 3G and H), expression levels of VvACS2 and VvACS6 were concurrent with ethylene production, peaking at 35 and 81 DAFB. The highest transcriptional accumulations of VvACS3 and VvACS7, VvACS8, and VvACS9 were at 64 DAFB. The highest expression levels of VvACS4 and VvACS5 were detected at harvest, right after its lowest level at 91 DAFB.

Expression patterns of the VvACO genes in berry and rachis were also analyzed during grape berry development. In ‘Thompson Seedless’ rachis (Fig. 4A), expression of VvACO1 was highest at 57 DAFB. The expression of VvACO2 peaked at harvest, which was concurrent with the ethylene peak. Little change in expression of VvACO3 was observed during berry development. In ‘Thompson Seedless’ berry (Fig. 4B), expression levels of VvACO1 and VvACO2 were the highest at 21 DAFB and then decreased gradually with berry development, and the patterns were consistent with ethylene production in berry. Slight changes of VvACO3 expression were observed in ‘Thompson Seedless’ berry (Fig. 4B).

Fig. 4.
Fig. 4.

Expression patterns of ACO genes in berry and rachis of ‘Thompson Seedless’ and ‘Ruby Seedless’ during berry development. Expression of ACO genes in (A) rachis of ‘Thompson Seedless’, (B) berry of ‘Thompson Seedless’, (C) rachis of ‘Ruby Seedless’, and (D) berry of ‘Ruby Seedless’. The value of sample at harvest was set as “1” for ACO relative gene expression analysis. H = harvest date.

Citation: HortScience horts 52, 3; 10.21273/HORTSCI11050-16

In ‘Ruby Seedless’ rachis (Fig. 4C), expression levels of VvACO1 were steady throughout grape berry development. The expression levels of VvACO2 and VvACO3 peaked at 91 and 81 DAFB, respectively. In ‘Ruby Seedless’ berry (Fig. 4D), the highest expression level of VvACO1 was observed at 35 DAFB, and the lowest at harvest, an expression pattern consistent with ethylene production. Expression of VvACO2 had an obvious peak at 81 DAFB, and was relatively lower at other stages. Little expression changes of VvACO3 were observed in the berry of either ‘Thompson Seedless’ (Fig. 4B) or ‘Ruby Seedless’ (Fig. 4D).

Postharvest expression of ACS and ACO genes in berry and rachis treated with exogenous ethephon during development.

Grape clusters were treated with ethephon at 3 d before harvest, and there were no obvious changes in postharvest ethylene production from berries in either cultivar, whether they were treated with exogenous ethephon or not during development (Fig. 1C and D). Therefore, expression of the nine ACS and three ACO genes were analyzed only in rachis during storage. The expression of several ACS and ACO genes in rachis was modulated by ethephon treatment (Figs. 5 and 6). In ‘Thompson Seedless’ rachis (Fig. 5A–C), expression of VvACS1 generally decreased during storage, and this trend was almost consistent with ethylene production in rachis. The expression levels of VvACS2, VvACS4, VvACS6, and VvACS7 increased at the 3rd DAH, and then generally decreased during storage. Expression levels of VvACS5 and VvACS9 peaked at 6 DAH. In response to ethephon treatment, expression levels of VvACS1, VvACS2, VvACS5, and VvACS6 were mostly downregulated during storage, except 4 DAH. Expression levels of VvACS3, VvACS7, VvACS8, and VvACS9 were downregulated during storage, except 6 DAH. Generally, expression of ACS in rachis was downregulated under ethephon treatment (Fig. 5A–C).

Fig. 5.
Fig. 5.

Expression of VvACS genes in rachis of Vitis vinifera ‘Thompson Seedless’ and ‘Ruby Seedless’ during storage and in response to ethephon treatment. Expression of ACS genes in rachis of (AC) ‘Thompson Seedless’ and (DF) ‘Ruby Seedless’. The value for each tissue type at harvest (Day 0) was set as “1” for ACS relative gene expression analysis. CK = untreated control; ETH = samples that were treated with ethephon at harvest.

Citation: HortScience horts 52, 3; 10.21273/HORTSCI11050-16

Fig. 6.
Fig. 6.

Expression of VvACO genes in rachis of Vitis vinifera ‘Thompson Seedless’ and ‘Ruby Seedless’ during storage and in response to ethephon treatment. Expression of ACO genes in rachis of (A) ‘Thompson Seedless’ and (B) ‘Ruby Seedless’. The value for each tissue type at harvest (Day 0) was set as “1” for ACO relative gene expression analysis. CK = untreated control; ETH = samples that were treated with ethephon at harvest.

Citation: HortScience horts 52, 3; 10.21273/HORTSCI11050-16

In ‘Ruby Seedless’ rachis during storage (Fig. 5D–F), slight expression changes for VvACS1 and VvACS6 were observed, except for dips in expression at 5 DAH. Two expression peaks of ACS2 were observed at 3 and 9 DAH. The expression levels of ACS3ACS5 and ACS7–ACS9 generally decreased during storage. In response to ethephon treatment, the expression levels of the VvACS genes were upregulated or downregulated in rachis of ‘Ruby Seedless’ in ways that did not seem to correlate with ethylene production.

The response of the three ACO genes during storage with or without treatment of ethephon at the 3rd d before harvest was also measured. In ‘Thompson Seedless’ rachis (Fig. 6A), VvACO1 expression peaked 3 and 6 DAH. VvACO2 and VvACO3 expressions generally increased during storage. Expression of all three ACO genes decreased in response to ethephon treatment, except for VvACO3 at 6 DAH.

In ‘Ruby Seedless’ rachis (Fig. 6B), expression of VvACO1 generally decreased and of VvACO2 gradually increased during storage, and both were downregulated in response to ethephon treatment. Expression VvACO3 peaked at 7 DAH and was upregulated in response to ethephon treatment, which was consistent with ethylene production.

Discussion

A previous study on the grape cultivar Thompson Seedless in Chile showed that the veraison stage at 8–9 weeks after full bloom (WAFB) was accompanied by a peak of ethylene production of berry at veraison (9 WAFB) during observations from 7 to 12 WAFB (Munoz-Robredo et al., 2013). The highest ethylene production during berry development was also observed at veraison in the grape cultivars Muscat Hamburg (Sun et al., 2010) and Cabernet Sauvignon (Chervin et al., 2004). In nonclimacteric fruits such as strawberry (Perkins-Veazie et al., 1996) and citrus (Katz et al., 2005), the pattern is typified by an ethylene production peak in young fruitlets that then decreases dramatically with fruit development. In our study, the similar ethylene production pattern as strawberry and citrus was observed in ‘Ruby Seedless’ and ‘Thompson Seedless’ berries, and the highest ethylene production of berries was observed at the beginning of berry development (3 and 5 WAFB), after which ethylene production sharply decreased before veraison. Munoz-Robredo et al. (2013) and Chervin et al. (2004) started to collect berry samples at 7 and 5 WAFB, respectively, and they may missed the earlier ethylene production peak of berry. The reason(s) behind high ethylene production at the beginning of berry development, whether physiological abscission of fruitlets or wounding stress resulted from sampling, and the mechanisms leading to ethylene changes both require further research.

Although leaves and other vegetative tissues are generally considered as nonclimacteric, ethylene production of detached citrus leaves was demonstrated a climacteric system II behavior during senescence or in response to ethephon treatment (Katz et al., 2004; Li et al., 2015; Morgan et al., 1992). A similar phenomenon was observed in the rachis during berry developing of ‘Thompson Seedless’ in our study, and there was a climacteric peak in ethylene production of rachis. In response to ethephon treatment, ethylene production in the rachis of both cultivars was induced, whereas ethylene production of the berry was unaffected, which suggested that rachis of grape may be a climacteric organ in ethylene production. After harvest, ethylene production in berry continuously decreased in ‘Ruby Seedless’, which is not susceptible to berry drop. However, ‘Thompson Seedless’ berry, which is more susceptible to berry drop (Cooper et al., 1993), showed a higher ethylene production without an obvious decreasing trend. Similar results were obtained in the rachis of the two cultivars, with higher ethylene production in ‘Thompson Seedless’ rachis compared with rachis of ‘Ruby Seedless’. Li et al. found that the average level of rachis ethylene production of ‘Thompson Seedless’ was significantly lower than that of ‘Mystery’ and ‘3003’, and rachis browning levels of ‘Thompson Seedless’ were much lower than those of the other two cultivars (Li et al., 2015). We also tested other four cultivars, and the results indicated that there were significant differences in ethylene production of rachis among these tested grape cultivars (data not shown). All the above results suggested that ethylene either may have a direct or indirect influence on rachis senescence and berry drop, or may have a reciprocal effect with ABA that results in berry senescence after harvest (Sun et al., 2010).

Organ-specific expression of ACS and ACO genes was observed in our study. For example, in ‘Thompson Seedless’, VvACS1 and VvACO2 in rachis, VvACO1 in tendril, and VvACO3 in berry were highly expressed. In ‘Ruby Seedless’, similar results were observed, with the highest expression of VvACS1 and VvACS3 in rachis, VvACO1 in tendril, and VvACO3 in berry. In addition, some organ-specific transcription levels highly correlated with ethylene production. For example, the highest expression levels of VvACS1 and VvACO2 in rachis were concurrent with a peak of ethylene production at harvest in ‘Thompson Seedless’, which may suggest that these organ-specific VvACS1 and VvACO2 expressions played important roles in rachis senescence. However, some organ-specific expression did not agree with ethylene production in the same organ. For example, VvACO3 was highly and specifically expressed in berry, but no obvious changes in its expression were observed in ‘Thompson Seedless’ or ‘Ruby Seedless’ berries, when ethylene production decreased during berry development. Although the highest transcriptional levels of VvACO1 were observed in the tendrils of ‘Thompson Seedless’ and ‘Ruby Seedless’, the lower expression pattern of ACO1 did coincide with ethylene production in berry of both cultivars. Similar results also suggest that VvACO1 highly correlates with ethylene production in berry development (Munoz-Robredo et al., 2013).

It is interesting to note that expression level of VvACS1 gene was about 40 times higher in rachis than that in other tested organs at 71 DAFB, and there is an obvious positive correlation between VvACS1 gene expression and ethylene production in rachis of ‘Thompson Seedless’ (r = 0.796) during berry development and in storage, which indicate that VvACS1 is one of the key genes synthesizing ethylene in the grape rachis and playing important roles in rachis senescence (Rosales et al., 2013). In apple, it was also found that MdACS1 gene was initiated at fruit ripening of apple when climacteric ethylene started to evolve, and the MdACS1 expression pattern paralleled the changes of ethylene production of fruit tissue in storage (Tan et al., 2013), which suggested that ACS1 gene seemed to operate in system II ethylene biosynthesis during berry ripening.

Conclusions

In our study on the grape cultivars Ruby Seedless and Thompson Seedless, ethylene production peaks of detached berries were observed at the beginning of berry development, and berry ethylene production was unaffected by ethephon treatments. Ethylene production of rachis was higher than that of berry and was greatly induced by exogenous ethephon treatment in both cultivars. Among nine ACS gene family members, VvACS1 was the only gene with the highest expression in rachis and showed expression levels coincident with ethylene production in rachis of ‘Thompson Seedless’ during berry development and in storage. We propose that this organ-specific VvACS1 expression played important roles in rachis senescence. In berry, the transcriptional level of VvACO1, ACS2, and ACS6 coincided with ethylene production in berry in both ‘Thompson Seedless’ and ‘Ruby Seedless’, which implies that VvACO1 correlates with ethylene production during berry development.

Literature Cited

  • Balic, I., Moreno, A., Sanhueza, D., Huerta, C., Orellana, A., Defilippi, B.G. & Campos-Vargas, R. 2012 Molecular and physiological study of postharvest rachis browning of table grape cv Red Globe Postharvest Biol. Technol. 72 47 56

    • Search Google Scholar
    • Export Citation
  • Barry, C.S., Blume, B., Bouzayen, M., Cooper, W., Hamilton, A.J. & Grierson, D. 1996 Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato Plant J. 9 525 535

    • Search Google Scholar
    • Export Citation
  • Cooper, T., Retamales, J. & Bangerth, F. 1993 Berry drop affected by GA3 and Promalin in Thompson seedless grapes Acta Hort. 329 134 136

  • Chervin, C., El-Kereamy, A., Roustan, J.P., Latché, A., Lamon, J. & Bouzayen, M. 2004 Ethylene seems required for the berry development and ripening in grape, a non-climacteric fruit Plant Sci. 167 1301 1305

    • Search Google Scholar
    • Export Citation
  • Chervin, C., Tira-umphon, A., Terrier, N., Zouine, M., Severac, D. & Roustan, J.P. 2008 Stimulation of the grape berry expansion by ethylene and effects on related gene transcripts, over the ripening phase Physiol. Plant. 134 534 546

    • Search Google Scholar
    • Export Citation
  • Gambino, G., Perrone, I. & Gribaudo, I. 2008 A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants Phytochem. Anal. 19 520 525

    • Search Google Scholar
    • Export Citation
  • Hedberg, P.R. & Goodwin, P.B. 1980 Factors affecting natural and ethephon-induced grape berry abscission Amer. J. Enol. Viticult. 31 109 113

  • Jiang, T.M., Wang, P., Yin, X.R., Zhang, B., Xu, C.J., Li, X. & Chen, K.S. 2011 Ethylene biosynthesis and expression of related genes in loquat fruit at different developmental and ripening stages Sci. Hort. 130 452 458

    • Search Google Scholar
    • Export Citation
  • Katz, E., Lagunes, P.M., Riov, J., Weiss, D. & Goldschmidt, E.E. 2004 Molecular and physiological evidence suggests the existence of a system II-like pathway of ethylene production in non-climacteric Citrus fruit Planta 219 243 252

    • Search Google Scholar
    • Export Citation
  • Katz, E., Riov, J., Weiss, D. & Goldschmidt, E.E. 2005 The climacteric-like behaviour of young, mature and wounded citrus leaves J. Expt. Bot. 56 1359 1367

    • Search Google Scholar
    • Export Citation
  • Larionov, A., Krause, A. & Miller, W. 2005 A standard curve based method for relative real time PCR data processing BMC Bioinformatics 6 62

  • Li, L., Kaplunov, T., Zutahy, Y., Daus, A., Porat, R. & Lichter, A. 2015 The effects of 1-methylcyclopropane and ethylene on postharvest rachis browning in table grapes Postharvest Biol. Technol. 107 16 22

    • Search Google Scholar
    • Export Citation
  • Lichter, A. 2016 Rachis browning in tablegrapes Austral. J. Grape Wine Res. 22 161 168

  • Mencarelli, F., Andrea, B. & Giancarlo, D. 2005 Grape: Post-harvest operations. University of Viterbo, Viterbo, Italy

  • Morgan, P.W., He, C.J. & Drew, M.C. 1992 Intact leaves exhibit a climacteric-like rise in ethylene production before abscission Plant Physiol. 100 1587 1590

    • Search Google Scholar
    • Export Citation
  • Munoz-Robredo, P., Gudenschwager, O., Chervin, C., Campos-Vargas, R., González-Agüero, M. & Defilippi, B.G. 2013 Study on differential expression of 1-aminocyclopropane-1-carboxylic acid oxidase genes in table grape cv Thompson Seedless. Postharvest Biol. Technol. 76 163 169

    • Search Google Scholar
    • Export Citation
  • Pech, J.C., Bouzayen, M. & Latché, A. 2008 Climacteric fruit ripening: Ethylene-dependent and independent regulation of ripening pathways in melon fruit Plant Sci. 175 114 120

    • Search Google Scholar
    • Export Citation
  • Perkins-Veazie, P.M., Huber, D.J. & Brecht, J.K. 1996 In vitro growth and ripening of strawberry fruit in the presence of ACC, STS or propylene Ann. Appl. Biol. 128 105 116

    • Search Google Scholar
    • Export Citation
  • Raban, E., Kaplunov, T., Zutahy, Y., Daus, A., Alchanatis, V., Ostrovsky, V., Lurie, S. & Lichter, A. 2013 Rachis browning in four table grape cultivars as affected by growth regulators or packaging Postharvest Biol. Technol. 84 88 95

    • Search Google Scholar
    • Export Citation
  • Reid, K.E., Niclas, O., James, S., Peng, F. & Lund, S.T. 2006 An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development BMC Plant Biol. 6 27

    • Search Google Scholar
    • Export Citation
  • Rosales, R., Fernandez-Caballero, C., Irene, R., Ma, I.E., Carmen, M. & Sanchez-Ballesta, M.T. 2013 Molecular analysis of the improvement in rachis quality by high CO2 levels in table grapes stored at low temperature Postharvest Biol. Technol. 77 50 58

    • Search Google Scholar
    • Export Citation
  • Rottmann, W.H., Peter, G.F., Oeller, P.W., Keller, J.A., Shen, N.F., Nagy, B.P., Taylor, L.P., Campbell, A.D. & Theologis, A. 1991 1-Aminocyclopropane-1-carboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence J. Mol. Biol. 222 937 961

    • Search Google Scholar
    • Export Citation
  • Ruijter, J.M., Ramakers, C., Hoogaars, W.M., Karlen, Y., Bakker, O., van den Hoff, J.M. & Moorman, A.F. 2009 Amplification efficiency: Linking baseline and bias in the analysis of quantitative PCR data Nucleic Acids Res. 37 e45

    • Search Google Scholar
    • Export Citation
  • Sun, L., Zhang, M., Ren, J., Qi, J., Zhang, G. & Leng, P. 2010 Reciprocity between abscisic acid and ethylene at the onset of berry ripening and after harvest BMC Plant Biol. 10 257

    • Search Google Scholar
    • Export Citation
  • Tan, D., Li, T. & Wang, A. 2013 Apple 1-Aminocyclopropane-1-carboxylic acid synthase genes, MdACS1 and MdACS3a, are expressed in different systems of ethylene biosynthesis Plant Mol. Biol. Rpt. 31 204 209

    • Search Google Scholar
    • Export Citation
  • Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. & Speleman, F. 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3:RESEARCH0034.1-0034.11

  • Xu, M. & Wang, M.H. 2012 Genome-wide analysis of 1-amino-cyclopropane-1-carboxylate synthase gene family in Arabidopsis, rice, grapevine and poplar Afr. J. Biotechnol. 11 1106 1118

    • Search Google Scholar
    • Export Citation
  • Yang, S.F. & Hoffman, N.E. 1984 Ethylene biosynthesis and its regulation in higher plants Annu. Rev. Plant Physiol. 35 155 189

  • Yip, W.K., Moore, T. & Yang, S.F. 1992 Differential accumulation of transcripts for four tomato 1-aminocyclopropane-1-carboxylate synthase homologs under various conditions Proc. Natl. Acad. Sci. USA 89 2475 2479

    • Search Google Scholar
    • Export Citation
Supplemental Table 1.

Quality trait of Vitis vinifera ‘Thompson Seedless’ during berry developing.

Supplemental Table 1.
Supplemental Fig. 1.
Supplemental Fig. 1.

Total soluble solid contents of grape during berry developing. DAFB = days after full bloom.

Citation: HortScience horts 52, 3; 10.21273/HORTSCI11050-16

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Contributor Notes

We thank Anita K. Snyder for critical reading of the manuscript. The work was supported by National Natural Science Foundation of China (31201605) and Major Science and Technology Project in Henan Province (151100110900).

These authors contributed equally to this work.

Corresponding author. E-mail: jcfeng@henau.edu.cn.

  • View in gallery

    Ethylene production rate from berry and rachis during berry development and in storage. Ethylene production rates in (A) rachis of Vitis vinifera ‘Thompson Seedless’ during berry development, (B) rachis of V. vinifera ‘Thompson Seedless’ in storage, (C) rachis of V. vinifera ‘Ruby Seedless’ during berry development, (D) rachis of V. vinifera ‘Ruby Seedless’ in storage, (E) berry of ‘Thompson Seedless’ during berry development, (F) berry of ‘Thompson Seedless’ in storage, (G) berry of ‘Ruby Seedless’ during berry development, and (H) berry of ‘Ruby Seedless’ in storage. CK, untreated control; ETH, samples that were treated with ethephon at harvest; H, harvest date. The veraison stage of ‘Thompson Seedless’ was at 49 d after full bloom (DAFB), and ‘Ruby Seedless’ was at 81 DAFB. Different letters indicated significant differences between transgenic lines and the wild type at P < 0.05.

  • View in gallery

    Expression patterns of ACS and ACO genes from different organs in Vitis vinifera ‘Thompson Seedless’ and ‘Ruby Seedless’. Expressions of ACS genes in (A) V. vinifera ‘Thompson Seedless’, (B) V. vinifera ‘Ruby Seedless’, (C) V. vinifera ‘Thompson Seedless’, and (D) V. vinifera ‘Ruby Seedless’. The value of shoot tip was set as “1” for ACS and ACO relative gene expression analysis.

  • View in gallery

    Expression patterns of ACS genes in berry and rachis of ‘Thompson Seedless’ and ‘Ruby Seedless’ during berry development. Expressions of ACS genes in (A, B) rachis of ‘Thompson Seedless’, (C, D) berry of ‘Thompson Seedless’, (E, F) rachis of ‘Ruby Seedless’, and (G, H) berry of ‘Ruby Seedless’. The value of sample at harvest was set as “1” for ACS relative gene expression analysis. H = harvest date.

  • View in gallery

    Expression patterns of ACO genes in berry and rachis of ‘Thompson Seedless’ and ‘Ruby Seedless’ during berry development. Expression of ACO genes in (A) rachis of ‘Thompson Seedless’, (B) berry of ‘Thompson Seedless’, (C) rachis of ‘Ruby Seedless’, and (D) berry of ‘Ruby Seedless’. The value of sample at harvest was set as “1” for ACO relative gene expression analysis. H = harvest date.

  • View in gallery

    Expression of VvACS genes in rachis of Vitis vinifera ‘Thompson Seedless’ and ‘Ruby Seedless’ during storage and in response to ethephon treatment. Expression of ACS genes in rachis of (AC) ‘Thompson Seedless’ and (DF) ‘Ruby Seedless’. The value for each tissue type at harvest (Day 0) was set as “1” for ACS relative gene expression analysis. CK = untreated control; ETH = samples that were treated with ethephon at harvest.

  • View in gallery

    Expression of VvACO genes in rachis of Vitis vinifera ‘Thompson Seedless’ and ‘Ruby Seedless’ during storage and in response to ethephon treatment. Expression of ACO genes in rachis of (A) ‘Thompson Seedless’ and (B) ‘Ruby Seedless’. The value for each tissue type at harvest (Day 0) was set as “1” for ACO relative gene expression analysis. CK = untreated control; ETH = samples that were treated with ethephon at harvest.

  • View in gallery

    Total soluble solid contents of grape during berry developing. DAFB = days after full bloom.

  • Balic, I., Moreno, A., Sanhueza, D., Huerta, C., Orellana, A., Defilippi, B.G. & Campos-Vargas, R. 2012 Molecular and physiological study of postharvest rachis browning of table grape cv Red Globe Postharvest Biol. Technol. 72 47 56

    • Search Google Scholar
    • Export Citation
  • Barry, C.S., Blume, B., Bouzayen, M., Cooper, W., Hamilton, A.J. & Grierson, D. 1996 Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato Plant J. 9 525 535

    • Search Google Scholar
    • Export Citation
  • Cooper, T., Retamales, J. & Bangerth, F. 1993 Berry drop affected by GA3 and Promalin in Thompson seedless grapes Acta Hort. 329 134 136

  • Chervin, C., El-Kereamy, A., Roustan, J.P., Latché, A., Lamon, J. & Bouzayen, M. 2004 Ethylene seems required for the berry development and ripening in grape, a non-climacteric fruit Plant Sci. 167 1301 1305

    • Search Google Scholar
    • Export Citation
  • Chervin, C., Tira-umphon, A., Terrier, N., Zouine, M., Severac, D. & Roustan, J.P. 2008 Stimulation of the grape berry expansion by ethylene and effects on related gene transcripts, over the ripening phase Physiol. Plant. 134 534 546

    • Search Google Scholar
    • Export Citation
  • Gambino, G., Perrone, I. & Gribaudo, I. 2008 A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants Phytochem. Anal. 19 520 525

    • Search Google Scholar
    • Export Citation
  • Hedberg, P.R. & Goodwin, P.B. 1980 Factors affecting natural and ethephon-induced grape berry abscission Amer. J. Enol. Viticult. 31 109 113

  • Jiang, T.M., Wang, P., Yin, X.R., Zhang, B., Xu, C.J., Li, X. & Chen, K.S. 2011 Ethylene biosynthesis and expression of related genes in loquat fruit at different developmental and ripening stages Sci. Hort. 130 452 458

    • Search Google Scholar
    • Export Citation
  • Katz, E., Lagunes, P.M., Riov, J., Weiss, D. & Goldschmidt, E.E. 2004 Molecular and physiological evidence suggests the existence of a system II-like pathway of ethylene production in non-climacteric Citrus fruit Planta 219 243 252

    • Search Google Scholar
    • Export Citation
  • Katz, E., Riov, J., Weiss, D. & Goldschmidt, E.E. 2005 The climacteric-like behaviour of young, mature and wounded citrus leaves J. Expt. Bot. 56 1359 1367

    • Search Google Scholar
    • Export Citation
  • Larionov, A., Krause, A. & Miller, W. 2005 A standard curve based method for relative real time PCR data processing BMC Bioinformatics 6 62

  • Li, L., Kaplunov, T., Zutahy, Y., Daus, A., Porat, R. & Lichter, A. 2015 The effects of 1-methylcyclopropane and ethylene on postharvest rachis browning in table grapes Postharvest Biol. Technol. 107 16 22

    • Search Google Scholar
    • Export Citation
  • Lichter, A. 2016 Rachis browning in tablegrapes Austral. J. Grape Wine Res. 22 161 168

  • Mencarelli, F., Andrea, B. & Giancarlo, D. 2005 Grape: Post-harvest operations. University of Viterbo, Viterbo, Italy

  • Morgan, P.W., He, C.J. & Drew, M.C. 1992 Intact leaves exhibit a climacteric-like rise in ethylene production before abscission Plant Physiol. 100 1587 1590

    • Search Google Scholar
    • Export Citation
  • Munoz-Robredo, P., Gudenschwager, O., Chervin, C., Campos-Vargas, R., González-Agüero, M. & Defilippi, B.G. 2013 Study on differential expression of 1-aminocyclopropane-1-carboxylic acid oxidase genes in table grape cv Thompson Seedless. Postharvest Biol. Technol. 76 163 169

    • Search Google Scholar
    • Export Citation
  • Pech, J.C., Bouzayen, M. & Latché, A. 2008 Climacteric fruit ripening: Ethylene-dependent and independent regulation of ripening pathways in melon fruit Plant Sci. 175 114 120

    • Search Google Scholar
    • Export Citation
  • Perkins-Veazie, P.M., Huber, D.J. & Brecht, J.K. 1996 In vitro growth and ripening of strawberry fruit in the presence of ACC, STS or propylene Ann. Appl. Biol. 128 105 116

    • Search Google Scholar
    • Export Citation
  • Raban, E., Kaplunov, T., Zutahy, Y., Daus, A., Alchanatis, V., Ostrovsky, V., Lurie, S. & Lichter, A. 2013 Rachis browning in four table grape cultivars as affected by growth regulators or packaging Postharvest Biol. Technol. 84 88 95

    • Search Google Scholar
    • Export Citation
  • Reid, K.E., Niclas, O., James, S., Peng, F. & Lund, S.T. 2006 An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development BMC Plant Biol. 6 27

    • Search Google Scholar
    • Export Citation
  • Rosales, R., Fernandez-Caballero, C., Irene, R., Ma, I.E., Carmen, M. & Sanchez-Ballesta, M.T. 2013 Molecular analysis of the improvement in rachis quality by high CO2 levels in table grapes stored at low temperature Postharvest Biol. Technol. 77 50 58

    • Search Google Scholar
    • Export Citation
  • Rottmann, W.H., Peter, G.F., Oeller, P.W., Keller, J.A., Shen, N.F., Nagy, B.P., Taylor, L.P., Campbell, A.D. & Theologis, A. 1991 1-Aminocyclopropane-1-carboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence J. Mol. Biol. 222 937 961

    • Search Google Scholar
    • Export Citation
  • Ruijter, J.M., Ramakers, C., Hoogaars, W.M., Karlen, Y., Bakker, O., van den Hoff, J.M. & Moorman, A.F. 2009 Amplification efficiency: Linking baseline and bias in the analysis of quantitative PCR data Nucleic Acids Res. 37 e45

    • Search Google Scholar
    • Export Citation
  • Sun, L., Zhang, M., Ren, J., Qi, J., Zhang, G. & Leng, P. 2010 Reciprocity between abscisic acid and ethylene at the onset of berry ripening and after harvest BMC Plant Biol. 10 257

    • Search Google Scholar
    • Export Citation
  • Tan, D., Li, T. & Wang, A. 2013 Apple 1-Aminocyclopropane-1-carboxylic acid synthase genes, MdACS1 and MdACS3a, are expressed in different systems of ethylene biosynthesis Plant Mol. Biol. Rpt. 31 204 209

    • Search Google Scholar
    • Export Citation
  • Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. & Speleman, F. 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3:RESEARCH0034.1-0034.11

  • Xu, M. & Wang, M.H. 2012 Genome-wide analysis of 1-amino-cyclopropane-1-carboxylate synthase gene family in Arabidopsis, rice, grapevine and poplar Afr. J. Biotechnol. 11 1106 1118

    • Search Google Scholar
    • Export Citation
  • Yang, S.F. & Hoffman, N.E. 1984 Ethylene biosynthesis and its regulation in higher plants Annu. Rev. Plant Physiol. 35 155 189

  • Yip, W.K., Moore, T. & Yang, S.F. 1992 Differential accumulation of transcripts for four tomato 1-aminocyclopropane-1-carboxylate synthase homologs under various conditions Proc. Natl. Acad. Sci. USA 89 2475 2479

    • Search Google Scholar
    • Export Citation
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