Isoprothiolane advances improvement of color through gibberellic acid metabolism in satsuma mandarin (Citrus unshiu Marc.).

doi:10.21203/rs.3.rs-2430198/v1

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Isoprothiolane advances improvement of color through gibberellic acid metabolism in satsuma mandarin (Citrus unshiu Marc.).

https://doi.org/10.21203/rs.3.rs-2430198/v1

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published 13 Mar, 2023

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The functions of isoprothiolane (IPT), which is a fungicide against rice blast, in carotenoid accumulation were examined in satsuma mandarin fruit (Citrus unshiu Marc). Whole trees were treated with IPT at 25 days before harvest. Concentrations of gibberellic acid-1 (GA₁)and GA₄ in the flavedo of IPT-treated trees were significantly decreased at 5 days after treatment (DAT) compared to the untreated control. The expressions of CitGA20ox1 in IPT-treated flavedo were lower than those in the untreated controls at 5 and 25 DAT. The CitGA3ox expressions in IPT-treated flavedo were lower than those in the untreated control at 5 DAT. ABA concentrations in IPT-treated flavedo were significantly higher than those in the untreated control at 25 DAT. The concentrations of b-cryptoxanthin in IPT-treated flavedo were higher than those in the untreated control at 25 DAT. The chlorophyll concentrations of IPT-treated flavedo were lower than those in the untreated control at 5 and 20 DAT. These results suggest that IPT advances b-cryptoxanthin accumulation through the regulation of endogenous GA₁ and GA₄ based on the inhibition of CitGA20ox1 and CitGA3ox expressions. It is possible that IPT can be utilized to improve coloration in other citrus fruit.

ABA

carotenoid

color

Citrus

In general, fruit coloration is an important factor in fruit quality. The primary pigments in citrus fruits are carotenoids and chlorophyll (Alos et al., 2006). It has been shown that environmental factors influence the accumulation of pigment in satsuma mandarin (Citrus unshiu Marcow.); for example, temperatures above 30°C inhibit coloration (Utsunomiya et al., 1982). Our previous report also showed that in pummelo (Citrus maxima), temperatures greater than 35°C four months before harvest retard lycopene synthesis with upregulation of phytoene synthase (CsPSY) and ζ-carotene desaturase (CsZDS) and downregulation of lycopene β-cyclase (CsβLCY), β-carotene hydroxylase (CsβCHX) andε-ring hydroxylase (CsεCHX) (Promkaew et al., 2020). In contrast, temperatures less than 20°C two months before harvest have been shown to inhibit lycopene accumulation in pummelo (Buaban et al., 2020). Light is also a crucial environmental factor in carotenoid accumulation in plant tissues (Pizarro and Stange, 2009). In tomatoes (Solanum lycopersicum), light can induce carotenoid accumulation and carotenoid biosynthetic genes (Azari et al., 2010). Besides environment conditions, it has been shown that plant hormones can affect coloration in citrus fruit. For example, prohexadione-calcium application, which is a gibberellic acid (GA) biosynthesis inhibitor, increased carotenoid concentrations with chlorophyll degradation (Barry and Roux, 2010). In papaya (Carica papaya L.), paclobutrazol application, which is a GA inhibitor, increased total carotenoids (Auxcilia et al., 2010). Ethephon (2-chloroethyl phosphonic acid) application, which is an ethylene-generating agent, was shown to induce degreening and improve coloration in orange peels (Citrus sinensis (L.) Osbeck) (Arzam et al., 2021). It has been shown that abscisic acid (ABA) is associated with maturation processes including coloration and sugar accumulation (Lin et al., 2018, Rehman et a., 2018). Pre-harvest spray of ABA improved fruit color from yellow to deep orange and increased total carotenoids in the flavedo of oranges (Rehman et al., 2018).

Isoprothiolane (diisopropyl-1, 3-dithiolan-2-ylidenemalonate: IPT) is a fungicide used for the control of rice blast (Tanaka 1980). We previously reported our more than ten years’ experience (2009–2019) that IPT application of 0.69 mM (200 ppm) 20 to 30 days before harvest improved peel color in satsuma mandarin (Aramaki et al., 2020). As a result, IPT was registered as Fuji-one® in 2019 and is being utilized to improve coloration in satsuma mandarins in Japan. However, the mechanism by which IPT improves peel coloration in satsuma mandarin is not clear. Our previous report showed that IPT also regulated maturation in grape berries, which like satsuma mandarin is a non-climacteric fruit (Lin et al., 2018). Therefore, this study examined the effects of IPT on plant hormone (GA₁, GA₄ and ABA), pigment (carotenoids and chlorophylls), and sugar concentrations.

Plant materials

Six randomly selected 12-year-old ‘Nichinan ichigou’ satsuma mandarins grafted onto trifoliate orange (Poncirus trifoliata (L.) Raf.) rootstocks in an open field in Sasebo city, located at 33°N, 129°E, and an altitude of 35 m, were used for the experiment. Each tree was trained as a modified open-center. Two test groups of three trees were created in 2019. In the first group, three trees were sprayed with a 0.69-mM (200 ppm) IPT solution at 25 days before harvest (DBH), which was shown to induce coloration in our previous experiment (Aramaki et al., 2020). The second group of three trees comprised the untreated control. These trees were sprayed at 25 DBH with water only. Fruits (30 per sampling date, 10 per tree) in each group were sampled at 0, 5, 19, and 25 days after treatment (DAT). 0 DAT was prior to coloration, and 25 DAT was the commercial harvest time. After analysis of total soluble solids (Brix) and titratable acidity in juice sacs (30 fruits per treatment: 10 fruits per tree), flavedo samples were frozen by liquid nitrogen and stored at -80°C until analysis of GAs, ABA, carotenoids, chlorophyll, and gene expressions.

Analysis of endogenous GAs

The extraction of endogenous GAs was performed according to Manzai et al. (2015) and Opio et al. (2020) with slight modification. Flavedo samples weighing 2 grams (three replications of 10 fruits) were homogenized with 200 ng [²H₂]-GA₁and[²H₂]-GA₄as internal standards in 80% (v/v) methanol. After homogenization, the samples were filtered and then purified using 0.1 M phosphate buffer at pH 8.5 and n-hexane. The sample solution was purified with Sep-Pak C₁₈ cartridges (Waters Co., Milford, MA, USA). The solution including GAs was eluted with 80% methanol containing 1% acetic acid. The residue was dissolved with 1 M formic acid. The sample was loaded into an Oasis MCX column (Waters Co.). GA₁ and GA₄ were eluted through the column using 5 mL methanol and collected for further purification. The quantification of GA₁ and GA₄ was performed using a LC/MS-2010EV (Shimadzu, Kyoto, Japan) with a mass spectrometer equipped with an electron spray ionization (ESI) source. The operation was performed by selected ion monitoring in positive mode. The compounds were separated by an ODS Mightysil RP-18 column (150 mm╳2.0 mm i.d., 5mm) isocratically with methanol/water containing 20 mM formic acid (80:20 v/v) at a flow rate of 0.3 mL. LC-MS conditions were as follows: ESI spray voltage, 4.5 kV; nebulizer gas (N₂) flow, 1.5 mL min^-1. The ions of m/z 347 and 349 for GA₁ and 331 and 333 for GA₄ were used for quantification.

Analysis of endogenous ABA

ABA was analyzed based on our previous report (Tomiyama et al., 2021). Two grams of frozen samples (three replications of 10 fruits) were homogenized with 200 ng [²H₆]-ABA as an internal standard in 80% methanol (v/v) containing 0.1% butylated hydroxytoluene, 0.1% L-ascorbic acid, and 0.5 g polyvinylpolypyrrolidone (PVPP). The homogenized samples were concentrated to an aqueous phase and extracted by ethyl acetate. The sample was dried and re-dissolved by one mL 100% (v/v) methanol. As with GAs, quantification of ABA was performed using LC-MS. ABA was quantified in selected ion monitoring (SIM) in negative ion mode. ABA concentrations were calculated from the ratio of peak areas for m/z 263 (ABA)/ 269 ([²H₆]-ABA). Data collection and peak analysis were performed using Labsolutions v.3 (Shimadzu).

Carotenoid analysis and chlorophyll measurement

The extraction of carotenoid (b-carotene and b-cryptoxanthin) was carried out according to the method of Matsumoto et al. (2007) with slight modifications. Three-gram flavedo samples were homogenized with 0.3 mmol b-apo-8’-carotenal as an internal standard in 25 mL of 40% methanol (v/v) with 0.3 g magnesium carbonate. After filtration, the samples were extracted using diethyl ether/methanol (7:3, v/v) with 0.1% (w/v) 2,6-di-tert-butyl-4-methylphenol (BHT). The filtrate was then extracted using diethyl ether and NaCl-saturated water. After the ether phase was evaporated, the residue was re-dissolved by methyl tert-butyl ether (MTBE)/methanol (1:1, v/v) containing BHT to 2.5 mL and 30 mL. The extract was determined by HPLC (model LC-2010, Shimadzu) with a C₃₀ carotenoid column (250 ×4.6 mm i.d., 5 mm, YMC, Kyoto, Japan): column temperature, 25 °C; mobile phase, methanol/MTBE (1:1) at flow rate of 0.3 mL/min. 　

Calculations of total chlorophyll concentrations were performed according to Porra et al (1989). Frozen flavedo samples of 0.5 g were immersed in 3 mL of N, N-dimethylformamide and kept at 4 °C in the dark for 24 h. Absorbances at 647 nm and 664 nm were measured by spectrophotometer (model: U-2910, Hitachi, Tokyo, Japan).

RNA extraction, cDNA synthesis, and quantitative real-time RT-PCR analysis

Total RNA from flavedo (1 g FW: three replications of 10 fruits) was extracted as previously decreased by Kondo et al. (2014). A 2-mg aliquot of total RNA was treated with 5 U of DNaseⅠ(Takara Bio, Inc., Otsu, Japan) and used for first-stand cDNA synthesis. RT-qPCR was carried out on the StepOnePlus Real-Time PCR system (Applied Biosystems, CA, USA) with the KAPA SYBR FAST qPCR Master Mix ABI Prism (Kapa Biosystems, MA, USA). Gene-specific primers for each gene were designed as shown in Table 1. The expression levels of genes were determined by the comparative cycle threshold (2^-^△△^Ct) values and normalized to be Ct values of CuActin (Kotoda et al., 2016).

GA₁ and GA₄ were detected in the flavedo (Fig. 1). GA₁ and GA₄ concentrations in IPT-treated flavedo were significantly decreased at 5 DAT compared to the untreated control. The concentrations of GA₁ and GA₄ in the IPT-treated and untreated control flavedos were not significantly different at 20 and 25 DAT. The expressions of CitKO in the flavedos were not significantly different at any measurement date (Fig. 2). The expression of CitKAO in IPT-treated flavedo at 25 DAT was lower than that of the untreated control while those at 5 and 19 DAT were not significantly different. The expressions of CitGA20ox1 in IPT-treated flavedo at 5 and 25 DAT were lower than those in the untreated controls. The CitGA3ox expression in the IPT-treated flavedo was lower than that in the untreated control at 5 DAT while those at 19 and 25 DAT showed no significant difference between treatments.

The ABA concentration in the IPT-treated flavedo at 25 DAT was significantly higher than that in the untreated control while those at 5 and 19 DAT were not different between treatments (Fig. 3). The expression of CitNCED3 in IPT-treated flavedo at 5 DAT was higher than that in the untreated control while those at 19 and 25 DAT showed no significant difference between treatments.

The concentrations of b-carotene in both treatments gradually decreased toward maturation (Fig. 4). The b-carotene concentration in the IPT-treated flavedo at 19 DAT was lower than that in the untreated control. The b-carotene concentrations at 5 and 25 DAT showed no significant differences between treatments. The concentration of b-cryptoxanthin in the IPT-treated flavedo at 25 DAT was higher than that in the untreated control, although those at 5 and 19 DAT showed no significant differences between treatments. Chlorophyll concentrations in both treatments were gradually decreased with maturation. At 5 and 20 DAT, the chlorophyll concentrations of IPT-treated flavedo were lower than those in the untreated control.

Total soluble solids (Brix) and titratable acidity were not significantly different between IPT-treated and untreated control fruit (data not shown).

It has been shown that color break in C. clementina mandarin fruit is characterized by the increase of b-cryptoxanthin and degradation of b-carotene and chlorophyll (Also et al., 2006; Alquezar et al., 2008). b-carotene is catabolized to b-cryptoxanthin in the carotenoid pathway (Kato et al., 2004). Our study agrees with those previous results. For example, b-carotene concentrations in IPT-treated fruit decreased at 19 DAT and b-cryptoxanthin concentrations increased at 25 DAT compared to the untreated control. The chlorophyll concentrations in IPT-treated fruit also decreased at 5 and 19 DAT. These results suggest that IPT treatment can advance the coloration process in citrus fruit. In general, accumulation of carotenoids correlates with maturation in citrus fruit, parallel to sugar increase (Kato et al., 2004; Promkaew et al., 2020). However, IPT treatment did not affect sugar or acid concentrations. Therefore, the influence of whole-tree IPT spraying may be limited to the flavedo of the satsuma mandarin fruit. The relationship between maturation such as carotenoid accumulation, GAs, and ABA in citrus fruit maturation has been previously investigated. GA treatment delayed the reduction of 1-deoxy-D-xyluose 5-phosphate synthase (DXS) expression, resulting in the delay of de-greening (Alos et al., 2006). Furthermore, a combination spray of GA and prohydrojasmon, which is a jasmonic acid analog, reduced peel puffing in Satsuma mandarins (Ma et al., 2021). These facts suggest that GA retards the maturation process in citrus fruit. Therefore, the low concentrations of GAs at 19 DAT may also be associated with the decrease in chlorophyll concentrations. A previous report showed similar results in Satsuma mandarins (Garcia-Luis, et al., 1985). In our study, IPT treatment significantly decreased endogenous GA₁ and GA₄ concentrations at 5 DAT. Some kinds of GA retardants have been developed to regulate plant growth. For example, mepiquat chloride and paclobutrazol have been utilized to regulate plant growth by the inhibition of copalyl-diphosphate synthase or ent-kaurene synthase in the GA synthesis pathway (Wilhelm, 2000). We investigated four genes in the GA synthesis pathway to clarify the effect of IPT on coloration improvement. Although IPT did not affect gene expressions of CitKO, which catabolizea ent-Kauren to ent-Kaurenic acid, or CitKAO, which catabolizes ent-Kaurenic acid to GA_12, the expression of CitGA20ox1, which catabolizes GA₁₂ to GA₂₀ and GA₉, decreased in IPT-treated fruit. Furthermore, the expressions of CitGA₃ox, which catabolizes GA₂₀ to GA₁ and GA₉ to GA₄, significantly decreased in IPT-treated fruit. These facts suggest that IPT can regulate GA₁ and GA₄ biosynthesis by retarding the expressions of CitGA20ox1 and CitGA3ox in citrus fruit.

Abscisic acid (ABA) is associated with the maturation process in non-climacteric fruit (Kondo et al., 2014). In sweet orange (Citrus sinensis (L.) Osb.), GA₃ application reduced ABA concentrations in the flavedo and delayed chlorophyll degradation and b-cryptoxanthin accumulation (Gambetta et al., 2014). Furthermore, it has been shown that ABA is a key signal that induces peel maturation in sweet orange (Kato et al., 2006; Romero et al., 2019). We also observed an increase of ABA in IPT-treated fruit at 25 DAT. These facts suggest that IPT may influence ABA metabolism because CitNCED expression levels in IPT-treated fruit increased at 5 DAT or that IPT may increase ABA as a result of the decrease of GA concentrations and the Cit20ox1 and CitGA3ox expressions at 5 DAT.

In summary, IPT decreased GA concentrations based on inhibition of the expression of Cit20ox1 and Cit3ox, resulting in the decrease of chlorophyll and increase of b-cryptoxanthin as shown in Fig. 5.

Acknowledgments. This work was supported by Nihon Nohyaku Co. (Tokyo).

Competing interests. The authors have no potential conflicts of interest to report.

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Table 1 is available in the Supplementary Files section.

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Isoprothiolane advances improvement of color through gibberellic acid metabolism in satsuma mandarin (Citrus unshiu Marc.).

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