The Horticulture Journal
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ORIGINAL ARTICLES
Sheet-mulching Cultivation Promotes the Number of Floral Buds via Up-regulation of Citrus Flowering Locus T Expression in Two Citrus Cultivars, ‘Haraguchi-wase’ (Citrus unshiu Marcow.) and ‘Harehime’ (C. spp.)
Mitsunori IwasakiFumie NishikawaHiroshi Fukamachi
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2019 Volume 88 Issue 2 Pages 164-171

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Abstract

Sheet-mulching cultivation during the fruit developmental stage is often carried out to produce high-quality Satsuma mandarin (Citrus unshiu Marc.) and ‘Harehime’ ((‘Kiyomi’ × ‘Osceola’) × ‘Miyagawa-wase’) fruits because they show high Brix% by exposure to drought stress conditions. In this study, we investigated the effect of drought stress on the number of floral buds in ‘Haraguchi-wase’ Satsuma mandarins and ‘Harehime’. To clarify the relationship between drought stress and the number of floral buds, we applied four different drying treatments to the fruit trees, (i) first-half drying (drying treatment during the first-half of the fruit development stage), (ii) second-half drying (drying treatment during the second-half of the fruit development stage), (iii) all-drying (drying during the full fruit development stage), and (iv) well-watered (non-drought stress during the fruit development stage). The drying treatment was applied to the fruits at ψ max −0.7 to −1.2 MPa at an intensity comparable to proper drought stress for high-quality fruit production according to our previous studies. In ‘Haraguchi-wase’, the all-drying treatment produced a higher number of floral buds in the following spring season compared with the well-watered treatment, in which the increase in the number of floral buds took place concomitant with the enhancement of CiFT expression in December. As expected, the fruits after all-drying were smaller with a significantly higher Brix% and a similar level of citric acid content, suggesting that drought stress of suitable intensity resulted not only in high-quality fruit production, but also in an increased number of floral buds in the following spring season. The results also demonstrated that the first-half drying caused higher CiFT expression and more inflorescences than in the well-watered plants, while there were no differences in CiFT expression or number of floral buds between the second-half drying and well-watered treatments. These tendencies observed in ‘Haraguchi-wase’ were the same as in ‘Harehime’. Therefore, the drying treatment during the first-half of fruit development could be an effective method to increase the number of floral buds.

Introduction

In Japan, most consumers prefer mandarins with a high sugar content and suitable acidity in the juice; therefore, such mandarins are traded as high-quality fruits at a higher price than those with low sugar content. Since it is known that drought stress increases the sugar content of fruit juice, sheet-mulching cultivation, by which drought stress can be imposed on fruit trees, is generally utilized in Satsuma mandarin (Citrus unshiu Marc.) and ‘Harehime’ ((‘Kiyomi’ × ‘Osceola’) × ‘Miyagawa-wase’) production (Iwasaki et al., 2011, 2012). However, it is assumed that drought stress for high-quality fruit production also affects the number of floral buds in the following spring season. Southwick and Davenport (1986) reported that both continuous and cyclical water-stress treatments increased the number of floral buds in ‘Tahiti’ lime, and Chica and Albrigo (2013), suggesting that water deficit and cool temperatures produced more inflorescences in potted sweet oranges. On the other hand, fewer floral buds were observed in Satsuma mandarins when they were exposed to severe water stress conditions (Katayama et al., 1989; Koshita and Takahara, 2004). Therefore, it has been reported that application of various drought stresses caused the increase/decrease in the number of floral buds in the following spring season. However, it has not yet been well documented how drought stress for high-quality fruit production affects the number of floral buds.

Large differences in the number of floral buds over two successive years could be the cause of alternate bearing in citrus (Monselise and Goldschmidt, 1982), which results in unstable annual income for citrus farmers. As a result, the important factor for stable citrus production could be optimization of the number of floral buds each year. However, despite the fact that drought stress has been practically applied at production sites for high-quality fruit production, there is no available literature showing the relationship between the number of floral buds and such drought stress. Therefore, we investigated the effect of the drying treatment for high-quality fruit production on the number of floral buds in ‘Haraguchi-wase’ Satsuma mandarin and ‘Harehime’. To do this, we applied four drying treatments to the fruit trees, (i) first-half drying (drying treatment during the first half of fruit development stage), (ii) second-half drying (drying during the second-half of the fruit development stage), (iii) all-drying (drying during throughout the fruit developmental stage), and (iv) well-watered (non-drought stress during the developmental stage), and monitored the number of floral buds, along with fruit quality parameters such as fruit size, Brix%, and citric acid content.

Upon evaluation of the number of floral buds the following spring, we focused on the expression level of FLOWERING LOCUS T (FT), which is involved in floral induction (Kobayashi et al., 1999). Extensive studies of FT in citrus (CiFT) showed that the transgenic trifoliate orange nursery, which caused ectopic overexpression of CiFT indicates the early flowering feature (Endo et al., 2005). Nishikawa et al. (2007, 2009) also reported that the endogenous expression of CiFT increased in the fall and winter, concurrently with seasonal floral induction. These results in citrus indicated the positive relationships between CiFT expressions in fall and winter and the number of floral buds the following spring. Therefore, we investigated CiFT expression levels as an indicator of the number of floral buds the following spring using trees exposed to various drought stress regimes.

Materials and Methods

Plant materials and drought stress treatments

First, 21-year-old (2011) ‘Haraguchi-wase’ Satsuma mandarin trees grafted onto trifoliate orange (Poncirus trifoliata (L.) Raf.) trees in a field at the National Agriculture and Food Research Organization (NARO) Institute of Fruit Tree Science, Kuchinotsu Station (currently Kyushu Okinawa Agricultural Research Center, NARO, Kuchinotsu Citrus Research Station) were used to clarify the effect of drought stress on the number of floral buds. To clarify the influence of stress stage, four treatments with different water stresses were applied to the trees: (i) first-half drying (drought stress from mid-July to mid-September), (ii) second-half drying (drought stress from mid-September to mid-November of the harvest season), (iii) all-drying (drought stress from mid-July to mid-November), and (iv) well-watered (non-drought stress from mid-July to mid-November). Four trees with similar flower/shoot ratios in the latest spring and without alternate bearing features were used for each treatment. Drought stress was applied using moisture-permeable plastic sheeting (Tyvek700AG; DuPont-Asahi Flash Spun Products Co., Ltd. Japan), and the intensity was adjusted using a plastic watering-tube (MistAce20 Saiteki 04L03; Sumika Agrotech Co., Ltd. Japan). Irrigation during the drying treatment was carried out based on water potential as described below, and irrigation during the non-drought stress treatment was conducted twice a week. The irrigation amount each time was set at 50 to 100 L/tree. The measurement of water stress adopted was the maximum leaf water potential (ψ max) using a pressure chamber instrument (model 600; PMS, USA). Two spring leaves were sampled from each tree (total of four trees), and the ψ max was measured from midnight to 2 AM once a week throughout the fruit developmental stage. The ψ max level of trees with applied drought stress was adjusted to about −0.7 to −1.2 MPa, which is considered suitable drought stress for high-quality fruit production (Iwasaki et al., 2012). Since the ψ max changes with low temperature, except for drought stress, the daily average temperature was measured with a thermometer installed in the field. To adjust the fruit bearing, the number of leaves per fruit was set to about 20 at the end of July. The full bloom stage in ‘Haraguchi-wase’ was early May and the harvesting date was November 12. Other cultivation practices were performed according to conventional methods.

To obtain more information on reproducibility of the results obtained in ‘Haraguchi-wase’, we also used 10-year-old (2010) and 12-year-old (2012) ‘Harehime’ trees grafted on trifoliate orange trees cultivated in a field at Kuchinotsu Station. In 2010, all-drying treatment (drought stress from August to December of the harvest season) and well-watered treatment (non-drought stress from August to December) were performed. In 2012, three different stress treatments were used: (i) first-half drying (drought stress from August to September), (ii) second-half drying (drought stress from October to December of the harvest season), and (iii) well-watered (non-drought stress from August to December). Seven and four trees with similar flower/shoot ratios in the latest spring and without alternate bearing features were used for each drying treatment in 2010 and 2012, respectively. The methods for applying and measuring drought stress were the same as in the ‘Haraguchi-wase’ experiment. In all treatments, the full bloom stage took place in early May, and the number of leaves per fruit was set to about 35 at the end of July. The harvesting date of ‘Harehime’ was December 10 in both years.

Sampling for CiFT transcript analysis

For CiFT expression analysis in ‘Haraguchi-wase’, three vegetative shoots (15 cm long) were collected from each treated tree three times during the floral bud differentiation period (November 15, December 14 in 2011, and January 12 in 2012). Based on the results showing the highest CiFT expression in ‘Haraguchi-wase’, samplings of ‘Harehime’ were conducted on December 14 in 2010 and 2012, when three vegetative shoots (20 cm long) were collected from each treated tree. In both cultivars, leaves and branches were separated from the vegetative shoots, and only the branches were used for CiFT expression analysis. The branches were immediately frozen in liquid nitrogen and powdered with a homogenizer (Shake Master; BMS, Japan) before storage at −80°C.

Total RNA extraction and real-time quantitative PCR

For real-time reverse transcription (RT)-PCR analysis, total RNA was extracted with an RNeasy Mini Kit (Qiagen, Germany) and cleaned by on-column DNase digestion. RT reactions were performed with 0.4 μg purified total RNA and a random hexamer at 37°C for 2 h using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, USA). A TaqMan MGB probe and sets of primers for total CiFT were designed using Primer Express software (Applied Biosystems) (Nishikawa et al., 2007). These probe and primers detect mRNAs for three CiFTs, CiFT1, CiFT2, and CiFT3, without discrimination. As an endogenous control, the TaqMan Ribosomal RNA Control Reagent VIC Probe (Applied Biosystems) was used. TaqMan real-time PCR was carried out with the TaqMan real-time PCR Master Mix (Applied Biosystems) using an ABI PRISM 7000 system (Applied Biosystems) according to the manufacturer’s instructions. Each reaction contained 900 nM primers, a 250 nM TaqMan MGB Probe, and 2.5 μL template cDNA. The thermal cycling conditions were 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. Gene expression levels were analyzed with an ABI PRISM 7000 Sequence Detection System Software (Applied Biosystems) and normalized to the results for 18S ribosomal RNA. Real-time quantitative RT-PCR was performed in three replicates for each sample.

Estimation of floral bud and fruit quality

To count the floral buds the following spring, 15 vegetative shoots per tree from each drying treatment were randomly selected in late April, and the numbers of current vegetative shoots, leafless inflorescences, and leafy inflorescences per sprouting node were recorded. To investigate the effect of drought stress on fruit quality, fruit weight, fruit shape index, Brix%, and citric acid content were estimated using five fruits per tree from each treatment at the harvesting stage. The Brix% and citric acid content were analyzed by NH-2000 (HORIBA, Japan).

Results

Drying treatments and their effect on fruit quality, CiFT expression, and the number of floral buds in ‘Haraguchi-wase’

In ‘Haraguchi-wase’, the ψ max in the well-watered treatment remained at approximately −0.5 MPa until late October and then decreased to approximately −1.0 MPa toward the winter season (Fig. 1). The ψ max of the first-half drying was from −0.7 to −1.2 MPa from late July to mid-September, and that of the second-half drying was from −0.7 to −1.7 MPa from mid-September to the harvesting period. The ψ max of the all-drying treatment was ordinarily 0.3 to 0.7 MPa lower than that of the well-watered treatment throughout the fruit developmental stage. Regarding the fruit quality at harvest, the fruit weights with the all-drying treatment were the lowest among all treatments, while those of the well-watered treatment were the highest (Table 1). There was no significant difference in the fruit shape index among all treatments. The Brix% in the fruit juice with the all-drying treatment was 14.1%, which was the highest among all treatments. The Brix% values in fruit juice with first- and second-half drying treatments were higher than in the well-watered treatment. The citric acid content with both all-drying and second-half drying was 0.94%, which was 1.4 times higher than that of the well-watered treatment.

Fig. 1

Change in maximum leaf water potential under different water stress treatments in ‘Haraguchi-wase’ Satsuma mandarins in 2011. Temperatures in the figure represent the daily average temperature in the field. Vertical bars indicate standard error (n = 4). Drying periods in each treatment were mid-Jul. to mid-Sep. in first-half drying, mid-Sep. to mid-Nov. in second-half drying, mid-Jul. to mid-Nov. in all-drying and no drying in the well-watered treatment.

Table 1

Effect of different water stresses on ‘Haraguchi-wase’ Satsuma mandarin fruit quality in 2011.

In ‘Haraguchi-wase’, there was no difference in CiFT expression between the four treatments on November 15, and the CiFT levels were considerably lower than those of samplings after that (Fig. 2). On December 14, CiFT expressions were significantly higher in first-half drying and all-drying than in well-watered treatment, while there was no difference between second-half drying and well-watered treatment. On January 12, the CiFT expression level in the all-drying treatment was twice as high as that in other treatments. In the following spring season, the number of vegetative shoots of the trees in well-watered and second-half drying treatments were higher than other treatments, and that with the all-drying treatment was the lowest among all treatments (Table 2). The numbers of leafless and leafy inflorescences with the all-drying treatment were the highest among the treatments, and the number of leafy inflorescences with the first-half drying treatment was twice as high as that with second-half drying and well-watered treatments.

Fig. 2

Change in CiFT expression levels under different water stresses over three time points during the floral bud differentiation period (November 15, December 14, and January 12) in ‘Haraguchi-wase’ Satsuma mandarins in 2011. Vertical bars in the figure indicate standard error (n = 4). Different letters indicate significance at the 5% level according to Tukey’s HSD test. Drying periods in each treatment were mid-Jul. to mid-Sep. in first-half drying, mid-Sep. to mid-Nov. in second-half drying, mid-Jul. to mid-Nov. in all-drying and no drying in the well-watered treatment.

Table 2

Effect of different water stresses on the number of vegetative shoots and floral buds in ‘Haraguchi-wase’ Satsuma mandarins in 2011.

Drying treatments and their effect on fruit quality, CiFT expression, and the number of floral buds in ‘Harehime’

To obtain more information on the reproducibility of results obtained in ‘Haraguchi-wase’, we also used ‘Harehime’ under all-drying and well-watered treatments in 2010 and three different stress treatments: first-half drying, second-half drying, and well-watered treatments in 2012. In 2010, the ψ max of the well-watered treatment remained flat at approximately −0.5 MPa until mid-November and then decreased to about −0.9 MPa toward the winter season, whereas the ψ max with the all-drying treatment was ordinarily 0.1 to 0.6 MPa lower than that of the well-watered treatment from early August to mid-December (Fig. 3A). In 2012, the ψ max with the first-half drying treatment was −0.7 to −1.0 MPa from late July to early October, which was lower than in the well-watered treatment (Fig. 3B). The ψ max with second-half drying was from −0.7 to −1.4 MPa from early October to the harvest season; these values were also lower than in the well-watered treatment. Regarding the fruit quality of ‘Harehime’ in 2010, the fruit weight with all-drying was 148.8 g, which was 10% lower than that in the well-watered treatment (Table 3). The Brix% with all-drying was 14.2%, which was 1.3 times higher than that in the well-watered treatment, and the citric acid content with all-drying was 0.89% (1.3 times higher than in the well-watered treatment). In 2012, the fruit weights with the well-watered treatment were higher than those with the second-half drying treatment (Table 3). The Brix% values with first-half drying and second-half drying were 13.4% and 12.4%, respectively, and these values were significantly higher than that in the well-watered treatment. The citric acid content with first-half drying was 0.86%, which was higher than that in the well-watered treatment. There was no difference in the fruit shape index among treatments in either year.

Fig. 3

Change in the maximum leaf water potential under different water stresses in ‘Harehime’. Temperatures in the figure represent the daily average temperature in the field. The top (A) and bottom (B) panels represent 2010 and 2012, respectively. Vertical bars indicate the standard error (n = 4). Drying periods in each treatment were Aug. to Dec. in all-drying, Aug. to Sep. in first-half drying, Oct. to Dec. in second-half drying and no drying in the well-watered treatment.

Table 3

Effect of different water stresses on ‘Harahime’ fruit quality.

In ‘Harehime’ (2010), the CiFT expression level with all-drying was 4 times higher than that in the well-watered treatment (Table 4). In the following spring season, the numbers of leafless and leafy inflorescences with all-drying treatment were 2.8 and 5.1 times higher than those of the well-watered treatment, respectively (Table 5). There was no difference in the number of vegetative shoots between the all-drying and well-watered treatments. In 2012, the CiFT level with first-half drying was 5 times higher than that in the well-watered treatment (Table 4). There was no difference in CiFT expression levels between second-half drying and well-watered treatment. The number of leafless inflorescences in the following spring with first-half drying was 2.5 times higher than that in the well-watered treatment, but that with second-half drying was not significantly different from that in the well-watered treatment (Table 5). There was no difference in the numbers of vegetative shoots and leafy inflorescences among the three treatments. Therefore, the trends in ‘Harehime’ showed patterns similar to those in ‘Haraguchi-wase’ Satsuma mandarin, indicating that drying in the first half of the fruit development stage increases the number of floral buds by promoting CiFT expression.

Table 4

CiFT expression levels under different water stresses on December 14 in ‘Harehime’.

Table 5

Effect of different water stresses on the number of vegetative shoots and floral buds in ‘Harehime’.

Discussion

Effect of drought stress intensity on high-quality fruit production and the number of floral buds in terms of CiFT expression (comparison of all-drying and well-watered treatment)

High-quality mandarin fruit is defined as medium sized fruits with a high sugar content (12% or more) and appropriate acid (about 1%) in Japan (Morinaga et al., 2005). In this study, since ‘Haraguchi-wase’ Satsuma mandarin and ‘Harehime’ fruits with all-drying treatment conformed to this fruit size and had a high Brix% and citric acid content, we considered that the drought stress intensity used in this study was appropriate for high quality fruit production. Cassin et al. (1969) and Moss (1969) showed that citrus floral buds were induced by prolonged exposure to either cool temperatures or water stress. On the other hand, Koshita and Takahara (2004) reported that severe water stress depressed floral bud induction as compared to moderate water stress. In this study, since the number of inflorescences with all-drying was significantly higher than that in well-watered treatment in each cultivar, it was inferred that the drought stress intensity to produce high-quality fruits induced floral buds without any negative side effects such as leaf fall. Regarding regulation of the vegetative and reproductive phases, the involvement of three major factors in floral bud induction has been considered: nitrogen metabolism, assimilate partitioning, and interactions of phytohormones (Davenport, 1990). In addition, the FT gene that plays a key role in floral bud induction was recently found in Arabidopsis, and the protein encoded by FT is a mobile signal synthesized in the phloem of leaves (An et al., 2004; Mathieu et al., 2007); furthermore, FT is transported to the shoot apical meristem (Corbesier et al., 2007), where it activates the expression of floral meristem identity genes (Michaels et al., 2005; Wigge et al., 2005). Chica and Albrigo (2013) demonstrated that the water deficit simultaneously up-regulated FT expression in the potted sweet orange. Nishikawa et al. (2017) suggested that CiFT levels around November, which is the period of floral induction, were closely correlated with the number of floral buds the following spring season. In ‘Haraguchi-wase’ in this study, the CiFT level with all-drying was significantly higher than that of well-watered treatment in December and January, but not in November. Okuda et al. (2004) suggested that the time of floral induction fluctuates year by year because of the environment or the fruit load in the Satsuma mandarin. Therefore, it seems that the sampling in November of this experiment was earlier than the appropriate period due to the effect of annual fluctuation, since the CiFT level in November was much lower than in December and January. It was assumed that the CiFT expression due to drought stress stably increased in December, which is the peak period of expression, including the results for ‘Harehime’. In conclusion, these results suggest that the drought stress intensity used for high-quality fruit production can increase the number of floral buds in the following spring season, concomitant with the induction of CiFT expression. Ono et al. (2010) demonstrated that water stress promoted first-flush flowering in the kumquat because of the increase in total sugars in the roots and a continuously high ABA level. Koshita and Takahara (2004) reported that water stress affects endogenous GA1/3, IAA, and ABA concentrations in leaves. Detailed elucidation of the relationship between CiFT expression, nutrients, and phytohormones, all of which are affected by drought stress, could open a new avenue to understand the mechanism underlying floral induction/initiation/development.

Relationship between drought stress stage in high quality fruit production and the number of floral buds in terms of CiFT expression

Cooling temperatures clearly promote floral induction in citrus (Davenport, 1990), and Satsuma mandarin trees remain in a vegetative growth phase until exposed to temperatures lower than 25°C (Inoue and Harada, 1988). However, Suzuki et al. (1967) demonstrated that a water deficit in the summer season increased the number of floral buds in potted Satsuma mandarins the following spring season. Additionally, Inoue (1989) demonstrated that high drought stress induced floral buds in potted Satsuma mandarin trees despite the high temperature (25°C) condition. In this study, the number of floral buds with first-half drying was higher than that in the well-watered treatment despite the temperature in the field exceeding 25°C. On the other hand, the number of floral buds with second-half drying was not different from that in the well-watered treatment. It is worth noting that in this study, first-half drying promoted CiFT expression, while second-half drying did not. Suzuki et al. (1967) suggested that a water deficit, which suppressed vegetative tree growth in the summer season, induced floral buds by promoting reproductive growth. Therefore, we suggested that summer drought stress induced floral buds particularly by suppressing vegetative growth and promoting CiFT expression, which could be a good index for reproductive growth. Iwasaki et al. (2012) suggested that the drought stress from July to September is highly correlated with the increase in sugar content in Satsuma mandarin juice. In this study, the Brix% with first-half drying was significantly higher than that with second-half drying in ‘Harehime’. Therefore, we suggest that drying during the first half of the fruit development stage not only achieves high-quality fruit production, but also increases the number of floral buds the following spring season.

In the alternate bearing cultivars of the sweet orange, the relationship between the number of floral buds and yield follows a curve in two phases: the first with between 0 and 20 flowers per 100 nodes, in which the yield increases with the number of flowers, and a second with more than 20 flowers per 100 nodes, in which the yield is independent of the number of floral buds (Becerra and Guardiola, 1984). Therefore, raising the number of floral buds above this threshold is essential to adjust alternate bearing (Agusti et al., 1992). Since the drought stress used for high quality fruit production also increased the number of floral buds, sheet-mulching cultivation may also improve alternate bearing. On the other hand, Suzuki et al. (1967) demonstrated that a marked increase in the number of floral buds caused alternate bearing because of the decrease in the number of floral buds the following spring. Since sheet-mulching cultivation is usually carried out every year at the production site, we should clarify the long-term effect of drought stress on alternative bearing and yield.

Acknowledgements

We are grateful to Dr. Takaya Moriguchi, director of the Institute of Fruit Tree and Tea Science, NARO, for helping to improve this paper.

Literature Cited
 
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