Effects of Gibberellin Biosynthetic Inhibitors on Oil, Secoisolaresonolodiglucoside, Seed Yield and Endogenous Gibberellin Content in Flax

Abstract

Flax (Linum usitatissimum L.) has been used for the edible oil in Korea. The evaluate the effect of plant growth retardants on flaxseed yield, oil content, and gibberellin level, chloromequat chloride (CMC), paclobutrazol (PBZ), and prohexadione-Ca (PHC) were used as plant growth retardants in this study. These plant growth retardants were foliar-sprayed to flax plant at 55 days after seeding. The concentrations of each plant growth retardant were as followed: CMC 250, 500, 1000 ppm, PBZ 40, 80, 160 ppm and PHC 500, 1000, 2000 ppm. PHC treatment to forming bolls was more stimulated than CMC and PBZ. The highest ripened seed rate was observed in PHC treatment at 2000ppm. The high see yields were obtained in PHC treatment following PBZ and CMC, in turn. Seed yield that significantly increased in PBZ and PHC was found to be increased 12.4 ~ 23.9% as compared to the control. The PHC showed higher flaxseed production and oil yield than that of CMC and PBZ. The results obtained in the present study suggest that higher concentration of plant growth retardant (PHC) increased flaxseed yield and oil content. The optimal concentration of PHC treatment was observed in 2000 ppm. It concludes that the foliar application of PHC 2000 ppm may be useful for the increasing oil and higher seed production in flax plants.

Introduction

Flax (Linum usitatissimum L.) is used for fiber and oil is an annual crop belonging to the family Linaceae (Simmonda, 1976). Flaxseed meal is 35 to 40% proteins, and together with cottonseed and sunflower supplies about 23% of the world’s oilcake and meal (Hatje, 1989). In flax culture, it has been tried to raise seed and oil productivity. Application of plant growth regulators (PGR), particularly growth retardants, may maintain internal hormonal balance, i.e., efficient sink–source relationship, thus enhancing crop productivity (Singh et al., 1987). Mepiquat chloride and chloromequat chloride were found by Dippenaar et al. (1990) and Pípolo et al. (1993) to restrict vegetative growth, thus enhancing reproductive organs (Wang et al., 1995). Mepiquat chloride as one of gibberellins biosynthetic inhibitors has been found to restrict the vegetative growth in the cost of enhanced reproductive organs (Wang et al., 1995). Fan et al. (1999) reported that mepiquat chloride improved photosynthetic efficiency. In addition, the good population type and canopy structure for dwarf plants, smaller leaves and bigger bolls could be achieved by mepiquat chloride application. Mepiquat chloride and trinexapac-ethyl tend to be shorter and more compact than untreated plants (Hodges et al., 1991; Jung et al., 1975; Kerby, 1985; Reddy et al., 1992; Stuart et al., 1984; Willard et al., 1976).

Mepiquat chloride, chloromequat chloride, and daminozide also have been associated with increased photosynthesis through increased total chlorophyll concentration in plant leaves (Nepomuceno et al., 1997; Wu et al., 1985).

Sawan et al. (1991) indicated that application of mepiquat chloride to cotton plants increased protein and oil yields and also caused a general decrease in the oil saturated fatty acids, associated with an increase in unsaturated fatty acids. Al-Gharbi and Yousif (1989) observed that application of chloromequat chloride increased sunflower oil seed content. Osman and Abu-Lila (1985) showed that spraying flax plants with chloromequat chloride increased the levels of unsaturated fatty acids. Osman and Ahmed (1985) found that spraying sesame plants with daminozide caused an increase in the oil unsaturated fatty acids. Zafirova et al. (1987) reported that Alar increased seed and oil yields of sunflower. We have focused on changes of endogenous gibberellins to get a better understanding together with better agronomic practices. In a previous study, it was revealed that foliar application of mepiquat chloride increases seed oil content and promotes unsaturated fatty acids in flax (Kim et al., 2011). The present study aimed to evaluate the optimal concentration of plant growth retardants in order to increase seed yield and oil content in flax plants.

 

Materials and Methods

Field experiment was conducted at the experimental field of Gyeongsangbuk-do Provincial Agricultural Research & Extension Services, Daegu, Republic of Korea. Flax (Linum usitatissimum L. cv. Hwabaek) seeds were planted in 4.8 m (16 rows) with 6.0 m (row length) plots covered with black polyethylene vinyl on March 12 2011 and March 11 2012, respectively. Row spacing (wide) was 0.3 m and plant density was 11,111/ha. Prior to seeding, fertilizer was supplied with nitrogen, phosphorus, and potassium at the 100, 25, and 90kg/ha, incorporating as basal and top dressing (7:3, w/w) to the soil, respectively.

Three plant growth retardants, chloromequat chloride (CMC), paclobutrazol (PBZ), and prohexadione-Ca (PHC), were treated to flax plants. The concentrations of each plant growth retardant were as followed: CMC 250, 500, and 1000 ppm, PBZ 40, 80, 160 ppm and PHC 500, 1000, 2000 ppm. Each plant growth retardant was foliar-sprayed at 55 days after seeding. The each solution volume applied was also 930 L ha-1. The untreated check was sprayed with distilled water. All foliar spray was done early hours of the day to reduce evaporation in the morning. A randomized complete block design with four replications was used.

Flax seeds were harvested on 10 August in 2011 and 9 August in 2012, depending on seed ripening degree. Oil content and fatty acid composition were determined from matured flax seed. Crushed flax seed (5 g fresh weight) was extracted by percolating with diethyl ether 100 ml using soxhlet apparatus. For analysis of fatty acid, the extracted solvent derived from soxhlet was evaporated and concentrated. Fatty acid methyl esters were prepared from in 200 ml of a 1% (w/v) solution of sodium methoxide in methanol as described previously (Hitz et al., 1994). After 20 min of incubation at room temperature, fatty acid methyl esters were recovered by the addition of 250 ml of 1 M sodium chloride and extraction with 250 ml of heptane and analyzed using a gas chromatogram (Model HP 5890, USA). Fatty acid methyl esters were resolved using an Omegawax 320 column (Supelco, PA, USA), and the oven temperature was programmed from 185°C to 215°C at a rate of 2.5°C min-1.

Extraction and analysis of SDG (secoisolariciresinol diglucoside) followed the reference (Meagher et al., 1999). Defatted flax powder was extracted with 50 ml of 80% methanol for 5h at 55°C in a shaking water bath. The methanolic extract was filtered and concentrated by rotary evaporation. The resulting aqueous extract was hydrolyzed with 0.8 ml of 1M hydrochloric acid for 1 h at 100°C. The acid hydrolysate was diluted with water and extracted twice ethyl acetate/hexanes (1:1). The dry samples were redissolved in methanol, filtered and applied to the HPLC. HPLC semipreparative separations were carried out on a Beckman 114M HPLC system with a Microsorb semipreparative C18 column. The HPLC system was equipped with an HP 1100 DAD, with detection set at 280 nm and 400 nm for identification. Elution was carried out with a flow rate of 0.6 ml/min using following solvent systems: solvent A=water/glacial acetic acid (99.8:0.2 v/v) and solvent B=acetonitrile. An initial ratio of 70A:30B was followed by a linear gradient to 50A:50B, over 55 min, then back to 70A:30B, for equilibration of the system over 5 min.

Extraction and analysis of gibberellin metabolites followed the reference (Lee et al., 1998). The seeds harvested were immediately frozen in liquid nitrogen and stored at -80ºC. When all the required materials for GA analysis had been collected, the samples were lyophilized for 48 h. The extraction of endogenous gibberellins was followed as described by Lee et al. (1998). The GAs were chromatographed on a 3.9×300 mm μBonda-Pak C18 column (Waters) and eluted at 1.5 ml min-1 with following gradient: 0 to 5 min, isocratic 28% MeOH in 1% aqueous acetic acid; 5 to 35 min, linear gradient from 28 to 86% MeOH; 35 to 36 min, 86 to 100% MeOH; 36 to 40 min, isocratic 100% MeOH. Up to 50 fractions of 1.5 ml each were collected. Small aliquots (15μl) from each fraction were taken, and radioactivity was measured with liquid scintillation spectrometry (Beckman, LS 1801) to determine accurate retention times of each GA based upon the elution of 3H-GA standards. The fractions were dried on a Savant Speedvac and combined according to the retention times of 3H-GA standards and previously determined retention times of the labeled (deuterated) GA standards. GAs were quantified using [17, 17-2H2]-GAs (20 ng each) as internal standards (purchased from Prof. L. N. Mander, Australian National University, Canberra, Australia). The three or five prominent ions were analyzed by GC-MS-SIM (Finnigan Mat GCQ) with dwell times of 100 ms. Endogenous GA contents were calculated from the peak area ratios respectively and retention time was determined by the hydrocarbon standards to calculate the KRI value.

The collected data for endogenous gibberellins and tuber yield were analyzed by using SAS package for Duncan’s multiple range tests.

 

Results and Discussion

The study was performed to test the effects of three gibberellin biosynthetic inhibitors (chloromequat chloride: CMC, paclobutrazol: PBZ, and prohexadione-calcium: PHC) on changes of oil, lignan, seed yield and gibberellins in flax plant. Table 1 shows influences of three plant growth retardants on growth characteristics and seed yield of flax plant. Three plant growth retardants significantly reduced plant height. Plant height reduction was related to shortening of the internodes. The CMC, PBZ, and PHC are used widely in agriculture and horticulture. It acts by inhibiting gibberellins biosynthesis resulting in shorter internode length and it inhibits gibberellin production much later in the biosynthetic pathway than MC, chloromequat, and triazole compounds (Hafner, 2001; Rademacher, 2000).

Table 1Note: CMC = chloromequat chloride; PBZ = paclobutrazol; PHC = prohexadione-ca. Different letters in the column indicate significant difference at α = 0.05.

Qian (1998) reported that TE reduces stem elongation and mowing requirements stimulating a favorable vertical shoot growth in turfgrass.

Kim et al. (2011) reported that single MC treatment had a higher seed ripening than TE treatment. CMC and PBZ had a similar effect in reducing plant height, otherwise PHC showed more significant reduction than CMC and PBZ in plant height. There was significant difference in number of branch per plant treated with CMC, PBZ and PHC. As compared with the control, the incidence of branch occurrence in flax plant was significantly increased by treatment with PHZ and PHC, and it was same tendency by treatment with CMC. PHZ and PHC also proved to be more effective than PHC treatment. In comparison with control, number of boll per branch was also increased by plant growth retardants. However, two plant growth retardants both CMC and PBZ did not alter the number of boll per branch as compared with control. PHC-treated plant showed higher capsule than that of CMC and PBZ treatment. PHC treatment to ability forming bolls was more stimulated than that of CMC and PBZ. The highest capsule productions were observed in PHC at 2000 ppm. The ripened seed rate was significantly increased with PBZ and PHC except for CMC. The highest ripened seed rate was observed in PHC treatment at 2000 ppm. The high see yields were obtained in PHC treatment following PBZ and CMC, in turn. Seed yield was significantly increased in PBZ and PHC. These treatments were found to be increased 12.4 ~ 23.9% as compared to the control.

Change of oil and fatty acid contents in flax seeds treated with three plant growth retardants are presented in Table 2. The treatment of flax plant with foliar growth retardants in oil content resulted in an increase in the proportion of concentrations. Among these plant growth retardants, highest oil content was observed in plants treated with PHC, whereas lowest oil content was observed in plants treated with CMC. Kim et al. (2011) reported that the foliar application with mepiquat chloride causes higher oil content than trinexapac-ethyl. Furthermore, it was also revealed that the treatment of mepiquat chloride (MC) causes increased oil content when the higher concentration of mepiquat chloride was applied (Kim et al., 2011).

Table 2Note: CMC = chloromequat chloride; PBZ = paclobutrazol; PHC = prohexadione-ca. Different letters in the column indicate significant difference at α = 0.01.

However, in our study, CMC that has same inhibiting point of gibberellin biosynthesis like a MC showed low oil content. This was probably due to the cultivars of flax and applied concentration. Two saturated fatty acids both palmitic acid and stearic acid were not changed by three plant growth retardants. In change of linolenic acid, the highest content was found in plants treated with PHC. In particular, PHC treatment gave the higher unsaturated fatty acid content than that of CMC and PBZ. Meanwhile, the oleic and linoleic acid contents were slightly decreased to increased concentration of PBZ and PHC. Low content of saturated fatty acids is desirable for edible uses. The bioactive and total gibberellin content was determined by GC-MS from flax seeds treated with CMC, PBZ and PHC (Table 3).

Table 3Note: CMC = chloromequat chloride; PBZ = paclobutrazol; PHC = prohexadione-ca. ECH = Early C-13 hydroxylation group including GA53, GA44, GA19, GA20 and GA1; NCH = Non C-13 hydroxylation group including GA12, GA15, GA24, GA9 and GA4. Total GA indicates sum of ECH and NCH. Different letters in the column indicate significant difference at α = 0.01.

In our previous study, it was first reported that eight gibberellins were identified and quantified operating two different gibberellin biosynthetic pathways, early C-13 hydroxylation and non C-13 hydroxylation routes (Kim et al., 2009). Two endogenous bioactive gibberellins (GA1 and GA4) in flax seeds were decreased by CMC, PBZ and PHC. Although higher concentration of these plant growth retardants in bioactive gibberellin (GA1) showed lower content as compared to the control, there was no statistically significant difference between concentration and plant growth retardants

Bioactive gibberellin (GA1) content was always 3.8 ~ 4.7 times higher than that of GA4 in all treatments. Bioactive gibberellin (GA1) was less suppressed than GA4 by plant growth retardants. It showed that gibberellin content of the early C-13 hydroxylation pathway including GA53, GA44, GA19, GA20 and GA1 was not significantly changed by plant growth retardants. However, it was tended to decrease with increased concentration of plant growth retardants

We also revealed that gibberellin content of the non C-13 hydroxylation pathway including GA12, GA15, GA24, GA9 and GA4 was significantly decreased by plant growth retardants. In particular, it was observed that PHC treatment with 2000 ppm caused dramatic decrease in gibberellin content of the group of non C-13 hydroxylation pathway. It is known that PHC (prohexiadione-Ca) as acylcyclohexanediones like a trinexapacethyl, block particularly 3ß-hydroxylation, thereby strongly inhibiting the formation of highly active GAs from inactive precursors. Lignans, secondary plant metabolites, are widely distributed in the plant kingdom. Flaxseed is known to be rich source of lignans such as secoisolariciresinol (SECO) and matairesinol (MATAI).

In the present study, this is the first report of the lignans being determined in flaxseed treated with plant growth retardants. Fig. 1 shows SDG content of whole flaxseed as affected by different concentrations of three plant growth retardants. The results showed that the flax plants applied with CMC, PBZ and PHC except for PBZ 400ppm produced much higher SDG content than that of control. The SDG contents in plant growth retardant treated flaxseeds were ranged from 12.5 to 14.8 mg. Among these treatments, the flaxseed treated with highest concentration of PBZ recorded a highest SDG content. The SDG content from defatted flaxseed was also determined (Fig. 2). The SDG content of defatted flaxseeds treated with three plant growth retardants were 21.5 to 26.1 mg. The higher concentration applied to flax plants, the more SDG content increased. The more significant increase of SDG content was observed in much higher concentration of PHC treatment. Our results obtained in the present study suggest that plant growth retardants (CMC, PBZ, and PHC) enhanced flaxseed yield, oil and SDG content.

Fig. 1.SDG contents of whole flaxseed. Note: ctr = distilled water applied; T1 = CMC 250ppm; T2 = CMC 500ppm; T3 = CMC 1000ppm; T4 = PBZ 40ppm; T5 = PBZ 80ppm; T6 = PBZ160ppm; T7 = PHC 500ppm; T8 = PHC 1000ppm; T9 = PHC 2000ppm; CMC = chloromequat chloride; PBZ = paclobutrazol; PHC = prohexadione-ca. Vertical bars means SE (n=3). Asterisk on a vertical bar means significant difference at P = 0.01.

Fig. 2SDG contents of defatted flaxseed. Note: ctr = distilled water applied; T1 = CMC 250ppm; T2 = CMC 500ppm; T3 = CMC 1000ppm; T4 = PBZ 40ppm; T5 = PBZ 80ppm; T6 = PBZ160ppm; T7 = PHC 500ppm; T8 = PHC 1000ppm; T9 = PHC 2000ppm; CMC = chloromequat chloride; PBZ = paclobutrazol; PHC = prohexadione-ca. Vertical bars means SE (n=3). Asterisk on a vertical bar means significant difference at P = 0.01.