Effects of GA3 and NAA on Fatty Acid Degradation in Germination and Seedling Growth of Cassia obtusifolia L. Seed

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1. Key Laboratory for Information System of Mountainous Area and Protection of Ecological Environment of Guizhou Province, Guizhou Normal University, Guiyang 550001, China, 2. School of Life Science, Guizhou Normal University, Guiyang 550001, China

Abstract [Objectives] To understand the content changes of fatty acid compositions and seedling growths during seed germinations of Cassia obtusifolia L. [Methods] The fatty acid compositions of germinations and seedling growths of C. obtusifolia seeds treated with different concentrations of gibberellin (GA3) and naphthalene acetic acid (NAA) were analyzed using gas chromatograph-mass spectrometer (GC-MS), and the germination rates, root lengths, plant heights and fresh weights of their seedlings were analyzed. [Results] The contents of eicosenoic acid, α-linoleic acid, arachidic acid, eicosadienoic acid and eicosapentaenoic acid of C. obtusifolia seeds treated with different concentrations of GA3 and NAA decreased with germination time. There were significant differences between the ratios of germinations of C. obtusifolia seeds treated with different concentrations of GA3 and NAA. GA3 and NAA of different ratio treatments could promote seedling heights and fresh weights of C.obtusifolia, but increases of seedling heights and fresh weights were different. Eicosenoic acid, α-linoleic acid, arachidic acid, eicosadienoic acid and eicosapentaenoic acid were remarkably negative correlation with seedling heights and fresh weights. [Conclusions] The fatty acid components of C. obtusifolia seed germination and growth of their seedlings were affected. It indicated that GA3 and NAA treatments can affect fatty acid metabolism, the germination rates, root lengths, plant heights and fresh weights of C. obtusifolia. It is intended to provide a theoretical basis and technological reference for standardized cultivated techniques of C. obtusifolia.

Key words Cassia obtusifolia L., Seed germination, Germination percentage, Plant growth regulator, Fatty acid

1 Introduction

Plant growth regulators, which are commonly used in plant tissue culture, play an important role in breaking seed dormancy, promoting seed germination and accelerating plant growth and development[1], and commonly referred to as plant hormones, are trace organic substances that are synthesized in plants and that have a significant effect on plant growth and development. At present, there are five recognized plant hormones including auxins, gibberellins, cytokinins, ethylene and abscisic acid[2-3]. As an essential component of the organism and an important component of the cell membrane, fatty acids are important energy materials for plants and play an important physiological role in seed germination[4-6]. And studies have shown that the composition of fatty acids is related to plant seed germination rate and germination rate[7].

Cassiaobtusifoliaseed, as a dry mature seed of the leguminous plantCassiaobtusifoliaL., is a homologous plant of medicine and food[8], and contains rich nutrients such as amino acids, fatty acids, mineral elementsetc. Among them, mineral elements include iron (Fe), manganese (Mn), copper (Cu), zinc (Zn) and other important trace elements[9]. In addition,C.obtusifoliacontains some important active ingredients such as terpenoids, naphthopone, polysaccharidesetc.[10]. Terpenoids mainly include aurantio-obtusin, obtusifolin, emodin, chrysophanol, physcion, rhein, aloe-emodin and the like[11]. The naphthopyrone inC.obtusifoliaseed has erythromycin gentioside, cassiaside C[12], toralactone, isotoralaetone, torosachrysone, cassiaside B2, and so on[13]. Semen cassiae polysacharide are mainly composed of monosaccharides such as glucuronic acid, arabinose, xylose, mannose, glucose, galactose and aminogalactose[14].C.obtusifoliaseed had the functions of anti-constipation, anti-inflammatory, visual acuity, and hepato-protective activities[15]. In some areas,C.obtusifoliaseed had been used in the treatment of mycosis and its mode of action on antifungal activity had been described[16-17]. Other studies have shown thatC.obtusifolialeaves were expected to be an alternative to antidiabetic drugs[18], andC.obtusifoliamedical abdominal belt could be used clinically for hemostasis, and it works well after use, and could improve postoperative comfort[19].

C.obtusifoliafatty acids are not only closely related to tissue immunity, specificity and cell recognition, but also play an important role in seedling growth and development during seed germination[20]. The effects of exogenous phytohormone on the metabolism ofC.obtusifoliafatty acids directly affects its germinations and seedling growths. Therefore, it is of great theoretical and practical significance to carry out the research on fatty acids changes and seedling growth during the germination ofC.obtusifoliainduced by exogenous phytohormone. The impact of environmental factors (such as temperature, light and seed drying methods,etc.) on the germination and seedling growth ofC.obtusifoliaand a lot of the results achieved were reported[21-25]. The fatty acid metabolism of plant seeds also were affected by external factors[26-27], for examples, contents of seed fatty acids were affected by low temperature stress, seed germination rates were affected by the contents of oleic acid, linoleic acid and arachidic acid, in addition, the fatty acids composition of the seeds changes correspondingly after mildew[28]. At the same time, the effects of exogenous phytohormone on the germination and seedling growth ofC.obtusifoliahave been reported in related literatures[29-32]. The effects of exogenous hormones on fatty acid compositions and contents during germination ofC.obtusifoliawas poorly reported.C.obtusifoliaseeds was treated with gibberellin (GA3) and naphthaleneacetic acids (NAA) to determine the fatty acid compositions, contents and germination rates during germination, as well as, fresh weights, root lengths and plant heights of their seedling, and analyze the relationship between fatty acid changes and their seedling growth status, in order to provide a theoretical basis forC.obtusifoliacultivation techniques.

2 Materials and methods

2.1 Experimental materialsC.obtusifoliaseeds were purchased from the medicinal materials market in Yunyan District, Guiyang City, Guizhou.

2.2 Experimental instruments and reagents

2.2.1Instruments: gas chromatography-mass spectrometry (GCMS-QP2010, Shimadzu Corporation, Japan); soxhlet extractor; constant temperature water bath (Tianjin Sterling Instrument Co., Ltd.); rotary evaporator (BUCHI); constant temperature drying oven (Tianjin City Stewart Instrument Co., Ltd.); AL104 electronic analytical balance (Mettler Instruments).

2.2.2Reagents: methyl palmitate (16∶0), palmitoleic acids methyl ester (16∶1), methyl stearate (18∶0), methyl oleate (18∶1), methyl linoleate (18∶2),α-linoleic acid methyl ester (18∶3), methyl aramate (20∶0), octadecyl methyl ester (20∶1), docosaenoic acid dimethyl ester (20∶2), trimethyl sesquienoate (20∶3), tetramethyl eicosenoate (20∶4) and pentamethyl eicoate (20∶5) were purchased by J & K Scientific Limited company; gibberellin (GA3) and naphthaleneacetic acid (NAA) were purchased from Aladdin; petroleum ether, methanol, potassium hydroxide, n-hexane, anhydrous sodium sulfate, and formalin were purchased from local chemical reagent suppliers.

2.3 Experimental designThe experimental individuals with full and uniform size selected fromC.obtusifoliaseeds by water selection were soaked for 20 min with 0.15% formalin solution, then, rinsed with deionized water for 2 or 3 times. The above-mentioned seeds were divided into five portions, and soaked with the mixed solutions of GA3(0, 25, 50, 100 and 200 mg/L) and NAA (0, 25, 50 and 100 mg/L) for 24 h, respectively. The specific ratio of the mixed solutions was listed in Table 1. The 50 seeds soaked were randomly selected, and placed on the bottom of double-layer moist filter paper in a petri dish indoor at 25 ℃, and the light conditions were 14 h in day time and 10 h at night. The number of germinations were recorded every day. The germinated seeds were placed in another petri dish. And the fatty acids of the seeds picked at 14:00 every day for the corresponding germination days were determined. The seedlings ofC.obtusifoliaseeds (about 1 g) were randomly picked in each dish. Their root lengths and plant heights were measured, then 10 strains of their fresh weights were weighed.

Table 1 Proportions of NAA and GA3forCassiaobtusifoliaseeds treated

mg/L

2.4 Extraction and methylation of fatty acidsThe germinated seeds picked were immediately placed in an oven at 105 ℃ for 5 min, then dried to constant weight at 50 ℃. The dried seeds are pulverized and then extracted with a petroleum ether as a solvent using a Soxhlet extractor. The extract solutions were concentrated to 2 mL using a rotary evaporator. The extracts concentrated were transferred to a 10 mL tube, and evaporated until dry, and then added 3 mL of 0.5% potassium hydroxide-methanol solution, 0.5 g anhydrous sodium sulfate and 2 mL n-hexane, shaken to make it evenly dispersed, and put it overnight. Added 2 mL of purified water at the next day, shaken to make it evenly dispersed, centrifuged for 10 min, and taken the organic layer place a refrigerator at 4 ℃ for analysis of the fatty acids. The reference substances of fatty acid methyl esters were formulated into a stock solution, stored at -20 ℃ for use, and diluted into a working solution according to the experiments.

2.5 Analysis conditions of GC-MS

2.5.1GC conditions. Column: VF-WAXms (30 m×0.25 mm×0.25 μm); injector temperature: 220 ℃; carrier gas: He (99.999%); flow rate: 1.05 mL/min; column temperature program: 50 ℃ (1 min), 40 ℃/min to 150 ℃, 4 ℃/min to 250 ℃ (10 min).

2.5.2MS conditions. Ion source and ionization voltage: EI and 1.2 KV, respectively; interface temperature 260 ℃; ion source temperature: 200 ℃; solvent delay time: 5 min; scan range: 33-450 m/z. A typical total ion chromatogram of the fatty acids inC.obtusifoliaseed germinated shown in Fig.1.

2.6 Data statistics and analysisData mapping and statistical analysis were performed using Excel 2013 and SPSS 19.0. Qualitative analysis was carried out according to the retention time of the control sample and the peak of the test sample, and the peak area of the test sample and the reference substance was quantified, and the formula of the fatty acids concentration (cx) of the samples was calculated as follows:cx=Ax/As×cs/mx/1 000, where:Asrepresented the area of the control peak,Axrepresented the peak area of the sample,cswas the solubility of the reference substance of the fatty acids, andmxwas the mass of the sample (g).

3 Results and analysis

3.1 The fatty acid compositions ofC.obtusifoliaSeedsTable 2 showed that the fatty acids of ungerminatedC.obtusifoliaseeds were palmitic acids (C16∶0), stearic acid (C18∶0), arachidic acid (C20∶0), palmitoleic acid (C16∶1), oleic acid (C18∶1), linoleic acid (C18∶2),α-linoleic acid (C18∶3), eicosanoic acid (C20∶1), eicosadienoic acid (C20∶2) and eicosapentaenoic acid (C20∶5). The relative content of the unsaturated fatty acids was 69.04% of the total content. The relative content of oleic acid and linoleic acid was 22.52% and 43.56%, respectively. While the relative contents of palmitoleic acid,α-linoleic acid, eicosanoic acid, eicosadienoic acid and eicosapentaic acid was 0.39%, 1.04%, 0.64%, 0.04% and 0.85%, respectively. the relative content of saturated fatty acids was 30.96% of the total content. The relative contents of palmitic acid, stearic acid and arachidic acid was 19.84%, 9.16% and 1.93%, respectively.

Fig.1 Total ion chromatograms of fatty acid comparisons, BD (2 d) and CD (7 d) fatty acids(1=16∶0, 2=10∶1, 3=18∶0, 4=18∶1, 5=18∶2, 6=18∶3, 7=20∶0, 8=20∶1, 9=20∶2, 10=20∶3, 11=20∶4, 12=20∶5)

Table 2 GC-MC analysis of fatty acids inCassiaobtusifoliaseeds

SequenceNo.Fatty acidsMolecularformulaRelativecontents∥%1Palmitic acid (C16∶0)C16H32O219.842Stearic acid (C18∶0)C18H36O29.163Arachidic acid (C20∶0)C20H40O21.934Palmitoleic acid (C16∶1)C16H30O20.395Oleic acid (C18∶1)C18H34O222.526Linoleic acid (C18∶2)C18H32O243.567α-Linolenic acid (C18∶3)C18H30O21.048Eicosanoic acid (C20∶1)C20H38O20.649Eicosadienoic acid (C20∶2)C20H36O20.0710Eicosapentaenoic cid (C20∶5)C20H30O20.85

3.2 Fatty acids changes ofC.obtusifoliaseeds during germinationFig.2 showed the content changes of major fatty acids in the germination process ofC.obtusifoliaseeds. The contents of palmitic acid, stearic acid, oleic acid, linoleic acid and arachidic acid decreased with the germination time, and the contents decreased significantly in the 4th d after seed germination took place. The contents of palmitic acid, stearic acid, oleic acid, linoleic acid and arachidic acid ofC.obtusifoliaseed treated with A decreased with the germination time. In addition to arachidic acid, it was significantly lower than CK in the 4 d after seed germination took place. The contents of palmitic acid, stearic acid, oleic acid, linoleic acid and arachidic acid inC.obtusifoliaseeds treated with B decreased with the germination time. And the decrease of palmitic acid content between B and CK was not significantly different in the 4th d after germination took place. The stearic acid content changed from significantly lower to significantly higher than CK. The contents of palmitic acid, stearic acid, oleic acid, linoleic acid and arachidic acid inC.obtusifoliaseed treated with C decreased with the germination time, and the levels of their reductions were significantly greater than that of CK. The palmitic acid content of C. cassia seed treated with D decreased with germination time, and the difference between D and CK was not significant. The content of stearic acid decreased with germination time, and the decrease of the content changed from less than gradually to higher than CK, and the content of oleic acid decreased with germination time, and the reduction level was significantly lower than that of CK. The content of linoleic acid decreased with the germination time, the reduction level was not significant with CK. The content of arachidic acid decreased with the germination time, and the reduction level was significantly higher than that of CK.

Fig.2 Fatty acids content ofCassiaobtusifoliaseeds treated with different treatments at 8 d after germination

3.3 Correlation of GA3, NAA the and fatty acidsTable 3 showed the correlation between fatty acids ofC.obtusifoliaand GA3and NAA during germination. Stearic acid (C18∶0) and oleic acid (18∶1) were significantly positive correlation with GA3. Oleic acid (18∶1) was significantly positively correlated with NAA, and arachidic acid (C20∶0) was significantly negatively correlated with GA3,α-linoleic acid (18∶3), eicosenoic acid (20∶1), eicosadienoic acid (20∶2) and eicosapentaenoic acid (20∶5) were extremely significantly negative correlation with GA3, palmitoleic acid (C16∶1),α-linoleic acid (18∶3), eicosenoic acid (20∶1) and eicosapentaenoic acid (20∶5) were significantly negative correlation with NAA, arachidic acid (C20∶0) and eicosadienoic acid (20∶2) were significantly negatively correlated with NAA, indicating that the germination ofC.obtusifoliaseed treated by different concentrations of GA3and NAA has an effect on the conversion of fatty acids.

Table 3 Analysis of correlation between fatty acids composition and GA3, NAA inCassiaobtusifoliaafter germination

AuxinC (16∶0)C (16∶1)C (18∶0)C (18∶1)C (18∶2)C (18∶3)C (20∶0)C (20∶1)C (20∶2)C (20∶5)GA30.334∗-0.159-0.0430.338∗0.306-0.466∗∗-0.418∗-0.475∗∗-0.545∗∗-0.511∗∗NAA0.215-0.372∗0.1930.556∗∗-0.046-0.399∗-0.556∗∗-0.326∗-0.461∗∗-0.414∗

Note:*denotes significant correlation at the 0.05 level,**denotes significant correlation at the 0.001 level.

3.4C.obtusifoliagermination ratesFig.3 showed the germination status ofC.obtusifoliaseeds in each treatment. The percentage of germination in each treatment was increased with time. The germination rates ofC.obtusifoliaseeds treated with A, C and D were significantly lower than that of CK, while the germination rate ofC.obtusifoliaseeds treated with B was significantly higher than that of CK. The order of germination rate of C.obtusifolia seeds at different treatments was C

Fig.3 Germination percentages ofCassiaobtusifoliaseeds treated with different ratio of NAA and GA3

3.5 Root length and plant height of C.obtusifolia seedling

Fig.4 showed the growth status ofC.obtusifoliaseedlings treated with different concentrations of GA3and NAA. Fig.5a showed the changes in root lengths and plant heights of seedlings ofC.obtusifoliaseeds induced at different ratio of GA3and NAA. The root lengths ofC.obtusifoliaseedlings treated with A, B, C and D decreased with the increase of the growth time, while CK reversed. In the treatment A, the root lengths ofC.obtusifoliaseedlings decreased from 4.22 to 2.81 cm on the 2nd to 8th day. The root lengths ofC.obtusifoliaseedlings in B treatment decreased from 4.18 to 3.08 cm, and the root lengths ofC.obtusifoliaseedlings in C treatment decreased from 4.18 to 3.63 cm. The root lengths ofC.obtusifoliaseedlings in D treatment decreased from 4.20 to 3.14 cm, while in CK, the root lengths ofC.obtusifoliaseedlings increased from 4.95 to 7.15 cm. There was a significant difference in root length between CK and C.obtusifolia seedlings treated with A, B, C and D. Fig.5b showed the variations of plant heights of seedlings ofC.obtusifoliaseeds treated with A, B, C and D. The plant heights of seedlings ofC.obtusifoliaseeds treated with A, B, C and D increased with the growth time. The plant heights of otherC.obtusifoliaseedlings was significantly higher than that of CK, except that the plants height ofC.obtusifoliaseedlings in D treatment changed from higher than gradual to lower than CK and gradually changed to higher than CK. For example, the plant height ofC.obtusifoliaseedlings with A, B, C and D treatment on the 8th day increased by 16.95%, 11.73%, 15.55% and 22.49%, respectively, compared with CK. It indicated that GA3and NAA could promote the growth ofC.obtusifoliaseedlings, but their growth induced at different concentrations of GA3and NAA were different.

Fig.4 Representative germination seedlings ofCassiaobtusifoliaat different treatments (on 6th d)

Fig.5 Variation of root lengths (a) and plant heights (b) of seedlings ofCassiaobtusifoliaat different treatments for 8 d

3.6C.obtusifoliaseed fresh weight changesFig.6 showed the fresh weight changes ofC.obtusifoliaseedlings treated with different concentrations of GA3and NAA. On the 4th day, the fresh weight of C.obtusifolia seedlings treated with CK, A, B, C and D increased by 20.2%, 20.0%, 42.4%, 53.1% and 26.8%, respectively. Compared with CK, except for the treatment A, the other treatments increased by 1.10, 1.65, and 0.32 times, respectively. On the 8th day, the fresh weight ofC.obtusifoliaseedlings treated with CK, A, B, C and D increased by 29.6%, 74.7%, 81.9%, 57.7% and 82.7%, respectively. Compared with CK, the weight increased 1.52, 1.77, 0.95 and 1.79 times, respectively. It indicated that GA3and NAA could promote significantly the fresh weights ofC.obtusifoliaseedlings.

Fig.6 Variation of fresh weight of seedlings ofCassiaobtusifoliain different time

3.7 Correlation between fatty acids and root length, plant height and fresh weightTable 4 showed the correlation between fatty acids and root length, plant height and fresh weight. Palmitic acid, stearic acid, oleic acid, linoleic acid and arachidic acid were significantly negatively correlated with fresh weight and plant height, and not significantly correlated with root length. the growth of seedlings after the germination ofC.obtusifoliaseeds were strongly correlated with the fatty acids. The fatty acids were continuously consumed to provide seedling growth, which could explain the correlation between the fresh weight and plant height and the fatty acids.

Table 4 Analysis of correlation between the fatty acids and the root length, plant height and fresh weight

Fatty acidsRoot lengthPlant heightFresh weightAalmitic acid0.022-0.783∗∗-0.738∗∗Octadecanoic acid0.087-0.754∗∗-0.723∗∗Oleic acid-0.085-0.750∗∗-0.710∗∗Linoleic acid0.138-0.779∗∗-0.736∗∗Arachidic acid 0.314-0.787∗∗-0.780∗∗

Note:*denotes significant correlation at the 0.05 level,**denotes significant correlation at the 0.001 level.

4 Discussions

4.1 Effects of different concentrations of GA3and NAA on fatty acids inC.obtusifoliagerminationSeed germination is an important stage in the growth process of plants, and its stored substances, which play an important role in seed germination, are the source of energy in the early stages of seed germination. The metabolism of storage materials are various with the diversely typical plant seed. The fat, as an important storage material of seeds, which of metabolism leads to the change contents of fatty acids in the process of seed germination. The fatty acid contents ofC.obtusifoliaseed treated with GA3and NAA in the process of the germination were more significant decrease than that of CK. There were differences in the fatty acid contents ofC.obtusifoliatreated with different concentrations of GA3and NAA, and some differences were significant. This indicated that the metabolism ofC.obtusifoliafatty acids were affected at the contents of GA3and NAA, and the degree of metabolism was related to the contents of exogenous hormones. The fatty acid compositions were significantly correlated with GA3and NAA, indicating that the metabolism process ofC.obtusifoliafatty acids might be affected by plant growth regulators, suggesting that GA3and NAA can change the regulation mechanism of fatty acid compositions in the growth process ofC.obtusifoliaseedlings. This makes the difference in the growth status ofC.obtusifoliaseedlings. The fatty acid contents ofC.obtusifoliaseeds were gradually reduced after germination, indicating that the energy required for growth ofC.obtusifoliaseedlings was partly provided by the fatty acids carried by the seeds.

4.2 Effects of different concentrations of GA3and NAA on the growthC.obtusifoliaseedlingsPlant exogenous hormones have an important impact on seed germination and seedling growth, and affect the germination rate and germination speed of seeds. Root lengths ofC.obtusifoliaseedlings treated with different GA3and NAA decreased with growth time, contrary to CK, indicated that the GA3and NAA inhibited the development of root length. The degrees of the root lengths ofC.obtusifoliaseedlings inhibited at different concentrations of GA3and NAA treatment were partly significantly different. GA3and NAA could promote the growth of plant heights ofC.obtusifoliaseedlings, the plant heights were different with their different concentrations induced. GA3and NAA could promote the increase of fresh weight ofC.obtusifoliaseedlings, which were significantly different. The changes of fatty acid compositions and contents could result in the growth of root lengths, plant heights and fresh weights affected. Palmitic acid, stearic acid, oleic acid, linoleic acid and arachidic acid were significantly negatively correlated with fresh weight and plant height, indicating that the growth of seedlings after germination ofC.obtusifoliawas inextricably linked with the presence of fatty acids. Fatty acids are continuously consumed to provideC.obtusifoliaseedling growth, which could explain the correlation between fresh weight and plant height and fatty acids.C.obtusifoliafatty acids had an important impact on seed vigor and germination. TheC.obtusifoliaseeds soaked with GA3and NAA diluted solution could break their dormancy, eliminate the active ingredients that inhibit the germination, and will help the seeds absorb water and promote their germination. TheC.obtusifoliaseeds treated with exogenous phytohormone is one of the effective methods to improve the germination rate and strong seedling of seeds.

In this experiment,C.obtusifoliaseeds were treated with different concentrations of GA3and NAA, and the fatty acid contents and compositions were monitored before and after germination. The contents of fatty acids ofC.obtusifoliaseed before and after germination was different. The fatty acid contents ofC.obtusifoliaseedlings were affected by GA3and NAA, and different at their diverse ratios. Thus, metabolism ofC.obtusifoliafatty acid was affected by plant growth regulators and affected the growth ofC.obtusifoliaseedlings.

5 Conclusions

The fatty acid compositions and growth ofC.obtusifoliaseedlings by different concentrations of GA3and NAA treatments were analyzed. The relative contents of unsaturated fatty acids in the ungerminatedC.obtusifoliaseeds were higher, the total content was 69.03%, among which the content of linolenic acid was 43.4%, followed by oleic acid (22.52%). The contents of saturated fatty acids in ungerminatedC.obtusifoliaseeds was 30.97%, and the highest content in saturated fatty acids was palmitic acid. The preliminary results showed that the fatty acid compositions and seedling growths ofC.obtusifoliaseeds were affected, suggesting GA3and NAA affected the metabolism of the fatty acids, the germination rate, fresh weight, root length and plant height ofC.obtusifolia. The results obtained will provide theoretical basis and technical reference forC.obtusifoliastandard cultivation techniques.