Exogenous Gibberellin Promotes the Germination of Phellodendron Chinense Schneid Seeds by Regulating the Expression of Phytohormone Related Genes and Anabolic Proteins

doi:10.21203/rs.3.rs-133103/v1

Download PDF

Research Article

Exogenous Gibberellin Promotes the Germination of Phellodendron Chinense Schneid Seeds by Regulating the Expression of Phytohormone Related Genes and Anabolic Proteins

https://doi.org/10.21203/rs.3.rs-133103/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background: Phellodendron chinense Schneid is an important Chinese herb that contains berberine, phellodendrine, palmatine, and medicinal compounds. The germination rate of Phellodendron chinense Schneid seeds is lower after storage, and the exogenous gibberellin3 (GA₃) hormone promotes seed germination, but the mechanism is not cleared.

Results: Exogenous GA₃ hormone promoted germination of Phellodendron chinense Schneid seeds, elevated germination rates. It also increased the levels of activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Moreover, it enhanced the contents of berberine and endogenous GA₃, and increased the expression levels of Pc(S)-GA2ox, Pc(S)-GA3ox, and Pc(S)-THBO. However, it reduced the expression level of Pc(S)-ABI5. Furthermore, exogenous GA₃ up-regulated the protein levels of DNA guides RNA polymerase β'-subunits, Coffee coenzyme A oxymethyl transferase, and Histone H1.

Conclusion: These findings indicated that exogenous GA₃ promoted germination of Phellodendron chinense Schneid seeds by regulating the expression levels of phytohormone related genes and anabolic proteins.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Phellodendron chinense Schneid

gibberellin3 (GA3)

phytohorme

medicinal component

gene expression

proteomics.

Phellodendron chinensis is a deciduous perennial tree species of Rutaceae, also known as Huangbai [1]. In China, there are two species and one variety, Phellodendron amurense Rupr, Phellodendron Chinense Schneid Var glabriusculum Schneid and Phellodendron chinense var meiguense Stian-lun Dai et Hong Wu [1]. The Phellodendron amurense Rupr is mainly distributed in the three eastern provinces, and the Phellodendron chinense Schneid is mainly distributed in Hubei, Hunan, Chongqing, Sichuan, and Yunnan provinces [2]. Phellodendron chinensis contains alkaloids and other medicinal compounds, which have been used for more than 2000 years in china [2]. The levels of the medicinal ingredients in the stem stark of Phellodendron chinense Schneid are higher than Phellodendron amurense Rupr. Phellodendron chinense Schneid mainly contains alkaloids, flavonoids, phenols, and terpenoids, with the alkaloids being the key medicinal compounds, which include berberine and phellodendrine [3, 4]. These two compounds have important physiological functions, such as immune modulation, anti-inflammatory, antimicrobial, antibacterial, anticancer, hypotensive, antiarrhythmic, antioxidant, antigastric ulcer, and antipyretic agents [5, 6].

Seed germination marks the beginning of plant growth and development, and is affected by the availability of water, oxygen, temperature, light and plant growth regulators [7]. Among them, the gibberellin3 (GA₃) plant growth regulator hormone belongs to diterpenoids that plays a key role during seed germination, vegetative growth, carbohydrate transformation, prevention of abscission, abloom period and accumulation of medicinal compounds [8, 9]. It has been documented that exogenous GA₃ promotes the seed germination of F. hupehensis, increases the levels of endogenous trans-Zetain (ZT) and GA₃ contents, but decreases the levels of endogenous abscisic acid (ABA) [10]. Another study showed that exogenous GA₃ (1 µM) enhances the germination rate of Stevia rebaudiana, and improves the development of seedlings [11]. In Phellodendron chinense Schneid, low temperature storage gradually decreases the seed germination rate [12, 13]. However, the physiological mechanism through which exogenous GA₃ regulates Phellodendron chinense Schneid seed germination remains unclear. Herein, we analyzed the seed germination rates, endogenous phytohormone contents, gene expression levels, and protein expression levels, in order to clarify the physiological mechanism underlying the regulation of seed germination in Phellodendron chinense Schneid by exogenous GA3.

Materials

The seeds of Phellodendron chinense Schneid were picked from Xiangxi Autonomous Prefecture (Hunan province), and identified by professor Jiaxiang Li (College of Forestry, Central South University of Forestry and Technology).

Seeds germination

Seeds germination referred to published methods[14]. The full seeds were selected, and immersed in concentrated sulfuric acid (Sinopharm, CHN) for 30 min for shelling, than disinfected in 75% alcohol (Sinopharm, CHN) for 45 s, and washed three times using sterile distilled water. Subsequently, the seeds were sterilized in 0.1% HgCl (w/v, Sinopharm, CHN) for 20 min, and washed five times using sterile distilled water. Finally, one batch of the seeds was transferred to sterile distilled water (H₂O, control group) and another batch into 1 mg·L^-1 Gibberellin3 (GA₃) solution (experimental group) for 24 h, respectively. The seeds were cleaned with sterilized filter paper, and transferred to Murashige and Skoog’s (Murashige and Skoog, 1962, MS) medium (agar 0.8%, sugar 3%, pH5.8, Sinopharm, CHN) on a sterile ultra-clean table for culturing and germination. Each group had 20 bottles (20 seeds/bottle), photos were taken, and the germinating seeds were counted, and then collected materials of seeds at 0 d, 3 d, 6 d, and 9 d. At the same time, the seeds were collected and frozen in -80℃refrigerator for the analyses of enzyme activities, determination of endogenous phytohormone contents, detection of gene expression levels, and proteomic analysis. All the seeds were cultured in the tissue culture room and the culturing conditions were; in tissue culture room with temperature 25±1℃, light period 12 h light/12 h dark, light intensity 1000-1500 Lux. All the experiments were conducted in three replicates (n=400/20 bottles).

Enzyme activity assay

The collected seeds (0.2 g) were placed into a pre-cooled mortar containing 1 mL pre-cooled phosphate buffer (0.05 mol·L^-1), and then ground to pulp. Finally, the volume was adjusted to a fixed volume of 8 mL using phosphate buffer. The extracts were transferred to the centrifugal tube, and centrifuged at 12000 g at 4℃ for 20 min; the supernatants were the enzyme extracts. The analysis of the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were performed as previously described [15-17]. All the experiments were conducted in three replicates (n=3).

Berberine and phellodendrine analyses

The extraction and determination of berberine and phellodendrine in Phellodendron chinense Schneid seeds was conducted as previously described [18, 19]. The standards of berberine and phellodendrine were purchased from Beijing Solarbio Science & Technology Company of China. All the experiments were performed in three replicates (n=3).

Endogenous phytohormones assay

Endogenous phytohormones assay referred to published methods [20]. Firstly, the collected seeds were ground into powder in liquid nitrogen, and then 100 mg samples were placed into pre-cooled centrifuge tubes containing 1.5 mL extraction solution (50% acetonitrile containing 0.3 ng ABA isotope as the internal standard, Sigma, USA). Afterwards, the samples were ultrasonicated for 3 mins under ice bath conditions, then placed into the rotating instrument overnight at 4℃. Thereafter, the samples were centrifuged at 13000 rpm at 4℃ for 10 min, and the supernatants were transferred into new centrifuge tubes (2 mL). Subsequently, the precipitation were re-dissolved by 1.0 mL extraction solution, and extracted again at 4℃ for 60 min, and then two supernatants were combined after centrifugation for determination. Then, the Oasis HLB RP (1 cc/30 mg, Merck, GER) column was activated by washing with 1mL methanol and 1mL water, followed by equilibration with 50% acetonitrile (ACN). The extracts were then added to Oasis HLB RP column, and the filtrates collected, followed by washing of the column with 30% (v/v) can. The filtrates were combined. The filtrates were dried with nitrogen at low temperature, then resuspended in 300 μL 30% acetonitrile (Sigma, USA) and filtered with 0.22 μm nylon membrane. The filtrates were analyzed by mass spectrometry. The ExionLC AD System of SCIEX company (USA) chromatographic system was used in the study. According to the properties of phytohormones, the Waters BEH C18 RP Column (1.7 μm, 2.1×150 mm) was used. The mobile phase was 0.01% formic acid water (A) - acetonitrile (B) (Sigma, USA). The elution gradient was 0-1 min, 5% B; 1-7 min, 5% -100% B; 7-9 min, 100% B; 9-9.3 min, 100% -5% B; 9.3-12 min, 5% B. The flow rate was 0.3 mL·min^-1, the injection volume 10 μL, and the column temperature was 45℃.

UHPLC-ESI-MS/MS was used for qualitative and quantitative detection of plant hormones in the samples [21]. Mass spectrometry system (AB SCIEX QTRAP 5500, USA) detection system with electrospray ion source (ESI) in multi response monitoring (MRM) mode analysis. Mass spectrometric analysis conditions were as follows: air curtain gas (PSI): 40; temperature (centigrade): 600; spray gas 1 (PSI): 60. Gas 2 (PSI): 60; ionization voltage (V): -4500 or +5500.

Gene Expression analysis

The conserved regions of the sequences of the GA2-OXIDASE (GA2ox), GA3-OXIDASE (GA3ox), ABSCISIC ACID-INSENSITIVE 5 (ABI5), and TETRAHYDROPROTOBERBERINE OXIDASE (THBO) genes were cloned by polymerase chain reaction (PCR) from leaves of Phellodendron chinense Schneid, and then primers designed and their stability for quantitative reverse transcription PCR (qRT-PCR) determined on an ABI 7500 (ABI, USA) detection system. The total RNA was extracted from the collected seeds using the RNA extraction Kit (OMEGA, USA). Afterwards, cDNA synthesis was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). Then, q-PCR reactions were performed with the SYBR Green Premix Pro Taq HS qPCR Kit (without ROX) (Accurate Biotechology(Hunan)Co.,Ltd, CHN). The expression levels of Pc(S)-GA2ox, Pc(S)-GA3ox, Pc(S)-ABI5 and Pc(S)-THBO were determined on the ABI 7500 detection system according to operation procedure. Each qRT-PCR detection was standardized using the expression level of the 18S rRNA gene. All experiments were conducted in three replicates (n=3). Expression levels were calculated using the Livak calculation method (2^-ΔΔCt) [22]. The gene specific primers used are listed in table 1.

Table 1 Primer names and sequences in experiments of gene expression

Primer names	Sequences (5’-3’)	Lengths (bp)
qPc(S)-GA2ox-F	TAATGGTGATGTTGGTTGGA	20
qPc(S)-GA2ox-R	TAAAGAGGTGGTGTGTGTAGA	21
qPc(S)-GA3ox-F	GGTAAGTTCCGACCCGCA	18
qPc(S)-GA3ox-R	AATACGAGGAGCACAAAGCC	20
qPc(S)-ABI5-F	CGCAGGAATCGTAAGTGAGC	20
qPc(S)-ABI5-R	CGAAGAAAACCAAGCCGTC	19
qPc(S)-THBO-F	TCTGTTGCGGTCCGATTTC	19
qPc(S)-THBO-R	ATAACCCAAGGTATGCCCA	19
18S rRNA-F	GTGTTGCTTACCCACGAAA	19
18S rRNA-R	AAGGGCACAAGGCGGAT	17

Proteomics analysis

The proteins from the collected seeds of Phellodendron chinense Schneid were homogenized in PBS (pH 7.6) containing 65 mM K₂HPO₄, 2.6 mM KH₂PO₄, 400 mM NaCl, and 3 mM NaN₃ (Sinopharm, CHN) by using a glass pestle and mortar. Subsequently, the proteins were separated using two-dimensional electrophoresis and identified by mass spectrometry referred to published method [23].

Data analysis

The Origin 2018 and IBM SPSS Statistics 22 softwares were used to perform data analyses.

Exogenous GA₃promotes seed germination

With the prolongation of culturing periods, the germination rates of the Phellodendron chinense Schneid seeds treated with H₂O and exogenous GA₃ increased gradually (Fig. 1I). The seeds treated with H₂O (control) began to germinate on the third day, and had few hypocotyls (Fig. 1B). By the 6^th day of culturing, most of the seeds had germinated, and had many short hypocotyls (Fig. 1C). Furthermore, the cotyledons were observed on the 9^th day; however they were short (Fig. 1D). The germination rates of the seeds under H₂O treatment were 0%, 13.27%, 52.33%, 73.62% at 0, 3, 6, 9 days, respectively. The seeds treated with GA₃ also began to germinate on 3^th day, and the germination rate was 21.63%, i.e., GA₃enhanced germination by 61.37% compared with control (Fig. 1F). The seeds under the 6-day culturing, exhibited a germination rate of 75.26%, hence GA₃increased germination by 22.93%, and the hypocotyls were longer compared with control (Fig. 1G). On 9^th day of culturing, the seed germination rate was 83.36%, GA₃improving germination by 9.75% compared with control at the same period (Fig. 1H). Furthermore, the cotyledons and hypocotyls under GA₃ treatment were larger and longer than in the control group. These results showed that exogenous GA₃ promoted the germination of Phellodendron chinense Schneid seeds, and elevated its germination rates.

Exogenous GA₃ enhances enzyme activities

During the culturing times of 0-9 days, the SOD activities in the Phellodendron chinense Schneid seeds treated with H₂O and exogenous GA₃ all gradually increased, and then began to decrease after reaching the maximum on the 6^th day (Fig. 2A). Culturing on the 6^th day, the SOD activity in seeds under H₂O treatment was 6394.08 U·min^-1·g^-1FW, which was 1.47-fold compared with that of the culturing at the initial stage. On the 9^th day, the SOD activity began to decrease, and it was 1.34-fold compared with that of the initial period culturing. The highest peak of SOD activity of Phellodendron chinense Schneid seeds treated with GA₃ also appeared on the 6^th day. The activity was 8239.37 U·min^-1·g^-1FW, which was 1.39-fold and 1.29-fold compared with that of the initial stage and control group at the same stage, respectively. Culturing on the 9^th day, the SOD activity began to decrease, reducing by 42.38% compared with that of the initial period, and decreased by 41.69% compared with control group on 9^th day. Notably, the SOD activities in the seeds treated with GA₃ were higher compared with control group during the culturing period of 0-6 days.

During the entire culture periods, the POD activities in the Phellodendron chinense Schneid seeds treated with H₂O and exogenous GA₃ increased gradually, and reached the peak on the 9^th day (Fig. 2B). In the control group, culturing for 0, 3, 6 and 9 days the POD activities in the seeds were 2058.05, 26532.20, 50190.86 and 60533.00 U·min^-1·g^-1FW, respectively. Moreover, the POD activities at 3, 6, 9 days were 12.89-, 24.39-, and 29.41-fold, respectively compared with that at 0 day. When the seeds were treated with GA₃, the POD activities were 23303.05, 64946.67, and 77722.91 U·min^-1·g^-1FW at 3, 6, 9 days, respectively, which increased by 30.63-, 87.16-, and 104.50-fold compared with that at 0 day. Besides, the POD activities in the experimental group seeds at 0, 3, 6 and 9 days were 35.79%, 87.83%, 129.40% and 128.40%, respectively, compared with that of the control group over the same culturing times. Furthermore, the result showed that the POD activity in seeds under GA₃ treatment were higher than the control during late germination (6-9 days).

With the prolongation of the incubation time, the CAT activities of Phellodendron chinense Schneid seeds treated with H₂O and GA₃ also increased gradually, and reached the peal on the 9^th day (Fig. 2C). During culture period, the CAT activities in the seeds under H₂O treatment were, 1139.67, 1360.54 and 2058.58 U·min^-1·g^-1FW at 3, 6, 9 day, which were 2.21-, 2.64- and 3.99-fold, respectively, compared with that at 0 day. After GA₃ treatment, the CAT activities in the seeds were 1580.42, 2668.22 and 2774.72 U·min^-1·g^-1FW at 3, 6, 9 day, which were 2.30-, 3.88- and 4.04-fold, respectively, compared with initial stage. Compared with control group, the CAT activities were 1.33-, 1.39-, 1.96- and 1.35-fold at the same stage, respectively. The result indicated that the CAT activities of Phellodendron chinense Schneid seeds treated with GA₃were higher than those in the control during in all the culture periods.

Exogenous GA₃promotes the accumulation of berberine

During times of germination, the levels of berberine in the Phellodendron chinense Schneid seeds treated with H₂O reached their peak at the 3^rd day (19.87 ng·g^-1DW), which was 2.24-fold compared with the levels in the initial stage, then began to decrease gradually (Fig. 3). Culturing for 9 days, the berberine level was 15.89 ng·g^-1DW. This was 1.95-fold and 0.80-fold compared with that at 0 day and 3 day, respectively. Culturing at the 3^rd day, the content of berberine in seeds treated with GA₃ reached the maximum, which was 2.24-fold higher compared with that at 0 day, and then began to reduce gradually. Germination on the 9^th day, the berberine content was 2.15-fold compared with that of initial stage, but it was higher than that of control group at the same time. The contents of phellodendrine in the seeds treated with H₂O increased gradually, and reached the peak at the 9^th day. The phellodendrine content under the H₂O treatment was 2.61-, 1.70- and 1.52-fold on the 9^th day compared with that at 0, 3, 6 day, respectively (Fig. 3). The contents of phellodendrine in seeds under GA₃ treatment improved gradually, and reached its peak on the 9^th day, which was 3.35-, 1.26- and 1.26-fold compared with culturing for 0, 3, 6 days; however, the level of phellodendrine was lower than that of control group at the same culturing periods.

Exogenous GA₃enhances the content of endogenous GA₃

The results showed that the contents of endogenous GA₃ in seeds under H₂O and GA₃ treatment were 882.27 ng·g^-1FW and 14927.81 ng·g^-1FW at 24 h; the latter was enhanced 14.92-fold compared with former (Fig. 4A). Under the same conditions, the contents of ABA in seeds were 6.94 ng·g^-1FW and 7.18 ng·g^-1FW, with no significant difference reported (Fig. 4B). These results indicated that exogenous GA₃ significantly enhanced the contents of endogenous GA₃ during seed germination.

Exogenous GA₃ regulates gene expression

During 0-6 day, the expression levels of Pc(S)-GA2ox in the seeds under GA₃ treatment gradually increased, which were 10.88-, 29.31-, 139.89-fold compared with those under the H₂O treatment, and were 2.69- and 12.86-fold, respectively, compared with those of the GA₃ treatment at 0 day. Then, the expression level of Pc(S)-GA2ox was reduced at 9 day, but it was obviously higher than in the control. During the entire periods, the expression levels of Pc(S)-GA3ox gradually increased, and reached the peak at 9 day, which were 438.96-fold compared with that under the H₂O treatment, and were 141.60-fold compared with that under the GA₃ treatment at 0 day. The expression levels of Pc(S)-ABI5 in the seeds treated with GA₃ were maximum and minimum on the 3^rd day and 9^th day, respectively, which were increased by 9.14-fold and reduced by 2.11-fold compared with the seeds in the GA₃ treatment at 0 day; however, they were higher than the H₂O treatment seeds at 0 day. During 0-6 day, the expression levels of Pc(S)-THBO in seeds under the GA₃ treatment gradually increased, and reached the maximum on the 6^th day, which were enhanced by 1397.54-fold compared with the control (Fig. 5). These results indicated that exogenous GA₃ increased the expression levels of Pc(S)-GA2ox, Pc(S)-GA3ox, and Pc(S)-THBO, but inhibited the expression levels of Pc(S)-ABI5.

Proteomics analysis

The results showed that 22 different protein points were obtained in the H₂O treatment group, among which 14 proteins were related to energy metabolism and substance synthesis, and 3 proteins were related to resistance to virus invasion (Fig. 6 and Table 2). The expression levels of 13 identified proteins were down-regulated except for the up-regulated expression of protein TIC 214 on the 6^th day in control group. However, among 3 proteins related to virus invasion, the expression level of ubiquitin folding modifier 1 was down-regulated on the 3^rd day, while the expression level of antiviral protein S was continuously up-regulated on 3^rd and 9^th days. Furthermore, the expression level of the proteinase-chymotrypsin inhibitor CI-1A was up-regulated on the 6^th day and down-regulated on the 9^th day. Under GA₃ treatment, 6 different protein points were obtained, among which 3 proteins were related to substance synthesis, and 1 protein was related to substance metabolism. The coffee coenzyme A oxymethyl transferase was down regulated on the 3^th day, but up-regulated on the 6^th and 9^th day. DNA guides RNA polymerase β'- subunit and 1, 4-alpha-glucan dismutase was identified on the 6^th day. Except Coffee coenzyme A oxymethyl transferase, all of other 3 proteins were down regulated on the 9^th day (Table 2).

The grouping of gels cropped from different parts of the different gels, using clear delineation either with white space

Table 2 Identification of differential proteins during seeds germination under exogenous GA₃ treatment

Dispose	Number	Protein Name	Germination time/d
Dispose	Number	Protein Name	0	3	6	9
H₂O	Energy
	1	Thylakoid body cavity 13.8kDa protein	○	↓	↓	↓
	2	Citrate dehydrogenase	○	↓	↓	↓
	7	Ctyochrome C	○	↓	×	×
	8	Phosphoenolpyruvate carboxylase	○	↓	×	×
	9	Protochlorophyllate reductase	○	↓	×	×
	13	Oxygen release enhances protein 3-2	○	↓	×	×
	14	Protein TIC 214	○	↓	↑	↓
	23	Chloroplast 50S ribosomal protein L14	×	×	×	○
	24	isocitrate dehydrogenase	×	×	×	○
	Anabolic metabolism
	3	9-diethylene ether synthase（DES）	○	↓	↓	×
	4	L-alanine-D/L-isomerase	○	↓	↓	×
	10	chalcone synthase	○	↓	↓	↓
	11	30S ribosomal protein S18	○	↓	↓	↓
	16	13- hydroxyl lupine O- transferase	○	↓	×	×
	Virus/Defense
	12	Ubiquitin folding modifier 1	○	↓	×	×
	17	Antiviral protein S	○	↑	↑	↑
	18	Corn subtilis proteinase-chymotrypsin inhibitor CI-1A	×	○	↑	↓
	unknown
	15	Unknown mitochondrial protein ymf11	○	↓	↓	×
GA₃	Anabolic metabolism
	5	DNA guides RNA polymerase β'-subunits	○	↑	↓	↓
	6	Coffee coenzyme A oxymethyl transferase	×	○	↑	↑
	20	DNA guides RNA polymerase β'- subunit	×	×	○	↓
	21	DNA guides RNA polymerase β'- subunit	○	×	↑	↓
	22	Histone H1	○	×	↑	↓
	19	1, 4-alpha-glucan dismutase	×	×	○	↓

The symbol "○" means detected protein, and the symbol "×" means no detected protein. The symbol "↑" means the protein content is increased, and the symbol "↓" means the protein content is reduced.

Seed germination is a very important stage during the processes of growth and development in plants, and is accompanied by a series of physiological and molecular changes, including enzyme activities, endogenous phytohormone contents, gene expression levels, and protein types [24]. This process is affected by plant growth regulators, water, and temperature, among others [24–27]. The plant growth regulators are divided into five classes, one of them being Gibberellin (GA), which is a diterpenoid compound, and the most active artificially synthesized hormone [28, 29]. A previous studies showed that the germination rate of Primula beesiana seeds under 15/5℃ treatment was less than 10%, but exogenous GA₃ significantly increased seed germination [30]. In this study, exogenous GA₃ significantly promoted seed germination, enhanced germination rate, promoted the growth and elongation of the hypocotyl in Phellodendron chinense Schneid (Fig. 1). At the same time, exogenous GA₃ increased the enzyme activity levels of POD and CAT, but reduced the enzyme activity level of SOD in the seeds at 9 day (Fig. 2). The exogenous GA₃ increases the permeability of the seed coat and the membrane through osmosis, accelerates lipid peroxidation of the cell membrane [31], and enhances the antioxidant enzyme activities to reduce the damage caused by reactive oxygen species, explaining the enhanced seed germination in Phellodendron chinense Schneid seeds. Similarly, exogenous GA₃ could promote the transformation and utilization of starch and lipid through inducing or increasing the activities of amylase and other enzymes related to the glyoxylic acid cycle and tricarboxylic acid cycle [32, 33] further promoting seed germination. Furthermore, exogenous GA₃ increased the content of berberine through elevating the expression level of Pc(S)-THBO, but reduced the phellodendrine content in seeds (Fig. 3). These results indicated that exogenous GA₃ promoted seed germination in Phellodendron chinense Schneid.

Seed germination is a complex and ordered reaction process, which is regulated by endogenous phytohormones, and the phytohormone contents are controlled by key synthesis enzymes and genes [34, 35]. The ratio of GA₃ and ABA in seeds is an important factor in seed germination, and their synthesis and accumulation are regulated by genes [31, 36]. Previous studies have shown that exogenous GA₃ promotes seed germination in Fraxinus by increasing the content of endogenous GA₃, reducing the content of ABA, and regulating the expression levels of genes related to the synthesis of GA₃ and ABA [10]. In the present study, the content of GA₃ in seeds under exogenous GA₃ treatment was significantly higher than the control group, but there was no significant change in the content of ABA (Fig. 4). The contents of GA₃ and ABA are related to the expression levels of Pc(S)-GA2ox, Pc(S)-GA3ox, and Pc(S)-ABI5 genes. Our results showed that exogenous GA₃ obviously enhanced the expression levels of Pc(S)-GA2ox and Pc(S)-GA3ox, but significantly reduced the expression level of Pc(S)-ABI5, consistent with the contents of endogenous GA₃ and ABA in seeds under the GA₃ treatment (Fig. 5), as well as with the findings of a previous study [37]. It was further confirmed that exogenous GA₃ promotes seed germination by increasing the levels of endogenous GA₃ and the expression levels of Pc(S)-GA2ox and Pc(S)-GA3ox.

The protein kinases [38, 39], protein phosphatases, and transcription factor [40, 41] classes of protein serve to regulate many physiological processes [42]. Seed germination marks the starting point of plant growth and development, which is regulated by some proteins [43]. These proteins regulate catabolism [44], energy metabolism [45], anabolism, and defense reactions [46]. A previous study showed that rate of seed germination in Arabidopsis midasin1-1 (mdn1-1) was decreased by increasing the expression levels of SEED STORAGE ALBUMIN (SESA)1/3/4/5, EMBRYO SAC DEVELOPMENT ARREST4, and ABI5 compared with wild type (Col-0)[47]. In the present study, 17 proteins were identified in the Phellodendron chinense Schneid seeds during germination under normal conditions. The expression levels of most of these proteins were decreased, especially ctyochrome C, phosphoenolpyruvate carboxylase, 30S ribosomal protein S18, and chalcone synthase, which are related to oxidative phosphorylation, seed bloating, ribosome assembly, and flavonoid synthesis, further reducing the germination rate of Phellodendron chinense Schneid seeds. Under exogenous GA₃ treatment, 4 proteins were identified in Phellodendron chinense Schneid seeds. These proteins regulated the RNA synthesis, nucleosome assembly, and catabolism of lipids and sugars. These results indicated that exogenous GA₃ enhances the expression levels of key proteins during the processes of energy metabolism and RNA metabolism (Fig. 6 and Table 2). It was further confirmed that exogenous GA₃ promotes Phellodendron chinense Schneid seed germination through some key proteins that accelerate energy metabolism and RNA synthesis.

Herein, we demonstrated that exogenous GA₃ promotes seed germination in Phellodendron chinense Schneid by increasing endogenous GA₃ levels, increasing the expression levels of Pc(S)-GA2ox and Pc(S)-GA3ox and proteins related to energy metabolism and RNA synthesis. However, seed germination is a complex and orderly process, with the molecular mechanism underlying the regulation seed germination in Phellodendron chinense Schneid by exogenous GA₃ remaining unclear. Therefore, further study using transcriptomics and yeast two hybrid technologies should be conducted in future to elucidate this mechanism.

ABA

abscisic acid; ABI5:ABSCISIC ACID-INSENSITIVE 5; ACN:acetonitrile; CAT:catalase; GA:Gibberellin;GA₃:gibberellin3; GA2ox:GA2-OXIDASE; GA3ox:GA3-OXIDASE; mdn1-1:midasin1-1; PCR:polymerase chain reaction; POD:peroxidase; qRT-PCR:quantitative reverse transcription PCR; SESA:SEED STORAGE ALBUMIN; SOD:superoxide dismutase; THBO:TETRAHYDROPROTOBERBERINE OXIDASE; ZT:trans-Zetain.

Acknowledgments

We would like to thank Mr. Zhencheng LV (Huizhou universtiy) for his help with technical support.

Authors’ contributions

Wenkai SUN and Xuejing ZHANG performed the experiments, wrote the first draft; Yahan WANG and Huanhuan LI analyzed the data, prepared the figures; Wende YAN, Hanjie HE, Lili CHEN and Gongxiu HE conceived and designed the experiments, reviewed and corrected the paper, approved the final draft. All authors read and approved the manuscript.

Funding

This research was financially supported by the Key Research and Development Project of Hunan Province (No.2018NK2041; 2020NK2018), Key Project of Hunan Education Department (No.18A158; 19B590), Science and Technology Project of Changsha city (No.kq1901144), and Student Innovation Project of Hunan province (2018105338004). These funding bodies had no influence on the design of the study, data collection, analysis, interpretation of data, or manuscript writing.

Availability of data and materials

All data generated or analyzed during this study are available by contacting the corresponding author ([email protected]).

Ethics approval and consent to participate

The authors declare that the study was carried out following scientific ethics and conduct.

Consent for publication

Not applicable.

Conflict of interest

The authors declare that they have no competing interests.

Author details

¹National Engineering Laboratory for Applied Technology of Forestry ＆ Ecology in South China/Hunan Provincial Key Laboratory of Forestry Biotechnology, College of Life Science and Technology, ²College of Forestry, Central South University of Forestry and Technology, Hunan Changsha 41004, China

Zhang ZP, Wang XY, Zhang Z, Yao H, Zhang XM, Zhang Y, Zhang BG: The impact of genetic diversity on the accuracy of DNA barcoding to identify species: A study on the genus Phellodendron. Ecol Evol 2019, 9(18):10723–10733.
Sun Y, Lenon GB, Yang AWH: Phellodendri Cortex: A Phytochemical, Pharmacological, and Pharmacokinetic Review. Evid Based Complement Alternat Med 2019, 2019:7621929.
Sun ML, Xu LJ, Peng YL, Liu T, Zhang YH, Zhou ZQ: Multiscale analysis of the contents of palmatine in the Nature populations of Phellodendron amurense in Northeast China. J Forestry Res 2016, 27(2):265–272.
Hu YM, Su GH, Sze SC, Ye W, Tong Y: Quality assessment of Cortex Phellodendri by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Biomed Chromatogr 2010, 24(4):438–453.
Cao X, Sun L, Li D, You G, Wang M, Ren X: Quality Evaluation of Phellodendri Chinensis Cortex by Fingerprint(-)Chemical Pattern Recognition. Molecules 2018, 23(9).
Ryuk JA, Zheng MS, Lee MY, Seo CS, Li Y, Lee SH, Moon DC, Lee HW, Lee JH, Park JY et al: Discrimination of Phellodendron amurense and P. chinense based on DNA analysis and the simultaneous analysis of alkaloids. Arch Pharm Res 2012, 35(6):1045–1054.
Vercellino RB, Pandolfo CE, Cerrota A, Cantamutto M, Presotto A: The roles of light and pericarp on seed dormancy and germination in feral Raphanus sativus (Brassicaceae). Weed Res 2019, 59(5):396–406.
Murcia G, Fontana A, Pontin M, Baraldi R, Bertazza G, Piccoli PN: ABA and GA3 regulate the synthesis of primary and secondary metabolites related to alleviation from biotic and abiotic stresses in grapevine. Phytochemistry 2017, 135:34–52.
Ravindran P, Kumar PP: Regulation of Seed Germination: The Involvement of Multiple Forces Exerted via Gibberellic Acid Signaling. Mol Plant 2019, 12(1):24–26.
Song Q, Cheng S, Chen Z, Nie G, Xu F, Zhang J, Zhou M, Zhang W, Liao Y, Ye J: Comparative transcriptome analysis revealing the potential mechanism of seed germination stimulated by exogenous gibberellin in Fraxinus hupehensis. BMC Plant Biol 2019, 19(1):199.
Simlat M, Skrzypek E, Warchol M, Maciaszek I, Ptak A: Evaluation on Stevia rebaudiana Bertoni seed germination and seedling development under phytohormones treatment. Sci Hortic-Amsterdam 2019, 257.
Xu SC, Zhou Y, Wang PM, Wang F, Zhang XM, Gu RT: Salinity and temperature significantly influence seed germination, seedling establishment, and seedling growth of eelgrass Zostera marina L. Peerj 2016, 4.
Mildaziene V, Pauzaite G, Malakauskiene A, Zukiene R, Nauciene Z, Filatova I, Azharonok V, Lyushkevich V: Response of perennial woody plants to seed treatment by electromagnetic field and low-temperature plasma. Bioelectromagnetics 2016, 37(8):536–548.
Moon YH, Cha YL, Lee JE, Kim KS, Kwon DE, Kang YK: Investigation of suitable seed sizes, segregation of ripe seeds, and improved germination rate for the commercial production of hemp sprouts (Cannabis sativa L.). J Sci Food Agric 2020, 100(7):2819–2827.
Woith E, Stintzing F, Melzig MF: SOD activity and extremophilicity: a screening of various plant species. Pharmazie 2017, 72(8):490–496.
Kong WW, Liu F, Zhang C, Bao YD, Yu JJ, He Y: Fast detection of peroxidase (POD) activity in tomato leaves which infected with Botrytis cinerea using hyperspectral imaging. Spectrochim Acta A 2014, 118:498–502.
Luna CM, Pastori GM, Driscoll S, Groten K, Bernard S, Foyer CH: Drought controls on H2O2 accumulation, catalase (CAT) activity and CAT gene expression in wheat. Journal of experimental botany 2005, 56(411):417–423.
He HJ, Qin JM, Ma ZT, Sun WK, Yan WD, He GX, Yang MX, Zhang DQ: Highly efficient regeneration and medicinal component determination of Phellodendron chinense Schneid. In Vitro Cell Dev-Pl 2020.
Xu K, He G, Qin J, Cheng X, He H, Zhang D, Peng W: High-efficient extraction of principal medicinal components from fresh Phellodendron bark (cortex phellodendri). Saudi journal of biological sciences 2018, 25(4):811–815.
Zhou C, Luo Y, Lei Z, Wei G: UHPLC-ESI-MS Analysis of Purified Flavonoids Fraction from Stem of Dendrobium denneaum Paxt. and Its Preliminary Study in Inducing Apoptosis of HepG2 Cells. Evid Based Complement Alternat Med 2018, 2018:8936307.
Liang J, Guo XD, Han F: UHPLC-ESI-MS/MS Quantitative Analyses of Multicomponent Hu Gan Tablets. Molecules 2019, 24(23).
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25(4):402–408.
Afroz A, Hashiguchi A, Khan MR, Komatsu S: Analyses of the proteomes of the leaf, hypocotyl, and root of young soybean seedlings. Protein Pept Lett 2010, 17(3):319–331.
He H, Qin J, Cheng X, Xu K, Teng L, Zhang D: Effects of exogenous 6-BA and NAA on growth and contents of medicinal ingredient of Phellodendron chinense seedlings. Saudi journal of biological sciences 2018, 25(6):1189–1195.
Chen YM, Huang JZ, Hou TW, Pan IC: Effects of light intensity and plant growth regulators on callus proliferation and shoot regeneration in the ornamental succulent Haworthia. Bot Stud 2019, 60(1):10.
Salinas-Cornejo J, Madrid-Espinoza J, Ruiz-Lara S: Identification and transcriptional analysis of SNARE vesicle fusion regulators in tomato (Solanum lycopersicum) during plant development and a comparative analysis of the response to salt stress with wild relatives. Journal of plant physiology 2019, 242:153018.
Xia Q, Ponnaiah M, Thanikathansubramanian K, Corbineau F, Bailly C, Nambara E, Meimoun P, El-Maarouf-Bouteau H: Re-localization of hormone effectors is associated with dormancy alleviation by temperature and after-ripening in sunflower seeds. Sci Rep 2019, 9(1):4861.
He J, Xin P, Ma X, Chu J, Wang G: Gibberellin Metabolism in Flowering Plants: An Update and Perspectives. Front Plant Sci 2020, 11:532.
Daviere JM, Achard P: Gibberellin signaling in plants. Development 2013, 140(6):1147–1151.
Yang LE, Peng DL, Li ZM, Huang L, Yang J, Sun H: Cold stratification, temperature, light, GA3, and KNO3 effects on seed germination of Primula beesiana from Yunnan, China. Plant Divers 2020, 42(3):168–173.
Bicalho EM, Pinto-Marijuan M, Morales M, Muller M, Munne-Bosch S, Garcia QS: Control of macaw palm seed germination by the gibberellin/abscisic acid balance. Plant Biol (Stuttg) 2015, 17(5):990–996.
Sun J, Jia H, Wang P, Zhou T, Wu Y, Liu Z: Exogenous gibberellin weakens lipid breakdown by increasing soluble sugars levels in early germination of zanthoxylum seeds. Plant science: an international journal of experimental plant biology 2019, 280:155–163.
Zheng XK, Hou HL, Zhang H, Yue MX, Hu Y, Li LJ: Histone acetylation is involved in GA-mediated 45S rDNA decondensation in maize aleurone layers. Plant cell reports 2018, 37(1):115–123.
Cao Y, Liang L, Cheng B, Dong Y, Wei J, Tian X, Peng Y, Li Z: Pretreatment with NaCl Promotes the Seed Germination of White Clover by Affecting Endogenous Phytohormones, Metabolic Regulation, and Dehydrin-Encoded Genes Expression under Water Stress. Int J Mol Sci 2018, 19(11).
Wang Y, Htwe YM, Li J, Shi P, Zhang D, Zhao Z, Ihase LO: Integrative omics analysis on phytohormones involved in oil palm seed germination. BMC Plant Biol 2019, 19(1):363.
Chen J, Yan B, Tang Y, Xing Y, Li Y, Zhou D, Guo S: Symbiotic and Asymbiotic Germination of Dendrobium officinale (Orchidaceae) Respond Differently to Exogenous Gibberellins. Int J Mol Sci 2020, 21(17).
Linkies A, Muller K, Morris K, Tureckova V, Wenk M, Cadman CS, Corbineau F, Strnad M, Lynn JR, Finch-Savage WE et al: Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana. The Plant cell 2009, 21(12):3803–3822.
Lu K, Zhang YD, Zhao CF, Zhou LH, Zhao QY, Chen T, Wang CL: The Arabidopsis kinase-associated protein phosphatase KAPP, interacting with protein kinases SnRK2.2/2.3/2.6, negatively regulates abscisic acid signaling. Plant molecular biology 2020, 102(1–2):199–212.
Roskoski R, Jr.: Cyclin-dependent protein serine/threonine kinase inhibitors as anticancer drugs. Pharmacol Res 2019, 139:471–488.
Gil RS, Vagnarelli P: Protein phosphatases in chromatin structure and function. Biochim Biophys Acta Mol Cell Res 2019, 1866(1):90–101.
Ruta V, Longo C, Lepri A, De Angelis V, Occhigrossi S, Costantino P, Vittorioso P: The DOF Transcription Factors in Seed and Seedling Development. Plants (Basel) 2020, 9(2).
Rodriguez-Iglesias A, Schmoll M: Protein phosphatases regulate growth, development, cellulases and secondary metabolism in Trichoderma reesei. Sci Rep 2019, 9(1):10995.
He D, Li M, Damaris RN, Bu C, Xue J, Yang P: Quantitative ubiquitylomics approach for characterizing the dynamic change and extensive modulation of ubiquitylation in rice seed germination. The Plant journal: for cell and molecular biology 2020, 101(6):1430–1447.
de la Ballina LR, Munson MJ, Simonsen A: Lipids and Lipid-Binding Proteins in Selective Autophagy. Journal of molecular biology 2020, 432(1):135–159.
Ke R, Xu Q, Li C, Luo L, Huang D: Mechanisms of AMPK in the maintenance of ATP balance during energy metabolism. Cell Biol Int 2018, 42(4):384–392.
Hong JK, Hwang IS, Hwang BK: Functional roles of the pepper leucine-rich repeat protein and its interactions with pathogenesis-related and hypersensitive-induced proteins in plant cell death and immunity. Planta 2017, 246(3):351–364.
Li PC, Ma JJ, Zhou XM, Li GH, Zhao CZ, Xia H, Fan SJ, Wang XJ: Arabidopsis MDN1 Is Involved in the Establishment of a Normal Seed Proteome and Seed Germination. Frontiers in Plant Science 2019, 10.

originalgels.docx
Additional file 1. Proteomics analysis of Phellodendron chinense Schneid seeds under water and GA3 treatment full-length gels.

Download PDF

Version 1

posted

You are reading this latest preprint version

Exogenous Gibberellin Promotes the Germination of Phellodendron Chinense Schneid Seeds by Regulating the Expression of Phytohormone Related Genes and Anabolic Proteins

Status:

Version 1

Abstract

Figures

Background

Methods

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1