McMYB4 improves temperature adaptation by regulating phenylpropanoid metabolism and hormone signaling in apple

Temperature changes affect apple development and production. Phenylpropanoid metabolism and hormone signaling play a crucial role in regulating apple growth and development in response to temperature changes. Here, we found that McMYB4 is induced by treatment at 28 °C and 18 °C, and McMYB4 overexpression results in flavonol and lignin accumulation in apple leaves. Yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs) further revealed that McMYB4 targets the promoters of the flavonol biosynthesis genes CHS and FLS and the lignin biosynthesis genes CAD and F5H. McMYB4 expression resulted in higher levels of flavonol and lignin biosynthesis in apple during growth at 28 °C and 18 °C than during growth at 23 °C. At 28 °C and 18 °C, McMYB4 also binds to the AUX/ARF and BRI/BIN promoters to activate gene expression, resulting in acceleration of the auxin and brassinolide signaling pathways. Taken together, our results demonstrate that McMYB4 promotes flavonol biosynthesis and brassinolide signaling, which decreases ROS contents to improve plant resistance and promotes lignin biosynthesis and auxin signaling to regulate plant growth. This study suggests that McMYB4 participates in the abiotic resistance and growth of apple in response to temperature changes by regulating phenylpropanoid metabolism and hormone signaling.


Introduction
As an important environmental factor, temperature often impacts plant growth, development and productivity during the growing season 1 . Therefore, increasing attention has been directed toward the regulation of plants in response to temperature changes 2 . Plants are believed to activate protective mechanisms during their adaptation to temperature changes by balancing resistance and growth. For example, under high-temperature conditions, SLG1 (Slender Guy 1) could encode cytosolic tRNA 2-thiolation protein 2 (RCTU2) by regulating its promoter and coding regions, which led to an increase in thiolated tRNA and enhanced resistance and thus improved rice yields 3 . In Arabidopsis thaliana, the photosystem II (PSII)-associated protein PSB27 downregulated PSII electron transport under low-temperature conditions to affect plant photosynthesis and growth 4 .
Apple (Malus domestica) is frequently exposed to challenges from various environmental factors. Temperature has a serious impact on growth and development processes, which affects apple yields 5 . At a low temperature (15°C), McMYB10 bound to the promoter of flavonoid 3'-hydroxylase (McF3'H) and promoted its expression to enhance anthocyanin accumulation in crabapple leaves and calli, which promoted apple coloration 6 . In apple, MdERF1B-MdCIbHLH1 upregulated the expression of the ethylene biosynthesis genes MdACO1 and MdERF3 and promoted ethylene production, which positively modulated the response of apple to 4°C 7 . Therefore, substance metabolism and hormone signaling in apple plants play a crucial role in their response to temperature changes.
Phenylpropanoid metabolism is an important secondary metabolic process in the plant kingdom 8 . Flavonol, a key component in this metabolic pathway, can increase plant resistance and regulate plant growth. For instance, the flavonol contents in leaves of a hybrid Arabidopsis line are higher than those in leaves of Columbia plants, and this difference is accompanied by greater accumulation of soluble sugars and a higher proline content after a 14-day low-temperature treatment, which increases plant freezing tolerance 9 . Moreover, the flavonol contents in Arabidopsis thaliana were also correlated with a shortened plant stature. The flavonoid 3-O-glucosyltransferase mutant ugt78d2 regulated the flavonol glycoside pattern and reduced polar auxin transport (PAT) in shoots, which resulted in a dwarf-type stature 10 . Lignin, as a complex phenolic polymer, can affect the cell wall and vascular structure and thereby regulate plant growth and resistance 11,12 . For example, expression of the p-coumaroyl ester 3-hydroxylase gene C3'H promoted lignin biosynthesis and changed the cell wall structure, thereby regulating plant growth and dwarfing in rice 13 . In maize roots, the fraction of the lignocellulosic complex in the cell wall was significantly increased, which resulted in lignin deposition and root growth promotion 14 . Furthermore, chilling temperatures (8°C/4°C) also promoted lignified xylem deposition in cell walls by regulating the transcriptional levels of secondary cell wall (SCW) genes in Eucalyptus gunnii × Eucalyptus dalrympleana hybrids. The expression levels of most genes involved in lignin biosynthesis were upregulated and contributed to the cold tolerance of the hybrids 15 . These results indicate that flavonol and lignin play an important role in plant growth and resistance. However, their effects on apple in response to temperature changes need further study.
Phytohormone signaling can also regulate plant growth and resistance in response to temperature changes. Under low-temperature conditions, RNA sequencing (RNA-seq) revealed that the C-repeat binding transcription factor (CBF1) promoted the expression of genes involved in auxin, GA, and cytokinin signaling in peach and affected hormone homeostasis and growth 16 . In apple, MdPIF4 could transactivate the MdYUCCA8a promoter, which promoted indole-3-acetic acid (IAA) accumulation and further improved plant height, thus affecting apical dominance and silique malformation under high-temperature conditions 17 . Moreover, high temperatures also promoted the expression of auxin biosynthesis pathway genes (GmYUCCA3, GmYUCCA5 and GmYUCCA7) to increase IAA accumulation and promoted soybean hypocotyl elongation 18 . However, under freezing conditions, overexpression of BYPASS1-LIKE (B1L) decreased hypocotyl length and fresh weight in Arabidopsis by regulating the BR signaling pathway. Furthermore, the TRANSTHYRETIN-LIKE (TTL) ttl-1 mutant promoted freezing tolerance. Therefore, B1L overexpression promoted plant freezing tolerance by interacting with TTL 19 . BR could also positively regulate the net photosynthetic rate and stomatal conductance under lowtemperature treatment (8°C), which promoted the growth and development of tung trees 20 . These results suggest that phytohormones have a major impact on the regulation of plant growth and resistance in response to temperature changes.
MYB transcription factors act as key regulators and affect plant growth and resistance by regulating target genes related to substance synthesis and metabolism 21 . More recent studies in Arabidopsis have shown that overexpression of AtHY5 and AtMYB12 enhanced flavonol accumulation under low-temperature conditions and promoted plant resistance to freezing conditions 22 . In apple plants, MdMYB308L could interact with MdbHLH33 and undergo MdMIEL1-mediated protein degradation to regulate cold tolerance 23 . In maize, sorghum and rice, MYB31 and MYB42 also promoted the expression of the key genes 4CL2, F5H, and CSE in the lignin biosynthesis pathway, thus altering lignin production and promoting plant growth 24 . The R2R3-MYB transcription factor MYB15 could accelerate lignification and thereby affect plant growth and resistance in energy-related and agricultural crops by regulating MYB-responsive elements in genes involved in secondary wall biosynthesis 25 . Therefore, investigating the involvement of MYB transcription factors in the response to temperature changes by regulating substance synthesis in apple would be interesting.
In this study, we overexpressed McMYB4 in Malus domestica "Golden Delicious" apple tissue culture seedlings. The results showed that flavonol and lignin accumulation in McMYB4-overexpression (OE) lines is correlated with high McMYB4 expression under different temperature conditions. Y1H assays and EMSAs revealed that McMYB4 not only targets the promoters of the flavonol biosynthesis genes CHS and FLS but also binds to the promoters of the lignin biosynthesis genes CAD and F5H to promote more flavonol and lignin biosynthesis at 28°C and 18°C than at 23°C. McMYB4 also promotes IAA and BR signaling pathways by upregulating the expression of AUX/ARF and BRI/BIN. Our findings suggest that McMYB4 improves the temperature adaptation of apple by regulating phenylpropanoid metabolism and hormone signaling.

McMYB4 cloning and temperature response analysis
The novel gene McMYB4, which was identified in the Malus crabapple cultivar "Royalty", contains conserved R2 and R3 DNA-binding domains, and a 33 amino acid deletion was found in the C-terminus of McMYB4 compared with that of M. domestica MdMYB22 (Fig. 1a). The phylogenetic tree showed that the similarity between McMYB4 and MdMYB22 was 95% (Fig. 1b), and a subcellular localization analysis revealed that McMYB4 is located in the nucleus (Fig. 1c).
MdMYB22 was significantly induced in the "Gala" apple cultivar by low temperature exposure, and its expression also correlated strongly with anthocyanin accumulation 21 .
To explore the temperature response of McMYB4 in "Royalty", we cultivated tissue culture seedlings of "Royalty" under 28°C and 18°C conditions with 23°C as the control. qRT-PCR indicated that 28°C treatment increased McMYB4 expression by 2.4-fold in young leaves and 2.1-fold in mature leaves. In addition, 18°C treatment increased McMYB4 expression by 60-fold in young leaves and 21-fold in mature leaves (Fig. 1d). Therefore, McMYB4 expression is responsive to temperature changes in Malus crabapple.

McMYB4 promotes flavonol and lignin biosynthesis in response to temperature changes
To further explore the temperature response of McMYB4 in "Golden Delicious" apple, we transformed the McMYB4 overexpression vector into "Golden Delicious" apple tissue culture seedlings. We selected higherexpressing McMYB4-OE transgenic plants (lines 1, 10, and 12) and "Golden Delicious" apple tissue culture seedlings (used as the WT control) and grew them at 28°C, 18°C, and 23°C (with 23°C as the control) for seven days. The results showed that the WT lines wilted, and McMYB4 overexpression alleviated the severity of wilting (S- Fig. 2). At 18°C, the survival rate of the WT lines was 27%, and that of the McMYB4-OE lines was 67%. The survival rate of the WT and McMYB4-OE lines was~100% at 28°C (Fig. 2a). qRT-PCR analysis indicated that McMYB4 expression in the McMYB4-OE lines was 1.3-fold and 4.1-fold higher at 28°C and 18°C than at 23°C, respectively (Fig. 2b). McMYB4 overexpression promoted flavonol and lignin accumulation. HPLC results showed that the flavonol contents in the McMYB4-OE lines grown at 28°C and 18°C were 493 μg/g and 2715 μg/ g higher than those at 23°C, respectively, which were accompanied by upregulated CHS and FLS expression (Fig. 2c, e, g). The lignin content in the McMYB4-OE lines grown at 28°C and 18°C was increased by 19200 μg/g and 5370 μg/g, respectively, compared with that in the McMYB4-OE lines grown at 23°C, and these increases were accompanied by upregulated CAD and Y1H assays also showed that the AbAi reporter gene was activated in yeast transformants containing the AD-McMYB4 vector and vectors harboring the promoters of the target genes CAD and F5H (Fig. 4b), and EMSAs demonstrated that McMYB4 binds to the biotin-labeled promoters of CAD and F5H (Fig. 4d, f). Therefore, these results show that McMYB4 can also bind to the promoters of CAD and F5H to promote lignin biosynthesis.
McMYB4 transgenic "Golden Delicious" apple responds to temperature changes by regulating IAA and BR signaling To analyze the function of McMYB4 in phytohormone signaling pathways in response to temperature changes, we subsequently evaluated the hormone contents of IAA and BR in the WT and McMYB4-OE lines. The results showed that McMYB4 overexpression promotes IAA and BR accumulation. Compared with that at 23°C (which served as the control), the IAA content was increased by 14.08 μg/L and 10 μg/L in the McMYB4-OE lines grown at 28°C and 18°C, respectively, and this increase was accompanied by upregulated AUX and ARF expression (Fig. 5a, c, e). In addition, the BR content was increased by 14 μg/L and 18 μg/L in the McMYB4-OE lines grown at 28°C and 18°C, respectively, compared with that at 23°C, and this change was accompanied by upregulated BRI and BIN expression (Fig. 5b, d, f). Moreover, Y1H assays and EMSAs indicated that McMYB4 mainly activates the promoters of AUX, ARF, BRI, and BIN and promotes the IAA and BR signaling pathways (Fig. 6)  lines exhibited lower DAB and NBT staining intensities, which reflected lower levels of O 2 and H 2 O 2 accumulation (S- Fig. 3a, b). Therefore, in the McMYB4-OE lines, the 28°C and 18°C treatments increased the O 2 content by 37 μmol/g and 18 μmol/g, respectively (S- Fig. 3c), and increased the H 2 O 2 content by 0.62 μmol/g and 0.29 μmol/g, respectively, compared with the levels found with the control treatment (23°C; S- Fig. 3d). The

Discussion
Temperature changes can regulate apple growth and influence apple production and quality. For example, silencing of the MADS-box gene affected the dormancy and bud break of apple trees in response to chilling temperatures, resulting in an ever-growing or evergreen phenotype and decreased fruit production 26 . In "Fuji" apple, the expression of a B-box transcription factor, MdCOL4, was promoted by high temperatures. MdCOL4 downregulated MdMYB1 expression by interacting with MdHY5 under high-temperature conditions, resulting in decreases in apple color intensity and growth 27 . Therefore, understanding the response mechanisms for adapting to temperature changes is crucial for improving apple yields.

McMYB4 promotes flavonol and lignin accumulation in apple plants adapting to temperature changes
Plants can activate a temperature response by regulating phenylpropanoid metabolism 28 . In tea plants, lowtemperature treatment induced high CsHCT transcription, resulting in the production of chlorogenic acid or acylated flavonol glycosides 29 . In mulberry leaves, low temperatures could also promote the expression of the UFGT gene and lead to flavonoid accumulation 30 . However, the function of McMYB4 in flavonol biosynthesis pathways in response to temperature changes is unclear. In our study, the expression of CHS and FLS, which have been verified as target genes of McMYB4, was increased in the McMYB4-OE transgenic lines at 28°C and 18°C (Figs. 2, 4). In addition, MYB TFs also regulate lignin biosynthesis in plant growth and development processes 31 . In loquat fruit, EjAP2-1 interacted with EjMYB1 and EjMYB2 and transrepressed the promoter of the lignin biosynthesis gene Ej4CL1 to regulate postharvest lignification during low-temperature storage 32 . In maize, ZmMYB31 downregulated the expression of genes involved in monolignol biosynthesis to significantly reduce the lignin content and increase cell wall degradability 33 . In addition, PvMYB4 acts as a master repressor  34 . However, MdUGT88F1-mediated phloridzin biosynthesis could increase the levels of lignin and cell wall polysaccharides in apple trees, resulting in increases in internode length and stem and adventitious root numbers and improved growth 35 . In our study, the lignin content was increased in the McMYB4-OE lines grown at 28°C and 18°C compared with the plants grown at 23°C, and this increase was accompanied by upregulated CAD and F5H expression (Figs. 2, 4). Therefore, McMYB4 coordinates the temperature response in apple plants by promoting flavonol and lignin biosynthesis at 18°C and 28°C.

The response of McMYB4 to temperature changes is related to the IAA and BR signaling pathways in apple plants
Phytohormone signaling can regulate plant resistance and growth in response to temperature changes 36 . In Arabidopsis thaliana, the chromatin-modifying enzyme HDA9 mediated histone deacetylation of a rate-limiting enzyme involved in auxin biosynthesis under high-temperature conditions and promoted auxin accumulation and thermomorphogenesis 37 . Under 28°C conditions, the precursor of bioactive GA12 promoted the degradation of DELLAs and induced PIF4-induced upregulation of IAA19/29 expression and hypocotyl elongation in Arabidopsis 38 . Similarly, under 28°C conditions, COP1 could regulate BZR in the BR signaling pathway and promote hypocotyl elongation and petiole growth in Arabidopsis 39 . Under low-temperature conditions, RNA-sequencing analysis showed that BR treatment upregulated the expression of several key genes involved in chlorophyll biosynthesis, promoting the photosynthetic capacity and growth of wucai 40 . Moreover, Arabidopsis thaliana SCD1 (stomatal cytokinesis defective 1), a temperature-sensitive allele, could regulate auxin transport and auxin-induced gene expression and affect the gravitropic growth of Arabidopsis in response to different temperatures (25°C and 18°C) by regulating PIN protein trafficking 41 . In our study, McMYB4 overexpression increased not only the IAA and BR contents but also the transcript levels of AUX/ARF and BRI/BIN in the IAA and BR signaling pathways (Figs. 5, 6), suggesting that the response of McMYB4 to temperature changes is related to the IAA and BR signaling pathways in apple plants.
McMYB4 might participate in abiotic resistance and growth by promoting phenylpropanoid biosynthesis and phytohormone signaling in response to temperature changes Phenylpropanoids are a large class of secondary metabolites involved in plant resistance and growth in coordination with phytohormone signaling 42 . For example, p35S:F3H transgenes increased flavonol levels, and flavonol accumulation reduced ROS accumulation, promoting antioxidant activity in tomato under high-temperature conditions 43,44 . In addition, cytokinin could also induce flavonol accumulation in the root transition zone, and a high flavonol content decreased the superoxide radical content, promoting root resistance in Arabidopsis 45 . Moreover, in red mango fruit, RNA-seq analysis suggested that the genes involved in the brassinosteroid signaling pathway were upregulated and increased plant resistance to light 46 . In addition, in brassinosteroid signaling, SERK2, as a BR signaling component, could interact with OsBRI1 to promote early BR signaling and enhance grain size and salt tolerance 47 . Therefore, flavonol biosynthesis and BR signaling have important functions in plant resistance, and during this process, ROS act as plant signals to respond to environmental changes 48. In watermelon, the H 2 O 2 content increased and improved adaptation to 4°C treatment 49 . In our study, the flavonol levels in the McMYB4-OE lines grown at 28°C and 18°C were increased by 493 μg/g and 2715 μg/g, respectively, compared with those in the control treatment, and the BR contents were increased by 14 μg/L and 18 μg/L in these lines, respectively. The increases in the contents of these metabolites facilitate scavenging of the ROS induced by temperature changes (S- Fig. 3).
As an important component of the cell wall, lignin can affect plant growth. In tea plants, melatonin treatment modified the expression of enzyme genes in the lignin synthesis pathway, increased the lignin content, and promoted plant growth and development 50 . Similarly, in plant growth and development processes, auxin can also affect cell division and elongation to regulate plant growth. For instance, in Arabidopsis, the auxin transcriptional repressor IAA3 interacted with lightcontrolled PIF transcription factors to regulate hypocotyl growth, and disruption of IAA3 led to an elongated hypocotyl under different light intensity conditions 51 . In our study, McMYB4 also promoted lignin biosynthesis and IAA signaling at 28°C and 18°C. The lignin content in the McMYB4-OE lines grown at 28°C and 18°C was increased by 19200 μg/g and 5370 μg/g, respectively, compared with that found in the plants treated at 23°C, and the IAA content was increased by 14.08 μg/L and 10 μg/L in these lines, respectively. Taken together, the results demonstrated that McMYB4 upregulates flavonol biosynthesis and brassinolide signaling to improve plant resistance during growth at 18°C. Moreover, McMYB4 upregulates lignin biosynthesis and auxin signaling and thereby promotes plant growth at 28°C.
In conclusion, McMYB4 increases flavonol and lignin accumulation by promoting the expression of CHS/FLS and F5H/CAD in apple at 18°C and 28°C. Moreover, McMYB4 promotes AUX/ARF and BRI/BIN expression and is associated with IAA and BR signaling. Therefore, McMYB4 plays a role in plant resistance and growth by regulating phenylpropanoid metabolism and hormone signaling in response to temperature changes in apple (Fig. 7).

Plant materials and growth conditions
Tissue culture seedlings of the Malus crabapple cultivar "Royalty", M. domestica "Golden Delicious" apple (WT), and transgenic "Golden Delicious" apple (lines 1, 10, and 12) were cultivated at the Tissue Culture Center of Beijing University of Agriculture under a 16/8-h photoperiod (1800-2000 lx) and 70% humidity at 23°C for one month.  Table 1.

Ectopic expression of McMYB4 in "Golden Delicious" apple seedlings
We cloned McMYB4 from the Malus crabapple cultivar "Royalty", constructed a pH7FWG2-McMYB4-GFP-overexpression vector containing XbaI and SacI sites and transformed it into Agrobacterium tumefaciens GV3101. After the A. tumefaciens populations grew to saturation (OD 600 = 0.8) in Luria-Bertani media, the culture was centrifuged. The thallus was resuspended in a solution of 10 mM MgCl 2 , 10 mM 2-(N-morpholino) ethanesulfonic acid (MES), and 150 mM acetosyringone and maintained at room temperature for 2 h. The tissue culture seedlings of "Golden Delicious" apple were infected with Agrobacterium containing the corresponding construct using the leaf blade scratch method. Buds were then induced, and positive transgenic buds were transplanted into Murashige and Skoog (MS) media after hygromycin screening and PCR testing. The stable transformation lines (1, 10, and 12) were selected by qRT-PCR tests (S- Fig. 1a, b). Subculture and expanded propagation of lines 1, 10, and 12 were conducted for follow-up assays.

Subcellular localization of McMYB4
We transformed the pH7FWG2-McMYB4-GFP vector into Agrobacterium tumefaciens GV3101. The A. tumefaciens populations were cultivated to saturation (OD 600 = 1.0) in Luria-Bertani media, and the culture was then centrifuged. The thallus was resuspended in a solution of 10 mM MgCl 2 , 10 mM 2-(N-morpholino) ethanesulfonic acid (MES), and 150 mM acetosyringone. Nicotiana benthamiana was selected for four weeks and infected via the back of the leaves with a needle-free syringe. The leaves were cultivated for two days and then subjected to laser confocal microscopy (LEICA SP8) observation.

Transcriptome analysis
We selected tissue culture seedlings of McMYB4-OE transgenic "Golden Delicious" apple (line 1) and "Golden Delicious" apple (WT) for transcriptome sequencing analysis and utilized three sampling times for each plant. The samples were ground immediately in liquid nitrogen, and total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Determination of RNA quantity and purity, the addition of adapters, size selection, and RNA-seq were performed by Novogene (Beijing, China). An RNA-sequencing library was prepared and sequenced using the Illumina HiSeq TM platform. Quality control (QC) was performed by analyzing the error rate distribution along reads and the classification of raw reads. DEGseq 1.12.0 was used to analyze the DEGs in each sample based on the criteria |log2(Fold change)|>1 and q < 0.005. Based on the DEG analysis, a KEGG analysis was conducted using KOBAS v2.0 52 .

Measurement of the flavonol, lignin and hormone contents
HPLC was used to measure the flavonol content. Approximately 0.8-1.0 g (fresh weight) of each sample was placed in a test tube containing 10 mL of extract solution (methanol:water:formic acid:trifluoroacetic acid = 70:27:2:1). The samples were then shaken every 6 h during incubation at 4°C in the dark for 72 h, and the samples were filtered and analyzed by HPLC 6 . The lignin content was measured as described previously and is expressed as OD 280 μg·g −1 (DW) 34 . The contents of endogenous IAA/BR were detected by enzyme-linked immunoassay. The data are presented as the means ± SDs from three repeated experiments.

Microscopic examination
After staining with DPBA (25 mg of DPBA dissolved in 10 mL of methyl alcohol) for 30 min, the leaves of tissue culture seedlings of WT and transgenic "Golden Delicious" apple were subjected to microscopy, and the samples were transferred to an objective table for color observations under UV light (Eclipse 80i fluorescence microscope). Cross-sections of the petioles of tissue culture seedlings of WT and transgenic "Golden Delicious" apple were observed under UV light, and images were collected and analyzed using digital imaging software.

Y1H assays
The McMYB4 CDS was cloned into a pJG4-5 vector (Clontech) with the galactokinase 1 (GAL1) promoter, which served as an effector construct. The flavonol biosynthesis gene (CHS/FLS) promoter sequences were cloned into a pLacZi vector with the LacZ reporter gene. The vectors were transformed into competent cells of the yeast strain EGY48, which yielded the following yeast strains: pJG4-5-McMYB4/pLacZi-promoter of flavonol regulation genes and pJG4-5/pLacZi-promoter of flavonol regulation genes. The cells were selected on SD-Trp/-Ura media, and positive colonies were spotted onto glucose plates (2%) containing X-gal at 28°C for two days to confirm the development of a blue color.

EMSAs
The McMYB4 CDS was cloned into a pMAL-C2X expression vector, and the resulting vector was subsequently transformed into Escherichia coli Rosetta (DE3) competent cells. The MBP tag was also cloned in the pMAL-C2X vector to purify the recombinant protein.
Isopropyl β-D-1-thiogalactopyranoside (IPTG; 0.3 mM) was used to induce McMYB4 expression at 170 rpm for 6 h at 28°C. The recombinant protein was purified using a One-Step MBP-Tagged Protein Mini-prep Pack (BioLab Co., Ltd., Beijing, China). EMSAs were conducted using the Light Shift ® Chemiluminescent EMSA Kit (Thermo Fisher Scientific) according to the manufacturer's protocol with 10 μg of purified McMYB4 protein and CHS/FLS/ CAD/F5H and AUX/ARF/BRI/BIN probes with biotinlabeled oligonucleotides 53 .

ROS and enzyme activity assays
Leaf tissues (0.1 g) were ground to powder in liquid nitrogen and then added to an extraction solution. The

Data analysis
Statistical analyses and figure preparation were performed with OriginPro 8 (Origin Lab Corporation, USA) and Photoshop. The error bars for each symbol indicate the means ± SDs of three biological replicate reactions. The statistical analyses in the study were performed using Student's t test, where * 0.01 < P < 0.05 and **P < 0.01.