LlU Wei-guo, REN Meng-lu, LlU Ting, DU Yong-li, ZHOU Tao, LlU Xiao-ming, LlU Jiang, Sajad Hussain,YANG Wen-yu

Institute of Ecological Agriculture, Sichuan Agricultural University/Sichuan Engineering Research Center for Crop Strip Intercropping System, Chengdu 611130, P.R.China

1. lntroduction

The maize-soybean relay intercropping system has been expanded in the South China and has contributed greatly to soybean production (Liu et al. 2015). However, in this intercropping system, light is an important limiting factor,and soybean seedlings are shaded by maize during the co-growth stage (Malézieux et al. 2009). Low levels of photosynthetically active radiation (PAR) and low ratios of red light to far-red light (R:FR) are determined as characteristics of shade conditions (Vandenbussche et al. 2005; Franklin et al. 2008; Yang et al. 2014). When exposed to shade condition, the soybean stem morphology characteristics, including main stem length, internode length,and plant height increase, while stem diameter and stem breaking strength decrease (Ruberti et al. 2012; Liu et al.2015). These morphological characteristics leave soybean susceptible to lodging, which seriously influences soybean production. Most of the literature confirm that stem strength is a pivotal factor in improving the lodging resistance of soybean (Luo et al. 2007; Liang and Guo 2008; Zou et al. 2015). Lignin, which is frequently a major structural component of the secondary cell walls in plants, is not only associated with plant growth and development, but also with conferring mechanical strength to the plant body (Malézieux et al. 2009). The lignin content determines the physical strength. Crop varieties with shade tolerance and lodging resistance have higher lignin contents in their stems (Kokubo et al. 1989; Zou et al. 2015). In addition, Zhang et al. (2009)found that as the lignin content decreases, the mechanical strength of the rice stem decreases and is easy to lodge.The genes PAL, 4CL, C4H, C3H, CAD, CCR, COMT,CCOAOMT, and POD are involved in the lignin synthesis process (Humphreys et al. 2003; Raes et al. 2003; Acker et al. 2013). When these genes are inhibited, the plant’s lignin content decreases (Zhong et al. 2000; John et al.2012). Additionally, Ren et al. (2016) found by transcriptome analysis that the genes used in our study had differential expression levels in soybean stems under shade conditions,indicating that these genes are involved in lignin metabolism and closely related to lignin content. Some intermediates are generated in lignin synthesis, including the following:cinnamic, p-coumaric, caffeic, ferulic, and sinapinic acids(Vanholme et al. 2010). Furthermore, many studies on lignin biosynthesis have focused on Arabidopsis, tobacco,and poplar, but little is known about the effects of shade stress on the lignin metabolism in soybean stems, especially in soybean varieties with various shade tolerance levels.The objectives of this study were to investigate the effects of shade on lignin biosynthesis, including the expressions of key enzyme genes and the accumulations of metabolic intermediates in the lignin metabolism of soybean stems.A greater understanding can provide a theoretical basis for enhancing the lignin content to prevent soybean lodging and improve grain yield and quality in intercropping systems.

2. Materials and methods

2.1. Plant materials and growth conditions

Two soybean (Glycine max L.) varieties with different genetic origins were employed in this study: Nandou 12 and Nan 032-4. Both varieties were developed by the Nanchong Academy of Agricultural Sciences, Sichuan Province, China.Nandou 12 was the shade-tolerant variety and planted widely in Southwest China, because its stem had high physical strength and lodging resistance under shade stress.Nan 032-4 was shade-susceptible variety and easy to lodging when intercropped with maize (Liu et al. 2016). The experiment was conducted in 2016 at the No. 2 Laboratory Building of Sichuan Agricultural University, Chengdu, located in the western Sichuan Basin. A total of 6–10 seeds of the soybean were sown in plastic pots (12.5-cm diameter). In every pot, there were three holes, and finally, one seedling was maintained per hole. Each soybean variety was planted in 50 pots. Then, all of the pots were divided into two groups, each group included 25 pots of Nandou 12 and 25 pots of Nan 032-4. Soybeans were germinated and grown in a climate chamber with a light source (diode) emitting blue, red and far-red light (LED-41L2, Percival Scientific Inc., Boone, IA, USA). The photoperiod was 12 h light/12 h dark. In the daytime, the temperature was at (28±0.5)°C with a photosynthetically active radiation (PAR) of 440 μmol m?2s?1. At night, the temperature was (23±0.5)°C. When the first compound leaf of soybean expanded fully (～16 d after sowing), the soybean seedlings of the two varieties were separated into two groups. One group was the control and continued to grow in the above conditions; the other group grew under shade conditions (Table 1). The temperature,humidity, and photoperiod were kept the same in all of the treatments. The second internodes (from the base to the top)of plants were harvested at 0 h, 0.5 h, 1.5 h, 3.5 h, 5.5 h, 8.5 h,10 d, and 15 d after the start of the treatments. The samples were wrapped in foil and immediately frozen in liquid nitrogen for half an hour and stored at ?80°C until RNA extraction.After 10 d of shade treatment, the stems were harvested for the determination of metabolic intermediates. The samples for the lignin assay and morphological measurements were harvested after 15 d of treatments. The PAR in the climate chambers was monitored by a light sensor (LiCor SA190,LiCor Inc., Lincoln, NE, USA) and the ratio of R:FR was measured using a Field Scout Red/Far-Red Meter (Item 3414F, Spectrum, Plainfield, IL, USA).

2.2. Morphological measurements of soybean stems

After 15 d of treatments, the seedling heights and each internode length per plant were determined using a ruler,and each stem diameter per plant was measured using Vernier calipers at the middle position of each internode.Each treatment had four replications, and the mean values of each replicate were calculated.

2.3. Analysis of the lignin contents

The determination of lignin content was performed as described by Reddy et al. (1999). The stem tissues (0.3 g)were homogenized and ground in 6 mL of 99.5% ethanol, andthe crude extract was centrifuged at 5 000×g for 15 min. The solids were collected by centrifugation, and the supernatant was discarded. Then, the solids were washed three times by resuspension in 3 mL of 99.5% ethanol per wash and centrifuged. The final solids were air-dried overnight. The dry solids were placed in a screw-cap tube, and then 5 mL of 2 mol L–1HCl and 0.5 mL of thioglycolic acid were added.The tubes were capped and heated at 100°C for 4 h. After cooling, the contents were centrifuged at 6 000×g for 30 min at 4°C. The solids were washed by resuspension in 5 mL of water followed by centrifugation, then resuspended and incubated in 5 mL of 0.5 mol L–1NaOH at 25°C for 18 h.After this extraction, solids were removed by centrifugation,and the supernatant was transferred to a 10-mL centrifuge tube. Then, 1 mL of concentrated HCl was added to the centrifuge tube, and the acidified solution was held at 4°C for 4 h to aid in lignin thioglycolic acid precipitation. After centrifugation at 5 000×g for 30 min, the precipitated lignin thioglycolic acid was collected. The pellet was washed three times by resuspension and centrifugation in 3 mL of 0.1 mol L–1HCl per wash. The final pellet was dissolved in 2.5 mL of 0.5 mol L–1NaOH. The final solution was centrifuged at 5 000×g for 5 min to remove any insoluble material prior to the 280 nm absorbance measurement of the solution. The absorbance was measured against a NaOH blank at 280 nm.

Table 1 Light conditions in the climate chamber (μmol m?2 s?1)1)

2.4. Real-time quantitative PCR analysis

Total RNA was extracted using RNAqueousTMphenol-free total RNA Reagent (Cat# AM1912, Ambion, USA) following the manufacturer’s instructions, and the RIN number was checked to determine the RNA integrity using an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA,USA). Then, cDNA was generated using a TUREscript 1st-Stand cDNA Synthesis Kit (Aidlab, Beijing, China) according to manufacturer’s instructions. The resulting cDNA was diluted 4× for use as the template in the expression analyses.Based on the transcriptomic data (https://soybase.org/soyseq/) and our transcriptome analysis of soybean stems under shade stress (Ren et al. 2016), 10 key genes that participate in lignin synthesis were chosen for the analysis of gene expression. Primer sequences for real-time quantitative PCR are shown in Table 2. The reference gene was actin11, which served as an internal control to normalize the amount of total RNA presented in each reaction. Expression analyses were conducted using an ABI 7900 HT Sequence Detection System (ABI, Foster City, CA,USA) with a reaction system containing forward and reverse primers (0.5 μL), 2×SYBR Green PCR buffer (5 μL), cDNA(1 μL), and ddH2O to a final 10-μL volume. The real-time quantitative PCR amplification was performed using the following cycling parameters: 95°C for 3 min, 95°C for 10 s,58°C for 30 s, 95°C for 10 s (39 cycles), with the addition of 1°C per cycle from 60 to 95°C. Three technical replicates of the real-time quantitative PCR were performed for each sample on the same plate to ensure the reproducibility of results, and the relative expression levels of genes were calculated using the 2?ΔΔCTmethod.

2.5. Analysis of phenolic acids by high-performance liquid chromatography (HPLC)

The phenolic acids were identified by HPLC (Agilent 1100,Agilent) following the method of Cai et al. (2010) and Chen et al. (2003). To prepare standards, a 10-mg standard sample of each acid was dissolved in 10 mL of HPLC-grade methanol under sonication. The solutions were stepwise diluted and combined to make a series of mixed standard solutions. Quantifications were performed by external standardization with each acid, using the established regression equations (peak area). The chemicals were purchased from Sigma (St. Louis, MO, USA). The solvents were obtained from Fisher Chemicals (Fair Lawn, NJ, USA).Analytical grade chemicals were used, and all solvents usedin sample preparation and HPLC were of HPLC grade.Water was prepared using a Milli-Q Deionization System(Millipore, Bedford, MA, USA).

Table 2 Primers used for real-time quantitative RT-PCR analysis

The phenolic acids were extracted and purified following the method of Chen et al. (2003). The samples were ground into a powder with a mortar and pestle under liquid nitrogen and freeze-dried. Extractions of freeze-dried soybean stems (50 mg) were placed in screw-capped centrifuge tubes (10 mL). Methanol (0.8 mL) and hexane (0.8 mL)were added to the tubes, and samples were extracted by sonication for 40 min in an ultrasonic bath (Branson CPX3800H-C, Branson Ultrasonic Corporation, Danbury,CT, USA). Then, 0.3 mL of water was added, and the samples were sonicated for an additional 40 min. Next,after a brief centrifugation at 5 000×g at room temperature,the hexane and aqueous phases were separated using a microsyringe. The aqueous fractions were used directly for soluble phenolic profiling.

Phenolic acids were assayed by HPLC using a Diamonsil C18reversed phase column (5 μm particle, 250 mm×4.6 mm). The mobile phases consisted of two solvents:phase A was 10 mmol L–1ammonium formate in water(pH 3.7), and phase B was 10 mmol L–1ammonium formate in acetonitrile/water (80/20, v/v). The gradient elution parameters were as follows: 10% B for 2 min, increasing to 35% B from 2 to 52 min, 100% B from 52 to 62 min, and a final elution with 100% B for 5 min. The flow rate was 1.0 mL min–1, the injection volume was 10 μL, the detection wavelength was 280 nm, and the column temperature was 25°C in all of the experiments.

2.6. Statistical analysis

Each treatment was carried out in triplicate in a completely randomized design. The data analysis were performed using an analysis of variance (ANOVA) with SPSS 19.0 for Windows. The significance of treatments was tested at the P＜0.05 level. Standard errors were provided in all figures as appropriate. All the statistical analyses were analyzed using the SPSS software package (SPSS Inc., Chicago, IL,USA) and Microsoft Excel 2007. Sequence data from this article can be found in the NCBI data libraries under gene names, which are shown in Table 2.

3. Results

3.1. Effect of shade on soybean stem growth

The shade treatment promoted the elongation of soybean stems. Compared with the control treatment, both Nandou 12 and Nan 032-4 showed greater plant heights and internode lengths with smaller stem diameters under the shade treatment (Fig. 1). Moreover, the lengths and diameters of the third internodes were significantly different between the two varieties. Under normal light conditions, the plant heights and stem diameters of Nandou 12 were greater than those of Nan 032-4. Under shade conditions, Nandou 12 showed lower plant heights and smaller internode lengths than Nan 032-4, but the stem diameter was thicker. Thus,compared with Nandou 12, Nan 032-4 was more sensitive to shade stress.

3.2. Effect of shade on lignin content

Fig. 1 Stem diameter, internode length, and height per plant of soybean Nandou 12 and Nan 032-4, grown under normal light conditon (NL) and shade conditon (SC). Data are means of three replicates±SE. Values with different letters in the column diagram indicate significant differences at P=0.05 according to Duncan’s test.

Under shade conditions, the stem lignin contents of Nandou 12 and Nan 032-4 decreased significantly by 13.8 and 9.75%,respectively (Fig. 2). However, Nandou 12 had a higher lignin content than Nan 032-4 under both normal light and shade conditions. Thus, light environment and variety had significant effects on the lignin content. In addition, there was a significant light environment-soybean variety interaction on the lignin content of soybean stems (F=6.346＊). Because of higher accumulation of ligin in the stem of Nandou 12, it was shade tolerant and lodging resistant.

Fig. 2 The lignin contents of both Nandou 12 and Nan 032-4 grown under shade condition (SC) and normal light condition(NL). Data are means of three replicates±SE. Values with different letters in the column diagram indicate significant differences at P=0.05. Values followed by the same letter within a row are not significantly different at P=0.05 according to Duncan’s test.

3.3. Effect of shade on the expression levels of key enzyme genes of lignin biosynthesis in soybean stem

Our data demonstrated that the expression levels of key enzyme genes in lignin synthesis changed significantly under shade stress (Fig. 3). This significant change was also found at different time points during the shade treatment. Compared with normal light conditions, in Nandou 12, all 10 genes had down-regulated expression levels at nearly all the different shade time points. However,in Nan 032-4, the expression of C3H, CCR, CCoAOMT,and POD were down-regulated, while the remaining six genes (PAL, 4CL, C4H, CAD, COMT, and LAC) were upregulated at nearly all the different shade time points. Under shade stress, although C3H, CCR, CCoAOMT, and POD were down-regulated expression both in Nandou 12 and Nan 032-4, the decrease degree of shade-tolerant soybean(Nandou 12) was less significant than shade-sensitive one(Nan 032-4). This suggested that the shade treatment inhibited the expression levels of four genes (C3H, CCR,CCoAOMT, and POD) and had a long-lasting effect on the lignin biosynthesis. Shade-tolerant soybean had greater gene expression of C3H, CCR, CCoAOMT, and POD.

Fig. 3 Expression analysis of 10 key enzyme genes in the lignin biosynthesis of Nandou 12 and Nan 032-4 grown under shade condition (SC) and normal light condition (NL). Line charts indicate time courses of the key enzyme gene (PAL, C4H, C3H, 4CL,CAD, CCR, COMT, CCoAMOT, POD, and LAC) expression levels in the soybean stems of Nandou 12 and Nan 032-4 at various time intervals during the shade treatment. Each circle represents the mean±SE of three replicates.

Time-course studies were designed to investigate the gene expression levels in shade-treated and control tissues.The same genes under normal light and shade conditions showed similar changes, based on gene-response curves, in both Nandou 12 and Nan 032-4, except for LAC in Nan 032-4.Compared with normal light conditions, the same genes had differential expression levels at different time points under the shade condition in Nandou 12 and Nan 032-4. However,the differential expression levels were not stable. Nandou 12 showed greater differential expression levels than Nan 032-4 after 0.5 h of shade treatment, while Nan 032-4 expressed higher differential expression levels later. As the treatment time reached 8.5 h, there were no significant differences in the gene expression levels between Nandou 12 and Nan 032-4 under shade condition. The different genes in Nandou 12 also showed similar response curves under both light environments, except for CCoAOMT. The different genes in Nan 032-4, except for CCoAOMT and LAC, also performed similarly in gene-response curves.

Comparing the light environment-variety interactions on gene expression levels after 8.5 h of treatment, we found that nearly all of the genes in Nandou 12 exhibited stronger expression levels than those in Nan 032-4 under normal light condition. However, under shade stress, three genes (PAL,C4H, and COMT) in Nandou 12 showed weaker expression levels than those in Nan 032-4. Additionally, for the variance analysis of genes expression levels, the interaction of light environment, variety, and gene were extremely significant(Table 3). A more detailed analysis showed that the expression levels of three genes (PAL, 4CL, and CAD)varied significantly based on the light environment, and five genes (C3H, COMT, CCoAOMT, CCR, and POD)varied extremely significantly. For variety, all of the gene expression levels were influenced significantly, except for POD. In addition, the expression levels of eight genes(PAL, C4H, C3H, 4CL, COMT, CCR, CAD, and LAC) were extremely influenced significant by the interaction of light environment and soybean variety.

3.4. Effect of shade on metabolic intermediates in the lignin biosynthesis pathway

The metabolic pathway of lignin is complex, and the biosynthetic pathway of lignin in the soybean stem remains unclear. Thus, we analyzed five important intermediates in the metabolic pathway of lignin: cinnamic, p-coumaric,caffeic, sinapic, and ferulic acids. The phenolic acid contents in the stems of both soybean varieties were affected significantly by the shade stress (Fig. 4). Compared with under normal light conditions, shade condition promoted the synthesis of cinnamic and p-coumaric acids in the two soybean varieties. Under normal light condition, the cinnamic in the stem of Nandou 12 was higher than that of Nan 032-4, but there was no difference between the two varieties under shade stress. The content of p-coumaric acid in Nandou 12 was lower than that of Nan 032-4.Shade stress accelerated p-coumaric acid biosynthesis,and there was no difference between varieties. On the contrary, the contents of caffeic, sinapic, and ferulic acids decreased dramatically under shade stress. In the stem of Nandou 12, the contents of caffeic, sinapic, and ferulic acids decreased by 30.0, 20.1, and 20.2%, respectively,and in Nan 032-4 by 10.7, 24.1, and 24.0%, respectively.Compared with Nandou 12, the contents of p-coumaric,sinapic and ferulic acids in Nan 032-4 decreased by 9.8,64.5, and 64.5%, respectively, under shade condition.Both under light and shade condition, the contents of caffeic, sinapic, and ferulic acids in Nandou 12 were always higher than those in Nan 032-4 (except ferulic acids under shade stress).

4. Discussion

4.1. Effect of shading on stem strength and genotype differences for lignin biosynthesis

Light is an abiotic factor that is particularly important for plants. Kurepin et al. (2007) reported that the quality and quantity of irradiance can trigger plant morphological responses. Yan et al. (2010) and Casal (2012) found that shading conditions increase plant heights and reduce the stem diameters. In this study, conspicuous differences in the seedling heights, internode lengths and stem diameters were observed under shade conditions when compared with under normal light conditions (Fig. 1). These results wereconsistent with the previous reports which researched in the intercropping conditions (Nagasuga and Kubota 2008; Yang et al. 2014; Liu et al. 2015). Thus, our research suggested that by decreasing the PAR and the ratio of R:FR in the plant growth chamber using a LED light source, we successfully simulated shade conditions, and the soybean seedlings were stressed by the conditions. Under such conditions,the lignin content in the soybean stem also decreased significantly (Fig. 2). According to the results of Zou et al.(2015), the lignin contents of intercropping soybean stems were significantly lower than those of monocropping soybean. Reis et al. (2013) thought that shaded plants had lower physiological ages, which result in lower levels of lignin, but plants grown in shade tend to have higher lignin contents compared with those experiencing no restriction of light in forages. This may be related to the different species.Independent of stem morphology or lignin content, the two soybean varieties showed different responses under shade conditions: Nandou 12 produced lower plant heights, shorter internode lengths, thicker stem diameters, and higher lignin contents than Nan 032-4 (Figs. 1 and 2). Thus, we inferred that Nandou 12 was relatively less sensitive to shade stress than Nan 032-4.

Table 3 Variance analysis of each key enzyme gene expression levels after 8.5 h of shade treatment1)

Fig. 4 Phenolic acid contents in the soybean stem under shade condition (SC) and normal light condition (NL). Values with different letters in the same column diagram indicate significant differences at P=0.05. Values followed by the same letters within a column diagram are not significantly different at P=0.05 according to Duncan’s test.

4.2. Effect of shading on relative expression of lignin biosynthesis genes

Lignin is a polymer of phenylpropanoid compounds formed through a complex biosynthesis pathway, which is represented by a metabolic network involving genes that have been identified in several plants, such as Arabidopsis,poplar, and tobacco (Jullyana et al. 2010). The genes PAL, 4CL, C4H, C3H, CAD, CCR, COMT, CCOAOMT,and POD have important effects on the lignin synthesis process (Humphreys et al. 2002; Raes et al. 2003;Acker et al. 2013). In our study, the expression levels of four genes (C3H, CCR, CCoAOMT, and POD) were down-regulated under shade stress (Fig. 3). John et al.(2012) found that the lignin level steadily declines with the suppression of the C3H level, which is a limiting enzyme that governs the carbon flux though the pathway to the ultimate monolignols. Abdulrazzak et al. (2006) inserted a mutant into Arabidopsis to inhibit CYP98A3, a C3H(Franke et al. 2002), which resulted in as much as a 40%reduction in total cell wall lignin. This phenomenon was supported by the results of Reddy et al. (2005). Meanwhile,according to Coleman et al. (2008), reductions in C3H,and consequently lignification, result in varying effects on growth properties. The lignin contents of plants having mutant alleles in CCR or CCoAOMT decreases (Acker et al. 2013). The antisense suppression of CCR can lead to decreases in the total lignin content (Chabannes et al.2001; Goujon et al. 2003; Leple et al. 2007). Coincidentally,Arabidopsis knockout mutants of the CCR1-gene show a reduced lignin formation (Derikvand et al. 2008). Zhao et al. (2002) and Zhong et al. (2000) studied transgenic tobacco and found that the inhibition of CCoAOMT activity significantly reduced the lignin content in that species.Similarly, the jute lignin content is reduced through CcCCoAOMT1 gene inhibition as reported by Zhang et al. (2014). For POD, tobacco plants transformed with a chimeric tobacco anionic peroxidase gene have higher lignin contents (Lagrimini 1991). Analogously, Ipekci et al.(1999) studied transgenic poplar and found that a reduced leaf peroxidase activity is associated with a reduced lignin content. In conclusion, there were important effects caused by the four genes (C3H, CCR, CCoAOMT, and POD) on the lignin content of the soybean seedlings. The reduction in the soybean lignin content correlated with the downregulation of these genes in shade-treated soybean. The down-regulation of these genes is key in reducing the lignin content under shade conditions. Nevertheless, Nandou 12 had greater expression levels for the four genes than Nan 032-4. This resulted that Nandou 12 having a higher lignin content than Nan 032-4 and reflected a stronger response to shading. Thus, we inferred that Nandou 12 had a relatively stronger ability to respond to shade stress than Nan 032-4.

4.3. Molecular mechanism of lignin biosynthesis in relation to weak stem under shading

The direct carbon flow from the shikimate pathway to the various branches of the general phenypropanoid metabolism is well known, and the pathway of lignin biosynthesis is one branch of the general phenypropanoid metabolism (Davin et al. 2008). p-Coumaric acid is derived from the hydroxylation of cinnamic acid, and p-coumaric acid may transform into caffeic, ferulic, and sinapinic acids. The five phenolic acids are important intermediates in lignin synthesis (Vanholme et al. 2010).In our study, both cinnamic and p-coumaric acids showed significant increases under shade conditions (Fig. 4).Thus, the pathway of phenypropanoid metabolism might be reinforced. Camera et al. (2004) thought that abiotic stress could promote the secondary metabolism of plants. Cai et al. (2010) thought lignin monomers were synthesized through two routes. The first route being from phenylalanine to cinnamic acid, p-coumaric acid,coumaroyl-CoA, and coumaric aldehyde, which then forms coniferyl and sinapinic alcohols. The second route being from p-coumaric acid sequentially to coumaroyl-CoA,caffeoyl-CoA, or caffeic acid, coniferyl aldehyde, coniferyl alcohol and sinapinic alcohol. In this study, we found that caffeic, sinapic, and ferulic acids were significantly reduced under shade conditions compared with under normal light conditions (Fig. 4). Thus, we hypothesized that the biosynthesis of soybean lignin may involve the second route, and the metabolism may follow the phenylpropanoid metabolic pathway from phenylalanine to cinnamic acid and then to acyl-CoA ester, aldehyde, and alcohol, followed by the synthesis of G-S lignin, because soybean is a dicotyledonous plant, which mainly includes G-S lignin (Whetten et al. 1998). The decreased lignin content and the down-regulation of the C3H gene could support this hypothesis, but further studies are needed.Under poor photosynthetic conditions, when plants are stressed, low concentrations of shikimate pathway intermediates may re-direct the entire phenylpropanoid pathway towards the production of phytoalexins, volatiles,flavonoids, and anthocyanins, as well as the de novo synthesis of proteins (Abdulrazzak et al. 2006; Schoch et al. 2006). Thus, the secondary metabolism is first fortified, and then, the initial enzymes (PAL, C4H, and 4CL)are encoded by multiple genes, which have activities that are frequently presented in excess (Sewalt et al. 1997;Rohde et al. 2004; Li et al. 2015), resulting in the increase of cinnamic and p-coumaric acids. This conclusion was supported by Wang et al. (2010), who studied tea and found marked increases in phenolic acid concentrations in shaded leaves. Then, cinnamic and p-coumaric acids could derive some metabolites, such as flavonoids,catechins, alkaloids, and lignin. We hypothesize that some of the metabolites were increased, other than lignin.For instance, under the shading conditions, the alkaloids of both Adenostyles alliariae and Adenostyles alpina increase, and one sesquiterpene, cacalol-trimer, is also present in a high concentration, as reported by Haegele and Rowell-Rahier (1999). Coincidentally, Wang et al.(2004) found that the camptothecin content of Camptotheca acuminata increased in shaded seedlings. Thus, even if the cinnamic and p-coumaric acids had increased under shade stress, the subsequent acids would not greatly accumulate. In addition, the down-regulation of genes(C3H, CCR, CCoAOMT, and POD) suppressed the activity levels of homologous enzymes, which made the upstream acids (cinnamic acid and p-coumaric acid) accumulate,whereas the downstream acids (caffeic, sinapic and ferulic acids) were reduced. Thus, the inhibition of C3H was stronger than that of the others. Finally, the lignin content decreased significantly in shaded stems. In this study, compared with under normal light conditions, the content of each acid in Nandou 12 was more than that in Nan 032-4 under shade stress, which was attributed to their relatively high expression levels. Additionally,Nandou 12 had a higher final lignin content than Nan 032-4(Fig. 5).

5. Conclusion

Fig. 5 Effect of shade condition on lignin biosynthesis in soybean stem. R:FR, red light:far-red light; PAR, photosynthetically active radiation; C3H, p-coumarate 3-hydroxylase gene; CCR, cinnamoyl-CoA reductase gene; CCoAOMT, caffeoyl-CoA O-methyltransferase gene; POD, peroxidase gene.

Shading affects the normal growth of soybean seedlings.Under shade conditions, the expression levels of C3H, CCR,CCoAOMT, and POD were down-regulated, which resulted in decreases in caffeic, sinapic, and ferulic acids. Finally, the lignin content decreased significantly under shade stress.Thus, the lignin content is closely related to the expression of the above genes and metabolite levels. Nevertheless,Nandou 12 had greater gene expression levels and higher metabolite contents than Nan 032-4, which contributed to lignin biosynthesis, and thus, Nandou 12 showed a relatively greater shade tolerance.

Acknowledgements

The research was supported by the National Natural Science Foundation of China (31671626).

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