Mild shading promotes sesquiterpenoid synthesis and accumulation in Atractylodes lancea by regulating photosynthesis and phytohormones

Mild shading facilitates sesquiterpenoid accumulation and growth in Atractylodes lancea rhizome

To determine a concrete shading value for the production of high-quality and high-yielding AR, we examined the major compounds, including the sesquiterpenoids hinesol (Hin), β-eudesmol (Edu), and atractylone (Atl), and the polyacetylene atractylodin (Atd), as well as the biomass of AR at different growth stages (Fig. 1A–C) under various light intensities. The sum of these four volatile oils as the total volatile oil content was subsequently analyzed. The results revealed that the accumulation of volatile oils was significantly different (p < 0.05) across seedling, expansion, and harvesting stages under different light intensities. The common features at the three life stages were that the total volatile oil content was the highest under 80% mild shading and the lowest under 7% low light intensity. At 80% mild shading, the total volatile oil content in seedling, expansion, and harvest stages increased by 58%, 52%, and 35%, respectively, compared to 100% strong light. Thus, excessive light inhibited the accumulation of volatile oils. Compared to 7% low light, the increased accumulation of volatile oils at 80% mild shading reached 144%, 178%, and 94% from seedling to harvesting stages. We found that the weaker the light intensity (20% and 7%), the lower the volatile oil accumulation. In addition, the biomass indicators of fresh and dry weights (Fig. 1G,H) indicated that the response to different light intensities yielded trends that were consistent with volatile oil accumulation. Thus, both quality and yield of AR can be achieved under 80% mild shading.

To further investigate the component that contributes the most to the total volatile oil accumulation due to changes in light intensity, individual compounds were analyzed at different life stages (Fig. 1D–F). The results revealed that each compound was significantly affected by changes in light intensity at different stages (Fig. 1D–F). Statistically, the sum of the difference contribution ratio (DCR) of the three sesquiterpenoids Hin, Edu, and Atl was greater than 90% of the total variation at any stage of growth, with less than 10% of Atd. Of these, Hin and Edu contributed approximately 70% to the total variation (Table S-1). Therefore, the effect of different light intensities on the accumulation of Hin and Edu was higher than that of Atl and Atd. Sesquiterpenoids, especially Hin and Edu, could reliably determine the variations in the total volatile oil content. Compared to 100% strong light, Hin increased by 60%, 52%, and 30% and Edu increased by 40%, 60%, and 45% at 80% mild shading, respectively, from seedling to harvesting stages. Compared to 7% low light, Hin increased by 111%, 185%, and 92%, and Edu increased by 182%, 224%, and 81% at 80% mild shading, respectively, from seedling to harvesting stages. In addition, we also measured the levels of the key enzyme genes of sesquiterpenoid biosynthesis during the expansion stage of AR. The expression levels of HMGR, DXR, and FPPS in the mevalonic acid (MVA) pathway and methylerythritol phosphate (MEP) pathway were measured by qRT-PCR. The results revealed that the expression of these genes under different light intensities significantly differed among the groups (Fig. 1I). The expression of each gene was the highest under 80% mild shading, that is, greater than 100% strong light, and the lowest under 7% low light. The differential expression patterns of these genes under different light intensities is consistent with sesquiterpenoid accumulation. Thus, the expression analysis further confirmed that light intensity affects sesquiterpenoid accumulation.

80% mild shading enhances photosynthetic efficiency in A. lancea, whose trend was affected by changes in light intensity, consistent with sesquiterpenoid accumulation

The intercellular CO₂ concentration (C_i), transpiration rate (T_r), net photosynthetic rate (P_n), and stomatal conductance (G_S) as gas exchange parameters were significantly different at the different light intensities. The C_i, T_r, P_n, and G_s values of 80% mild shading were the highest (Fig. 2), increasing by 30%, 33%, 27%, and 27%, respectively, compared to 100% strong light and increasing by 45%, 369%, 347%, and 81%, respectively, compared to 7% low light. The results showed that between 100% strong light and 80% mild shading, the differences of the four photosynthetic parameters are close to 30%. While between 7% low light and 80% mild shading, the changes of T_r, P_n are far higher than C_i, G_s. As the degree of shade increased, light utilization decreased starting at 80% light intensity. The results showed that mild shading promotes photosynthetic efficiency in A. lancea, and the trend of variation is consistent with sesquiterpenoid accumulation, which is affected by changes in light intensity.

80% mild shading significantly enhances the phytohormones ABA and GA₃ in A. lancea, whose trend was affected by changes in light intensity, consistent with sesquiterpenoid accumulation

Phytohormones play important roles in signal transduction and secondary metabolism in plants. We measured the levels of common phytohormones in root (R) and leaf (L), as shown in Fig. 3. Data analysis found that the levels of JA, salicylic acid (SA), ABA, and gibberellin (GA₃) under 80% mild shading were significantly (p > 0.05) higher than that of 100% and 7% light intensities, regardless of the plant part (Fig. 3). Furthermore, JA, SA, ABA, and GA₃ levels in 80% mild shading were 1.7, 2.9, 3.1, and 2.2-fold higher than those in 100% strong light, and 4.2, 3.2, 22.9, 19.8-fold higher than those in 7% low light in root (Fig. 3A). And JA, SA, ABA, and GA₃ levels in 80% mild shading were 4.2, 3.2, 21.2, and 19.8-fold higher than those in 100% strong light, and 7.8, 2.9, 11.8, 2.2-fold higher than those in 7% low light in leaves (Fig. 3B). The results showed that between 100% strong light and 80% mild shading, the phytohormone change folds of ABA and GA₃ are higher than other phytohormones in leaves. While between 7% low light and mild shading, in root the phytohormone change folds of ABA and GA₃ are higher than other phytohormones. Therefore, under strong light stress, ABA and GA₃ in leaves were more responsive, while under weak light stress, ABA and GA₃ in roots were more responsive. Other phytohormones content also fluctuated with different light intensity changes, but the fluctuation range was relatively small. In addition, all phytohormone levels gradually decreased with weakening light intensity starting at 80% light intensity (Fig. 3). The results indicated that the changes in the phytohormone levels are consistent with those of sesquiterpenoid accumulation.

In addition, we compared the phytohormone response levels in root and leaf. When the phytohormone change fold (HCF) of roots to leaves was equal to 1, the hormone response level was similar in both root and leaf. When the HCF was higher than 1, the hormone response level was higher in root than in leaf, indicating that hormonal stress in roots is more sensitive to changes in light intensity. By contrast, when the HCF was lower than 1, the hormone response level was lower in root than in leaf, indicating that hormonal stress in leaves is more sensitive to changes in light intensity. Table 1 shows that JA was more sensitive to light intensity changes in leaves than in roots, while ABA was higher responseive to light intensity changes in roots than in leaves.

Ci, Tr, and Pn in photosynthesis; GA₃ and ABA in root; and JA in leaf were strongly correlated with the accumulation of each sesquiterpenoid

The relationships among various physiological and biochemical factors and volatile oils were assessed by Pearson correlation coefficients under different light intensities. As shown in Table S-2, most indexes were significantly correlated with the volatile oil content (p < 0.01). The correlation coefficient represents the strength of the correlation between the two indexes. Correlation coefficients greater than 0.85 were used as the screening criteria. As shown in Fig. 4, the values of C_i, T_r and P_n were strongly correlated with the sesquiterpenoids hinesol and β-eudesmol (r > 0.85). From the correlation network, we found that R-ABA, R-GA₃ and L-JA showed the highest correlation with the accumulation of sesquiterpenoids. Based on the results of photosynthetic efficiency test, phytohormone test and Pearson correlation analysis, Tr and Pn in photosynthesis, GA3 and ABA in root, and JA in leaf that are not only significantly responsive to light intensity changes but also significantly related to sesquiterpenes are screened out.

Promoter regions of HMGR, DXRs, and FPPS contain both light and phytohormone cis-acting regulatory elements

HMGR, DXR, and FPPS have been reported as key enzyme genes involved in biosynthesis of sesquiterpenoids. Therefore, we analyzed the cis-acting elements within the upstream promoter regions (2000 bp) of the three genes which had been selected for qRT-PCR analysis. The relevant sequences are shown in Fig. S-1. The results indicated that HMGR, DXR, and FPPS promoter regions included several light and phytohormone regulatory elements (Table 2), confirming that sesquiterpenoid genes may co-regulated by light and phytohormones.

Source: Ecology - nature.com