The combined formulation of brassinolide and pyraclostrobin increases biomass and seed yield by improving photosynthetic capacity in Arabidopsis thaliana

Abstract

In the context of global food crisis, applying the phytohormone-brassinosteroids (BRs) in combination with the fungicide-pyraclostrobin (Pyr) was beneficial for plant quality and productivity in several field trials. However, in addition to the benefits of disease control due to the innate fungicidal activity of Pyr, it remains to be understood whether the coapplication of BL+ Pyr exerts additional growth-promoting effects. For this purpose, the effects of BL treatment, Pyr treatment, and BL+ Pyr treatment in Arabidopsis thaliana were compared. The results showed that the yield increased at a rate of 25.6% in the BL+Pyr group and 9.7% in the BL group, but no significant change was observed in the Pyr group. Furthermore, the BL+Pyr treatment increased the fresh weight of both the leaves and the inflorescences. In contrast, the Pyr and BL treatments only increased the fresh weight of leaves and inflorescences, respectively. Additionally, the BL + Pyr treatment increased the P_n, G_s, T_r, V_{c, max}, J_max, V_TPU, ETR, F_v'/F_m', ΦPSII, Rd, AYE and Rubisco enzyme activity by 26%, 38%, 40%, 16%, 19%, 15%, 9%, 10%, 17%, 179%, 18% and 32%, respectively. While, these paraments did not change significantly by the BL or Pyr treatments. Treatment with BL + Pyr and Pyr, rather than BL, improved the chlorophyll a and chlorophyll b contents by upregulating genes related to chlorophyll biosynthesis and downregulating genes related to chlorophyll degradation. Additionally, according to transcriptomic and metabolomic analysis, the BL+ Pyr treatment outperformed the individual BL or Pyr treatments in activating the transcription of genes involved in photosynthesis and increasing sugar accumulation. Our results first validated that the combined usage of BL and Pyr exerted striking synergistic effects on enhancing plant biomass and yield by increasing photosynthetic efficiency. These results might provide new understanding for the agricultural effects by the co-application of BL and Pyr, and it might stimulate the efforts to develop new environment-friendly replacement for Pyr to minimize the ecotoxicology of Pyr.

Keywords: Arabidopsis thaliana; brassinosteroids; metabolomics; photosynthesis; pyraclostrobin; transcriptomics; yield.

Figure 1

BL+Pyr increased the major axis and the fresh weight of rosette leaves during the vegetative growth period. Photos were taken on the 7^th day after the first-round application (A, 27-day-old seedlings) and 11^th day after the first-round application (B–D, 31-day-old seedlings). CK, untreated seedlings; Pyr, seedlings treated with 3 μM pyraclostrobin; BL+Pyr, seedlings treated with 1 μM BL and 3 μM pyraclostrobin; BL, seedlings treated with 1 μM BL.

Figure 2

BL+Pyr increased the major axis and the fresh weight of rosette leaves and inflorescence during the reproductive growth period. Photos were taken on the 4^th day after the second-round application (A, B, 39-day-old seedlings), 16^th day after the second-round application (C, D, 51-day-old seedlings), and 14^th day after the third-round application (E, 69-day-old seedlings). CK, untreated seedlings; Pyr, seedlings treated with 3 μM pyraclostrobin; BL+Pyr, seedlings treated with 1 μM BL and 3 μM pyraclostrobin; BL, seedlings treated with 1 μM BL.

Figure 3

BL+Pyr showed a synergistic effect on increasing the photosynthetic efficiency by enhancing the gas exchange process. (A) Net photosynthetic rate (P_n); (B) Transpiration rate (T_r); (C) Stomatal conductance (G_s); (D) Stomatal restriction value (L_s); (E) Apparent mesophyll conductance (AMC); (F) Intercellular CO₂ concentrations (C_i). Data are presented as the mean ± SD of three independent replicate experiments. Different letters indicate significant differences (p< 0.05) according to ANOVA followed by Tukey’s test. 31-day-old seedlings (the 11^th day after the first-round application) were used. CK, untreated seedlings; Pyr, seedlings treated with 3 μM pyraclostrobin; BL+Pyr, seedlings treated with 1 μM BL and 3 μM pyraclostrobin; BL, seedlings treated with 1 μM BL.

Figure 4

BL+Pyr showed a synergistic effect on increasing CO₂ assimilation efficiency. (A) Net photosynthetic rate (P_n) at different CO₂ concentration (400, 300, 200, 100, 400, 400, 600, 800, 1000, 1200, 1500, 1800 µmol mol⁻¹) under saturated light intensity (600 µmol m⁻² s⁻¹); (B) Rubisco enzyme activity (U/g FW); (C) Maximum in-vivo Rubisco carboxylation rates (V_c,max); (D) the maximum rate of electron transport driving regeneration of RuBP (J_max); (E) Maximum rate of photosynthetic product triose-phosphate utilization (V_TPU. Data are presented as the mean ± SD (n=3, measured at random in 3 separate replicate experiments). Data are presented as the mean ± SD of three independent replicate experiments. Different letters indicate significant differences (p< 0.05) according to ANOVA followed by Tukey’s test. 31-day-old seedlings (the 11^th day after the first-round application) were used. CK, untreated seedlings; Pyr, seedlings treated with 3 μM pyraclostrobin; BL+Pyr, seedlings treated with 1 μM BL and 3 μM pyraclostrobin; BL, seedlings treated with 1 μM BL.

Figure 5

BL+Pyr and Pyr increased chlorophyll contents by regulating the transcript levels of genes in the chlorophyll metabolism pathway. (A) The chlorophyll a and chlorophyll b contents (mg/g FW); (B) Hierarchical cluster analysis of the differentially expressed genes (DEGs) associated with chlorophyll biosynthesis and degradation, and FPKM was Z-scole normalised prior to analysis. Data are presented as the mean ± SD of three independent biological replicates. Different letters indicated significant differences (p< 0.05) according to ANOVA followed by Tukey’s test. The red line represents positive correlation and the blue line represents negative correlation. 31-day-old seedlings (the 11^th day after the first-round application) were used. CK, untreated seedlings; Pyr, seedlings treated with 3 μM pyraclostrobin; BL+Pyr, seedlings treated with 1 μM BL and 3 μM pyraclostrobin; BL, seedlings treated with 1 μM BL.

Figure 6

BL+Pyr activated the transcription of genes related to photosynthesis. (A) Hierarchical cluster analysis of the differentially expressed genes (DEGs) involved in photosynthesis according to the average FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) of 3 biological repeats; (B) The number of photosynthesis-related DEGs among the comparisons. ‘up’ represented up-regulated and ‘down’ represented down-regulated. (C-F) Venn diagrams showing the overlapping and non-overlapping (DEGs) related to photosynthesis among the comparisons. The genes with an FDR (the false discovery rate)<0.05 were assigned as DEG. CK, untreated; Pyr, Treated with 3 μM pyraclostrobin; BL+Pyr, Treated with 1 μM BL in combinations with 3 μM pyraclostrobin; BL, Treated with 1 μM BL.

Figure 7

BL+Pyr treatment activated transcription of genes in photosynthesis and carbon fixation pathway. (A) Photosynthesis pathway tagged with DEGs of BL+Pyr-treated group versus untreated group; (B) Carbon fixation pathway tagged with DEGs of BL+Pyr-treated group versus untreated group. The heat map close to the enzyme showed the expression level of the gene encoding the corresponding enzyme. The different colored boxes on the protein names indicate that the gene encoding the protein is either up- or down-regulated by the BL+Pyr-treated group versus the untreated group. CK, untreated; Pyr, Treated with 3 μM pyraclostrobin; BL+Pyr, Treated with 1 μM BL in combinations with 3 μM pyraclostrobin; BL, Treated with 1 μM BL. SBPase, Sedoheptulose-1,7-bisphosphatase; FBPA, Fructose-1,6-bisphosphate aldolase; FBPase, Fructose-1,6-bisphosphatases; TK, Transketolase; RPE, Ribulose-phosphate 3-epimerase; TPI, Triosephosphate isomerase; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; PGK, Phosphoglycerate kinase; RPI, Ribose 5-phosphate isomerase A; PRK, Phosphoribulokinase.

Figure 8

BL+Pyr showed a synergistic effect on increasing the accumulation of photosynthates. (A) Number of DAMs in the CO₂ fixation pathway and belonging to sugars of the three treatments (BL + Pyr, BL and Pyr) versus the untreated group; (B) Hierarchical cluster analysis of the sugars content of the three treatments (BL + Pyr, BL and Pyr) versus the untreated group; (C) Hierarchical cluster analysis of the content of DAMs in the CO₂ fixation pathway of the three treatments (BL + Pyr, BL and Pyr) versus the untreated group. The Metabolites with variable importance in projection (VIP) ≥ 1, fold change (FC) ≤ 0.8 or ≥1.2, and P-value< 0.05 were classified as DAMs (differential accumulation metabolites). Values shown in (B, C) are Z-scores normalized to concentrations, and ‘*’ represent significant differences versus the untreated group.

Figure 9

Correlation analysis of the transcriptome and metabolome profiles related to photosynthesis. (A) Pearson correlation analysis between photosynthesis-related DAMs and DEGs of the three treatments (BL + Pyr, BL and Pyr) versus the untreated group with correlation coefficient >0.99 and p-value<0.01. (B) Pearson correlation analysis between the unique photosynthesis-related DEGs and unique photosynthesis-related DAMs of the BL+Pyr group with correlation coefficient >0.95 and p-value<0.05. (C) The correlation network plot displaying the highly significant correlation pairs between the unique photosynthesis-related DEGs and unique photosynthesis-related DAMs of the BL+Pyr group with Pearson’s correlation coefficient >0.99 and p-value<0.01. Metabolite and transcriptome data were log2-transformed prior to correlation analysis. “*” represented 0.01< p-value<0.05, “**” represented 0.001< p-value<0.01 and “***” represented p-value ≤ 0.001.

Figure 10

A potential model for the combined application of BL+Pyr in synergistically increasing biomass and yield through improving photosynthetic efficiency.