Proteostatic Regulation of MEP and Shikimate Pathways by Redox-Activated Photosynthesis Signaling in Plants Exposed to Small Fungal Volatiles

Abstract

Microorganisms produce volatile compounds (VCs) with molecular masses of less than 300 Da that promote plant growth and photosynthesis. Recently, we have shown that small VCs of less than 45 Da other than CO₂ are major determinants of plant responses to fungal volatile emissions. However, the regulatory mechanisms involved in the plants' responses to small microbial VCs remain unclear. In Arabidopsis thaliana plants exposed to small fungal VCs, growth promotion is accompanied by reduction of the thiol redox of Calvin-Benson cycle (CBC) enzymes and changes in the levels of shikimate and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway-related compounds. We hypothesized that plants' responses to small microbial VCs involve post-translational modulation of enzymes of the MEP and shikimate pathways via mechanisms involving redox-activated photosynthesis signaling. To test this hypothesis, we compared the responses of wild-type (WT) plants and a cfbp1 mutant defective in a redox-regulated isoform of the CBC enzyme fructose-1,6-bisphosphatase to small VCs emitted by the fungal phytopathogen Alternaria alternata. Fungal VC-promoted growth and photosynthesis, as well as metabolic and proteomic changes, were substantially weaker in cfbp1 plants than in WT plants. In WT plants, but not in cfbp1 plants, small fungal VCs reduced the levels of both transcripts and proteins of the stromal Clp protease system and enhanced those of plastidial chaperonins and co-chaperonins. Consistently, small fungal VCs promoted the accumulation of putative Clp protease clients including MEP and shikimate pathway enzymes. clpr1-2 and clpc1 mutants with disrupted plastidial protein homeostasis responded weakly to small fungal VCs, strongly indicating that plant responses to microbial volatile emissions require a finely regulated plastidial protein quality control system. Our findings provide strong evidence that plant responses to fungal VCs involve chloroplast-to-nucleus retrograde signaling of redox-activated photosynthesis leading to proteostatic regulation of the MEP and shikimate pathways.

Keywords: : Clp protease system; MEP pathway; PQC system; chloroplast-to-nucleus retrograde signaling; plant–microbe interaction; proteostatic regulation; redox regulation.

FIGURE 1

Fungal VCs do not promote growth of cfbp1 plants. (A) External phenotypes and (B) rosette FW of WT, cfbp1, and cfbp2 plants cultured in the absence or continuous presence of small volatile compounds (VCs) released by adjacent Alternaria alternata cultures for 1 week. Values in “B” are means ± SE for three biological replicates (each comprising a pool of 12 plants) from four independent experiments. Asterisks indicate significant differences relative to plants not cultured with small VCs released by adjacent fungal cultures based on Student’s t-test (P < 0.05).

FIGURE 2

Fungal VCs do not increase photosynthetic capacities of exposed cfbp1 plants. Curve of net CO₂ assimilation rate (A_n) vs. intercellular CO₂ concentration (C_i) in leaves of WT and cfbp1 plants cultured in the absence or continuous presence of small VCs released by adjacent A. alternata cultures for 3 days. Treatment started 28 days after sowing plants. Values are means ±SE for four plants. Asterisks indicate significant differences relative to WT plants not cultured with small VCs released by adjacent fungal cultures according to Student’s t-test (P < 0.05).

FIGURE 3

Fungal VCs do not alter the contents of MEP and shikimate pathway-derived compounds in leaves of cfbp1 plants. The graphics represent the contents of MEP and shikimate pathway-derived compounds in leaves of WT and cfbp1 plants cultured in agar solidified MS medium without sucrose supplementation in the absence or continuous presence of small VCs released by adjacent A. alternata cultures for 3 days. Values are means ±SE for three biological replicates (each comprising a pool of 12 plants) from three independent experiments. ^aSignificant differences, according to Student’s t-test (P < 0.05), between WT and cfbp1 plants cultured without fungal VC treatment. ^bSignificant differences, according to Student’s t-test (P < 0.05), between VC-treated and non-treated WT plants.

FIGURE 4

Fungal VCs alter the expression of enzymes of the MEP and shikimate pathways and of proteins involved in the plastidial proteostasis in WT plants. The graphic represents the functional categorization of differentially expressed proteins (DEPs) in leaves of WT plants cultured in the presence of small VCs emitted by adjacent A. alternata cultures for 3 days. The proteins that were significantly down- and up-regulated following VC exposure were arranged according to the putative functional category assigned by MapMan software. The numbers of up- and down-regulated proteins in each categorical group are indicated by gray and black bars, respectively. Proteins discussed here are boxed, and putative Clp clients are indicated with asterisks. Data obtained from Supplementary Table 3.

FIGURE 5

Fungal VCs alter the expression of genes encoding proteins of the PQC system in WT plants. Relative abundance of transcripts encoding PQC system proteins differentially expressed following exposure to small fungal VCs in WT leaves. Fold change values are differences in levels of transcripts (measured by quantitative RT-PCR) in leaves of WT plants cultured in the presence of small fungal VCs for 3 days relative to those in leaves of plants cultured in the absence of VCs. Gray dashed lines indicate the threshold of log2-fold change = 1 (twofold change) used to identify genes significantly regulated by fungal VCs. Values are means ± SE for three biological replicates.

FIGURE 6

Plants with disrupted plastidial protein homeostasis weakly respond to fungal VCs. (A) External phenotypes and (B) rosette FW of WT, clpc1, and clpr1-2 plants cultured in the absence or continuous presence of small fungal VCs for 1 week. Total contents of (C) chlorophylls and (D) carotenoids in leaves of WT, clpc1, and clpr1-2 plants cultured in the absence or continuous presence of adjacent A. alternata cultures for 3 days. Values in “B” and “C” and “D” are means ± SE for three biological replicates (each comprising a pool of 12 plants) from four independent experiments. Letters “a,” “b,” and “c” indicate significant differences, according to Student’s t-test (P < 0.05), between (a) WT plants and mutants cultured without fungal VC treatment, (b) VC-treated and non-treated plants, and (c) VC-treated WT and mutant plants.

FIGURE 7

Suggested model of regulation of the plant response to small fungal VCs involving proteostatic modulation of MEP and shikimate pathways by redox-activated photosynthesis signaling. According to this model, the response of plants to small fungal VCs involves post-translational regulatory mechanisms wherein signaling of VC-promoted redox activation of photosynthesis and subsequent PQC system-mediated proteostatic up-regulation of enzymes of the MEP and shikimate pathways play important roles. Thiol redox activation of photosynthesis-related proteins promoted by microbial VCs increases the production of GAP and E4P, which enter the MEP and shikimate pathways, respectively, to fuel the production of compounds that initiate a cascade of signaling reactions leading to changes in the expression of nuclear encoded plastidial PQC system functions. Reduced protease (e.g., FTSH and Clp) activity and enhanced expression of chaperones in the chloroplast increase the stability and activity of enzymes of the MEP, shikimate, and tetrapyrrole biosynthesis pathways, creating a high metabolic flux toward the production of secondary metabolites important for growth and development including hormones, photosynthetic pigments, and ROS scavengers. The high chaperone-to-protease balance also promotes the accumulation of active thioredoxins that redox activates enzymes of the CBC, shikimate, and tetrapyrrole biosynthesis pathways. Enzymatic activities up-regulated by small fungal VCs are highlighted with red letters, while enzymatic activities and pathways down-regulated by fungal VCs are highlighted with blue letters. Multistep enzymatic reactions and signaling cascades are indicated by dashed arrows.