Arabidopsis Responds to Alternaria alternata Volatiles by Triggering Plastid Phosphoglucose Isomerase-Independent Mechanisms

Abstract

Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin-Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms.

Figure 1.

A. alternata VCs promote growth and the accumulation of exceptionally high levels of starch in the mesophyll cells of pgi1-2 plants. Rosette fresh weight (FW; A) and time course of starch content in the leaves (B) are shown for wild-type (WT) and pgi1-2 plants cultured in the absence or continuous presence of adjacent cultures of A. alternata. Values represent means ± se determined from four independent experiments using 12 plants in each experiment. The asterisks in A indicate significant differences between microbial VC-treated plants and controls (nontreated plants) according to Student’s t test (P < 0.05). The plants in A were exposed to VCs for 1 week.

Figure 2.

A. alternata VCs promote the accumulation of starch in mesophyll cells of pgi1-2 leaves. A, B, I, and J, Iodine staining of whole wild-type (WT; A and B) and pgi1-2 (I and J) plants cultured in the absence or continuous presence of VCs emitted by A. alternata for 14 h. C, D, K, and L, Iodine staining of cross sections of leaves of wild-type (C and D) and pgi1-2 (K and L) plants cultured in the absence or continuous presence of VCs emitted by A. alternata for 14 h. E, F, M, and N, Light microscopy of the mesophyll cells of leaves of wild-type (E and F) and pgi1-2 (M and N) plants cultured in the absence or continuous presence of VCs emitted by A. alternata for 14 h. Bars = 10 μm. G, H, O, and P, Electron microscopy of the mesophyll cells of leaves of wild-type (G and H) and pgi1-2 (O and P) plants cultured in the absence or continuous presence of VCs emitted by A. alternata for 14 h. St, Starch. Bars = 2 μm.

Figure 3.

A. alternata VCs enhance photosynthesis in the leaves of pgi1-2 plants. Levels of photosynthetic pigments (A), A_n curves (B), and photosynthetic electron transport rate (ETR) versus C_i (C) are shown in leaves of pgi1-2 plants cultured in the absence or continuous presence of adjacent cultures of A. alternata for 3 d. Treatment with VCs started at 18 d after the sowing growth stage of plants. Values in A represent means ± se determined from four independent experiments using 12 plants in each experiment. Asterisks indicate significant differences between the leaves of VC-treated and control (nontreated) plants according to Student’s t test (P < 0.05). FW, Fresh weight.

Figure 4.

A. alternata VCs increase photosynthate levels in pgi1-2 leaves. Carbohydrate contents (A) and free amino acid contents (B) are shown in leaves of plants grown in the absence or continuous presence of adjacent cultures of A. alternata for 3 d. Leaves were harvested at the end of the light period. Values represent means ± se determined from four independent experiments using 12 plants in each experiment. Asterisks indicate significant differences between the leaves of VC-treated and control (nontreated) plants according to Student’s t test (P < 0.05). FW, Fresh weight.

Figure 5.

Functional categorization of the differentially expressed proteins in leaves of pgi1-2 plants cultured in the presence of VCs emitted by A. alternata. Proteins that were both significantly down- and up-regulated following VC exposure were sorted according to the putative functional category assigned by MapMan software. The number of up- and down-regulated proteins in each categorical group is indicated by gray and black bars, respectively. Proteins discussed here are boxed. Proteins encoded by genes that are differentially expressed in the leaves of VC-treated plants are indicated in gray, and CK-regulated proteins are indicated with asterisks.

Figure 6.

Scheme illustrating the metabolic adjustment that occurs in leaves of pgi1-2 plants in response to A. alternata VC exposure. The VC-promoted up-regulation of PSII reaction center proteins and enzymes involved in the synthesis of sulfolipid and photosynthetic pigments results in enhanced photosynthetic activity. The resulting augmentation of GAP fuels the production of MEP-derived compounds, such as photosynthetic pigments and plastidic CKs, which initiate a cascade of reactions that cause various responses, such as the production of proteins involved in photoprotection, cell wall production and modification, and amino acid biosynthesis (Sánchez-López et al., 2016). Gln is metabolized to Glu, Asp, Asn, Met, and aliphatic glucosinolates. Also, cytosolic Glu is metabolized to Ala through the γ-aminobutyrate (GABA) shunt. According to this model, VC-promoted starch overaccumulation in pgi1-2 leaves is the consequence of the stimulation of starch biosynthetic pathways that bypass pPGI through the transport of cytosolic hexoses (e.g. Glc and/or Glc-6-P) and/or ADP-Glc (ADPG) into the chloroplast. The high levels of 3PGA and NADPH resulting from VC-enhanced photosynthesis activate AGP (Li et al., 2011), which, in turn, facilitates the synthesis of starch. VCs also increase the expression of starch breakdown enzymes to prevent the excessive accumulation of starch and establish a starch substrate cycle. Starch breakdown products can be exported to the cytosol for their subsequent conversion into UDP-Glc, which is necessary for the synthesis of Suc and/or cell wall polysaccharides. Enzymes that are up-regulated by VCs are indicated with thick arrows. Multistep reactions are depicted with dashed arrows.