Specialized Plastids Trigger Tissue-Specific Signaling for Systemic Stress Response in Plants

Abstract

Plastids comprise a complex set of organelles in plants that can undergo distinctive patterns of differentiation and redifferentiation during their lifespan. Plastids localized to the epidermis and vascular parenchyma are distinctive in size, structural features, and functions. These plastids are termed "sensory" plastids, and here we show their proteome to be distinct from chloroplasts, with specialized stress-associated features. The distinctive sensory plastid proteome in Arabidopsis (Arabidopsis thaliana) derives from spatiotemporal regulation of nuclear genes encoding plastid-targeted proteins. Perturbation caused by depletion of the sensory plastid-specific protein MutS HOMOLOG1 conditioned local, programmed changes in gene networks controlling chromatin, stress-related phytohormone, and circadian clock behavior and producing a global, systemic stress response in the plant. We posit that the sensory plastid participates in sensing environmental stress, integrating this sensory function with epigenetic and gene expression circuitry to condition heritable stress memory.

Figure 1.

Sorting of sensory plastids and chloroplasts. A, A chart of the experimental procedure. Sensory plastids were tagged with MSH1::GFP expressed under the control of the MSH1 native promoter. FACS was used to separate GFP-tagged sensory plastids from mesophyll chloroplasts. Mass spectrometry allowed proteome analysis of the individual fractions. B, Plants expressing an MSH1::GFP fusion in mutant msh1 background (DUAL) were used to extract plastids. C and D, Cross sections of floral stems showing GFP-associated sensory plastids confined to vascular parenchyma and epidermal cells (C; scale bar, 50 µm) and a nontransgenic plant for comparison (D; scale bar, 50 µm). E and F, Confocal laser-scanning image of sorted sensory plastids (E; scale bar, 5 µm) and chloroplasts (F; scale bar, 5 µm). Electron microscopy image of sorted sensory plastid (G; scale bar, 2 µm) and chloroplast (H; scale bar, 2 µm). I, Planar area of sensory plastids and chloroplasts. Values are means ± sd (n = 4). J, Plastoglobule count in sensory plastids and chloroplasts. Values are means ± sd (n = 4).

Figure 2.

Features distinguishing sensory plastids and chloroplasts. A, Chlorophyll mean fluorescence intensity (MFI) in sensory plastids and chloroplasts. B, Normalized chlorophyll MFI per planar area (chloroplast MFI as 100%). C, Distribution of proteins identified in sensory plastids and chloroplasts. D, GO enrichment analysis of combined data sets obtained in this study (“sensory + chloroplast”) in comparison to the plastid data set curated in PPDB. E, GO terms (% of counts) in the total proteome of chloroplasts. F, GO terms (% of counts) in the total proteome of sensory plastids. Fuchsia color in E and F denotes the ranking for the category “photosynthesis” in each plastid population.

Figure 3.

Sensory plastids in tobacco. A, Trichome of transgenic tobacco line expressing MSH1::GFP in sensory plastids. Inset is an enlargement of sensory plastids in the trichome stalk taken from an independent experiment in the region indicated by the white arrow. B, Trichome of nontransgenic tobacco. C, Trichome of stable transgenic Arabidopsis line expressing MSH1::GFP in sensory plastids. Inset is a zoom image of sensory plastids from the region indicated by the white arrow. D, Trichome of nontransgenic Arabidopsis. Inset is a zoom image of sensory plastids. E, Epidermal sensory plastids are positive for MSH1::GFP. F, On and off modulation of MSH1 in different tissues of tobacco and Arabidopsis. VP, Vascular parenchyma; M, mesophyll; E, epidermis; T, trichome. All scale bars are 25 µm.

Figure 4.

Cell-specific translatome profiling. A, Representative plants expressing a FLAG::RPL-18 fusion in both the wild type (WT; Col-0 RPL-18) and the msh1 mutant (msh1 RPL-18) backgrounds. B, Principal component analysis plot for translatome (IP) and transcriptome (total) reads. C, Prominent GO categories for biological function in the translatome and the transcriptome (this study) and total transcriptome (T; total plant data set from Shao et al., 2017). D, Network enrichment analysis of KEGG-derived pathways for the sensory plastid-associated translatome output.

Figure 5.

Cell-specific influence on chromatin remodeling, DNA methylation, and calcium signaling gene expression in msh1 T-DNA mutants based on translatome data. Panels show gene expression changes in nucleosome assembly (A), chromatin silencing (B), production of siRNA (C), methylation machinery (D), mRNA splicing (E), and calcium-mediated signaling (F). Gene expression changes are measured by log₂ fold-change (log₂FC).

Figure 6.

Phenotypes mediated in msh1 by sensory plastid perturbation. A, Graph of transcription factor changes. B, Differential accumulation of H₂O₂ in floral stem transversal cuts from msh1 and wild-type (WT) control by nitro-tetrazolium blue chloride (NBT) staining. Arrows indicate areas with differential staining relative to the wild type. C, Accumulation of O₂⁻ in transversal cuts of floral stems from msh1 and wild-type control by 3,3′-diaminobenzidine (DAB) staining. Arrows indicate areas with differential staining relative to the wild type.

Figure 7.

Proposed model for plastid-specific modulation of chromatin remodeling and stress response pathways mediated by MSH1. We postulate that experimental or stress-associated depletion of MSH1 from the thylakoid/nucleoid site induces ROS/redox changes and release of calcium from the plastid, binding calmodulins (CaMs) to activate calmodulin-binding protein kinases (CBKs) and protein kinase C to activate MAPKs via phosphorylation. Chromatin remodeling events may unleash a localized stress response, involving changes in translation of stress-related transcription factors, ABA regulators, and circadian clock components. A subset of methylome changes that accompany this process display transgenerational heritability for the msh1 memory effect. Emboldened gene classes reflect observed gene expression changes in the sensory plastid translatome data set.