Retrograde signaling in plants: A critical review focusing on the GUN pathway and beyond

Abstract

Plastids communicate their developmental and physiological status to the nucleus via retrograde signaling, allowing nuclear gene expression to be adjusted appropriately. Signaling during plastid biogenesis and responses of mature chloroplasts to environmental changes are designated "biogenic" and "operational" controls, respectively. A prominent example of the investigation of biogenic signaling is the screen for gun (genomes uncoupled) mutants. Although the first five gun mutants were identified 30 years ago, the functions of GUN proteins in retrograde signaling remain controversial, and that of GUN1 is hotly disputed. Here, we provide background information and critically discuss recently proposed concepts that address GUN-related signaling and some novel gun mutants. Moreover, considering heme as a candidate in retrograde signaling, we revisit the spatial organization of heme biosynthesis and export from plastids. Although this review focuses on GUN pathways, we also highlight recent progress in the identification and elucidation of chloroplast-derived signals that regulate the acclimation response in green algae and plants. Here, stress-induced accumulation of unfolded/misassembled chloroplast proteins evokes a chloroplast-specific unfolded protein response, which leads to changes in the expression levels of nucleus-encoded chaperones and proteases to restore plastid protein homeostasis. We also address the importance of chloroplast-derived signals for activation of flavonoid biosynthesis leading to production of anthocyanins during stress acclimation through sucrose non-fermenting 1-related protein kinase 1. Finally, a framework for identification and quantification of intercompartmental signaling cascades at the proteomic and metabolomic levels is provided, and we discuss future directions of dissection of organelle-nucleus communication.

Keywords: chloroplast; chloroplast unfolded protein response; genomes uncoupled; gun; retrograde signaling; subcellular metabolomes; subcellular proteomes.

Figure 1

The plastid-localized tetrapyrrole (tetpy) biosynthetic pathway. GUN-related enzymes of the tetpy pathway and the subplastidial localization of the two isoforms of protoporphyrinogen oxidase (PPO) and ferrochelatase (FC) are shown. In the tetpy pathway, glutamate is converted into 5-aminolevulinic acid at the rate-limiting step. Eight 5-aminolevulinic acid molecules are required to form porphyrins. Protoporphyrin IX is the substrate for the two metal-chelating chelatases, Mg chelatase (MgCh) and FC, at the branching point between the pathways leading to chlorophyll and heme synthesis, respectively. The MgCh complex consists of the subunits CHLH/GUN5, CHLI1, CHLI2, and CHLD. GUN4 is a regulator of MgCh. The isoforms PPO1 and FC2 presumably provide the heme used in plastids. Heme is further converted into phytochromobilin by the heme oxygenases HO1/GUN2, HO3, and HO4 and phytochromobilin synthase (HY2/GUN3). PPO2 and FC1/GUN6 are thought to produce the heme required for heme-dependent proteins outside of the plastids; furthermore, the heme pool is postulated to act as a retrograde signal. A heme transporter is assumed to export heme into the cytoplasm. It is of note that, in Arabidopsis, mitochondrial localization of PPO and FC can be excluded (see main text). Therefore, here heme is exclusively produced in chloroplasts.

Figure 2

Hypothetical model for the cpUPR in Chlamydomonas and Arabidopsis chloroplasts, respectively. (A) In Arabidopsis, lincomycin (LIN) treatment and other putative factors repress plastid gene expression and, thereby, inhibit expression of the plastid-encoded CLPP1 subunit, which results in reduced Clp protease activity. When Clp protease activity is compromised by LIN or directly by mutation of its constituents, misfolded proteins cannot be properly degraded and form aggregates. These aggregates then induce a cpUPR, and an unknown retrograde signal mediates upregulation of HEAT STRESS TRANSCRIPTION FACTOR A2 (HSFA2) in the nucleus. This TF, in turn, induces the expression of target genes, including those encoding chaperones HSP21 and CLPB3, to alleviate protein-folding stress in chloroplasts. Modified after Llamas et al. (2017). (B) In Chlamydomonas, external cues (shown in the box) lead to the accumulation of unfolded/misassembled proteins (orange) that can form aggregates. Their accumulation may lead to SCE stress in thylakoid membranes, which is recognized by the proteins VIPP2 and VIPP1 and may trigger retrograde signaling relayed by the cytosolic MARS1 kinase. The signaling pathway leads to transcriptional activation of genes encoding VIPP1/2, chloroplast-targeted molecular chaperones and proteases, and other proteins to attenuate oxidative stress (light green). VIPP1/2 and HSP22 E/F form a platform that allows stromal DEG1C and CLPB3 to degrade and refold unfolded and misassembled proteins, respectively. Dashed arrows indicate the need for further experimental evidence.

Figure 3

Biogenic and operational retrograde signaling pathways that control FB. Following conversion of plastid-derived phenylalanine to p-coumaroyl coenzyme A, chalcone synthase catalyzes formation of naringenin chalcone. In subsequent steps, the aglycones of flavonols and anthocyanins are synthesized. While structural genes involved in precursor formation for all flavonoids (e.g., CHS, CHALCONE ISOMERASE, and FLAVANONE 3-HYDROXYLASE) are grouped as early biosynthetic genes, FLAVONOL SYNTHASE catalyzes formation of flavonols. Gene products encoded by anthocyanin biosynthetic genes (ABG), including DIHYDROFLAVONOL 4-REDUCTASE and LEUCOCYANIDIN DIOXYGENASE, function in the anthocyanin branch of FB (for a more comprehensive overview, we refer to the review by Saito et al., 2013). Among others, early biosynthetic gene expression is controlled by the partially redundant transcription factors (TFs) MYB11, MYB12, and MYB111 in different tissues (Stracke et al., 2007). Transcript accumulation for ABGs in different tissues is under the control of a TF complex consisting of MYB, bHLH, and a WD40 component (MBW complex). The composition and activity of the MBW and the accumulation of anthocyanins are adjusted through concurrent interaction of components of the MBW complex with repressive factors, including MYB-LIKE 2 (MYBL2) and factors involved in phytohormone-dependent sequestration of members of the MBW complex (reviewed in LaFountain and Yuan, 2021). Different “operational signals” are proposed to regulate FB in response to external stimuli when fully developed plants face adverse environmental conditions. In contrast, during establishment of photoautotrophic chloroplasts, “biogenic signals” involving plastid-localized GENOMES UNCOUPLED (GUN) proteins are essential to regulate FB. GUN-dependent activation of FB involves HY5 and MYBL2 functioning downstream of the plastids to regulate anthocyanin accumulation when plastid biogenesis is perturbed. Further information is provided in the text. PS, photosystem; E, energy; e⁻, electron; CBB, Calvin–Bassham–Benson cycle; JA, jasmonate; GA, gibberellic acid; JAZ, JA zinc-finger inflorescence meristem domain.

Figure 4

Typical experimental workflows for identification and quantification of intercompartmental signaling cascades at the proteomic and metabolomic levels. Specific extraction and enrichment procedures are necessary for robust compound detection and quantification by methods that couple chromatography to MS. Quantification of compartment-specific (marker) enzyme activities and fluorescence signals enables validation of subcellular signals. Created with BioRender.