Chloroplasts overproduce reactive oxygen species (ROS) under unfavorable environmental conditions, and these ROS are implicated in both signaling and oxidative damage. There is mounting evidence for their roles in translating environmental fluctuations into distinct physiological responses, but their targets, signaling cascades, and mutualism and antagonism with other stress signaling cascades and within ROS signaling remain poorly understood. Great efforts made in recent years have shed new light on chloroplast ROS-directed plant stress responses, from ROS perception to plant responses, in conditional mutants of Arabidopsis thaliana or under various stress conditions. Some articles have also reported the mechanisms underlying the complexity of ROS signaling pathways, with an emphasis on spatiotemporal regulation. ROS and oxidative modification of affected target proteins appear to induce retrograde signaling pathways to maintain chloroplast protein quality control and signaling at a whole-cell level using stress hormones. This review focuses on these seemingly interconnected chloroplast-to-nucleus retrograde signaling pathways initiated by ROS and ROS-modified target molecules. We also discuss future directions in chloroplast stress research to pave the way for discovering new signaling molecules and identifying intersectional signaling components that interact in multiple chloroplast signaling pathways.

Singlet oxygen (¹O₂) is an excited state of molecular oxygen with an electron spin shift in the molecular orbitals, which is extremely unstable and highly reactive. In plants, ¹O₂ is primarily generated as a byproduct of photosynthesis in the photosystem II reaction center (PSII RC) and the light-harvesting antenna complex (LHC) in the grana core (GC). This occurs upon the absorption of light energy when the excited chlorophyll molecules in the PSII transfer the excess energy to molecular oxygen, thereby generating ¹O₂. As a potent oxidant, ¹O₂ promotes oxidative damage. However, at sub-lethal levels, it initiates chloroplast-to-nucleus retrograde signaling to contribute to plant stress responses, including acclimation and cell death. The thylakoid membranes comprise two spatially separated ¹O₂ sensors: β-carotene localized in the PSII RC in the GC and the nuclear-encoded chloroplast protein EXECUTER1 (EX1) residing in the non-appressed grana margin (GM). Finding EX1 in the GM suggests the existence of an additional source of ¹O₂ in the GM and the presence of two distinct ¹O₂-signaling pathways. In this review, we mainly discuss the genesis and impact of ¹O₂ in plant physiology.

Environmental information perceived by chloroplasts can be translated into retrograde signals that alter the expression of nuclear genes. Singlet oxygen (¹O₂) generated by photosystem II (PSII) can cause photo-oxidative damage of PSII but has also been implicated in retrograde signaling. We previously reported that a nuclear-encoded chloroplast FtsH2 metalloprotease coordinates ¹O₂-triggered retrograde signaling by promoting the degradation of the EXECUTER1 (EX1) protein, a putative ¹O₂ sensor. Here, we show that a ¹O₂-mediated oxidative post-translational modification of EX1 is essential for initiating ¹O₂-derived signaling. Specifically, the Trp643 residue in DUF3506 domain of EX1 is prone to oxidation by ¹O₂. Both the substitution of Trp643 with ¹O₂-insensitive amino acids and the deletion of the DUF3506 domain abolish the EX1-mediated ¹O₂ signaling. We thus provide mechanistic insight into how EX1 senses ¹O₂ via Trp643 located in the DUF3506 domain.

Significance: Singlet oxygen ( ¹ O ₂ )- and EXECUTER1 (EX1)-dependent signaling triggers programmed cell death in seedlings and inhibits growth of mature plants of the fluorescent ( flu ) mutant of Arabidopsis . The EX1 protein has been located in chloroplasts to the grana margins close to where chlorophyll is synthesized and the disassembly of damaged photosystem II (PSII) and reassembly of active PSII take place. With the onset of ¹ O ₂ -mediated signaling there is a rapid decline of EX1 that depends on the ATP-dependent zinc metalloprotease FtsH. Generation of ¹ O ₂ without the decline of EX1 is not sufficient to activate ¹ O ₂ signaling. As FtsH cleaves also the D1 reaction center protein of damaged PSII, EX1-dependent signaling seems not only spatially but also functionally linked to the repair of PSII.

Flowering plants sense various environmental and endogenous signals to trigger the floral transition and start the reproductive growth cycle. CONSTANS (CO) is a master transcription factor in the photoperiod floral pathway that integrates upstream signals and activates the florigen gene FLOWERING LOCUS T (FT). Here, we performed comprehensive structural and biochemical analyses to study the molecular mechanism underlying the regulation of FT by CO in Arabidopsis thaliana. We show that the four previously characterized cis-elements in the FT promoter proximal region, CORE1, CORE2, P1, and P2, are all direct CO binding sites. Structural analysis of CO in complex with NUCLEAR FACTOR-YB/YC (NF–YB/YC) and the CORE2 or CORE1 elements revealed the molecular basis for the specific recognition of the shared TGTG motifs. Biochemical analysis suggested that CO might form a homomultimeric assembly via its N-terminal B-Box domain and simultaneously occupy multiple cis-elements within the FT promoter. We suggest that this multivalent binding gives the CO–NF–Y complex high affinity and specificity for FT promoter binding. Overall, our data provide a detailed molecular model for the regulation of FT by the master transcription factor complex CO–NF–Y during the floral transition.