Investigating the mechanism of chloroplast singlet oxygen signaling in the Arabidopsis thaliana accelerated cell death 2 mutant.

As sessile organisms, plants have evolved complex signaling mechanisms to sense stress and acclimate. This includes the use of reactive oxygen species (ROS) generated during dysfunctional photosynthesis to initiate signaling. One such ROS, singlet oxygen (1O2), can trigger retrograde signaling, chloroplast degradation, and programmed cell death. However, the signaling mechanisms are largely unknown. Several proteins (e.g. PUB4, OXI1, EX1) are proposed to play signaling roles across three Arabidopsis thaliana mutants that conditionally accumulate chloroplast 1O2 (fluorescent in blue light (flu), chlorina 1 (ch1), and plastid ferrochelatase 2 (fc2)). We previously demonstrated that these mutants reveal at least two chloroplast 1O2 signaling pathways (represented by flu and fc2/ch1). Here, we test if the 1O2-accumulating lesion mimic mutant, accelerated cell death 2 (acd2), also utilizes these pathways. The pub4-6 allele delayed lesion formation in acd2 and restored photosynthetic efficiency and biomass. Conversely, an oxi1 mutation had no measurable effect on these phenotypes. acd2 mutants were not sensitive to excess light (EL) stress, yet pub4-6 and oxi1 both conferred EL tolerance within the acd2 background, suggesting that EL-induced 1O2 signaling pathways are independent from spontaneous lesion formation. Thus, 1O2 signaling in acd2 may represent a third (partially overlapping) pathway to control cellular degradation.

Plant U-Box 4 regulates chloroplast stress signaling and programmed cell death via Salicylic acid modulation.

In response to environmental stress, chloroplasts generate reactive oxygen species, including singlet oxygen (1O2), which regulates nuclear gene expression (retrograde signaling), chloroplast turnover, and programmed cell death (PCD). Yet, the central signaling mechanisms and downstream responses remain poorly understood. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant conditionally accumulates 1O2 and involves Plant U-Box 4 (PUB4), a cytoplasmic E3 ubiquitin ligase, in propagating these signals. To gain insights into 1O2 signaling pathways, we compared transcriptomes of fc2 and fc2 pub4 mutants. The accumulation of 1O2 in fc2 plants broadly repressed genes involved in chloroplast function and photosynthesis, while 1O2 induced genes and transcription factors involved in abiotic and biotic stress, the biosynthesis of jasmonic acid (JA), and Salicylic acid (SA). Elevated JA and SA levels were observed in stressed fc2 plants, but were not responsible for PCD. pub4 reversed the majority of 1O2-induced gene expression in fc2 and reduced the JA content, but maintained elevated levels of SA even in the absence of 1O2 stress. Reducing SA levels in fc2 pub4 restored 1O2 signaling and light sensitivity. Together, this work demonstrates that SA plays a protective role during photo-oxidative stress and that PUB4 mediates 1O2 signaling by modulating its levels.

A genetic screen for dominant chloroplast reactive oxygen species signaling mutants reveals life stage-specific singlet oxygen signaling networks.

Introduction: Plants employ intricate molecular mechanisms to respond to abiotic stresses, which often lead to the accumulation of reactive oxygen species (ROS) within organelles such as chloroplasts. Such ROS can produce stress signals that regulate cellular response mechanisms. One ROS, singlet oxygen (1O2), is predominantly produced in the chloroplast during photosynthesis and can trigger chloroplast degradation, programmed cell death (PCD), and retrograde (organelle-to-nucleus) signaling. However, little is known about the molecular mechanisms involved in these signaling pathways or how many different signaling 1O2 pathways may exist. Methods: The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant conditionally accumulates chloroplast 1O2, making fc2 a valuable genetic system for studying chloroplast 1O2-initiated signaling. Here, we have used activation tagging in a new forward genetic screen to identify eight dominant fc2 activation-tagged (fas) mutations that suppress chloroplast 1O2-initiated PCD. Results: While 1O2-triggered PCD is blocked in all fc2 fas mutants in the adult stage, such cellular degradation in the seedling stage is blocked in only two mutants. This differential blocking of PCD suggests that life-stage-specific 1O2-response pathways exist. In addition to PCD, fas mutations generally reduce 1O2-induced retrograde signals. Furthermore, fas mutants have enhanced tolerance to excess light, a natural mechanism to produce chloroplast 1O2. However, general abiotic stress tolerance was only observed in one fc2 fas mutant (fc2 fas2). Together, this suggests that plants can employ general stress tolerance mechanisms to overcome 1O2 production but that this screen was mostly specific to 1O2 signaling. We also observed that salicylic acid (SA) and jasmonate (JA) stress hormone response marker genes were induced in 1O2-stressed fc2 and generally reduced by fas mutations, suggesting that SA and JA signaling is correlated with active 1O2 signaling and PCD. Discussion: Together, this work highlights the complexity of 1O2 signaling by demonstrating that multiple pathways may exist and introduces a suite of new 1O2 signaling mutants to investigate the mechanisms controlling chloroplast-initiated degradation, PCD, and retrograde signaling.