Biology subjects

Woodson, J. D.

Publications and source records attributed to Woodson, J. D..

11 recordsLinked to original sources

bioRxiv2025

Cryptochrome 2 is necessary for promoting a blue light-independent chloroplast signal to control cellular degradation

To thrive in dynamic environments, plants can use their photosynthetic plastid (chloroplast) organelles to sense environmental changes and relay this information to the cell. This signaling can be initiated within chloroplasts by the accumulation of reactive oxygen species (ROS) such as singlet oxygen (1O2), which can accumulate under stress and initiate retrograde (chloroplast-to-nucleus) signaling, chloroplast degradation, and programmed cell death (PCD). However, the mechanisms driving these signals, and the relationship between gene expression and cellular degradation, are unknown. To understand this physiology, here we use the Arabidopsis thaliana plastid ferrochelatase 2 (fc2) mutant, which conditionally produces chloroplast 1O2 and initiates signaling. We show that the blue light photoreceptor cryptochrome 2 (CRY2) is necessary to promote 1O2-induced chloroplast degradation and PCD in the fc2 mutant and that cry2 blocks cellular degradation without affecting bulk 1O2 levels, suggesting that signaling, rather than photo-oxidative stress, has been blocked. Furthermore, a global RNA transcript analysis demonstrated that the impact of the cry2 mutation on retrograde signaling is limited and that the effect of 1O2 on nuclear gene expression and cellular degradation can be uncoupled. Under permissive conditions, fc2 cry2 mutants exhibited constitutive expression of photo-oxidative stress genes, suggesting that cry2 may lead to transcriptional priming to photo-oxidative stress. Finally, we demonstrate that this function of CRY2 is independent of blue light-signaling and that CRY2 isoforms lacking canonical blue light signaling capabilities restore 1O2-induced PCD to fc2 cry2. Together, these results reveal a blue light-independent role for CRY2 in chloroplast signaling and stress acclimation pathways.

plant biology↗

bioRxiv2025

Heme acts a chloroplast-to-nucleus retrograde signal to regulate intercellular trafficking via plasmodesmata

Intercellular communication via plasmodesmata (PD) is essential for plant growth, development, and defense, yet its regulation remains poorly understood. Chloroplasts communicate information about the environment and the physiological state of the plant cell to the nucleus. It has been proposed that chloroplast-generated signals also modulate the expression of nuclear genes related to PD to regulate the trafficking of photosynthetic products and metabolites along with other molecules that act non-cell-autonomously. In this study, we set out to identify the chloroplast retrograde signals that regulate intercellular trafficking via PD. Using a combination of Arabidopsis thaliana mutants and gene silencing in Nicotiana benthamiana, we found that the metabolites of the tetrapyrrole biosynthetic pathway, most likely heme, can act to modulate PD-mediated intercellular trafficking. We also identified genes that are potentially regulated by the heme signal to modify plasmodesmata function. Together, these findings strengthen the link between chloroplasts and PD in coordinating intercellular communication for optimal plant development and resource allocation.

plant biology↗

bioRxiv2024

A unified framework for hydromechanical signaling: Do plant signals go with the flow?

Local wounding in plants triggers signals that travel locally within the wounded leaf or systemically through the vasculature to distal leaves. The transmission mechanisms of this ubiquitous class of signals remain poorly understood. Here, we develop a unifying framework based on poroelastic dynamics to study two coupled biophysical processes - propagation of pressure changes and transmission of chemical elicitors via mass flows driven by these pressure changes - as potential mechanisms for the initiation and propagation of wound-induced signals. We show that rapid pressure changes in the xylem can transmit mechanical information across the plant, while their coupling with neighboring non-vascular tissue drives swelling and mass flow that can transport chemical elicitors to distal leaves. We confront our model predictions with signaling dynamics measurements in several species, and show that the poroelastic model captures observed mechanical, biochemical, and electrophysiological signals. This framework provides a valuable foundation for assessing mechanisms of signal transmission and for designing future experiments to elucidate factors involved in signal initiation, propagation, and target elicitation.

plant biology↗

bioRxiv2024

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

BackgroundIn response to environmental stresses, chloroplasts generate reactive oxygen species, including singlet oxygen (1O2), an excited state of oxygen that regulates chloroplast-to-nucleus (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 Plant U-Box 4 (PUB4), a cytoplasmic E3 ubiquitin ligase, is involved in propagating 1O2 signals for chloroplast turnover and cellular degradation. Thus, the fc2 and fc2 pub4 mutants are useful genetic tools to elucidate these signaling pathways. Previous studies have focused on the role of 1O2 in promoting cellular degradation in fc2 mutants, but its impact on retrograde signaling from mature chloroplasts (the major site of 1O2 production) is poorly understood. ResultsTo gain mechanistic insights into 1O2 signaling pathways, we compared transcriptomes of adult wt, fc2, and fc2 pub4 plants. The accumulation of 1O2 in fc2 plants broadly repressed genes involved in chloroplast function and photosynthesis, while inducing genes and transcription factors involved in abiotic and biotic stress, the biosynthesis of jasmonic acid (JA) and salicylic acid (SA), microautophagy, and senescence. Elevated JA and SA levels were observed in 1O2-stressed fc2 plants. pub4 reversed most of this 1O2-induced gene expression and reduced the JA content in fc2 plants. The pub4 mutation also blocked JA-induced senescence pathways in the dark. However, fc2 pub4 plants maintained constitutively elevated levels of SA even in the absence of bulk 1O2 accumulation. ConclusionsTogether, this work demonstrates that in fc2 plants, 1O2 leads to a robust retrograde signal that may protect cells by downregulating photosynthesis and ROS production while simultaneously mounting a stress response involving SA and JA. The induction of microautophagy and senescence pathways indicate that 1O2-induced cellular degradation is a genetic response to this stress, and the bulk of this transcriptional response is modulated by the PUB4 protein. However, the effect of pub4 on hormone synthesis and signaling is complex and indicates that an intricate interplay of SA and JA are involved in promoting stress responses and programmed cell death during photo-oxidative damage.

plant biology↗

bioRxiv2024

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 biology↗

bioRxiv2023

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

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. 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. While fc2 fas mutants all block 1O2-triggered PCD in the adult stage, only two fc2 fas mutants block such cellular degradation at the seedling stage, suggesting 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. 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.

plant biology↗

bioRxiv2022

Multiple pathways mediate chloroplast singlet oxygen stress signaling

Chloroplasts can respond to stress and changes in the environment by producing reactive oxygen species (ROS). Aside from being cytotoxic, ROS also have signaling capabilities. For example, the ROS singlet oxygen (1O2) can initiate nuclear gene expression, chloroplast degradation, and cell death. To unveil the signaling mechanisms involved, researchers have used several 1O2-producing Arabidopsis thaliana mutants as genetic model systems, including plastid ferrochelatase two (fc2), fluorescent in blue light (flu), chlorina 1 (ch1), and accelerated cell death 2 (acd2). Here, we compare these 1O2-producing mutants to elucidate if they utilize one or more signaling pathways to control cell death and nuclear gene expression. Using publicly available transcriptomic data, we demonstrate fc2, flu, and ch1 share a core response to 1O2 accumulation, but maintain unique responses, potentially tailored to respond to their specific stresses. Subsequently, we used a genetic approach to determine if these mutants share 1O2 signaling pathways by testing the ability of genetic suppressors of one 1O2 producing mutant to suppress signaling in a different 1O2 producing mutant. Our genetic analyses revealed at least two different chloroplast 1O2 signaling pathways control cellular degradation: one specific to the flu mutant and one shared by fc2, ch1, and acd2 mutants, but with life-stage-specific (seedling vs. adult) features. Overall, this work reveals chloroplast stress signaling involving 1O2 is complex and may allow cells to finely tune their physiology to environmental inputs.

plant biology↗

bioRxiv2021

Singlet oxygen leads to structural changes to chloroplasts during degradation in the Arabidopsis thaliana plastid ferrochelatase two mutant

During photosynthesis, chloroplasts can produce large amounts of reactive oxygen species (ROS), particularly under stressful conditions. Along with other nutrients, chloroplasts also contain 80% of a leafs nitrogen supply. For these reasons, chloroplasts are prime targets for cellular degradation to protect cells from photo-oxidative damage and to redistribute nutrients to sink tissues. Multiple chloroplast degradation pathways have been described and are induced by photo-oxidative stress and nutrient starvation. However, the mechanisms by which damaged or senescing chloroplasts are identified, transported to the central vacuole, and ultimately degraded are not well characterized. Here, we investigated the subcellular structures involved with degrading chloroplasts induced by the ROS singlet oxygen (1O2) in the Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant. Using a three-dimensional serial-block face electron microscopy analysis, we show up to 35% of degrading chloroplasts in fc2 mutants protrude into the central vacuole. While the location of a chloroplast within a cell had no effect on the likelihood of its degradation, chloroplasts in spongy mesophyll cells were degraded at a higher rate than those in palisade mesophyll cells. To determine if degrading chloroplasts have unique structural characteristics allowing them to be distinguished from healthy chloroplasts, we analyzed fc2 seedlings grown under different levels of photo-oxidative stress. A clear correlation was observed between chloroplast swelling, 1O2-signaling, and the state of degradation. Finally, plastoglobule enzymes involved in chloroplast disassembly were shown to be upregulated while plastoglobules increased their association with the thylakoid grana, implicating an interaction between 1O2-induced chloroplast degradation and senescence pathways.

plant biology↗

bioRxiv2021

Singlet oxygen-dependent chloroplast degradation is independent of macroautophagy in the Arabidopsis ferrochelatase two mutant

BackgroundChloroplasts respond to stress and changes in the environment by producing reactive oxygen species (ROS) that have specific signaling abilities. The ROS singlet oxygen (1O2) is unique in that it can signal to initiate selective degradation of damaged chloroplasts and then cell death. This chloroplast quality control pathway can be monitored in the Arabidopsis mutant plastid ferrochelatase two (fc2) that conditionally accumulates chloroplast 1O2 under diurnal light cycling conditions leading to rapid chloroplast degradation and eventual cell death. The cellular machinery involved in such degradation, however, remains unknown. Recently it has been demonstrated that whole damaged chloroplasts can be transported to the central vacuole via a process requiring autophagosomes and core components of the autophagy machinery. The relationship between this process, referred to as chlorophagy, and the degradation of 1O2-stressed chloroplasts and cells has remained unexplored. ResultsTo further understand 1O2-induced cellular degradation and determine what role autophagy may play, the expression of autophagy-related genes were monitored in 1O2-stressed fc2 seedlings and found to be induced. Although autophagosomes were present in fc2 cells, they did not associate with chloroplasts during 1O2 stress. Mutations blocking the core autophagy machinery (atg5, atg7, and atg10) were unable to suppress 1O2-induced chloroplast degradation or cell death in the fc2 mutant, suggesting autophagosome formation and macroautophagy are dispensable for 1O2-mediated cellular degradation. However, both atg5 and atg7 led to specific defects in chloroplast ultrastructure and photosynthetic efficiencies, suggesting macroautophagy may be involved in protecting chloroplasts from photo-oxidative damage. Finally, genes predicted to be involved in microautophagy were shown to be induced in stressed fc2 seedlings, indicating a possible role for an alternate form of autophagy in the dismantling of 1O2-damaged chloroplasts. ConclusionsOur results support the hypothesis that 1O2-dependent chloroplast degradation is independent from autophagosome formation, canonical macroautophagy, and chlorophagy. Instead, ATG-independent microautophagy may be involved in such degradation. However, canonical macroautophagy may still play a role in protecting chloroplasts from 1O2-induced photo-oxidative stress. Together, this suggests chloroplast function and degradation is a complex process that utilizes multiple autophagy and degradation machineries, possibly depending on the type of stress or damage incurred.

plant biology↗

bioRxiv2020

Cytidine triphosphate synthase two provides nucleotides for chloroplast development and stress responses

O_LIReactive oxygen species (ROS) produced in chloroplasts cause oxidative damage, but also signal to initiate chloroplast quality control pathways, cell death, and gene expression. The mechanisms behind these signals are largely unknown. C_LIO_LIThe Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant produces the ROS singlet oxygen in chloroplasts that activates such signaling pathways. Here we mapped one fc2 suppressor mutation to CYTIDINE TRIPHOSPHATE SYNTHASE TWO (CTPS2), which encodes one of five enzymes in Arabidopsis necessary for de novo cytoplasmic CTP (and dCTP) synthesis. C_LIO_LIThe ctps2 mutation reduces chloroplast transcripts and DNA content without similarly affecting mitochondria. Chloroplast nucleic acid content and singlet oxygen signaling are restored by exogenous feeding of the dCTP precursor deoxycytidine, suggesting ctps2 blocks signaling by limiting nucleotides for chloroplast genome maintenance. C_LIO_LIAn investigation of CTPS orthologs in Brassicaceae showed CTPS2 is a member of an ancient lineage distinct from CTPS3. Complementation studies confirmed this analysis; CTPS3 was unable to compensate for CTPS2 function in providing nucleotides for chloroplast DNA and signaling. C_LIO_LIOur studies link cytoplasmic nucleotide metabolism with chloroplast quality control pathways. Such a connection is achieved by a conserved clade of CTPS enzymes that may have evolved specialized functions in providing nucleotides to specific subcellular compartments. C_LI

plant biology↗

bioRxiv2020

Plastid gene expression is required for singlet oxygen-induced chloroplast degradation and cell death

Chloroplasts constantly experience photo-oxidative stress while performing photosynthesis. This is particularly true under abiotic stresses that lead to the accumulation of reactive oxygen species (ROS). While ROS leads to the oxidation of DNA, proteins, and lipids, it can also act as a signal to induce acclimation through chloroplast degradation, cell death, and nuclear gene expression. Although the mechanisms behind ROS signaling from chloroplasts remain mostly unknown, several genetic systems have been devised in the model plant Arabidopsis to understand their signaling properties. One system uses the plastid ferrochelatase two (fc2) mutant that conditionally accumulates the ROS singlet oxygen (1O2) leading to chloroplast degradation and eventually cell death. Here we have mapped three mutations that suppress chloroplast degradation in the fc2 mutant and demonstrate that they affect two independent loci (PPR30 and mTERF9) encoding chloroplast proteins predicted to be involved in post-transcriptional gene expression. Mutations in either gene were shown to lead to broadly reduced chloroplast gene expression, impaired chloroplast development, and reduced chloroplast stress signaling. In these mutants, however, 1O2 levels were uncoupled to chloroplast degradation suggesting that PPR30 and mTERF9 are involved in ROS signaling pathways. In the wild type background, ppr30 and mTERF9 mutants were also observed to be less susceptible to cell death induced by excess light stress. Together these results suggest that plastid gene expression (or the expression of specific plastid genes) is a necessary prerequisite for chloroplasts to activate 1O2 signaling pathways to induce chloroplast degradation and/or cell death. Significance summaryReactive oxygen species accumulate in the chloroplast (photosynthetic plastids) and signal for stress acclimation by inducing chloroplast degradation, cell death, and changes in nuclear gene expression. We have identified two chloroplast-localized proteins involved in gene regulation that are required to transmit these signals, suggesting that proper plastid gene expression and chloroplast development is necessary to activate chloroplast controlled cellular degradation and nuclear gene expression pathways.

plant biology↗