Host-Specific and Homologous Pairs of Melampsora larici-populina Effectors Unveil Novel Nicotiana benthamiana Stromule Induction Factors.

The poplar rust fungus Melampsora larici-populina is part of one of the most devastating group of fungi (Pucciniales) and causes important economic losses to the poplar industry. Because M. larici-populina is a heteroecious obligate biotroph, its spread depends on its ability to carry out its reproductive cycle through larch and then poplar parasitism. Genomic approaches have identified more than 1,000 candidate secreted effector proteins (CSEPs) from the predicted secretome of M. larici-populina that are potentially implicated in the infection process. In this study, we selected CSEP pairs (and one triplet) among CSEP gene families that share high sequence homology but display specific gene expression profiles among the two distinct hosts. We determined their subcellular localization by confocal microscopy through expression in the heterologous plant system Nicotiana benthamiana. Five out of nine showed partial or complete chloroplastic localization. We also screened for potential protein interactors from larch and poplar by yeast two-hybrid assays. One pair of CSEPs and the triplet shared common interactors, whereas the members of the two other pairs did not have common targets from either host. Finally, stromule induction quantification revealed that two pairs and the triplet of CSEPs induced stromules when transiently expressed in N. benthamiana. The use of N. benthamiana eds1 and nrg1 knockout lines showed that CSEPs can induce stromules through an eds1-independent mechanism. However, CSEP homologs shared the same impact on stromule induction and contributed to discovering a new stromule induction cascade that can be partially and/or fully independent of eds1. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

CHUP1 restricts chloroplast movement and effector-triggered immunity in epidermal cells.

Chloroplast Unusual Positioning 1 (CHUP1) plays an important role in the chloroplast avoidance and accumulation responses in mesophyll cells. In epidermal cells, prior research showed silencing CHUP1-induced chloroplast stromules and amplified effector-triggered immunity (ETI); however, the underlying mechanisms remain largely unknown. CHUP1 has a dual function in anchoring chloroplasts and recruiting chloroplast-associated actin (cp-actin) filaments for blue light-induced movement. To determine which function is critical for ETI, we developed an approach to quantify chloroplast anchoring and movement in epidermal cells. Our data show that silencing NbCHUP1 in Nicotiana benthamiana plants increased epidermal chloroplast de-anchoring and basal movement but did not fully disrupt blue light-induced chloroplast movement. Silencing NbCHUP1 auto-activated epidermal chloroplast defense (ECD) responses including stromule formation, perinuclear chloroplast clustering, the epidermal chloroplast response (ECR), and the chloroplast reactive oxygen species (ROS), hydrogen peroxide (H2O2). These findings show chloroplast anchoring restricts a multifaceted ECD response. Our results also show that the accumulated chloroplastic H2O2 in NbCHUP1-silenced plants was not required for the increased basal epidermal chloroplast movement but was essential for increased stromules and enhanced ETI. This finding indicates that chloroplast de-anchoring and H2O2 play separate but essential roles during ETI.

Stromule geometry allows optimal spatial regulation of organelle interactions in the quasi-2D cytoplasm.

In plant cells, plastids form elongated extensions called stromules, the regulation and purposes of which remain unclear. Here, we quantitatively explore how different stromule structures serve to enhance the ability of a plastid to interact with other organelles: increasing the effective space for interaction and biomolecular exchange between organelles. Interestingly, electron microscopy and confocal imaging showed that the cytoplasm in Arabidopsis thaliana and Nicotiana benthamiana epidermal cells is extremely thin (around 100 nm in regions without organelles), meaning that inter-organelle interactions effectively take place in 2D. We combine these imaging modalities with mathematical modelling and new in planta experiments to demonstrate how different stromule varieties (single or multiple, linear or branching) could be employed to optimise different aspects of inter-organelle interaction capacity in this 2D space. We found that stromule formation and branching provide a proportionally higher benefit to interaction capacity in 2D than in 3D. Additionally, this benefit depends on optimal plastid spacing. We hypothesize that cells can promote the formation of different stromule architectures in the quasi-2D cytoplasm to optimise their interaction interface to meet specific requirements. These results provide new insight into the mechanisms underlying the transition from low to high stromule numbers, the consequences for interaction with smaller organelles, how plastid access and plastid to nucleus signaling are balanced, as well as the impact of plastid density on organelle interaction.

Chloroplast redox state changes mark cell-to-cell signaling in the hypersensitive response.

Hypersensitive response (HR)-conferred resistance is associated with induction of programmed cell death and pathogen spread restriction in its proximity. The exact role of chloroplastic reactive oxygen species and its link with salicylic acid (SA) signaling in HR remain unexplained. To unravel this, we performed a detailed spatiotemporal analysis of chloroplast redox response in palisade mesophyll and upper epidermis to potato virus Y (PVY) infection in a resistant potato genotype and its transgenic counterpart with impaired SA accumulation and compromised resistance. Besides the cells close to the cell death zone, we detected individual cells with oxidized chloroplasts further from the cell death zone. These are rare in SA-deficient plants, suggesting their role in signaling for resistance. We confirmed that chloroplast redox changes play important roles in signaling for resistance, as blocking chloroplast redox changes affected spatial responses at the transcriptional level. Through spatiotemporal study of stromule induction after PVY infection, we show that stromules are induced by cell death and also as a response to PVY multiplication at the front of infection. Overall induction of stromules is attenuated in SA-deficient plants.

Plant environmental sensing relies on specialized plastids.

In plants, plastids are thought to interconvert to various forms that are specialized for photosynthesis, starch and oil storage, and diverse pigment accumulation. Post-endosymbiotic evolution has led to adaptations and specializations within plastid populations that align organellar functions with different cellular properties in primary and secondary metabolism, plant growth, organ development, and environmental sensing. Here, we review the plastid biology literature in light of recent reports supporting a class of 'sensory plastids' that are specialized for stress sensing and signaling. Abundant literature indicates that epidermal and vascular parenchyma plastids display shared features of dynamic morphology, proteome composition, and plastid-nuclear interaction that facilitate environmental sensing and signaling. These findings have the potential to reshape our understanding of plastid functional diversification.

Specificity of H2O2 signaling in leaf senescence: is the ratio of H2O2 contents in different cellular compartments sensed in Arabidopsis plants?

Leaf senescence is an integral part of plant development and is driven by endogenous cues such as leaf or plant age. Developmental senescence aims to maximize the usage of carbon, nitrogen and mineral resources for growth and/or for the sake of the next generation. This requires efficient reallocation of the resources out of the senescing tissue into developing parts of the plant such as new leaves, fruits and seeds. However, premature senescence can be induced by severe and long-lasting biotic or abiotic stress conditions. It serves as an exit strategy to guarantee offspring in an unfavorable environment but is often combined with a trade-off in seed number and quality. In order to coordinate the very complex process of developmental senescence with environmental signals, highly organized networks and regulatory cues have to be in place. Reactive oxygen species, especially hydrogen peroxide (H2O2), are involved in senescence as well as in stress signaling. Here, we want to summarize the role of H2O2 as a signaling molecule in leaf senescence and shed more light on how specificity in signaling might be achieved. Altered hydrogen peroxide contents in specific compartments revealed a differential impact of H2O2 produced in different compartments. Arabidopsis lines with lower H2O2 levels in chloroplasts and cytoplasm point to the possibility that not the actual contents but the ratio between the two different compartments is sensed by the plant cells.

Chloroplast behaviour and interactions with other organelles in Arabidopsis thaliana pavement cells.

Chloroplasts are a characteristic feature of green plants. Mesophyll cells possess the majority of chloroplasts and it is widely believed that, with the exception of guard cells, the epidermal layer in most higher plants does not contain chloroplasts. However, recent observations on Arabidopsis thaliana have shown a population of chloroplasts in pavement cells that are smaller than mesophyll chloroplasts and have a high stroma to grana ratio. Here, using stable transgenic lines expressing fluorescent proteins targeted to the plastid stroma, plasma membrane, endoplasmic reticulum, tonoplast, nucleus, mitochondria, peroxisomes, F-actin and microtubules, we characterize the spatiotemporal relationships between the pavement cell chloroplasts (PCCs) and their subcellular environment. Observations on the PCCs suggest a source-sink relationship between the epidermal and the mesophyll layers, and experiments with the Arabidopsis mutants glabra2 (gl2) and immutans (im), which show altered epidermal plastid development, underscored their developmental plasticity. Our findings lay down the foundation for further investigations aimed at understanding the precise role and contributions of PCCs in plant interactions with the environment.

On the move: redox-dependent protein relocation in plants.

Compartmentation of proteins and processes is a defining feature of eukaryotic cells. The growth and development of organisms is critically dependent on the accurate sorting of proteins within cells. The mechanisms by which cytosol-synthesized proteins are delivered to the membranes and membrane compartments have been extensively characterized. However, the protein complement of any given compartment is not precisely fixed and some proteins can move between compartments in response to metabolic or environmental triggers. The mechanisms and processes that mediate such relocation events are largely uncharacterized. Many proteins can in addition perform multiple functions, catalysing alternative reactions or performing structural, non-enzymatic functions. These alternative functions can be equally important functions in each cellular compartment. Such proteins are generally not dual-targeted proteins in the classic sense of having targeting sequences that direct de novo synthesized proteins to specific cellular locations. We propose that redox post-translational modifications (PTMs) can control the compartmentation of many such proteins, including antioxidant and/or redox-associated enzymes.

Plant-microbe interactions: organelles and the cytoskeleton in action.

Contents Summary 1012 I. Introduction 1012 II. The endomembrane system in plant-microbe interactions 1013 III. The cytoskeleton in plant-microbe interactions 1017 IV. Organelles in plant-microbe interactions 1019 V. Inter-organellar communication in plant-microbe interactions 1022 VI. Conclusions and prospects 1023 Acknowledgements 1024 References 1024 SUMMARY: Plants have evolved a multilayered immune system with well-orchestrated defense strategies against pathogen attack. Multiple immune signaling pathways, coordinated by several subcellular compartments and interactions between these compartments, play important roles in a successful immune response. Pathogens use various strategies to either directly attack the plant's immune system or to indirectly manipulate the physiological status of the plant to inhibit an immune response. Microscopy-based approaches have allowed the direct visualization of membrane trafficking events, cytoskeleton reorganization, subcellular dynamics and inter-organellar communication during the immune response. Here, we discuss the contributions of organelles and the cytoskeleton to the plant's defense response against microbial pathogens, as well as the mechanisms used by pathogens to target these compartments to overcome the plant's defense barrier.

Chloroplasts extend stromules independently and in response to internal redox signals.

A fundamental mystery of plant cell biology is the occurrence of "stromules," stroma-filled tubular extensions from plastids (such as chloroplasts) that are universally observed in plants but whose functions are, in effect, completely unknown. One prevalent hypothesis is that stromules exchange signals or metabolites between plastids and other subcellular compartments, and that stromules are induced during stress. Until now, no signaling mechanisms originating within the plastid have been identified that regulate stromule activity, a critical missing link in this hypothesis. Using confocal and superresolution 3D microscopy, we have shown that stromules form in response to light-sensitive redox signals within the chloroplast. Stromule frequency increased during the day or after treatment with chemicals that produce reactive oxygen species specifically in the chloroplast. Silencing expression of the chloroplast NADPH-dependent thioredoxin reductase, a central hub in chloroplast redox signaling pathways, increased chloroplast stromule frequency, whereas silencing expression of nuclear genes related to plastid genome expression and tetrapyrrole biosynthesis had no impact on stromules. Leucoplasts, which are not photosynthetic, also made more stromules in the daytime. Leucoplasts did not respond to the same redox signaling pathway but instead increased stromule formation when exposed to sucrose, a major product of photosynthesis, although sucrose has no impact on chloroplast stromule frequency. Thus, different types of plastids make stromules in response to distinct signals. Finally, isolated chloroplasts could make stromules independently after extraction from the cytoplasm, suggesting that chloroplast-associated factors are sufficient to generate stromules. These discoveries demonstrate that chloroplasts are remarkably autonomous organelles that alter their stromule frequency in reaction to internal signal transduction pathways.

Subcellular localization of Arabidopsis arogenate dehydratases suggests novel and non-enzymatic roles.

Arogenate dehydratases (ADTs) catalyze the final step in phenylalanine biosynthesis in plants. The Arabidopsis thaliana genome encodes a family of six ADTs capable of decarboxylating/dehydrating arogenate into phenylalanine. Using cyan fluorescent protein (CFP)-tagged proteins, the subcellular localization patterns of all six A. thaliana ADTs were investigated in intact Nicotiana benthamiana and A. thaliana leaf cells. We show that A. thaliana ADTs localize to stroma and stromules (stroma-filled tubules) of chloroplasts. This localization pattern is consistent with the enzymatic function of ADTs as many enzymes required for amino acid biosynthesis are primarily localized to chloroplasts, and stromules are thought to increase metabolite transport from chloroplasts to other cellular compartments. Furthermore, we provide evidence that ADTs have additional, non-enzymatic roles. ADT2 localizes in a ring around the equatorial plane of chloroplasts or to a chloroplast pole, which suggests that ADT2 is a component of the chloroplast division machinery. In addition to chloroplasts, ADT5 was also found in nuclei, again suggesting a non-enzymatic role for ADT5. We also show evidence that ADT5 is transported to the nucleus via stromules. We propose that ADT2 and ADT5 are moonlighting proteins that play an enzymatic role in phenylalanine biosynthesis and a second role in chloroplast division or transcriptional regulation, respectively.

Abscisic Acid Insensitive 4 transcription factor is an important player in the response of Arabidopsis thaliana to two-spotted spider mite (Tetranychus urticae) feeding.

Plants growing in constantly changeable environmental conditions are compelled to evolve regulatory mechanisms to cope with biotic and abiotic stresses. Effective defence to invaders is largely connected with phytohormone regulation, resulting in the production of numerous defensive proteins and specialized metabolites. In our work, we elucidated the role of the Abscisic Acid Insensitive 4 (ABI4) transcription factor in the plant response to the two-spotted spider mite (TSSM). This polyphagous mite is one of the most destructive herbivores, which sucks mesophyll cells of numerous crop and wild plants. Compared to the wild-type (Col-0) Arabidopsis thaliana plants, the abi4 mutant demonstrated increased susceptibility to TSSM, reflected as enhanced female fecundity and greater frequency of mite leaf damage after trypan blue staining. Because ABI4 is regarded as an important player in the plastid-to-nucleus retrograde signalling process, we investigated the plastid envelope membrane dynamics using stroma-associated fluorescent marker. Our results indicated a clear increase in the number of stroma-filled tubular structures deriving from the plastid membrane (stromules) in the close proximity of the site of mite leaf damage, highlighting the importance of chloroplast-derived signals in the response to TSSM feeding activity.

A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha.

Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity.

Chloroplast protrusions in leaves of Ranunculus glacialis L. respond significantly to different ambient conditions, but are not related to temperature stress.

The occurrence of chloroplast protrusions (CPs) in leaves of Ranunculus glacialis L. in response to different environmental conditions was assessed. CPs occur highly dynamically. They do not contain thylakoids and their physiological function is still largely unknown. Controlled in situ sampling showed that CP formation follows a pronounced diurnal rhythm. Between 2 and 27 °C the relative proportion of chloroplasts with CPs (rCP) showed a significant positive correlation to leaf temperature (TL; 0.793, P < 0.01), while irradiation intensity had a minor effect on rCP. In situ shading and controlled laboratory experiments confirmed the significant influence of TL. Under moderate irradiation intensity, an increase of TL up to 25 °C significantly promoted CP formation, while a further increase to 37 °C led to a decrease. Furthermore, rCP values were lower in darkness and under high irradiation intensity. Gas treatment at 2000 ppm CO2/2% O2 led to a significant decrease of rCP, suggesting a possible involvement of photorespiration in CP formation. Our findings demonstrate that in R. glacialis, CPs are neither a rare phenomenon nor a result of heat or light stress; on the contrary, they seem to be most abundant under moderate temperature and non-stress irradiation conditions.

Plastid Transient and Stable Interactions with Other Cell Compartments.

Plastids are organelles delineated by two envelopes playing important roles in different cellular processes such as energy production or lipid biosynthesis. To regulate their biogenesis and their function, plastids have to communicate with other cellular compartments. This communication can be mediated by metabolites, signaling molecules, and by the establishment of direct contacts between the plastid envelope and other organelles such as the endoplasmic reticulum, mitochondria, peroxisomes, plasma membrane, and the nucleus. These interactions are highly dynamic and respond to different biotic and abiotic stresses. However, the mechanisms involved in the formation of plastid-organelle contact sites and their functions are still far from being understood. In this chapter, we summarize our current knowledge about plastid contact sites and their role in the regulation of plastid biogenesis and function.

Plastid dynamism integrates development and environment.

In land plants plastid type differentiation occurs concomitantly with cellular differentiation and the transition from one type to another is under developmental and environmental control. Plastid dynamism is based on a bilateral communication between plastids and nucleus through anterograde and retrograde signaling. Signaling occurs through the interaction with specific phytohormones (abscisic acid, strigolactones, jasmonates, gibberellins, brassinosteroids, ethylene, salicylic acid, cytokinin and auxin). The review is focused on the modulation of plastid capabilities at both transcriptional and post-translational levels at the crossroad between development and stress, with a particular attention to the chloroplast, because the most studied plastid type. The role of plastid-encoded and nuclear-encoded proteins for plastid development and stress responses, and the changes of plastid fate through the activity of stromules and plastoglobules, are discussed. Examples of plastid dynamism in response to soil stress agents (salinity, lead, cadmium, arsenic, and chromium) are described. Albinism and root greening are described based on the modulation activities of auxin and cytokinin. The physiological and functional responses of the sensory epidermal and vascular plastids to abiotic and biotic stresses along with their specific roles in stress sensing are described together with their potential modulation of retrograde signaling pathways. Future research perspectives include an in-depth study of sensory plastids to explore their potential for establishing a transgenerational memory to stress. Suggestions about anterograde and retrograde pathways acting at interspecific level and on the lipids of plastoglobules as a novel class of plastid morphogenic agents are provided.

Chloroplast Envelopes Play a Role in the Formation of Autophagy-Related Structures in Plants.

Autophagy is a degradation process of cytoplasmic components that is conserved in eukaryotes. One of the hallmark features of autophagy is the formation of double-membrane structures known as autophagosomes, which enclose cytoplasmic content destined for degradation. Although the membrane source for the formation of autophagosomes remains to be determined, recent studies indicate the involvement of various organelles in autophagosome biogenesis. In this study, we examined the autophagy process in Bienertia sinuspersici: one of four terrestrial plants capable of performing C4 photosynthesis in a single cell (single-cell C4 species). We demonstrated that narrow tubules (stromule-like structures) 30-50 nm in diameter appear to extend from chloroplasts to form the membrane-bound structures (autophagosomes or autophagy-related structures) in chlorenchyma cells of B. sinuspersici during senescence and under oxidative stress. Immunoelectron microscopic analysis revealed the localization of stromal proteins to the stromule-like structures, sequestering portions of the cytoplasm in chlorenchyma cells of oxidative stress-treated leaves of B. sinuspersici and Arabidopsis thaliana. Moreover, the fluorescent marker for autophagosomes GFP-ATG8, colocalized with the autophagic vacuole maker neutral red in punctate structures in close proximity to the chloroplasts of cells under oxidative stress conditions. Together our results implicate a role for chloroplast envelopes in the autophagy process induced during senescence or under certain stress conditions in plants.

Membrane contacts with the endoplasmic reticulum modulate plastid morphology and behaviour.

Plastid behaviour often occurs in tandem with endoplasmic reticulum (ER) dynamics. In order to understand the underlying basis for such linked behaviour we have used time-lapse imaging-based analysis of plastid movement and pleomorphy, including the extension and retraction of stromules. Stable transgenic plants that simultaneously express fluorescent fusion proteins targeted to the plastid stroma, and the ER along with BnCLIP1-eGFP, an independent plastid envelope localized membrane contact site (MCS) marker were utilized. Our experiments strongly suggest that transient MCS formed between the plastid envelope and the ER are responsible for their concomitant behaviour.

Spatial chloroplast-to-nucleus signalling involving plastid-nuclear complexes and stromules.

Communication between chloroplasts and the nucleus in response to various environmental cues may be mediated by various small molecules. Signalling specificity could be enhanced if the physical contact between these organelles facilitates direct transfer and prevents interference from other subcellular sources of the same molecules. Plant cells have plastid-nuclear complexes, which provide close physical contact between these organelles. Plastid-nuclear complexes have been proposed to facilitate transfer of photosynthesis-derived H2O2 to the nucleus in high light. Stromules (stroma filled tubular plastid extensions) may provide an additional conduit for transfer of a wider range of signalling molecules, including proteins. However, plastid-nuclear complexes and stromules have been hitherto treated as distinct phenomena. We suggest that plastid-nuclear complexes and stromules work in a coordinated manner so that, according to environmental conditions or developmental state, the two modes of connection contribute to varying extents. We hypothesize that this association is dynamic and that there may be a link between plastid-nuclear complexes and the development of stromules. Furthermore, the changes in contact could alter signalling specificity by allowing an extended or different range of signalling molecules to be delivered to the nucleus. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Review: Morphology, behaviour and interactions of organelles.

High quality transmission electron micrographs have played a major role in shaping our views on organelles in plant cells. However, these snapshots of dead, fixed and sectioned tissue do not automatically convey an appreciation of the dynamic nature of organelles in living cells. Advances in the imaging of subcellular structures in living cells using multicoloured, targeted fluorescent proteins reveal considerable changes in organelle pleomorphy that might be limited to small regions of the cell. The fresh data and insights also challenge several existing ideas on organelle behaviour and interactivity. Here, using succinct examples from plastids, mitochondria, peroxisomes, and the endoplasmic reticulum I present an evolving view of subcellular dynamics in the plant cell.