skip to main content


Title: Two plastid POLLUX ion channel-like proteins are required for stress-triggered stromal Ca2+release
Abstract Two decades ago, large cation currents were discovered in the envelope membranes of Pisum sativum L. (pea) chloroplasts. The deduced K+-permeable channel was coined fast-activating chloroplast cation channel but its molecular identity remained elusive. To reveal candidates, we mined proteomic datasets of isolated pea envelopes. Our search uncovered distant members of the nuclear POLLUX ion channel family. Since pea is not amenable to molecular genetics, we used Arabidopsis thaliana to characterize the two gene homologs. Using several independent approaches, we show that both candidates localize to the chloroplast envelope membrane. The proteins, designated PLASTID ENVELOPE ION CHANNELS (PEC1/2), form oligomers with regulator of K+ conductance domains protruding into the intermembrane space. Heterologous expression of PEC1/2 rescues yeast mutants deficient in K+ uptake. Nuclear POLLUX ion channels cofunction with Ca2+ channels to generate Ca2+ signals, critical for establishing mycorrhizal symbiosis and root development. Chloroplasts also exhibit Ca2+ transients in the stroma, probably to relay abiotic and biotic cues between plastids and the nucleus via the cytosol. Our results show that pec1pec2 loss-of-function double mutants fail to trigger the characteristic stromal Ca2+ release observed in wild-type plants exposed to external stress stimuli. Besides this molecular abnormality, pec1pec2 double mutants do not show obvious phenotypes. Future studies of PEC proteins will help to decipher the plant’s stress-related Ca2+ signaling network and the role of plastids. More importantly, the discovery of PECs in the envelope membrane is another critical step towards completing the chloroplast ion transport protein inventory.  more » « less
Award ID(s):
1847382 1553506 1828266
NSF-PAR ID:
10312374
Author(s) / Creator(s):
; ; ; ; ; ;
Date Published:
Journal Name:
Plant Physiology
Volume:
187
Issue:
4
ISSN:
0032-0889
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract The inner-envelope K+ EFFLUX ANTIPORTERS (KEA) 1 and 2 are critical for chloroplast development, ion homeostasis, and photosynthesis. However, the mechanisms by which changes in ion flux across the envelope affect organelle biogenesis remained elusive. Chloroplast development requires intricate coordination between the nuclear genome and the plastome. Many mutants compromised in plastid gene expression (PGE) display a virescent phenotype, that is delayed greening. The phenotypic appearance of Arabidopsis thaliana kea1 kea2 double mutants fulfills this criterion, yet a link to PGE has not been explored. Here, we show that a simultaneous loss of KEA1 and KEA2 results in maturation defects of the plastid ribosomal RNAs. This may be caused by secondary structure changes of rRNA transcripts and concomitant reduced binding of RNA-processing proteins, which we documented in the presence of skewed ion homeostasis in kea1 kea2. Consequently, protein synthesis and steady-state levels of plastome-encoded proteins remain low in mutants. Disturbance in PGE and other signs of plastid malfunction activate GENOMES UNCOUPLED 1-dependent retrograde signaling in kea1 kea2, resulting in a dramatic downregulation of GOLDEN2-LIKE transcription factors to halt expression of photosynthesis-associated nuclear-encoded genes (PhANGs). PhANG suppression delays the development of fully photosynthesizing kea1 kea2 chloroplasts, probably to avoid progressing photo-oxidative damage. Overall, our results reveal that KEA1/KEA2 function impacts plastid development via effects on RNA-metabolism and PGE. 
    more » « less
  2. Abstract

    Plant development requires communication on many levels, including between cells and between organelles within a cell. For example, mitochondria and plastids have been proposed to be sensors of environmental stress and to coordinate their responses. Here we present evidence for communication between mitochondria and chloroplasts during leaf and root development, based on genetic and physical interactions between threeMechanosensitive channel ofSmall conductance‐Like (MSL) proteins fromArabidopsis thaliana.MSLproteins areArabidopsishomologs of the bacterialMechanosensitivechannel ofSmall conductance (MscS), which relieves cellular osmotic pressure to protect against lysis during hypoosmotic shock.MSL1 localizes to the inner mitochondrial membrane, whileMSL2 andMSL3 localize to the inner plastid membrane and are required to maintain plastid osmotic homeostasis during normal growth and development. In this study, we characterized the phenotypic effect of a genetic lesion inMSL1, both in wild type and inmsl2 msl3mutant backgrounds.msl1single mutants appear wild type for all phenotypes examined. The characteristic leaf rumpling inmsl2 msl3double mutants was exacerbated in themsl1 msl2 msl3triple mutant. However, the introduction of themsl1lesion into themsl2 msl3mutant background suppressed othermsl2 msl3mutant phenotypes, including ectopic callus formation, accumulation of superoxide and hydrogen peroxide in the shoot apical meristem, decreased root length, and reduced number of lateral roots. All these phenotypes could be recovered by molecular complementation with a transgene containing a wild type version ofMSL1. In yeast‐based interaction studies,MSL1 interacted with itself, but not withMSL2 orMSL3. These results establish that the abnormalities observed inmsl2 msl3double mutants is partially dependent on the presence of functionalMSL1 and suggest a possible role for communication between plastid and mitochondria in seedling development.

     
    more » « less
  3. Chloroplasts are endosymbiotic organelles derived from cyanobacteria. They have a double envelope membrane, including the outer envelope and the inner envelope. A complex membrane system, thylakoids, exists inside the chloroplast. It is the site of the light-dependent reactions of photosynthesis. The stroma is the main site of the carbon fixation reactions. Although photosynthesis is a very complicated process with many proteins involved, there are many other important processes that occur in chloroplasts, including the regulation of photosynthesis, the biogenesis and maintenance of the structures, carbohydrate, lipid, tetrapyrrole, amino acid, and isoprenoid metabolism, production of some phytohormones, production of specialized metabolites, regulation of redox, and interactions with other parts of the cell (Sabater, 2018). During evolution, most of the cyanobacterial genes were lost and many of them were transferred into the nuclear genome. A majority of chloroplast proteins are nuclear-encoded and possess an N-terminal transit peptide which helps the protein to be targeted into chloroplasts. Chloroplasts have their own highly reduced genome which works coordinately with the nuclear genome for the biogenesis and function of chloroplasts (Liebers et al., 2022). This Research Topic presents studies covering different aspects of chloroplast function, including photosynthesis, biogenesis, structure, and maintenance. These works push the frontiers of chloroplast research further in the field of plant biology. 
    more » « less
  4. SUMMARY

    Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols deliveringin situvolumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block‐face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild‐type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col‐0 wild‐type mesophyll chloroplasts is 93 μm3. Wild‐type guard cell plastids are approximately 18 μm3. Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment.

     
    more » « less
  5. Abstract

    Sigma factor (SIG) proteins contribute to promoter specificity of the plastid‐encodedRNApolymerase during chloroplast genome transcription. All six members of theSIGfamily, that is,SIG1–SIG6, are nuclear‐encoded proteins targeted to chloroplasts. Sigma factor 2 (SIG2) is a phytochrome‐regulated protein important for stoichiometric control of the expression of plastid‐ and nuclear‐encoded genes that impact plastid development and plant growth and development. AmongSIGfactors,SIG2 is required not only for transcription of chloroplast genes (i.e., anterograde signaling), but also impacts nuclear‐encoded, photosynthesis‐related, and light signaling‐related genes (i.e., retrograde signaling) in response to plastid functional status. AlthoughSIG2 is involved in photomorphogenesis in Arabidopsis, the molecular bases for its role in light signaling that impacts photomorphogenesis and aspects of photosynthesis have only recently begun to be investigated. Previously, we reported thatSIG2 is necessary for phytochrome‐mediated photomorphogenesis specifically under red (R) and far‐red light, thereby suggesting a link between phytochromes and nuclear‐encodedSIG2 in light signaling. To explore transcriptional roles ofSIG2 in R‐dependent growth and development, we performedRNAsequencing analysis to compare gene expression insig2‐2mutant and Col‐0 wild‐type seedlings at two developmental stages (1‐ and 7‐day). We identified a subset of misregulated genes involved in growth, hormonal cross talk, stress responses, and photosynthesis. To investigate the functional relevance of these gene expression analyses, we performed several comparative phenotyping tests. In these analyses, strongsig2mutants showed insensitivity to bioactiveGA3, high intracellular levels of hydrogen peroxide (H2O2) indicative of a stress response, and specific defects in photosynthesis, including elevated levels of cyclic electron flow (CEF) and nonphotochemical quenching (NPQ). We demonstrated thatSIG2 regulates a broader range of physiological responses at the molecular level than previously reported, with specific roles in red‐light‐mediated photomorphogenesis.

     
    more » « less