Plastid KEA-type cation/H+ antiporters are required for vacuolar protein trafficking in Arabidopsis

Arabidopsis plastid antiporters KEA1 and KEA2 are critical for plastid development, photosynthetic efficiency, and plant development. Here, we show that KEA1 and KEA2 are involved in vacuolar protein trafficking. Genetic analyses found that the kea1 kea2 mutants had short siliques, small seeds, and short seedlings. Molecular and biochemical assays showed that seed storage proteins were missorted out of the cell and the precursor proteins were accumulated in kea1 kea2. Protein storage vacuoles (PSVs) were smaller in kea1 kea2. Further analyses showed that endosomal trafficking in kea1 kea2 was compromised. Vacuolar sorting receptor 1 (VSR1) subcellular localizations, VSR–cargo interactions, and p24 distribution on the endoplasmic reticulum (ER) and Golgi apparatus were affected in kea1 kea2. Moreover, plastid stromule growth was reduced and plastid association with the endomembrane compartments was disrupted in kea1 kea2. Stromule growth was regulated by the cellular pH and K⁺ homeostasis maintained by KEA1 and KEA2. The organellar pH along the trafficking pathway was altered in kea1 kea2. Overall, KEA1 and KEA2 regulate vacuolar trafficking by controlling the function of plastid stromules via adjusting pH and K⁺ homeostasis.     

Retracted: Each of the chloroplast potassium efflux antiporters affects photosynthesis and growth of fully developed Arabidopsis rosettes under short‐day photoperiod

In Arabidopsis thaliana, the chloroplast harbors three potassium efflux antiporters (KEAs), namely KEA1 and KEA2 in the inner envelope and KEA3 in the thylakoid membrane. They may play redundant physiological roles as in our previous analyses of young developing Arabidopsis rosettes under long‐day photoperiod (16 h light per day), chloroplast kea single mutants resembled the wild‐type plants, whereas kea1kea2 and kea1kea2kea3 mutants were impaired in chloroplast development and photosynthesis resulting in stunted growth. Here, we aimed to study whether chloroplast KEAs play redundant roles in chloroplast function of older Arabidopsis plants with fully developed rosettes grown under short‐day photoperiod (8 h light per day). Under these conditions, we found defects in photosynthesis and growth in the chloroplast kea single mutants, and most dramatic defects in the kea1kea2 double mutant. The mechanism behind these defects in the single mutants involves reduction in the electron transport rate (kea1 and kea3), and stomata conductance (kea1, kea2 and kea3), which in turn affect CO₂ fixation rates. The kea1kea2 mutant, in addition to these alterations, displayed reduced levels of photosynthetic machinery. Taken together, our data suggest that, in addition to the previously reported roles in chloroplast development in young rosettes, each chloroplast KEA affects photosynthesis and growth of Arabidopsis fully developed rosettes.     

Golgi‐localized cation/proton exchangers regulate ionic homeostasis and skotomorphogenesis in Arabidopsis

Multiple transporters and channels mediate cation transport across the plasma membrane and tonoplast to regulate ionic homeostasis in plant cells. However, much less is known about the molecular function of transporters that facilitate cation transport in other organelles such as Golgi. We report here that Arabidopsis KEA4, KEA5, and KEA6, members of cation/proton antiporters‐2 (CPA2) superfamily were colocalized with the known Golgi marker, SYP32‐mCherry. Although single kea4,5,6 mutants showed similar phenotype as the wild type under various conditions, kea4/5/6 triple mutants showed hypersensitivity to low pH, high K⁺, and high Na⁺ and displayed growth defects in darkness, suggesting that these three KEA‐type transporters function redundantly in controlling etiolated seedling growth and ion homeostasis. Detailed analysis indicated that the kea4/5/6 triple mutant exhibited cell wall biosynthesis defect during the rapid etiolated seedling growth and under high K⁺/Na⁺ condition. The cell wall‐derived pectin homogalacturonan (GalA)₃ partially suppressed the growth defects and ionic toxicity in the kea4/5/6 triple mutants when grown in the dark but not in the light conditions. Together, these data support the hypothesis that the Golgi‐localized KEAs play key roles in the maintenance of ionic and pH homeostasis, thereby facilitating Golgi function in cell wall biosynthesis during rapid etiolated seedling growth and in coping with high K⁺/Na⁺ stress.     

Two plastid POLLUX ion channel-like proteins are required for stress-triggered stromal Ca2+release

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.     

Fine‐tuned regulation of the K+/H+ antiporter KEA3 is required to optimize photosynthesis during induction

KEA3 is a thylakoid membrane localized K⁺/H⁺ antiporter that regulates photosynthesis by modulating two components of proton motive force (pmf), the proton gradient (?pH) and the electric potential (?ψ). We identified a mutant allele of KEA3, disturbed proton gradient regulation (dpgr) based on its reduced non‐photochemical quenching (NPQ) in artificial (CO₂‐free with low O₂) air. This phenotype was enhanced in the mutant backgrounds of PSI cyclic electron transport (pgr5 and crr2‐1). In ambient air, reduced NPQ was observed during induction of photosynthesis in dpgr, the phenotype that was enhanced after overnight dark adaptation. In contrast, the knockout allele of kea3‐1 exhibited a high‐NPQ phenotype during steady state in ambient air. Consistent with this kea3‐1 phenotype in ambient air, the membrane topology of KEA3 indicated a proton efflux from the thylakoid lumen to the stroma. The dpgr heterozygotes showed a semidominant and dominant phenotype in artificial and ambient air, respectively. In dpgr, the protein level of KEA3 was unaffected but the downregulation of its activity was probably disturbed. Our findings suggest that fine regulation of KEA3 activity is necessary for optimizing photosynthesis.     

Albino midrib 1, encoding a putative potassium efflux antiporter,affects chloroplast development and drought tolerance in rice

Key message

Mutation of the AM1 gene causes an albino midrib phenotype and enhances tolerance to drought in rice

Abstract

K⁺ efflux antiporter (KEA) genes encode putative potassium efflux antiporters that are mainly located in plastid-containing organisms, ranging from lower green algae to higher flowering plants. However, little genetic evidence has been provided on the functions of KEA in chloroplast development. In this study, we isolated a rice mutant, albino midrib 1 (am1), with green- and white-variegation in the first few leaves, and albino midrib phenotype in older tissues. We found that AM1 encoded a putative KEA in chloroplast. AM1 was highly expressed in leaves, while lowly in roots. Chloroplast gene expression and proteins accumulation were affected during chlorophyll biosynthesis and photosynthesis in am1 mutants. Interestingly, AM1 was induced by salt and PEG, and am1 showed enhanced sensitivity to salinity in seed germination and increased tolerance to drought. Taken together, we concluded that KEAs were involved in chloroplast development and played important roles in drought tolerance.     

Studies on the System Regulating Proton Movement across the Chloroplast Envelope : Effects of ATPase Inhibitors, Mg, and an Amine Anesthetic on Stromal pH and Photosynthesis

Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H⁺ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg²⁺ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg²⁺ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg²⁺ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H⁺ fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg²⁺ modulated) H⁺ flux linked to monovalent cation antiport, and ATPase-dependent H⁺ efflux.     

PDV1 and PDV2 mediate recruitment of the dynamin-related protein ARC5 to the plastid division site

During plastid division, the dynamin-related protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5) is recruited from the cytosol to the surface of the outer chloroplast envelope membrane. In Arabidopsis thaliana arc5 mutants, chloroplasts arrest during division site constriction. Analysis of mutants similar to arc5 along with map-based cloning identified PLASTID DIVISION1 (PDV1), an integral outer envelope membrane protein, and its homolog PDV2 as components of the plastid division machinery. Similar to ARC5, PDV1 localized to a discontinuous ring at the division site in wild-type plants. The midplastid PDV1 ring formed in arc5 mutants and the ARC5 ring formed in pdv1 and pdv2 mutants, but not in pdv1 pdv2. Stromal FtsZ ring assembly occurred in pdv1, pdv2, and pdv1 pdv2, as it does in arc5. Topological analysis showed that the large N-terminal region of PDV1 upstream of the transmembrane helix bearing a putative coiled-coil domain is exposed to the cytosol. Mutation of the conserved PDV1 C-terminal Gly residue did not block PDV1 insertion into the outer envelope membrane but did abolish its localization to the division site. Our results indicate that plastid division involves the stepwise localization of FtsZ, PDV1, and ARC5 at the division site and that PDV1 and PDV2 together mediate the recruitment of ARC5 to the midplastid constriction at a late stage of division.     

RNR1, a 3'-5' exoribonuclease belonging to the RNR superfamily, catalyzes 3' maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana

Arabidopsis thaliana chloroplasts contain at least two 3′ to 5′ exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3′ processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5′ and 3′ ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3′ extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.     

Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein

The development of chloroplasts in Arabidopsis thaliana requires extensive lipid trafficking between the endoplasmic reticulum (ER) and the plastid. The biosynthetic enzymes for the final steps of chloroplast lipid assembly are associated with the plastid envelope membranes. For example, during biosynthesis of the galactoglycerolipids predominant in photosynthetic membranes, galactosyltransferases associated with these membranes transfer galactosyl residues from UDP-Gal to diacylglycerol. In Arabidopsis, diacylglycerol can be derived from the ER or the plastid. Here, we describe a mutant of Arabidopsis, trigalactosyldiacylglycerol4 (tgd4), in which ER-derived diacylglycerol is not available for galactoglycerolipid biosynthesis. This mutant accumulates diagnostic oligogalactoglycerolipids, hence its name, and triacylglycerol in its tissues. The TGD4 gene encodes a protein that appears to be associated with the ER membranes. Mutant ER microsomes show a decreased transfer of lipids to isolated plastids consistent with in vivo labeling data, indicating a disruption of ER-to-plastid lipid transfer. The complex lipid phenotype of the mutant is similar to that of the tgd1,2,3 mutants disrupted in components of a lipid transporter of the inner plastid envelope membrane. However, unlike the TGD1,2,3 complex, which is proposed to transfer phosphatidic acid through the inner envelope membrane, TGD4 appears to be part of the machinery mediating lipid transfer between the ER and the outer plastid envelope membrane. The extent of direct ER-to-plastid envelope contact sites is not altered in the tgd4 mutant. However, this does not preclude a possible function of TGD4 in those contact sites as a conduit for lipid transfer between the ER and the plastid.     

Stromal pH and Photosynthesis Are Affected by Electroneutral K and H Exchange through Chloroplast Envelope Ion Channels

Potassium movement across the limiting membrane of the chloroplast inner envelope is known to be linked to counterex-change of protons. For this reason, K⁺ efflux is known to facilitate stromal acidification and the resultant photosynthetic inhibition. However, the specific nature of the chloroplast envelope proteins that facilitate K⁺ fluxes, and the biophysical mechanism which links these cation currents to H⁺ counterflux, is not characterized. It was the objective of this work to elucidate the nature of the system regulating K⁺ flux linked to H⁺ counterflux across the chloroplast envelope. In the absence of external K⁺, exposure of spinach (Spinacia oleracea) chloroplasts to the K⁺ ionophore valinomycin was found to increase the rate of K⁺ efflux and H⁺ influx. These data were interpreted as suggesting that H⁺ counterexchange must be indirectly linked to movement of K⁺ across the envelope. Studies using the K⁺ channel blocker tetraethylammonium indicated that K⁺ likely moves, in a uniport fashion, into or out of the stroma through a monovalent cation channel in the envelope. Blockage of K⁺ efflux from the stroma by exposure to tetraethylammonium was found to restrict H⁺ influx, further substantiating an indirect linkage of these cation currents. Further studies comparing the effect of exogenous H⁺ ionophores and K⁺/H⁺ exchangers suggested that K⁺ uniport through this ion channel likely is the main endogenous pathway for K⁺ currents across the envelope. These experiments were also consistent with the presence of a proton channel in the envelope. Movement of H⁺ through this channel was speculated to be regulated and rate limited by an electroneutral requirement for K⁺ countercurrents through the separate K⁺ uniport pathway. K⁺ and H⁺ fluxes across the chloroplast envelope were envisioned to be interrelated via this mechanism. The significant effect of cation currents across the envelope, as mediated by these channels, on photosynthetic capacity of the isolated chloroplast was also demonstrated.     

Collaboration between NDH and KEA3 Allows Maximally Efficient Photosynthesis after a Long Dark Adaptation

In angiosperms, the NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around PSI (CET). K⁺ Efflux Antiporter3 (KEA3) is a putative thylakoid H⁺/K⁺ antiporter and allows an increase in membrane potential at the expense of the ∆pH component of the proton motive force. In this study, we discovered that the chlororespiratory reduction2-1 (crr2-1) mutation, which abolished NDH-dependent CET, enhanced the kea3-1 mutant phenotypes in Arabidopsis (Arabidopsis thaliana). The NDH complex pumps protons during CET, further enhancing ∆pH, but its physiological function has not been fully clarified. The observed effect only took place upon exposure to light of 110 µmol photons m⁻² s⁻¹ after overnight dark adaptation. We propose two distinct modes of NDH action. In the initial phase, within 1 min after the onset of actinic light, the NDH-dependent CET engages with KEA3 to enhance electron transport efficiency. In the subsequent phase, in which the ∆pH-dependent down-regulation of the electron transport is relaxed, the NDH complex engages with KEA3 to relax the large ∆pH formed during the initial phase. We observed a similar impact of the crr2-1 mutation in the genetic background of the PROTON GRADIENT REGULATION5 overexpression line, in which the size of ∆pH was enhanced. When photosynthesis was induced at 300 µmol photons m⁻² s⁻¹, the contribution of KEA3 was negligible in the initial phase and the ∆pH-dependent down-regulation was not relaxed in the second phase. In the crr2-1 kea3-1 double mutant, the induction of CO₂ fixation was delayed after overnight dark adaptation.

Photosynthesis consists of two sets of reactions, the light reactions and the Calvin-Benson cycle. It takes place in the chloroplast and fixes CO₂ into organic compounds using solar energy. In the light reactions, the absorption of photons activates electron transport in two photosystems. In linear electron transport (LET), PSII catalyzes the light-dependent oxidation of water, resulting in the release of oxygen and protons (H⁺) in the thylakoid lumen. The water-derived excised electrons are transferred to PSI through the cytochrome (Cyt) b₆f complex and ultimately to NADP⁺, producing NADPH. This electron transport is coupled with the translocation of H⁺ from the stroma to the thylakoid lumen via the quinone cycle at the Cyt b₆f complex, resulting in the formation of a proton concentration gradient across the thylakoid membrane. This ∆pH contributes to the formation of proton motive force (pmf) in addition to the membrane potential formed across the thylakoid membrane (∆ψ) that results from the uneven distribution of ions across the membrane. The pmf energizes ATP synthesis via F_oF₁-ATP synthase in chloroplasts (Kramer et al., 2003; Soga et al., 2017) and thus influences the efficiency of the light reactions.The Calvin-Benson cycle depends on NADPH and ATP produced by the light reactions. To fix a molecule of CO₂ into a carbohydrate, three molecules of ATP and two molecules of NADPH are needed. However, this ratio of ATP to NADPH (1.5) is not satisfied by LET (Shikanai, 2007). Photorespiration, which takes place due to the low specificity of Rubisco, the CO₂-fixing enzyme for CO₂, increases the energetic requirements in terms of ATP, raising the above ratio to 1.67. The additional ATP is thought to be supplied by cyclic electron transport around PSI (CET; Yamori and Shikanai, 2016). In contrast to LET, CET is driven solely by PSI and does not contribute to the net production of reducing power. CET recycles electrons from ferredoxin (Fd) to the plastoquinone (PQ) pool and contributes to the additional generation of ∆pH via the quinone cycle. As a result, CET balances the production ratio of ATP and NADPH. In angiosperms, CET has been proposed to consist of two pathways: the PROTON GRADIENT REGULATION5 (PGR5)/PGR5-like Photosynthetic Phenotype1 (PGRL1) protein-dependent, antimycin A-sensitive pathway and the NADH dehydrogenase-like (NDH) complex-dependent antimycin A-insensitive pathway (Munekage et al., 2004). The NDH complex pumps four protons, coupled with the movement of two electrons, from Fd to PQ, further increasing the efficiency of ∆pH formation (Strand et al., 2017).In addition to ATP synthesis, the ∆pH component of pmf also contributes to the down-regulation of electron transport (Shikanai, 2014). Acidification of the thylakoid lumen triggers the thermal dissipation of excessively absorbed light energy from the PSII antennae, a process that is monitored by nonphotochemical quenching (NPQ) of chlorophyll fluorescence (Müller et al., 2001). Low lumenal pH also down-regulates the activity of the Cyt b₆f complex, slowing down the rate of electron transport toward PSI (Stiehl and Witt, 1969). CET-dependent ∆pH formation is also necessary to induce the down-regulation of electron transport, as indicated by the phenotype of the pgr5 mutant. The Arabidopsis (Arabidopsis thaliana) pgr5 mutant cannot induce thermal dissipation under excessive light conditions (Munekage et al., 2002), suggesting that CET-generated ∆pH plays an important role in providing a sufficiently acidic lumen pH that can trigger NPQ. The pgr5 mutant is also defective in the down-regulation of Cyt b₆f activity, resulting in hypersensitivity of PSI to fluctuating light intensity (Tikkanen et al., 2010). Compared with the physiological function of the PGR5/PGRL1-dependent CET, the contribution of the NDH-dependent CET to photoprotection is somewhat minor, although clear phenotypes have been observed in these mutants at low light intensities and fluctuating light levels (Ueda et al., 2012; Yamori et al., 2015, 2016). Furthermore, the physiological function of the NDH complex has not been fully clarified.Both ∆pH and ∆ψ contribute to pmf, but only ∆pH down-regulates electron transport. To optimize the operation of the accelerator (ATP synthesis) and the brake on electron transport, it is necessary to precisely regulate the ratio of the two pmf components as well as the total size of pmf (Cruz et al., 2001; Kramer et al., 2003). Several channels and antiporters localized to the thylakoid membrane regulate the partitioning of the pmf components (Spetea et al., 2017). K⁺ Efflux Antiporter3 (KEA3) is thought to be an H⁺/K⁺ antiporter localized to the thylakoid membrane (Armbruster et al., 2014; Kunz et al., 2014), although its antiport activity has not been experimentally demonstrated (Tsujii et al., 2019). Based on its structure, topology, and the mutant phenotypes, KEA3 most likely moves H⁺ from the thylakoid lumen while taking up K⁺ as a counter ion. Consequently, KEA3 transforms ∆pH to ∆ψ and is necessary to rapidly relax the down-regulation of electron transport by raising the luminal pH (i.e. by alkalinizing the lumen). The C-terminal domain of KEA3, KTN (K⁺ transport/nucleotide binding), is exposed to the stroma (Wang et al., 2017) and is thought to regulate its activity by monitoring ATP or NADPH levels (Schlosser et al., 1993; Roosild et al., 2002). However, information on the regulation of KEA3 is limited. Armbruster et al. (2014) demonstrated that KEA3 contributes to efficient photosynthesis under fluctuating light conditions. The disturbed proton gradient regulation is a dominant mutant allele of KEA3, and its mutant phenotype is evident after a long period of dark adaptation (overnight; Wang et al., 2017). KEA3 is likely important during the induction of photosynthesis as well as under fluctuating light intensities. The similarity between the two conditions suggests that KEA3 is required for readjusting the ∆pH-dependent regulation immediately after any drastic change in light conditions.In this study, we characterized double mutants defective in the CET pathways and KEA3 to understand whether and how the synergy between CET and KEA3 in the regulatory network of photosynthesis affects this process. We focused on the contribution of NDH-dependent CET during the induction of photosynthesis after overnight dark adaptation in the kea3-1 mutant context. Based on our results, we propose a novel physiological function of the NDH complex: that of allowing flexibility of the regulatory network during the induction of photosynthesis.     

SOT1, a pentatricopeptide repeat protein with a small MutS‐related domain,is required for correct processing of plastid 23S–4.5S rRNA precursors in Arabidopsis thaliana

Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid‐localized pentatricopeptide repeat (PPR) protein with a small MutS‐related domain, is required for maturation of the 23S–4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5′ end of the 23S–4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA ‘footprint’ associated with this site in sot1 mutants. We found that more than half of the 23S–4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5′ and 3′ ends, and that the endonucleolytic cleavage product normally released from the 5′ end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5′ extremity of the 23S–4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5′ and 3′ ends.     

K stimulation of ATPase activity associated with the chloroplast inner envelope

Studies were conducted to characterize ATPase activity associated with purified chloroplast inner envelope preparations from spinach (Spinacea oleracea L.) plants. Comparison of free Mg²⁺ and Mg·ATP complex effects on ATPase activity revealed that any Mg²⁺ stimulation of activity was likely a function of the use of the Mg·ATP complex as a substrate by the enzyme; free Mg²⁺ may be inhibitory. In contrast, a marked (one- to twofold) stimulation of ATPase activity was noted in the presence of K⁺. This stimulation had a pH optimum of approximately pH 8.0, the same pH optimum found for enzyme activity in the absence of K⁺. K⁺ stimulation of enzyme activity did not follow simple Michaelis-Menton kinetics. Rather, K⁺ effects were consistent with a negative cooperativity-type binding of the cation to the enzyme, with the K_m increasing at increasing substrate. Of the total ATPase activity associated with the chloroplast inner envelope, the K⁺-stimulated component was most sensitive to the inhibitors oligomycin and vanadate. It was concluded that K⁺ effects on this chloroplast envelope ATPase were similar to this cation's effects on other transport ATPases (such as the plasmalemma H⁺-ATPase). Such ATPases are thought to be indirectly involved in active K⁺ uptake, which can be facilitated by ATPase-dependent generation of an electrical driving force. Thus, K⁺ effects on the chloroplast enzyme in vitro were found to be consistent with the hypothesized role of this envelope ATPase in facilitating active cation transport in vivo.     

Lidocaine and ATPase inhibitor interaction with the chloroplast envelope

Photosynthetic capacity of isolated intact chloroplasts is known to be sensitive to K⁺ fluxes across the chloroplast envelope. However, little is known about the system of chloroplast envelope proteins that regulate this K⁺ movement. The research described in this report focused on characterizing some of the components of this transport system by examining inhibitor effects on chloroplast metabolism. Digitoxin, an inhibitor of membrane-bound Na⁺/K⁺ ATPases, was found to reduce stromal K⁺ at a range of external K⁺ and inhibit photosynthesis. Scatchard plot analysis revealed a specific protein receptor site with a K_m for digitoxin binding of 13 nanomolar. Studies suggested that the receptor site was on the interior of the envelope. The effect of a class of amine anesthetics that are known to be K⁺ channel blockers on chloroplast metabolism was also studied. Under conditions that facilitate low stromal pH and concomitant photosynthetic inhibition, the anesthetic, lidocaine, was found to stimulate photosynthesis. This stimulation was associated with the maintenance of higher stromal K⁺. Comparison of the effects on photosynthesis of lidocaine analogs which varied in lipophilicity suggested a lipophilic pathway for anesthetic action. The results of experiments with lidocaine and digitoxin were consistent with the hypothesis that a K⁺ channel and a K⁺-pumping envelope ATPase contribute to overall K⁺ flux across the chloroplast envelope. Under appropriate assay conditions, photosynthetic capacity of isolated chloroplasts was shown to be much affected by the activity of these putative envelope proteins.     

Surface Charge-Mediated Effects of Mg on K Flux across the Chloroplast Envelope Are Associated with Regulation of Stromal pH and Photosynthesis

Studies of Spinacia oleracea L. were undertaken to characterize further how Mg²⁺ external to the isolated intact chloroplast interacts with stromal K⁺, pH, and photosynthetic capacity. Data presented in this report were consistent with the previously developed hypothesis that millimolar levels of external, unchelated Mg²⁺ result in lower stromal K⁺, which somehow is linked to stromal acidification. Stromal acidification directly results in photosynthetic inhibition. These effects were attributed to Mg²⁺ interaction (binding) to negative surface charges on the chloroplast envelope. Chloroplast envelope-bound Mg²⁺ was found to decrease the envelope membrane potential (inside negative) of the illuminated chloroplast by 10 millivolts. It was concluded that Mg²⁺ effects on photosynthesis were likely not mediated by this effect on membrane potential. Further experiments indicated that envelope-bound Mg²⁺ caused lower stromal K⁺ by restricting the rate of K⁺ influx; Mg²⁺ did not affect K⁺ efflux from the stroma. Mg²⁺ restriction of K⁺ influx appeared consistent with the typical effects imposed on monovalent cation channels by polyvalent cations that bind to negatively charged sites on a membrane surface near the outer pore of the channel. It was hypothesized that this interaction of Mg²⁺ with the chloroplast envelope likely mediated external Mg²⁺ effects on chloroplast metabolism.