Soil salinity is one of the most severe abiotic stresses affecting agricultural yield and productivity worldwide [1]. In plants, salinity induces ion imbalance, hyperosmotic stress, oxidative damage, and other subsequent disturbances. Plants have evolved sophisticated salt-responsive signaling and metabolic processes, including photosynthetic adjustment, ion exclusion, the synthesis of compatible products, the enhancement of reactive oxygen species (ROS) scavenging, phytohormone regulation, and cell structure modulation [2].

Chloroplasts are organelles for photosynthesis. Chloroplasts also participate in many other important metabolic processes, including the biosynthesis of amino acids, vitamins, isoprenoids, fatty acids, and lipids, as well as the reduction of nitrites and sulfates [3,4]. About 3000 distinct proteins are estimated to be localized to the Arabidopsis thaliana chloroplasts [5,6]. Most of these proteins are encoded by the nuclear genome, and less than 150 proteins are predicted to be encoded by the plastome [7]. Previous gene characterization studies have revealed a number of genes/proteins involved in various signaling and metabolic processes in chloroplasts [7,8]. However, the characterization of salinity-responsive genes encoding chloroplast-localized proteins is limited, although many genes/proteins have been proposed to be involved in salt tolerance using transcriptomic and proteomic approaches [9,10,11,12].

Over the past twenty years, only about 53 salt-responsive genes have been characterized that encode chloroplast-localized proteins from Arabidopsis, rice (Oryza sativa), maize (Zea mays), wheat (Triticum aestivum), beet (Beta vulgaris), cotton (Gossypium hirsutum), mangrove (Kandelia candel), tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), spinach (Spinacia oleracea), alkaligrass (Puccinellia tenuiflora), cowpea (Vigna unguiculata), pea (Pisum sativum), Thellungiella halophila, Mesembryanthemum crystallinum, Porteresia coarctata, Suaeda salsa, and Synechocystis sp. PCC 6803, respectively (Table 1). The proteins encoded by these salt-responsive genes are mainly involved in ROS scavenging, thylakoid membrane organization, photosystem II (PS II) activity, carbon dioxide (CO₂) assimilation, photorespiration, osmotic and ion homeostasis, abscisic acid (ABA) biosynthesis and signaling, and gene expression, as well as protein synthesis and turnover (Table 1). In this review, we summarize the genes encoding the chloroplast-localized proteins in response to salinity.

Chloroplasts are unique organelles due to their highly oxidizing metabolic activity and increased rate of electron flow, being especially prone to generating ROS, such as superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), and hydroxyl radical (OH•). The presence of ROS producing centers, such as triplet chlorophylls and the electron transport chain (ETC) in PS II and PS I, make chloroplasts a major site of ROS production [13]. Salinity stress enhances ROS production, leading to severe ROS-associated damage to chloroplasts. ROS seriously disrupt normal metabolism through oxidative damage to lipids, nucleic acids, and proteins, resulting in protein destruction and the peroxidation of membrane lipids [14]. Therefore, antioxidant enzyme systems and non-enzymatic antioxidants in chloroplasts offer greater protection from oxidative damage generated from salinity stress. Several salinity-responsive genes encoding antioxidant enzymes/proteins have been cloned (Table 1), which highlight specific ROS scavenging pathways in chloroplasts under salt stress.

Plants’ photosynthetic machinery respond to salt stress by regulating thylakoid membrane fluidity and remodeling membrane lipid composition, thus maintaining an environment suitable for the function of critical integral proteins during stress [69].

The modification of membrane fluidity is mediated by the changes in unsaturated fatty acid levels of thylakoid membranes. Fad6 encodes a chloroplast-localized ω-6 desaturase, which is a fatty acid desaturase that catalyzes the conversion of oleic acid (18:1) to linoleic acid (18:2) by inserting a double bond at the ω-6 position. The Arabidopsis fad6 mutant has an increased sensitivity to salt stress, implying that Fad6 is required for salt tolerance during early seedling development [36]. In addition, a chloroplast-localized glycerol-3-phosphate acyltransferase (GPAT) is one of the main factors that determine the content of cis-unsaturated fatty acids in the phosphatidylglycerol of thylakoid membranes (Figure 2A, Table 1) [37]. The overexpression of GPAT in tomato increased the cis-unsaturated fatty acid content of thylakoid membranes, and the transgenic plants exhibited higher activities of chloroplastic antioxidant enzymes, lower ROS contents, and a better photosynthetic performance, as well as an increased efficiency in alleviating PS II photoinhibition [37]. The increased unsaturation of fatty acids seems to enhance structural flexibility to the thylakoid membranes, which is favorable to thylakoid membrane binding to antioxidant enzymes for excess ROS scavenging [37]. Importantly, the unsaturation of fatty acids might boost the tolerance of PS II to salt stress by accelerating the repair of photodamaged D1. The PS II reaction center subunits D1 and D2 are enclosed by a belt of 11 lipids, which provides a flexible environment and fosters a high mobility of subunits. This would be beneficial to the degradation of damaged D1, the acceleration of de novo synthesis, and the insertion of the D1 protein [70]. However, the specific steps of the PS II repair that are regulated by the unsaturation of fatty acids still remain to be clarified.

Two galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactodiacylglycerol (DGDG), are major constituents of photosynthetic membranes in chloroplasts (Figure 2A, Table 1) [71]. MGDG has a conical shape with nonbilayer-forming characteristics, providing a high lateral pressure on the proteins embedded in the membrane. This permits the dense packing of membrane proteins, which facilitates the maintenance of the stability of the membrane structure. Furthermore, the non-bilayer lipid can easily segregate from the membrane and is crucial for the self-regulation of lipid content in the thylakoid membrane through the formation of osmiophilic lipid droplets in the chloroplasts. In addition, DGDG, a bilayer-prone lipid, is involved in lipid-mediated contact between adjacent trimers of the light harvesting complex II [71]. This is important for the stability of the lamellar structure of chloroplast membranes. Crystallization studies have revealed that MGDG was associated with the core of the reaction center of PS I, PS II, and the cytochrome b₆f complex, while DGDG was a component of PS I, PS II, and the light-harvesting complex of PS II [38,72,73]. Thus, they can provide a membrane environment to physically support the photosynthetic complex, and also contribute directly to various photosynthesis-related processes. The biosynthesis of the two galactoglycerolipids is catalyzed by MGDG synthase (MGD) and DGDG synthase, respectively [74]. A chloroplast outer envelope membrane-localized MGD encoding gene has been cloned in rice, and transgenic tobacco plants that overexpress OsMGD exhibited significantly higher levels of MGDG and DGDG, as well as higher DGDG/MGDG ratios than wild-type plants. Chloroplasts from salt-stressed OsMGD transgenic tobacco had well-developed thylakoid membranes and properly stacked grana lamellae, whereas the chloroplasts from salt-stressed wild-type plants were fairly disorganized and had large membrane-free areas [38]. Therefore, the increased levels of MGDG and DGDG could contribute to the organization of the plant photosynthetic membrane structure for the enhancement of salt resistance.

Some chloroplast-localized proteins are critical for the maintenance of PS II activity in response to salt stress. Rubredoxin (RUB) is a small, non-heme protein, which attaches to the thylakoid membranes and is exposed to the stroma, acting as an electron carrier in a variety of biochemical processes, such as the detoxification of ROS [75]. Mutant analysis of the RUB gene in the green alga Chlamydomonas reinhardtii suggests that RUB may be necessary for normal PS II activity in a diverse set of organisms that perform oxygenic photosynthesis. Knockout mutants of RUB orthologs in the cyanobacterium S. sp. PCC 6803 and Arabidopsis also imply that the activity and stability of PS II are specifically affected [76]. In addition, the overexpression of the PutRUB gene from alkaligrass in Arabidopsis increased the tolerance to NaCl and NaHCO₃ stress. This was probably due to decreasing H₂O₂ accumulation for chloroplast redox balance [39]. Similarly, a RCI gene encoding a chloroplast membrane protein has also been reported to be involved in the regulation of PS II activity. RCI is a homolog of a plasma membrane protein 3 family and contains two putative transmembrane domains. Arabidopsis plants overexpressing the wheat RCI performed better than the wild-type under salinity stress. Transgenic plants showed significantly higher PS II activity in terms of the maximum photochemical efficiency, and higher proline and chlorophyll contents than wild-type plants [40].

Under salt stress, the maintenance of stable CO₂ assimilation would be important to salinity tolerance. NADP⁺-dependent malate dehydrogenase (NADP-MDH) is responsible for the reduction of oxaloacetate (OAA) to malate in chloroplasts, which was suggested to be crucial for CO₂ fixation (Figure 2B, Table 1). In C3 plants, NADP-MDH is essential for the balance of reducing equivalents between chloroplasts and cytoplasm via the malate/oxaloacetate shuttle. In C4 plants, NADP-MDH is located exclusively in the mesophyll chloroplasts for C4-photosynthesis. NADP-MDH transcripts in the facultative Crassulacean acid metabolism (CAM) plant M. crystallinum significantly accumulated in response to salt stress. The salinity-induced expression of NADP-MDH suggests that CO₂ fixation is enhanced, which implies that the facultative, halophytic M. crystallinum shifts the photosynthesis carbon fixation mode from a C3 to a CAM in response to salinity [41]. In addition, the glyceraldehyde 3-phosphate dehydrogenase beta subunit (GAPB) is a key enzyme for the conversion of glycerate-3-phosphate (3-PGA) to glyceraldehyde-3-phosphate interacting with ATP and NADPH. 3-PGA can accept electrons from NADPH, preventing the ROS-induced deceleration of PS II repair (Figure 2B, Table 1). Arabidopsis overexpressing ThGAPB exhibited higher recycling rates of ADP and NADP⁺. This would reduce ROS production, contributing to the maintenance of photosynthetic efficiency under salinity conditions [42].

Additionally, salt stress-induced stomatal closure limits the CO₂ concentration in cells, which causes the over-reduction of photosynthetic ETC. Under such a condition, salinity-responsive ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) operates as an oxygenase, and photorespiration is activated in response to salt stress. Phosphoglycerate and CO₂ generated from photorespiration enter the Calvin cycle, and the consumption of NADPH and ATP may contribute to the dissipation of excess light energy or reducing power, for preventing the over-reduction of ETC. Interestingly, transgenic rice overexpressing a chloroplastic glutamine synthetase encoding gene GS2 increased their photorespiration capacity for improving salt tolerance (Figure 2B, Table 1) [43].

To withstand salinity-induced osmotic stress, plants synthesize and accumulate compatible solutes/osmoprotectants for stabilizing proteins, membranes, and even transcriptional and translational machineries in the cells. Major osmoprotectants include betaines, amino acids (e.g., proline), non-reducing sugars (e.g., trehalose and arabitol), and polyols [77]. Betaine is a quaternary ammonium compound found in a wide variety of plants, animals, and microorganisms [78]. Salinity-induced betaine accumulates in the chloroplasts of many halotolerant plants, stabilizing the quaternary structure of enzymes and protein complexes, as well as the highly-ordered structure of membranes in photosynthetic machinery [79]. For example, it can stabilize the Rubisco and PS II oxygen-evolving complex and accelerate the repair of photodamaged PS II under salt or other abiotic stress [80,81]. In plants, betaine is synthesized by several betaine-biosynthetic enzymes, such as choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) (Figure 2C, Table 1). Genes encoding CMO and BADH have been cloned and targeted to the chloroplast genome of various plant species (Table 1). In transgenic plants, the enzyme activities and betaine levels were increased, leading to an improved photosynthetic performance and enhanced tolerance to salt stress [44,45,46,47]. Importantly, trehalose and inositol also act as important osmolytes in chloroplasts, which can significantly enhance the salt tolerance of the transgenic plants. Chloroplast-localized trehalose-6-phosphate phosphatase (TPP) catabolizes trehalose-6-phosphate to generate trehalose, which is the final step of trehalose metabolism (Figure 2C, Table 1). Arabidopsis plants deficient in AtTPPD were hypersensitive, whereas plants overexpressing AtTPPD were tolerant to high salinity [48]. In addition, the co-expression of PcINO1 and McIMT1 in chloroplasts allowed the transgenic tobacco plants to perform better in terms of growth potential and photosynthesis rates with increased levels of myo-inositol and methylated inositol under salt stress [49].

Besides osmotic balance, ion homeostasis is also vital for plant cells to cope with salinity. Extra Na⁺ in chloroplasts destroys the thylakoid membrane structure, causing the inactivation of PS II and PS I and inhibiting the repair of photodamaged PS II, as well as decreasing photosynthesis electron transport, and therefore, the Na⁺ concentration in chloroplasts must be well-controlled. AtCHX23 encodes a putative Na⁺(K⁺)/H⁺ exchanger, which is localized in the chloroplast envelope and functions by putatively regulating the homeostasis of the cytoplasmic and stromal pH and Na⁺ concentration through the sequestering of Na⁺ entering into chloroplasts. Arabidopsis CHX23 mutants displayed a high sensitivity to NaCl [50]. Moreover, NHD1 encodes a sodium hydrogen antiporter, which is localized to the chloroplast envelope. Arabidopsis NHD1 functions as a chloroplast sodium exporter, protecting chloroplasts from deleterious Na⁺ accumulation after salt exposure. NHD1 T-DNA insertion mutants showed high Na⁺ levels in chloroplasts, resulting in a markedly impaired photosynthetic performance as revealed by a lower quantum yield of PS II and increased non-photochemical quenching [51]. In addition, several members of the putative K⁺-efflux antiporters (KEA), such as KEA1, KEA2, and KEA3, function as chloroplast K⁺/H⁺ antiporters for regulating the osmotic pressure, pH, and ion homeostasis of chloroplasts. KEA1 and KEA2 are chloroplast inner envelope membrane proteins, and function to release K⁺ from the chloroplasts in exchange for H⁺ influx, whereas KEA3 is a thylakoid membrane protein that uptakes K⁺ into the thylakoid lumen. Arabidopsis loss-of-function mutants of these three genes have been analyzed, and the higher-order mutants showed increasingly impaired photosynthesis, along with altered chloroplast pH homeostasis [52].

In addition, a chloroplast stroma-localized YL1 protein is an YqeH-type GTPase, involved in the regulation of Na⁺ delivery in response to salt stress. The expression of Arabidopsis YL1 was markedly reduced under high salinity. Shoots of the YL1 mutant accumulated significantly higher levels of Na⁺ than wild type under salt stress. The expression of Abscisic acid insensitive 4 (ABI4) was increased and high-affinity K⁺ transporter 1 (HKT1) was suppressed in the mutant shoots. HKT1, encoding a K⁺/Na⁺ symporter, is an important regulator that can directly retrieve Na⁺ from the xylem sap back to the phloem of the shoot and unload it in the root for ion homeostasis [82]. ABI4 has recently been reported to act as a negative regulator that could directly bind to the promoter region and inhibit HKT1 expression in Arabidopsis [83]. It is speculated that the reduction of YL1 and the induction of ABI4 may be an adaptive mechanism to achieve Na⁺ equilibrium in the entire plant, which needs to be further investigated [53].

The investigation of cellular ABA level fluctuation, ABA perception, and ABA-mediated signaling in response to salinity is pivotal for understanding plant stress tolerance. All except the last two steps of ABA de novo biosynthesis occur in plastids [84]. Most of the genes involved in ABA biosynthesis have been identified, among which, ABA1, ABA4, and NCED encode plastid-localized zeaxanthin epoxidase, neoxanthin synthase, and 9-cis-epoxycarotenoid dioxygenase, respectively (Figure 2D, Table 1) [54,55,85,86]. Salt stress has some influence on the cellular ABA content, and it was reported that AtABA1 and VuNCED1 were up-regulated by salt stress [54,55].

In addition, MDA1 encoding a transcription termination factor family protein, is likely required for a proper ABA response in chloroplasts. An Arabidopsis MDA1 mutant showed reduced sensitivity to ABA, enhanced salt tolerance, and some salt-responsive gene expression [56]. MDA1 deficit in plants may disrupt chloroplast homeostasis and negatively affect ABA retrograde signaling, which elicits the nuclear-encoded functions required for coping with salt stress [56]. Moreover, a gene encoding stroma-localized cold-responsive (COR) protein, COR15B, was up-regulated under salt stress in an ABA-dependent manner. Therefore, COR15B was proposed to be a potential member in the ABA signaling regulatory network in the Arabidopsis response to salt stress [57].

Protein kinases are major components of intracellular signal transduction, which mediate various signaling pathways that enable plant cells to rapidly acclimate and prevail in challenging environmental conditions. MsK4 is a plastid-localized novel alfalfa glycogen synthase kinase 3 like kinase, which was reported to be a novel signaling component in the regulation of starch metabolism and the salt stress response. Starch metabolism is highly sensitive to environmental changes and its accurate regulation is crucial for the adaptation of carbon and energy flow in response to stress conditions. Fluxes of carbon into and out of starch are extremely complex and must be highly controlled. Evidence is emerging that reversible protein phosphorylation is crucial for the regulation of starch-associated carbon metabolism, and the phosphorylation of starch metabolic enzymes has been shown to control their activities and protein complex formation. It has been found that MsK4 can bind to some isoforms of soluble and granule-bound starch synthases in vitro, suggesting that the enzymes involved in starch metabolism might be direct targets of the MsK4 action (Figure 2D, Table 1). High-salinity induced the activity of MsK4 kinase in alfalfa and Arabidopsis. Moreover, MsK4 overexpressing transgenic plants showed an enhanced salt tolerance and significantly increased starch content. This opens new perspectives as to how metabolic carbon flux can be regulated in response to salinity, and provides links between stress signaling and metabolic adaptation [58].

The salt-responsive gene expression of the chloroplast genome must be very precisely controlled, and the targeting of nuclear-encoded proteins into the chloroplasts is important in regulating chloroplast gene expression. Chloroplast gene expression is mainly regulated at the level of posttranscriptional RNA metabolism, including RNA processing, editing, splicing, decay, and translational control [87]. The functions of most RNA molecules rely on a well-defined three-dimensional structure, and the correct folding of RNA molecules requires the assistance of diverse RNA binding proteins (RBPs) [88]. A variety of nuclear-encoded RBPs are targeted to chloroplasts and play indispensable roles in the plant salt response through the posttranscriptional regulation of RNA metabolism and gene expression in chloroplasts, such as DEAD-box RNA helicases (RHs) and RNA-recognition motifs (RRMs)-containing proteins (Figure 2E, Table 1). Arabidopsis chloroplast-localized RH3 has been demonstrated to function in intron splicing and ribosome biogenesis. RH3 mutants displayed more sensitivity to salt stress and the splicing of several intron-containing chloroplast genes was inhibited [59]. Besides, Arabidopsis CRP1 and S-RBP11, two of the chloroplast-localized RRM containing proteins, were also shown to be involved in the salt stress response. Seed germination of the CRP1 mutant was delayed compared with that of the wild-type seeds under salt stress [60]. Similarly, transgenic Arabidopsis overexpressing S-RBP11 showed an increased salt tolerance, whereas S-RBP11 mutants were shown to be more sensitive to salt stress [61].

Moreover, protein synthesis, processing, and degradation in chloroplasts are also important for plant salt stress adaption (Figure 2E, Table 1). The expression of pea TufA, which encodes a chloroplast translation elongation factor (EF-Tu), was down-regulated in response to salinity [62]. EF-Tu is an essential component for polypeptide elongation during protein synthesis. It is reported that EF-Tu might have a chaperone-like property of refolding the denatured proteins or preventing their aggregation under heat stress [89].

Salt-induced excess excitation energy may cause the photodamage of PS II. The D1 protein is one of the core proteins in the PS II reaction center, which is the main target of oxidative damage. The de novo synthesis, assembly, and rapid degradation of the D1 protein are necessary for the efficient PS II repair in salt-stressed plants [63,90]. In cyanobacterium S. sp. PCC 6803, the transcription and translation of the D1 protein encoding gene psbA was decreased, and the repair of the photodamaged D1 protein was also inhibited under salt stress [63]. Moreover, a prokaryotic trypsin-type Deg/Htr serine protease (DegP2) was found to be vital for the photodamaged D1 protein degradation under salt stress [91]. DegP2 is peripherally associated with the outer surface of the thylakoid membrane, and involved in the primary cleavage of the photodamaged D1 protein on the stromal DE loop. The expression of Arabidopsis DegP2 was down-regulated under high salt stress (400 mM NaCl), while the amount of DegP2 protein was increased significantly under stress [64]. This would induce its molecular chaperone and proteolytic activities to enhance the repair of oxygen-evolving PS II in salinity-stressed plants.

Chloroplasts have evolved fine-tuned pathways for the salt response. In this paper, we reviewed and discussed the salt-responsive genes encoding chloroplast-localized proteins, which represent several crucial pathways in chloroplasts in response to salinity, such as chloroplast ROS scavenging (water–water cycle, stromal AsA-GSH cycle, Trx/Prx pathway, and non-enzymatic scavenging system), photosynthetic thylakoid membrane modulation, CO₂ assimilation, the synthesis of osmoprotectant, and ion homeostasis regulation, as well as chloroplast gene expression and protein turnover. These provide important molecular information for better understanding the chloroplast salt response, and allow us to expand our knowledge on the adaptation of this photosynthetic apparatus to salinity stress. However, the photosynthetic machinery and chloroplast metabolic pathways for salt tolerance are too complicated to be interpreted by the genes characterized so far. Although a large number of candidate genes/proteins have been identified in chloroplasts from plants under salt treatments using large-scale genomic, transcriptomic, and proteomic approaches [9,10,11,12,92], further investigations of their biological functions in salinity tolerance are needed. More importantly, a deeper analysis of the post-translational modifications and protein-protein interactions of these salt-responsive proteins will facilitate a thorough understanding of the complicated salt-responsive networks in chloroplasts.