Identification, cloning and expression analysis of chloroplast TOC-TIC complex components in a C₄ halophyte Suaeda aralocaspica without Kranz anatomy

Abstract

The translocon at the outer/inner chloroplast envelope membrane (TOC/TIC) complexes constitute the core machinery mediating the recognition and transmembrane translocation of nucleus-encoded precursor proteins, fulfilling essential functions in chloroplast biogenesis and photosynthesis. However, the TOC-TIC system remains poorly characterized in extremophytes, particularly in single-cell C₄ (SCC₄) halophytes such as Suaeda aralocaspica, which possess unique anatomical and physiological adaptations to saline and arid environments. In this study, we performed a genome-wide identification and comprehensive analysis of TOC-TIC components in S. aralocaspica. A total of 7 SaTOC and 19 SaTIC genes were identified, belonging to 4 and 11 distinct subfamilies, respectively. These genes exhibited conserved gene structures, protein motifs, and phylogenetic relationships with known homologs. Expression profiling analysis revealed that all SaTOC and SaTIC genes exhibited tissue-specific expression patterns. Notably, SaToc159, SaToc75, SaTic56, and SaTic110 were markedly upregulated during early germination, implying a potential role in seedling establishment. Most SaTOC and SaTIC genes were induced by light, while SaToc132 and SaToc34 displayed constitutive expression patterns. Furthermore, SaToc159, SaToc75, and SaTic100 were significantly upregulated in response to salt stress in germinating seedlings. Subcellular localization experiments confirmed that SaToc34, SaToc75, SaTic20-I, and SaTic56 are localized in the chloroplast. These findings provide the first systematic overview of TOC-TIC complex in a SCC₄ halophyte, establishing a foundation for elucidating chloroplast protein import mechanisms in plants adapted to extreme environments.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08640-5.

Background

Chloroplasts are indispensable organelles within plant cells, performing dual functions in photosynthesis and plant development [1]. Most chloroplast proteins are encoded in the nucleus and synthesized in the cytoplasm as precursor proteins before being imported into the chloroplast. This import is mediated by two multi-subunit translocon complexes: the translocon at the outer chloroplast membrane (TOC) and the translocon at the inner chloroplast membrane (TIC), which coordinate the translocation across the double-membrane envelope [2, 3]. The import machinery ensures the accurate recognition, targeting, and internalization of these nuclear-encoded precursor proteins, a process fundamental to chloroplast biogenesis and function. Moreover, this tightly regulated translocation process plays a critical role not only in chloroplast development but also in plant adaptive responses to environmental stresses [4, 5]. Dysregulation of protein import can compromise chloroplast function, thereby affecting overall plant growth and stress tolerance.

As the central component of the outer membrane transport system, the TOC complex has been extensively characterized in terms of its structure and functional mechanisms [6]. It has been suggested that Toc64 mediates initial substrate sorting by recognizing Hsp90-bound precursor proteins and transferring these substrates to the primary receptor Toc34 [7, 8]. The initiation of protein translocation requires the coordinated action of two homologous GTPases, Toc159 and Toc34, which form a precursor recognition interface on the chloroplast surface [9]. Toc75, a transmembrane channel protein, forms a protein-conducting pore in the outer membrane that facilitates precursor passage and directs substrates to the TIC complex, thereby initiating subsequent inner membrane transport events [10]. In contrast to the relatively well-defined TOC complex, the composition of the TIC complex has long been debated, with two principal theoretical models currently prevailing. The classical model proposes that the TIC complex consists of Tic110, Tic40, Tic22, and Tic20 [1], where Tic110 and Tic20 jointly form the transmembrane channel, and Tic40 and Tic22 act as chaperones to facilitate precursor protein folding. The revised model, a more recent framework, redefines the reconfigured core complex consisting of Tic20-I, Tic100, Tic56, and Tic214 [11]. Here, Tic20-I independently constitutes the translocation channel, Tic56 and Tic100 mediate substrate recognition, and Tic214 potentially participates in stabilizing complex assembly [12, 13]. Advancing experimental techniques have provided growing support for recent models. For instance, recent cryo-electron microscopy (cryo-EM) structural analyses of Arabidopsis thaliana and Chlamydomonas reinhardtii have confirmed that Tic20-I, Tic56, Tic100, and Tic214 form the core components of the 1-MDa TIC complex [12–15].

Current understanding of the TOC-TIC complexes function is mainly derived from studies on model plants. In Arabidopsis, systematic investigations have identified core components of this translocon, including the Toc159 family (AtToc159/132/120/90), Toc34 family (AtToc33/34), Tic20-I family (AtTic20-I/II/IV/V) and Tic110 [15–20]. Functional genetic studies reveal that mutations in AtToc159 and AtTic20-I impair chloroplast biogenesis and lead to an albino phenotype [21, 22], while complete knockout of AtToc75-III or AtTic110 results in embryonic lethality, confirming their indispensability for cell viability [19, 23]. In addition to chloroplast development, TOC-TIC components may also contribute critically to plant stress adaptation by regulating protein import and dynamic modification [5]. For instance, the expression of most Toc GTPase genes in tomato seedlings is upregulated under salt stress, which potentially promoting the import of stress-responsive proteins into chloroplasts, consequently enhancing salt tolerance [24]. The abundance of TOC complexes is precisely regulated by the ubiquitin-proteasome system (UPS) and autophagy. For instance, the E3 ubiquitin ligase SP1 mediates the degradation of TOC components to regulate stress responses: SP1 knockout lines exhibit increased sensitivity to salt, osmotic, and oxidative stresses, whereas overexpression lines display enhanced stress tolerance [5]. Furthermore, stress-induced K63-linked polyubiquitination triggers selective autophagy of TOC complexes via the autophagy adaptor NBR1, a pathway markedly induced under heat and UV-B stress [25]. These findings reveal how regulated degradation of TOC components modulates stress adaptation. However, the specific functions and adaptations of these complexes in non-model plants, particularly in desert-dwelling C₄ halophytes with unique photosynthetic mechanisms, remain largely unexplored. The recent availability of the Suaeda aralocaspica draft genome [26] now enables the first genome-wide identification and preliminary functional characterization of TOC-TIC components in this species.

Suaeda aralocaspica is an annual euhalophyte predominantly found in saline-alkaline deserts of Central Asia, noted for its exceptional environmental adaptability [26, 27]. Unlike Kranz-type C₄ plants, it performs single-cell C₄ (SCC₄) photosynthesis, which compartmentalizes both C₄ and C₃ cycles within a single chlorenchyma cell to achieve high photosynthetic efficiency through effective CO₂ concentration, while also conferring strong salinity and alkalinity tolerance [26–28]. Its chloroplast organization differs from that of other SCC₄ species such as Bienertia sinuspersici. In S. aralocaspica, dimorphic chloroplasts are arranged into distal and proximal compartments at the cell poles, whereas B. sinuspersici exhibits central and peripheral chloroplasts [9, 27–29]. The formation of these functionally distinct chloroplasts, analogous to the mesophyll and bundle sheath chloroplasts in Kranz-type plants, may rely on a precise protein sorting process to ensure nuclear encoded enzymes (such as PEPC, PPDK and NAD-ME, etc.) are correctly targeted to their respective chloroplast domains [9, 27, 30]. This suggests that the conserved TOC-TIC import complex in SCC₄ plants may not only serve as a general translocation gateway but could also act as a key regulator of selective protein import, potentially involving substrate-specific TOC isoforms that support plastid differentiation within the same cytoplasm [9, 27–32]. Given its unique integration of enhanced photosynthetic performance and superior stress resistance, S. aralocaspica serves as an ideal model for investigating the co-evolution of plant adaptive mechanisms. Consequently, this study aims to: (1) comprehensively identify TOC and TIC gene family members in the S. aralocaspica genome; (2) elucidate functional divergence among SaTOC and SaTIC isoforms through phylogenetic analysis, gene structure characterization, conserved motif identification, and subcellular localization prediction; and (3) clarify the roles of core SaTOC and SaTIC components in chloroplast development and their expression responses to light-dark transitions. The findings of this study are expected to advance our understanding of the coordinated mechanisms underlying photosynthetic enhancement and stress resistance in SCC₄ plants, thereby providing a theoretical foundation for crop genetic improvement.

Results

Identification and characterization of TOC and TIC gene family in S. aralocaspica

In this study, we systematically identified 7 SaTOC and 19 SaTIC transporter family members from the S. aralocaspica genome via a genome-wide identification approach. All identified members were annotated using a standardized nomenclature based on their Arabidopsis homologs. A comprehensive analysis of their physicochemical properties revealed significant variations within the SaTOCs and SaTICs family. Sequence lengths ranged from 310 (SaToc34) to 1284 (SaToc159) amino acids (aa), molecular weights spanned from 34.52 kDa (SaToc34) to 137.97 kDa (SaToc159), and theoretical isoelectric points (pI) varied from 4.68 (SaToc159) to 9.64 (SaToc34). Notably, SaToc159 and SaToc132/120 were classified as acidic proteins (pI < 7), SaToc90 as a neutral protein (pI ≈ 7), and the remaining members as alkaline proteins. The SaTICs family displayed sequence lengths from 201 aa (SaTic40) to 2145 aa (SaTic236), molecular weights ranging from 22.78 kDa (SaTic40) to 244.28 kDa (Tic100), and pI values ranging from 4.17 (SaTic40) to 10.16 (SaTic21). This family comprised six acidic proteins (SaTic40, SaTic56, SaTic62, SaTic100, SaTic110, SaTic236) and thirteen basic proteins. Detailed physicochemical parameters and structural features of these family members are systematically presented in Table 1 (SaTOC family) and Table 2 (SaTIC family).

Table 1.

Characteristics of TOC gene family in S. aralocaspica

Gene name	Genome ID	ORF (bp)	pI	MW (kDa)	Size (aa)	Subcellular location
SaToc34	GOSA_00020359-RA	933	9.64	34.52	310	Chloroplast/Cytoplasm
SaToc75	GOSA_00003239-RA	2454	7.66	89.94	817	Chloroplast
SaToc64-1	GOSA_00022171-RA	1845	9.26	69.85	614	Chloroplast/Nucleus
SaToc64-2	GOSA_00009650-RA	1908	8.69	66.79	635	Chloroplast/Nucleus
SaToc159	GOSA_00021403-RA	3855	4.68	137.97	1284	Chloroplast/Nucleus
SaToc90	GOSA_00011037-RA	2436	7.02	89.59	811	Chloroplast
SaToc132/120	GOSA_00020407-RA	3759	4.74	135.07	1252	Nucleus

Table 2.

Characteristics of TIC gene family in S. aralocaspica

Gene name	Genome ID	ORF (bp)	pI	MW (kDa)	Size (aa)	Subcellular location
SaTic20-I	GOSA_00002538-RA	801	8.95	30.29	266	Cell membrane/Chloroplast
SaTic20-II	GOSA_00007545-RA	669	10.14	24.77	222	Cell membrane
SaTic20-IV	GOSA_00000037-RA	786	9.72	30.38	261	Chloroplast/Nucleus
SaTic20-V	GOSA_00017334-RA	648	9.30	24.41	215	Cell membrane
SaTic21	GOSA_00005529-RA	861	10.16	30.23	286	Cell membrane/Nucleus
SaTic22	GOSA_00012127-RA	726	9.59	26.55	241	Nucleus
SaTic22-III	GOSA_00012823-RA	864	9.03	32.24	287	Cell membrane/Chloroplast
SaTic32-I	GOSA_00001180-RA	948	8.84	34.53	315	Chloroplast
SaTic32-II	GOSA_00006494-RA	948	8.91	34.27	315	Chloroplast
SaTic32-III	GOSA_00006496-RA	984	8.45	36.29	327	Chloroplast
SaTic32-IV	GOSA_00010696-RA	996	8.81	36.20	331	Chloroplast
SaTic32-V	GOSA_00026612-RA	987	9.50	36.10	328	Chloroplast
SaTic40	GOSA_00000704-RA	606	4.17	22.78	201	Nucleus
SaTic55	GOSA_00014654-RA	1665	8.45	62.74	554	Chloroplast
SaTic56	GOSA_00001460-RA	1539	5.67	60.57	517	Chloroplast/Cytoplasm/ Cell membrane
SaTic62	GOSA_00022408-RA	1530	4.40	54.73	510	Nucleus
SaTic100	GOSA_00009445-RA	6423	4.96	244.28	2140	Chloroplast
SaTic110	GOSA_00003019-RA	4146	5.46	152.91	1381	Chloroplast
SaTic236	GOSA_00025567-RA	6438	5.73	236.17	2145	Chloroplast/Cytoplasm

Phylogenetic analysis of SaTOC and SaTIC proteins in S. aralocaspica

Phylogenetic analysis of SaTOC and SaTIC proteins across multiple species was conducted using the neighbor-joining method to reveal their evolutionary relationships. The TOC protein phylogenetic tree (Fig. 1) showed that SaTOC family members share the closest phylogenetic affinity with Bienertia sinuspersici (BsTOCs). SaTOC members were classified into four subfamilies: I (SaToc75), II (SaToc64/OEP64), III (SaToc34), and IV. Subfamily IV are homologs of Toc159 and were further subdivided into SaToc159, SaToc132/120, and SaToc90, a pattern consistent with that reported in tomato [33]. Based on phylogenetic analysis with characterized TIC homologs from species including Arabidopsis thaliana, all identified SaTIC members clustered into distinct evolutionary clades, each with a bootstrap support value of no less than 70%. On this basis, the SaTIC family members were preliminarily classified into 11 subfamilies (Fig. 1). Subfamily I (SaTic32) containing five members, while 8 subfamilies-II (SaTic110), III (SaTic56), IV (SaTic40), V (SaTic100), VI (SaTic236), VII (SaTic62), X (SaTic21), and IX (SaTic55)-each comprised a single member. These results demonstrate significant evolutionary conservation of the SaTOC and SaTIC complexes and suggest potential functional divergence among subfamilies.

Fig. 1.

Phylogenetic analysis of TOC (A) and TIC (B) components of the chloroplast protein import machinery. A The TOC tree includes proteins from Arabidopsis thaliana (At), Bienertia sinuspersici (Bs), Chlamydomonas reinhardtii (Cr), Pisum sativum (Ps), Solanum lycopersicum (Sl), and Suaeda aralocaspica (Sa), with subfamilies I to IV indicated by distinct colored backgrounds. B The TIC tree includes proteins from Arabidopsis thaliana (At), Chlamydomonas reinhardtii (Cr), Pisum sativum (Ps), Suaeda aralocaspica (Sa), and Vitis vinifera (Vv), with subfamilies I to XI indicated by distinct colored backgrounds. In both trees, all proteins from S. aralocaspica (Sa) are highlighted in red font. A phylogenetic tree was constructed using the neighbor-joining method with the Jones-Taylor-Thornton model assuming uniform rates. Branch support was assessed with 1,000 bootstrap replications, and all other parameters were set to default values

Gene structure and conserved motif analysis of SaTOCs and SaTICs

Gene structure analysis revealed significant divergence in exon-intron architecture and conserved motif distribution between the SaTOC and SaTIC family members. The SaTOC genes exhibited intron numbers ranging from 0 to 12 (corresponding to 1–13 exons), with most members containing 1–7 introns. All SaTOC genes contain UTR regions; however, SaToc159 possesses only the 5’-UTR. Motif profiling identified twelve conserved motifs, with motifs 2 and 5 present in all members (Fig. 2A-C).

Fig. 2.

Phylogenetic relationships, conserved motifs, and gene structures of the SaTOC and SaTIC gene families in S. aralocaspica. A The neighbor-joining phylogenetic tree of SaTOC protein sequences. B Distribution of 12 conserved motifs (1–12) in SaTOC proteins, with motif sizes estimated using the scale at the bottom. C Exon-intron organization of SaTOC genes, where grey lines indicate introns, yellow boxes represent exons, and green boxes denote untranslated regions (UTRs). Intron phases are marked as 0, 1, or 2. D The neighbor-joining phylogenetic tree of SaTIC protein sequences. E Distribution of 12 conserved motifs (1–12) in SaTIC proteins, with motif sizes estimated using the scale at the bottom. F Exon-intron organization of SaTIC genes, where grey lines indicate introns, yellow boxes represent exons, and green boxes denote untranslated regions (UTRs). Intron phases are marked as 0, 1, or 2

The SaTIC family displayed greater structural diversity compared to SaTOC (Fig. 2D-F), with intron numbers varying from 0 to 23 (corresponding to 1–24 exons). Most SaTIC members contained 1–16 introns, and complete UTR regions (5’- and 3’-UTRs) were identified in all SaTIC genes. Analysis of conserved motif identified 12 motifs, with motif 5 conserved across all members. Most SaTIC members contained the highly conserved motifs 4 and 11, while Subfamily I uniquely possessed motifs 2 and 3. SaTic20-IV contained the fewest motifs among all family members. These results indicate that although the core motifs are highly conserved, forming the fundamental framework for their function, the composition and quantity of motifs vary among different subgroups, revealing their distinct functional specificities. Such functional divergence is likely driven by the addition, deletion, and specific combinations of subfamily-specific motifs.

Cis-acting regulatory element analysis of SaTOC and SaTIC promoters

Cis-acting element analysis was performed on the 2,000 bp promoter regions (upstream of the initiation codons) of SaTOC and SaTIC genes using the PlantCARE database. The results revealed that distinct compositions of functional cis-elements were observed between SaTOC and SaTIC families, reflecting their potential functional divergence in response to environmental and hormonal signals. In the SaTOC family (Fig. 3A), 14 categories of functional elements were distributed across 7 genes. The most abundant were light-responsive elements (6 types, including Box 4, I-box, GT1-motif, GATA-motif, L-box and G-Box), followed by hormone-responsive elements [ABA (ABRE), JA (TGACG/CGTCA-motif), IAA (AuxRR-core), GA3 (P-box/GARE), and SA (TCA)] and abiotic stress-responsive elements (MBS, LTR) (Fig. 3C). Notably, SaToc64-1 uniquely contained cis-elements related to circadian regulation and zein metabolism. In contrast, SaToc132 contained the highest number of cis-elements among all SaTOC members, indicating a potential core role in integrating hormonal signals and regulating stress resistance.

Fig. 3.

Analysis of cis-acting elements in the promoters of SaTOC and SaTIC genes in S. aralocaspica. A Cis-acting elements in SaTOC gene promoters. B Cis-acting elements in SaTIC gene promoters. C Statistical count of cis-acting elements in each SaTOC/SaTIC gene promoter. Numbers indicate the quantity of each element type presented

Compared to the SaTOC family, the SaTIC family contained a more diverse set of functional elements, which classified to 18 categories (Fig. 3B). Consistent with SaTOC, light-responsive elements were the most abundant in SaTIC promoters (including elements such as Box 4 and I-box), reflecting the conserved association of TOC/TIC complexes with photosynthetic regulation. Notably, the SaTIC family also exhibited specialized enrichment of development-related regulatory elements, highlighting its unique role in plant developmental programming. For instance, SaTic32-III contained cell cycle elements (MSA-like), SaTic32-IV had seed-specific expression elements (RY-element), SaTic32-I had mesophyll differentiation elements (HD-Zip 1), and several SaTIC members (e.g., SaTic20-II, SaTic21 and SaTic32-IV) contained endosperm expression elements (GCN4_motif) (Fig. 3C). Compared to SaTOC promoters, SaTIC promoters exhibited an expanded regulatory capacity in terms of tissue specificity and developmental staging, with a higher proportion of elements linked to organogenesis and cellular differentiation. These findings suggest functional divergence between the two gene families. Specifically, SaTOC family genes appear to coordinate plant growth and stress adaptation by responding to light, hormone, and stress signals, whereas SaTIC family members are more deeply involved in cellular differentiation and organ developmental precision.

Protein association network predictions and functional classification of SaTOCs and SaTICs

Based on the STRING interaction network prediction generated using Arabidopsis orthologous proteins as references (Fig. 4A), a total of 118 interaction relationships were identified among 26 SaTOC/TIC proteins (comprising 7 SaTOC and 19 SaTIC components), with an average of ~ 4.7 interactions per protein, reflecting a highly interconnected functional network. Among these, SaToc34, SaToc75, SaToc159, SaTic110, SaTic20-I, SaTic100, SaTic56, and SaTic40 formed a highly interconnected core in the interaction network. 3D conformational prediction of the core complex assemblies was further conducted using AlphaFold3. The results confirmed a highly compatible physical interaction interface between SaToc159 and SaToc75, notably, the transmembrane domain of SaToc159 is tightly associated with the β-barrel channel of SaToc75, supporting the formation of a functional heteromeric channel complex (Fig. 4B). For the TIC complex, both the classical (Fig. 4C) and revised (Fig. 4D) models are predominantly composed of α-helical structures, which confer greater structural flexibility in the membrane environment than β-barrel structures. This intrinsic property may enable dynamic conformational rearrangements of the TIC complex across different stages of preprotein translocation, thereby maintaining channel stability while providing the structural plasticity required to support the sophisticated process of chloroplast protein import [12, 13]. This observation further supports their putative functional roles as central components of the TOC-TIC translocon machinery, which is consistent with their conserved functions in chloroplast protein import across plant species.

Fig. 4.

Protein association network predictions and functional classification of SaTOC/SaTIC proteins. A Protein-protein interaction network of SaTOC and SaTIC in S. aralocaspica according to orthologs in Arabidopsis. Line and node colors indicate the different types and degrees of interactions, respectively. B AlphaFold3-based structure prediction of the core SaTOC complex. C AlphaFold3-based structure prediction of the classical model of the core SaTIC complex. D AlphaFold3-based structure prediction of the revised model of the core SaTIC complex. E, F Gene Ontology (GO) annotation of SaTOC and SaTIC proteins according to the three categories biological process, cellular component, and molecular function

GO annotation analysis revealed clear subcellular localization and functional specialization of SaTOC/SaTIC proteins (Fig. 4E, F), the 7 identified SaTOC components in S. aralocaspica were annotated as structural constituents of the chloroplast outer membrane (GO:0009707), while the 19 SaTIC components were annotated as structural constituents of the chloroplast inner membrane (GO:0009706), aligning with the known membrane localization of TOC/TIC complexes. Regarding biological processes, both groups of components are primarily involved in establishment of protein localization to chloroplast (GO:0072596), confirming their core function in mediating nuclear-encoded chloroplast protein import. KEGG pathway enrichment analysis revealed distinct functional biases between the two families. SaTOC-associated genes were significantly enriched in amino acid metabolic pathways (e.g., arginine, tryptophan, and proline metabolism; ko00330, ko00380), which may be linked to the processing or post-translational modification of precursor proteins before their translocation into target organelles. In contrast, SaTIC components were mainly implicated in signal transduction processes within the chloroplast (Supplementary Fig. S1).

Subcellular localization of SaTOC and SaTIC proteins

Predictions of subcellular localization indicated that the SaTOC and SaTIC proteins exhibited distinct compartmentalization patterns. SaTOC family members were predominantly localized to chloroplasts and the nucleus (Table 1). Furthermore, SaToc75 and SaTic20-I were predicted to contain a chloroplast transit peptide and transmembrane domains, as shown by TargetP and DeepTMHMM analyses (Supplementary Table 2). In contrast, SaTIC proteins were enriched in chloroplasts, cell membrane, and the nucleus (Table 2), reflecting their potential multifunctional roles across diverse subcellular compartments. To experimentally validate these predictions, two key components from the TOC family (SaToc34 and SaToc75) and two from the TIC family (SaTic20-I and SaTic56) were transiently expressed as GFP fusions in tobacco epidermal cells. In the empty vector control (35 S::eGFP), fluorescence signals were diffusely distributed throughout the cytoplasm, plasma membrane and nucleus. SaToc34, SaToc75, SaTic20-I and SaTic56 localized specifically to chloroplast (Fig. 5B). Overall, these experimentally determined localization patterns were consistent with the predictions generated by the Plant-mPLoc and Wolf PSORT online tools.

Fig. 5.

Subcellular localization of SaTOC and SaTIC proteins in tobacco epidermal cells. A Schematic diagram of recombinant vector construction; B Subcellular localization of SaTOC and SaTIC proteins in tobacco epidermal cells. DAPI, nuclear marker control; GFP, green fluorescent protein; Autofluorescence, chloroplast autofluorescence (shown as magenta). Scale bar = 25 μm. eGFP signal was excited at 488 nm with emission collected at 510–550 nm; chloroplast autofluorescence was excited at 640 nm and detected at the emission wavelength of 650–738 nm

Spatial and temporal expression patterns of SaTOC and SaTIC genes

To enable a global evaluation of SaTOC and SaTIC gene expression levels, based on available RNA-Seq data, the spatiotemporal expression profiles of SaTOC and SaTIC genes were analyzed across seed germination stages (dry, imbibed, and seedlings) and various tissues (leaf, fruit, stem, root, and mixed tissues) [27]. For the SaTOC gene family, SaToc34 and SaToc75 maintained consistently high expression levels across all the tissues and developmental stages, with peak expression during the seed and seedling phases, suggesting their critical roles in regulating chloroplast biogenesis and plant growth throughout early development. SaToc64-2 displayed tissue-specific enrichment in roots and stems, indicating its functional specialization in non-photosynthetic organs. SaToc132/120 maintained stable expression in all tested samples, reflecting its constitutive roles in basal biological processes. Notably, SaToc90, SaToc159, and SaToc64-1 showed significantly lower expression levels throughout all developmental stages and tissues, with the expression of SaToc90 approaching the detection threshold. Overall, SaTOC family genes exhibited pronounced spatiotemporal specificity in their expression patterns, and the majority displayed active transcriptional states during seed development and early seedling establishment (Fig. 6A, C).

Fig. 6.

RNA-Seq expression analysis of SaTOC and SaTIC genes in different tissues and different developmental stages. A SaTOC genes expression in different tissues and dimorphic seeds during germination. B SaTIC genes expression in different tissues and dimorphic seeds during germination. C SaTOC genes expression in leaves at different developmental stages. D SaTIC genes expression in leaves at different developmental stages. The color scale is shown at the right and higher expression levels are represented in red

Turning to the SaTIC gene family (Fig. 6B, D), SaTic56, SaTic110, SaTic22, and SaTic32-I displayed high expression across all seed germination stages and all examined tissues, suggesting their broad regulatory roles in developmental processes. Seven other SaTIC genes (SaTic22-III, SaTic100, SaTic32-V, SaTic40, SaTic20-II, SaTic20-I, and SaTic21) were also widely expressed throughout developmental stages and tissues, but their transcripts were predominantly enriched in mixed tissues, leaves, and fruits. Expression of SaTic20-IV was suppressed in roots and black dry seeds but markedly activated during other developmental stages. SaTic32-IV displayed specific upregulation in brown-seeded seedlings, while SaTic32-II transcripts preferentially accumulated in leaves and mixed tissues. SaTic55 and SaTic62 showed an expression polarity that favored mixed tissues, leaves, and fruits, with almost no detectable transcripts in black seeds. Both SaTic20-IV and SaTic236 displayed extremely low expression levels across all samples, leading to speculation that they have minimal involvement in developmental regulation. Collectively, SaTIC family members were ubiquitously and actively expressed throughout developmental progression and in photosynthetically active tissues (i.e., leaves), highlighting their pivotal roles in plant growth and development.

Transcriptomic analysis of S. aralocaspica leaves at different developmental stages revealed that most SaTOC genes were significantly more highly expressed in young leaves than in mature leaves, while SaToc132/120 expression remained stable. Among SaTIC genes, SaTic236, SaTic20-IV and SaTic32-IV maintained low expression levels throughout leaf development, whereas six SaTIC genes, including SaTic20-I, SaTic56, SaTic32-II, SaTic32-V, SaTic40 and SaTic21, exhibited relatively high expression. Comprehensive analysis suggested that several SaTOC and SaTIC gene are highly expressed in young leaves (Fig. 6), which may reflect their role in supporting active chloroplast biogenesis, as this process requires the import of numerous proteins. This hypothesis is supported by previous findings in Arabidopsis, where protein import components such as AtToc34 also show strongly expressed in young tissues containing undifferentiated plastids, likely due to the high demand for protein import in developing plastids [34].

Analysis of S. aralocaspica RNA-Seq data from root and shoot tissues under 0, 500 and 1000 mM NaCl stress revealed tissue-specific expression patterns among SaTOC and SaTIC gene family members. However, our preliminary observations indicate that their expression did not show a significant response to increasing salt concentrations. Within the SaTOC family, SaToc34 and SaToc132 exhibited constitutively high expression levels in both roots and shoots under all salinity levels. In contrast, SaToc64-2, SaToc159 showed markedly higher transcript abundance in shoots than in roots. Meanwhile, SaToc90 and SaToc64-1 maintained stable expression under all tested conditions (Fig. 7). The expression of the SaTIC gene family exhibited distinct tissue specificity (Fig. 7). Within this family, seven genes, namely SaTic56, SaTic20-I, SaTic110, SaTic62, SaTic32-V, SaTic40, and SaTic21, showed pronounced shoot-specific expression. Other members displayed varied patterns. SaTic20-IV was barely detectable across the tissues examined. In contrast, both SaTic236 and SaTic32-III were expressed stably in all tissues analyzed.

Fig. 7.

RNA-Seq expression analysis of SaTOC and SaTIC genes in different tissues under various (0, 500, 1000 mM) NaCl concentrations. A SaTOC genes expression in root. B SaTOC genes expression in shoot. C SaTIC genes expression in root. D SaTIC genes expression in shoot. The color scale is shown at the right and higher expression levels are represented in red

Developmental stage-dependent expression patterns of SaTOC and SaTIC genes

To elucidate the functional roles of SaTOC and SaTIC genes in S. aralocaspica development, this study selected core components of the TOC complex (SaToc159, SaToc132/120, SaToc34, and SaToc75) for expression analysis. For the TIC complex, given the lack of clear consensus regarding its core composition, the key representative genes from both the classical model (e.g., SaTic110) and the revised model (e.g., SaTic20-I, SaTic56, and SaTic100) were included. The expression patterns of these selected genes across different developmental stages of S. aralocaspica were subsequently analyzed using qRT-PCR.

During seed germination, the majority of SaTOC and SaTIC genes exhibited up-regulated expression, whereas SaToc34 showed a continuous decline in transcript levels as germination progressed. Specifically, SaToc159, SaToc75, SaTic110, and SaTic56 displayed a consistent and significant increase in transcript levels, peaking at either day 2 or day 7 of germination. Notably, SaToc159 demonstrated the most substantial up-regulation, with an approximately 10-fold increase in expression compared to dry seeds. These findings suggest that these genes may respond to the demand for cotyledon development and chloroplast biogenesis during early germination by participating in the transport of chloroplast-related proteins (Fig. 8A). During the pot-cultured developmental stages, the expression levels of most genes (e.g., SaToc159, SaToc75, and SaTic56) progressively increased with development, peaked at 15 days after potting, and subsequently declined. Among them, SaToc75, SaTic56, and SaTic110 exhibited a rebound in expression during the later developmental stage at 60 days. In contrast, SaTic20-I, SaTic100, and SaToc34 maintained consistently low expression levels throughout the entire developmental process (Fig. 8B).

Fig. 8.

qRT-PCR analysis of expression patterns of 4 SaTOC and 4 SaTIC candidate genes in S. aralocaspica at different developmental stages. A Expression profiles during seed germination stages. B Expression profiles in potted plant at different developmental phases. C Expression profiles in potted plant at different leaf lengths. Different lowercase letters above the bars indicate statistically significant differences (P < 0.05) in expression levels across developmental stages for the same gene. Data are presented as mean ± SD of three biological replicates (n = 6). Statistical significance was determined by one‑way ANOVA followed by Tukey’s HSD post hoc test (for homogeneous variances) or Welch’s ANOVA followed by the Games‑Howell post hoc test (for heterogeneous variances)

To systematically dissect the spatiotemporal expression dynamics of SaTOC/SaTIC genes and their potential roles in leaf development, qRT-PCR was performed to analyze the transcriptional profiles of these genes across a continuous developmental gradient of S. aralocaspica leaves. Given that leaves are the primary photosynthetic organs and chloroplast biogenesis is tightly coupled with leaf development, leaves of different lengths (3, 6, 9, and 15 mm) were selected to represent distinct developmental stages: 3 mm (young leaf primordia), 6 mm (blade expansion stage), 9 mm (rapid growth stage), and 15 mm (mature leaf stage). This analysis revealed distinct stage-specific expression patterns for key SaTOC/SaTIC genes (Fig. 8C). The genes SaToc159 and SaToc75 showed peak expression during the blade expansion stage (at leaf lengths of 6 mm) and rapid growth stage (at leaf lengths of 9 mm), indicating their pivotal roles in initiating leaf morphogenesis. In contrast, SaTic110 exhibited high expression levels at leaf lengths of 9 mm and 15 mm. However, SaToc34 expression gradually decreased during leaf elongation, while SaTic56 exhibited an opposing upregulation trend. Additionally, SaTic100 displayed a transient expression peak in 6 mm leaves, followed by a decline, suggesting a phase-specific function during blade expansion or early chloroplast differentiation.

Overall, these results demonstrate that SaTOC and SaTIC genes exhibit dynamic spatiotemporal regulation across S. aralocaspica developmental phases. Their coordinated upregulation during seed germination (2 to 7 days post-imbibition) likely facilitates chloroplast biogenesis through enhanced protein trafficking.

Differential expression patterns of SaTOC and SaTIC genes under varied light intensities and salt stress

To investigate the regulatory roles of the SaTOC and SaTIC gene family in leaf photosynthetic responses, the expression patterns of its members under different light intensities were examined by qRT‑PCR (Fig. 9A). The results showed that most genes (e.g., SaToc159, SaTic20‑I, SaTic56, SaTic100, and SaTic110) displayed a typical light‑inducible expression pattern. Their transcript levels increased in a dose‑dependent manner with rising light intensity from 0 to 200 μmol·m⁻²·s⁻¹, and peaking at 200 μmol·m⁻²·s⁻¹. In contrast, SaToc132/120 and SaToc34 expression remained stable across all light conditions, while SaToc75 maintained basal expression under low to medium light intensities (0-100 μmol·m⁻²·s⁻¹) but were significantly upregulated specially under high light intensity (200 μmol·m⁻²·s⁻¹).

Fig. 9.

Expression analysis of four SaTOC and four SaTIC candidate genes in S. aralocaspica under different conditions. A Light intensity-responsive expression patterns in seedlings. B Salt stress-responsive expression patterns in seedlings treated with different NaCl concentrations. Different lowercase letters indicate statistically significant differences (P < 0.05) among treatment groups within each panel for the same gene. Data are presented as mean ± SD of three biological replicates (n=6). Statistical significance was determined by one‑way ANOVA followed by Tukey’s HSD post hoc test (for homogeneous variances) or Welch’s ANOVA followed by Games-Howell post hoc test (for heterogeneous variances)

To further explore the response of this gene family to salt stress and assess potential spatiotemporal specificity, we analyzed the expression of SaTOC/TIC gene family members in germinating seedlings of S. aralocaspica under different NaCl concentrations via qRT-PCR. The results showed that the expression of SaToc159, SaToc75, and SaTic100 was progressively upregulated with increasing salt concentration, peaking at 300-500 mM NaCl (Fig. 9B), suggesting their potential involvement in protein transport under salt stress. In contrast, the expression of the remaining genes (SaToc132, SaToc34, SaTic20‑I, SaTic56, and SaTic110) did not exhibit a significant transcriptional response to salt stress.

Discussion

The TOC-TIC complex serves as the central channel for importing nucleus-encoded proteins into chloroplasts [19], a process pivotal to chloroplast biogenesis, functional establishment, and plant adaptive responses to environmental stresses [4, 5, 19, 35]. Recent cryo-EM studies in C. reinhardtii have revealed its architecture as a multi-subunit assembly spanning the outer and inner envelope membranes [12, 13]. However, current understanding of the characteristics of the TOC-TIC family is largely derived from Arabidopsis [15, 22, 34, 36], and the functional adaptation of this essential translocon machinery in non-model plants remains incompletely explored. Suaeda aralocaspica, a desert halophyte endowed with a unique SCC₄ photosynthetic system and remarkable tolerance to extreme stresses [26, 27], is hypothesized to depend on the precise regulation of chloroplast function to achieve its highly efficient photosynthesis. Nevertheless, the mechanisms underlying its unique photosynthetic metabolism and stress adaptation remain largely unknown. To identify the components and expression patterns of the TOC-TIC complex in S. aralocaspica, our study performed a genome‑wide identification and comprehensive analysis of this translocon system, providing a foundation for understanding chloroplast protein import in non-Kranz C₄ plants and offers new insights into the adaptive strategies of halophytes.

In the present study, we identified 7 TOC components and 19 TIC components in S. aralocaspica. In comparison, tomato has been reported to contain 10 TOC components [33]. Similar to tomato and Arabidopsis, the Toc GTPases in S. aralocaspica are classified into two subfamilies: Toc159 and Toc34 [33, 34, 36, 37]. Within the Toc159 subfamily, multiple isoforms including SaToc159 and SaToc132/120 were identified. This diversification within the Toc159 subfamily, which is also present in tomato and the closely related SCC₄ plant B. sinuspersici, suggesting its universal and essential regulatory role in chloroplast protein import across diverse plant species [9, 33, 37]. However, a notable distinction was observed in the Toc34 subfamily. Unlike Arabidopsis possesses two functionally divergent members AtToc34 and AtToc33, and tomato contains SlToc34-1 and SlToc34-2 [33, 34, 37], only one SaToc34 homolog was identified in S. aralocaspica. This genomic pattern is consistent with that reported in B. sinuspersici [9]. This finding suggests that SaTOC-SaTIC system may have undergone evolutionary simplification or functional consolidation, possibly as an adaptation to its specialized SCC₄ photosynthetic physiology [38].

Subcellular localization predictions combined with transient expression assays in tobacco indicated that most SaTOC/SaTIC components are predominantly targeted to chloroplasts (Fig. 5), which is consistent with observations in Arabidopsis and B. sinuspersici [9, 17, 39–41]. However, transient expression systems are susceptible to experimental artifacts such as protein overexpression or saturation [42], which may distort subcellular distribution, particularly critical for core translocon components (e.g., Toc75, Tic20-I) and putative precursor recognition factors (e.g., Toc34, Tic56) whose precise localization is essential for deciphering early regulatory steps in protein import [12–15]. Given the unique cellular architecture of the SCC₄ photosynthetic system, validation in a physiologically relevant native context is imperative. Therefore, further validation using B. sinuspersici protoplast assay remains necessary to determine the precise subcellular localization of individual SaTOC/SaTIC components [9, 43]. This approach is strongly justified by the close phylogenetic affinity, shared SCC₄ leaf anatomy, and highly conserved chloroplast ultrastructure, including dimorphic chloroplast differentiation and envelope membrane organization, between S. aralocaspica and B. sinuspersici (both Amaranthaceae) [9, 40, 43]. Such a homologous system may offer a biologically authentic platform to resolve the spatial dynamics of the SaTOC-SaTIC machinery and elucidate its functional regulation within the specialized SCC₄ cellular environment.

Cis-acting elements in promoter regions represent a key regulatory mechanism for plants to respond to environmental signals [44]. In tomato, SlTOC promoters are enriched with light-responsive elements as well as various hormone- and stress-responsive elements [33]. Similarly, analysis of SaTOC/SaTIC promoters identified abundant cis-acting elements associated with development, light signaling, hormones regulation, and stress response (Fig. 3), suggesting their potential functions in these processes. Notably, compared to the SaTOC family, the SaTIC family exhibits broader regulator potential in tissue-specific and developmental stage-dependent processes, and its promoters contain a greater abundance of relevant cis-elements linked to tissue specificity, development, and mesophyll differentiation (e.g., HD-Zip 1 and RY elements). Although current studies have not directly elucidated the specific role of the HD-Zip 1 promoter element in mesophyll cell differentiation, this element has been annotated in multiple plant species as associated with palisade tissue cell differentiation [45]. Given that SCC₄ photosynthesis relies on the coexistence of two functionally distinct chloroplast types within a single chlorenchyma cell, we propose that HD-Zip1 may contribute to this unique cellular differentiation program. The enrichment of such regulatory motifs implies that the SaTIC family has evolved more sophisticated transcriptional control to meet the complex demands of SCC₄ photosynthesis. Consequently, these specific results must be considered preliminary, and future improvements in genome assembly [26] will be necessary to validate the regulatory element predictions.

By integrating transcriptomic profiling with qRT-PCR validation, this study systematically characterized the expression patterns of SaTOC and SaTIC family members in S. aralocaspica across diverse tissues, developmental stages, and light conditions. Our analyses revealed that multiple core components exhibit pronounced tissue-specific and developmental stage-dependent expression, suggesting functional differentiation within the chloroplast protein import machinery. For example, SaToc34 and SaTic100 showed higher expression levels at the seedling stage or in young leaves (Fig. 8), likely to meet the demand for efficient protein import during early chloroplast biogenesis, a phase characterized by active chloroplast proliferation and photosynthetic apparatus assembly. In contrast, SaToc159 and SaTic20-I were predominantly expressed in photosynthetic shoot tissues (Fig. 7), with SaToc159 reaching highest expression level in mature leaves (Fig. 8), and displaying strong induction by light. These patterns closely mirror those of their Arabidopsis homologs AtToc159 and AtTic20-I [21, 39, 46, 47]. Notably, SaToc132 exhibited constitutive expression unaffected by light intensity and remained relatively stable in both roots and stems (Fig. 9), consistent with the expression characteristics of AtToc132 in Arabidopsis and BsToc132 in B. sinuspersici [9, 39, 40]. Functional studies in model plants provide critical context for these observations. In Arabidopsis, AtToc159 likely acts as a key import receptor for photosynthesis-related precursor proteins [36, 39, 47] Its loss-of-function mutant ppi2 exhibits an albino phenotype accompanied by defective photosynthesis and severe disruption of chloroplast lipid biosynthesis [22]. Moreover, biochemical and yeast two-hybrid (Y2H) assays in Arabidopsis and tomato have demonstrated substrate specificity between the Toc159 and Toc132 subfamilies: Toc159 shows higher affinity for photosynthesis-related precursors, whereas Toc132 preferentially binds non-photosynthetic precursors [48, 49]. Based on existing knowledge from these model plants and the expression differences observed in this study, we integrate scattered speculations regarding the functional specialization of SaTOC/SaTIC components into a clear and testable hypothesis. Specifically, we propose that S. aralocaspica possesses two functionally distinct chloroplast protein import modules. The first is a constitutive “basic transport platform”, composed of SaToc132/120 and SaToc34, which likely ensures the stable translocation of non-photosynthetic, housekeeping proteins, thereby maintaining essential cellular functions irrespective of light conditions. The second is a light-inducible “photosynthesis-specialized platform” likely centered on SaToc159 and SaTic20-I, which efficiently imports photosynthesis-related preproteins and may be specifically adapted to support the unique SCC₄ photosynthetic system of S. aralocaspica [11, 21, 22, 39, 50, 51]. On the other hand, SaToc64-1 exhibited low expression levels across all tissues. Although not considered a core component of the translocon machinery [52], it may serve as an initial receptor for Hsp90-associated precursors, assisting in their delivery to the main receptor Toc34. Therefore, these low-abundance members likely play auxiliary regulatory roles under specific conditions or for specific substrates [7, 8]. To test these functional hypotheses, future studies could employ Y2H and bimolecular fluorescence complementation (BiFC) assays to systematically validate the substrate specificity of SaTOC/SaTIC components, complemented by spatial validation via in situ hybridization. Additionally, structural biology approaches such as cryo-EM could resolve the high-resolution spatial architecture of the SaTOC-SaTIC complex in S. aralocaspica, thereby deepening our understanding of its molecular and structural regulatory mechanisms.

Moreover, expression heatmap analysis revealed that most SaTOC/SaTIC genes displayed no obvious transcriptional changes in roots and leaves of young plants treated with 500–1000 mM NaCl (Fig. 7), consistent with observations in Arabidopsis where TOC gene expression remains stable under stress [5]. To rigorously test whether this gene family participates in salt stress responses, we further performed qRT-PCR validation using seedlings at the germination stage (Fig. 9). Notably, SaToc159, SaToc75, and SaTic100 were significantly upregulated under salt stress, displaying expression trends similar to those of Toc GTPase genes in tomato seedlings. These findings suggest that specific members may mediate the import of stress-related proteins into chloroplasts during early establishment to promote stress tolerance [24]. We therefore hypothesize that the functional window of the SaTOC-SaTIC family is concentrated primarily in the vulnerable phase spanning seed germination to early seedling development, followed by transcriptional quiescence during subsequent growth stages. However, the observed “transcriptional insensitivity” from bulk-tissue RNA-seq does not preclude the existence of cell type-specific expression patterns or rapid, transient transcriptional responses that are readily obscured by tissue-level averaging. Therefore, future studies should employ single-cell RNA sequencing to decipher cell type-specific regulatory mechanisms and to capture rapid transient responses through dynamic time-course experiments. On the other hand, accumulating evidence indicates that the activity of the TOC/TIC complex is predominantly regulated at the post-translational level. For instance, the ubiquitin‑proteasome system (UPS) mediates the degradation of TOC proteins via E3 ligases [5], which may help limit chloroplast import under stress to reduce oxidative damage. Additionally, polyubiquitination, selective autophagy [25], and endogenous chloroplast proteases like the Clp system [53–55] contribute to the turnover and homeostasis of the import apparatus. These mechanisms may supplant transcriptional regulation in young plants, emerging as the core strategy for maintaining chloroplast functionality. In summary, we propose an integrated model in which S. aralocaspica employs a stage-specific strategy to combat salinity stress: it may rely on transcriptional activation of SaTOC/SaTIC genes during the seedling stage to ensure the import of critical proteins, while transitioning to an energy-efficient, post-translational regulatory mode during later growth phases. This dual regulatory paradigm is pivotal for balancing growth and stress resilience in extreme environments. Nevertheless, this study has certain limitations. Direct monitoring of SaTOC/SaTIC protein dynamics was not performed, and the specific contributions of autophagy or ubiquitination pathways remain to be conclusively validated. Future work should integrate Western blot analyses to assess protein abundance and employ autophagy inhibitors or proteasome inhibitors to systematically verify the contributions of post-translational regulation under salt stress. Such approaches will be essential for elucidating the full regulatory network and biological significance of SaTOC-SaTIC complex in salinity adaptation.

Conclusions

In this study, a total of 7 SaTOC and 19 SaTIC components were identified in Suaeda aralocaspica. Biochemical characterization of SaTOC and SaTIC proteins was performed, and based on conserved motifs, intron-exon structures, and phylogenetic analysis, the SaTOC and SaTIC families were classified into 4 and 11 subfamilies, respectively. Protein association network predictions revealed interactive relationships between SaTOC and SaTIC components, suggesting the formation of a supercomplex. Cis-element analysis identified numerous promoter elements related to light, hormones, and growth in both SaTOC and SaTIC genes. Transcriptomic and qRT-PCR analyses indicated that SaTOC and SaTIC genes are expressed in a tissue-specific manner during growth and development and respond transcriptionally to varying light conditions. Most SaTOC and SaTIC proteins were predicted to localize to chloroplasts, supporting their crucial roles in chloroplast function. The bioinformatic and transcriptomic data generated in this study establish a foundational resource for elucidating the composition and expression patterns of the SaTOC-SaTIC system in S. aralocaspica.

Materials and methods

Plant materials and treatments

Mature seeds of Suaeda aralocaspica were harvested from dry inflorescences of naturally grown individuals in October 2023. These plants were located in the Gurbantunggut Desert (Wujiaqu 103 regiment; 44°37′N, 87°26′E; 423 mH), Xinjiang Uygur Autonomous Region, China. After air-drying and the removal of impurities, seeds were stored at 4 °C until subsequent use. For germination assays, 100 seeds were evenly placed in 12-cm Petri dishes containing two layers of moist filter paper. Germination proceeded at 25 °C under a 16-h light/8-h dark photoperiod in an RXZ-5000 C growth chamber (Ningbo Jiangnan Instrument Factory, China). Samples were collected at 0 h (dry seed control), 8 h, 12 h, 24 h, 2 d, 5 d, and 7 d after imbibition. For pot cultivation experiments, 50 seeds were sown per treatment, and whole plants were harvested at 3 d, 15 d, 30 d, and 60 d after the initiation of pot cultivation. Light intensity gradient experiments (0, 20, 100, 200 µmol·m⁻²·s⁻¹) were performed under identical culture conditions, with sampling at 7 d. A leaf developmental series was established by collecting intact leaves at successive growth stages, defined by blade lengths of 3 mm, 6 mm, 9 mm, and 15 mm. Different salt stress treatments were applied by adding 20 mL of NaCl solutions at varying concentrations (0, 100, 300, and 500 mM) onto filter paper in Petri dishes. After 7 days of germination, entire seedlings with uniform growth status were collected. All samples were flash-frozen in liquid nitrogen immediately after collection and stored at − 80 °C. Three biological replicates were included for each treatment condition.

Sequence retrieval and identification of TOC and TIC genes in S. aralocaspica

To identify the TOC and TIC complex components in S. aralocaspica, a homology-based screening strategy was implemented using BLASTN and BLASTP alignment tools (E-value < 1e-5), with the known TOC complex components of Arabidopsis thaliana (https://www.arabidopsis.org/) as references (Supplementary Table 1). Genomic, protein sequence, and GFF3 files of S. aralocaspica were obtained from the GigaScience Giga DB database (http://gigadb.org/dataset/100646). Protein domains were annotated using the Pfam database (http://pfam.xfam.org/), and the corresponding protein sequences were extracted using TBtools [56]. The online CD-search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and SMART database (http://smart.embl-heidelberg.de/) were used to verify the presence of conserved TOC/TIC domains in the candidate proteins of S. aralocaspica. Sequence homology was further confirmed using NCBI BLASTP (E-value < 1e-5). Putative TOC/TIC components in S. aralocaspica were identified following manual curation to remove redundant sequences. The ExPASy ProtParam tool (https://web.expasy.org/protparam/) was used to predict the amino acid (aa) length, theoretical molecular weight (MW), and isoelectric point (pI) of the candidate proteins. Open reading frames (ORFs) of putative SaTOC and SaTIC genes were identified using the NCBI ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). Predictions of the plastid targeting signal and transmembrane domains were performed using TargetP (version 2.0) (https://services.healthtech.dtu.dk/services/TargetP-2.0/) and DeepTMHMM (version 1.0.5) ( http://www.cbs.dtu.dk/services/TMHMM/ ), respectively (Supplementary Table 2).

Phylogenetic analysis of TOC and TIC proteins in S. aralocaspica

To investigate the phylogenetic relationships of TOC and TIC proteins in S. aralocaspica, we performed evolutionary analyses using MEGA 11 [57]. The full-length amino acid sequences of putative SaTOC/TIC proteins were aligned via the ClustalW program with default parameters [57, 58]. A neighbor-joining (NJ) phylogenetic tree was constructed using homologous proteins from multiple species (Supplementary Table 1), including S. aralocaspica (Sa), Arabidopsis thaliana (At), Bienertia sinuspersici (Bs), Chlamydomonas reinhardtii (Cr), Pisum sativum (Ps), Solanum lycopersicum (Sl), and Vitis vinifera (Vv), with the Jones-Taylor-Thornton (JTT) model and Uniform Rates, 1,000 bootstrap replications, and other parameters at default values. The resulting Newick-format tree file was visualized and annotated using iTOL v.7.(https://itol.embl.de/).

Analysis of gene structures, conserved motifs, and cis-regulatory elements in SaTOC and SaTIC genes

The exon-intron structures of TOC and TIC genes in S. aralocaspica were analyzed and visualized using TBtools. Conserved protein motifs were identified using the MEME Suite v5.5.2 (https://meme-suite.org/meme/tools/meme) with the maximum number of motifs set to 12, and the results were exported in XML format. Promoter sequences (2000 bp upstream of the ATG start condon) of SaTOC and SaTIC genes were extracted using TBtools, and cis-acting elements were subsequently identified through the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). An integrated visualization of gene structures (Supplementary Table 3, 4, 5), conserved motifs, and promoter cis-regulatory elements was generated and arranged according to the topological order of the previously constructed phylogenetic tree.

Protein association network predictions and functional classification

The predicted TOC and TIC protein sequences of S. aralocaspica were analyzed using the STRING web server (http://string-db.org) under default parameters to construct a homologous gene network depicting functional interactions with A. thaliana. Genes with no predicted interactions were excluded from subsequent analysis. Conformational prediction of the complex was performed using the online AlphaFold3 server (https://alphafoldserver.com). The amino acid sequences of the proteins were submitted as input parameters to the prediction system, and complex modeling was conducted using default parameters. AlphaFold3 automatically generated five models (Model_0–4), and based on the confidence score (pLDDT value), Model_0 was selected as the final structure for visualization and analysis. The predicted results were visualized for three-dimensional structure and annotated for interaction sites using the open-source software PyMOL. Functional annotation of all identified SaTOC and SaTIC proteins was performed using the eggNOG-mapper online platform (http://eggnog-mapper.embl.de/). These annotations were subsequently visualized and analyzed with TBtools software [56], leading to comprehensive Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations.

Expression profile analysis of SaTOC and SaTIC genes in S. aralocaspica

The RNA-seq datasets of S. aralocaspica, which include dimorphic seeds during germination and various tissues were retrieved from the NCBI Sequence Read Archive (SRA; accession code: SRP128359) [26, 27]. Additionally, the transcriptome data of S. aralocaspica under salt stress were also incorporated (Liang ZC: Unraveling the stress response mechanisms in the halophyte Suaeda aralocaspica through transcriptome and metabolome analysis, unpublished). Raw reads were aligned to the S. aralocaspica reference genome using HISAT2 with default parameters. Transcript assembly and expression quantification were subsequently conducted with StringTie. Gene expression levels were normalized as fragments per kilobase of exon per million mapped reads (FPKM). To stabilize variance across expression values, FPKM measurements were log₂-transformed after adding a pseudocount of 1 [log₂(FPKM + 1)]. Differential gene expression patterns of SaTOC and SaTIC genes were visualized using the HeatMap module in TBtools. Hierarchical clustering of the differential expression data was performed using Euclidean distance and the complete linkage method.

Quantitative Real-Time PCR (qRT-PCR) analysis

Total RNA was isolated from S. aralocaspica using the FOREGENE Plant RNA Isolation Kit (Cat. No.RE-05021, FOREGENE, China) following the manufacturer’s instructions. cDNA was synthesis using the FOREGENE RT Easy™ II reverse transcription kit (Cat. No. RT-01022, FOREGENE, China) and stored at − 20 °C for later use. qRT-PCR was performed on a LineGene 9600 Real-Time PCR system (Model FQD-96 A, BIOER, China) in 20 µL reaction mixtures containing 1 µL cDNA template, 0.8 µL each of forward and reverse primers (Supplementary Table 6), 10 µL 2×Real PCR Easy™ SYBR Green I Mix (Cat. No. QP-01012, FOREGENE, China), 0.4 µL 50×ROX Reference dye, and 7 µL ddH₂O. The thermal cycling protocol began with an initial denaturation at 94 °C for 3 min, followed by 40 cycles of 94 °C for 10 s and 60 °C for 30 s. The β-Tubulin gene was employed as the reference gene for normalization [59]. Relative expression levels were calculated using the 2^−ΔΔCT method, where ΔΔCT = ΔCT_targetsample − ΔCT_{control sample}, and ΔCT_sample = CT_{test gene} − CT_{reference gene} [60]. The experiment included three biological replicates, each with two technical replicates (n = 6), and results are presented as the mean ± standard deviation (SD).

Determination of subcellular localization of SaTOC and SaTIC proteins

To validate the subcellular localization predictions for S. aralocaspica TOC/TIC proteins generated by Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) and Wolf PSORT (https://www.genscript.com/wolf-psort.html), four key TOC/TIC components (SaToc34, SaToc75,SaTic20-I, and SaTic56) were experimentally verified using a tobacco epidermal cell transient expression system. The ORFs of SaTOC and SaTIC genes without the termination codon were cloned into the pCAMBIA1300-MCS-eGFP plant expression vector. Recombinant plasmids were introduced into Agrobacterium tumefaciens strain GV3101 via the freeze-thaw method transformation method. Positive transformants were cultured in YEP medium supplemented with kanamycin (50 mg·L⁻¹), gentamicin (50 mg·L⁻¹), and rifampicin (50 mg·L⁻¹) at 28 °C with shaking at 220 rpm until the OD₆₀₀ value reached 0.8-1.0. Bacterial cells from 15 mL of culture were harvested by centrifugation at 5,000 rpm for 10 min and resuspended in infiltration buffer (10 mmol·L⁻¹ MES, 10 mmol·L⁻¹ MgCl₂, 150 µmol·L⁻¹ acetosyringone) to an OD₆₀₀ of 0.8. The recombinant Agrobacterium suspension (A) was mixed with a suspension of A. tumefaciens GV3101 harboring pCAMBIA1300-P19 (B) (expressing P19 protein to suppress gene silencing) at a volume ratio of 450 µL (A): 300 µL (B). This mixture was infiltrated into the abaxial epidermis of leaves from 5 to 6-week-old Nicotiana benthamiana, avoiding major veins. After infiltration, plants were kept in dark overnight and then transferred to normal growth conditions for 72 h. Finally, fluorescence signals were detected and imaged using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss, Jena, Germany). The eGFP signal was excited at 488 nm with emission collected at 510–550 nm, while chloroplast autofluorescence was detected using an excitation wavelength of 640 nm and an emission wavelength of 650–738 nm.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA) for data visualization and SPSS 26.0 (IBM Corp., Armonk, NY, USA) for statistical computations. Data that met the homogeneity of variance assumptions were analyzed by one-way ANOVA, followed by Tukey’s honest significant difference (HSD) post hoc test for multiple comparisons, with a significance level set at α = 0.05. When the assumption of homogeneity of variance was violated, Welch’s ANOVA with Games-Howell post hoc testing was applied [61].

Authors’ contributions

JC and RY designed the experiments and methodology. JC and RY wrote the manuscript. RY, MZ, XL, XY and FX conducted the experiments and collected the data. JC and RY analyzed the data. All authors contributed critically to the manuscript and gave final approval for publication.

Funding

This research was supported by the National Natural Science Foundation of China (Grant no. 32360066).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

All authors read and approved the manuscript.

Consent for publication

All authors agreed to publish this manuscript.

Competing interests

The authors declare no competing interests.