The mature seeds of S. aralocaspica were harvested from dry inflorescence of natural plants growing in the Gurbantunggut desert at Wujiaqu 103 regiment (44°37′N, 87°26′E; 423 mH) in October 2014, in the Xinjiang Uygur Autonomous Region, China. Seeds were air-dried indoor and cleaned and then stored at 4°C in a sealed brown paper bag. The brown seeds can germinate within 3 h upon contact with water, whereas the black seeds germinate much slower (Wang et al., 2008; He et al., 2013). However, the descendants from dimorphic seeds present no significant difference in morphological and physiological characteristics as well as gene expression patterns (Cao et al., 2015). Therefore, in this study, brown seeds were used in all the experiments.

To identify the potential members of PEPC gene family in S. aralocaspica genome, firstly, the amino acid sequences of the four PEPCs in Arabidopsis thaliana were used as a query to conduct a local BLASTP search by a cut-off E-value of 1 × 10^–5 (Supplementary Table 2). Subsequently, the Hidden Markov Model- (HMM-) based profile of the PEPCase domain (PF00311) obtained from the Pfam database¹ was used to verify the candidates of PEPC gene homologs by HMMER² and SMART³ searches. Finally, the candidates of PEPC homologs were further validated in the presence of a PEPC family domain (IPR021135), a lysine active site (IPR018129), and a histidine active site (IPR033129) on the InterProScan website⁴.

Multiple alignments were performed by FL amino acid sequences using the ClustalW program of MEGA X with the default settings (Kumar et al., 2018). The phylogenetic tree of PEPC proteins from 27 plant species was constructed using the unrooted neighbor-joining method of MEGA X with the following parameters: Poisson correction, pairwise deletion, and a bootstrap analysis with 1,000 replicates. The amino acid sequences of the other 26 representative species (Monocots: Brachypodium distachyon, Oryza sativa, Panicum virgatum, Setaria italica, Sorghum bicolor, and Zea mays; Dicots: A. thaliana, Arachis hypogaea, Brassica rapa, Chenopodium quinoa, Glycine max, Gossypium raimondii, Linum usitatissimum, Manihot esculenta, Medicago truncatula, Phaseolus vulgaris, Populus trichocarpa, Ricinus communis, and Solanum lycopersicum; Pteridophytes: Selaginella moellendorffii; Bryophyte: Physcomitrella patens; Photosynthetic algae: Chlamydomonas reinhardtii, Coccomyxa subellipsoidea, Micromonas pusilla, Ostreococcus lucimarinus, and Volvox carteri) were acquired from the Phytozome database⁵. Supplementary Table 2 provides a detailed description of the abovementioned proteins and their corresponding accession numbers.

For gene structure analysis, the exons and introns of PEPC genes were identified due to the alignment of cDNA sequences with the corresponding genomic DNA sequences and were illustrated using the GSDS 2.0 server⁶. The MEME program⁷ was employed to identify and analyze the conserved motifs of PEPC proteins with default parameters, and the maximum number of motifs to be detected was set as 10. The cis-regulatory elements in the promoter sequences (2,500 bp upstream of the start codon) of PEPC genes were identified using the PlantCARE database⁸. The Mfold RNA/DNA folding program⁹ was used to predict the secondary structure of a 5′-untranslated region (5′-UTR) of PEPC genes. The MEME and PlantCARE results were visualized using the TBtools software (Chen et al., 2020). Meanwhile, the theoretical molecular weight (MW), isoelectric point (pI), and grand average of hydropathicity (GRAVY) of PEPC candidates were predicted using the ExPASy website¹⁰.

Plant-mPLoc¹¹ and YLoc+¹² websites were used to predict the subcellular localization of candidate PEPCs in S. aralocaspica, which were further verified by a transient expression system in tobacco epidermal cells. The open reading frame (ORF) sequence of SaPEPC-1 or SaPEPC-2 (with the stop codon deletion) was fused to an enhanced green fluorescent protein (eGFP) ORF, and then inserted into the plant binary expression vector pCAMBIA1300, which resulted in the construct 35S::SaPEPCs-eGFP. The primers used for vector construction are presented in Supplementary Table 3.

The abovementioned recombinant vectors were transformed into Agrobacterium tumefaciens strain GV3101 through a CaCl₂ method. The correct single colony was inoculated in a YEB medium (50 mg⋅L^–1 kanamycin, 50 mg⋅L^–1 gentamicin, and 50 mg⋅L^–1 rifampicin) and cultivated with a shaking speed of 220 rpm at 28°C till the OD₅₉₅ value reached the range of 0.8–1.0. Then, 2 ml of cultures were removed for centrifugation at 12,000 rpm for 2 min to collect cells, which were then resuspended in an infiltration buffer (10 mmol⋅L^–1 MES, 10 mmol⋅L^–1 MgCl₂, and 150 μmol⋅L^–1 acetosyringone) at a final concentration of OD₅₉₅ = 0.8. A. tumefaciens suspension (A) of the abovementioned constructs was evenly mixed with 35S::CBL-RFP/GV3101 (B) [Calcineurin B-like protein 1 (CBL1), located on the plasma membrane, used as control] (Batistic et al., 2010) and 35S::P19/GV3101 (C) (P19 protein: promoted protein expression) suspensions with a volume ratio of 450 μl (A): 300 μl (B): 300 μl (C); for SaPEPC-2, 35S::ABI5-BFP/GV3101 (D) [abscisic acid insensitive 5 (ABI5), located in the nucleus, used as control] (Bensmihen et al., 2005) was also included in a volume ratio of 450 μl (A): 300 μl (B): 300 μl (C): 300 μl (D) of A. tumefaciens suspension. The mixture was held at room temperature for 2–3 h in the dark before use. About 5- to 6-week-old Nicotiana benthamiana plants were prepared for infiltration. The tip end of a syringe (without a needle) is placed against the underside of the leaf (in avoidance of the veins) with one finger supporting on the upper side, then gently pressing the syringe to infiltrate A. tumefaciens mixture into the fresh leaf and labeled the infiltration area for further recognition. The treated plants were held in the dark overnight, and were then transferred to the normal growth conditions for another 48 h. The fluorescent signals in the leaf of N. benthamiana were examined and photographed using the Zeiss LSM 800 confocal microscope (Carl Zeiss, Jena, Germany).

A series of 5′-deletions of SaPEPC-1 and SaPEPC-2 promoters were generated according to the predicted sites of light-response elements (Supplementary Figure 1). FL of each promoter was truncated into six fragments and labeled as fragments 1–6 in an order from the smaller to the larger upstream of ATG. Seven specific upstream primers (Ppc-FLF and Ppc-F1 to F6) and a single downstream primer (Ppc-FLR) were designed for each fragment in a SaPEPC promoter. In addition, another downstream primer Ppc-R_TSS was designed to combine with the upstream primer Ppc-F6 to amplify the 5′-flank regions from the transcription start site (TSS), which was labeled as TSS (Supplementary Table 3). Following the digestion with endonucleases HindIII and BamHI, the CaMV35S promoter sequence was replaced by the abovementioned fragments in the plant expression vector pBI121 to drive β-glucuronidase (GUS) gene. The recombinant constructs were transformed into A. tumefaciens strain EHA105 for the transient expression test in N. benthamiana, the manipulation was similar to that in the determination of subcellular localization except that the suspension of each construct was not necessary to mix with 35S::CBL1/GV3101, 35S::ABI5/GV3101, and 35S::P19/GV3101 strains. Treated tobacco plants were exposed to normal illumination (500 μmol⋅m^–2⋅s^–1) for 3 days in a growth chamber, and the darkness group was placed in the dark cabinet to avoid light until sampling. A GUS fluorometric assay was performed according to Jefferson et al. (1987). All leaves were ground in liquid nitrogen and homogenized in 1.0 ml of the freshly prepared GUS extraction buffer [200 mmol⋅L^–1 NaH₂PO₄, 200 mmol⋅L^–1 Na₂HPO₄, 500 mmol⋅L^–1 ethylenediaminetetraacetic acid (EDTA), 0.1% (v/v) Triton X-100, 0.1% (v/v) β-mercaptoethanol, and 10% (w/v) sodium dodecyl sulfate (SDS)]. After centrifugation at 12,000 rpm, 4°C for 15 min, the supernatant was employed to determine the GUS activity using 4-methylumbelliferyl glucuronide (4-MUG) as a substrate. The fluorescence of 4-methylumbelliferone (4-MU) produced by GUS-catalyzed hydrolysis was measured by the FLx800^TM Fluorescence Reader (BioTek, Winooski, VT, United States). The protein concentration of the supernatant was assessed by the method of Bradford (1976) using bovine serum albumin (BSA) as the standard. GUS activity was normalized to the protein concentration of each supernatant extract and calculated as pmol of 4-MU per microgram of soluble protein per minute.

Gene expression profile was analyzed based on public released data. Published gene expression data sets in different tissues (matured leaves, stems, roots, and fruits) of S. aralocaspica were downloaded from the NCBI (SRA: SRP128359; BioProject: JNA428881) (Wang et al., 2019). The RNA sequencing (RNA-Seq) data sets of dimorphic seeds in the germination of S. aralocaspica were obtained from the BioProject of PRJNA325861 (Wang et al., 2017). Gene expression levels were estimated by the fragments per kilobase of exon per million mapped reads (FPKM) values using the Cufflinks software (Trapnell et al., 2012). The heatmap was generated using the TBtools software (Chen et al., 2020), the color scale represents FPKM counts, and the ratios were expressed as log2 transformed.

To validate the transcriptomic data, we performed a quantitative real-time PCR (qRT-PCR) to analyze the expression of SaPEPC-1 and SaPEPC-2 genes. Total RNA was extracted from the collected plant samples using the E.Z.N.A.^® Plant RNA Kit (Cat. R6827, OMEGA, Norcross, GA, United States) according to the instructions of the manufacturer. RNA conc. and absorbance ratios (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀) were measured using the NanoDrop^® ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). Each reverse transcription reaction was performed with 1 μg of total RNA in a final volume of 20 μl using the M-MLV RTase cDNA Synthesis Kit (D6130, TaKaRa, Shiga, Japan) with a 2.5 μmol⋅L^–1 oligo (dT) primer following the instructions of the manufacturer. cDNA was stored at −20°C until use. qRT-PCR was carried out using GoTaqR^® qPCR Master Mix (Promega, Madison, WI, United States) in the GeneAmp^® 7500 Real-Time PCR System (ABI, Vernon, CA, United States). Gene-specific primers of SaPEPC-1 and SaPEPC-2 were designed using the Primer-Blast tools¹³ (Supplementary Table 3). To ensure the amplification of the desired product, a melt-curve analysis was performed to determine that only a single peak was present to represent a unique PCR product as per the MIQE guidelines (Bustin et al., 2009). Standard curves were generated for each primer to assess efficiency, and all primers had a value of efficiency between 1.9 and 2.1 (Supplementary Data). β-tubulin gene of S. aralocaspica was used as an internal reference (Cao et al., 2016). The reaction mixture consisted of 1 μl cDNA samples, 0.5 μl each of the forward and reverse primers (10 μmol⋅L^–1), 10 μl GoTaqR^® qPCR master mix, and 8 μl nuclease-free H₂O in a final volume of 20 μl. qRT-PCR was performed as follows: 2 min initial denaturation at 95°C, followed by 40 cycles at 95°C for 15 s, and 60°C for 1 min. Four biological replicates with two technical replicates for each treatment were applied, and the data were analyzed using the 2^–ΔΔCq method (Taylor et al., 2019). The final value of relative quantification was described as a normalized fold change in the gene expression of each target gene compared to the control. Data were expressed as geometric mean ± 95% CI of four biological replicates for each treatment.

The ORF of SaPEPC-1 or SaPEPC-2 was inserted into the prokaryotic expression vector pET28a. The primers used for vector construction are shown in Supplementary Table 3. The recombinant plasmids pET28a-SaPEPCs were transformed into Escherichia coli Transetta (DE3) strain. The positive clones were sequenced and cultivated in a liquid LB medium supplemented with 100 mg⋅L^–1 kanamycin and 0.8 mmol⋅L^–1 isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 4 h to induce the expression of SaPEPCs. The cell pellets of the recombinant strains were ultrasonically treated, and the total amount of proteins was harvested by centrifuging at 12,000 g, 4°C for 10 min. The precipitation was resuspended and resolved by –SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the recombinant protein was detected by an immunoblot according to the following steps: upon separation on 10% (w/v) PAGE, the proteins were electroblotted onto a polyvinylidene fluoride (PVDF) membrane, which was then blocked overnight at 4°C in a Tris-buffered saline (TBS) buffer (20 mmol⋅L^–1 Tris–HCl, pH 7.5; 150 mmol⋅L^–1 NaCl) containing 5% (w/v) powdered milk. After the incubation with mouse anti-His monoclonal antibody (1:1000 diluted) for 2 h at 37°C, the membrane was washed four times in a TBS buffer, and then incubated with a 1:1000 diluted goat anti-mouse IgG secondary antibody. The 3,3-diaminobenzidine (DAB) was added as a chromogen for staining.

The recombinant (Transetta: pET-28a-SaPEPCs) and control (Transetta: pET-28a) strains were inoculated in a fresh LB medium containing 100 mg⋅L^–1 kanamycin and cultured overnight at 37°C, which (1% of the culture) was then reinoculated to a fresh LB medium (in addition of 100 mg⋅L^–1 kanamycin) and cultivated for about 4 h till the OD₆₀₀ value reached 0.5. After the addition of 0.8 mmol⋅L^–1 IPTG, the cultures were incubated for another 4 h at 37°C. About 1% of the diluted culture (0.8 OD₆₀₀) was inoculated into a 50 ml fresh LB medium (in addition of 100 mg⋅L^–1 kanamycin) with the supplement of 400 mmol⋅L^–1 NaCl, 10% (w/v) PEG 6000, or 25 μmol⋅L^–1 methyl viologen (MV; to mimic the oxidative stress). For the test of acid or base response, the pH value of a LB medium was adjusted to 5.0. Except for the case of temperature assay (at 30°C), all other cultures were incubated at 37°C with a shaking speed of 220 rpm overnight. For the measurement of the time course of growth under different abiotic stresses, cultures (10 ml) were harvested at an interval of 3 h to a total of 12 h to measure the enzyme activity.

The recombinant proteins SaPEPC-1 and SaPEPC-2 were purified under native conditions with an Ni-NTA agarose resin (Qiagen, Hilden, Germany) according to the protocol of the manufacturer. After the determination of the concentration of the purified proteins by using PEP as a substrate, the enzyme activity was determined by the addition of the different conc. of PEP (0.5, 1, 3, 4, and 5 mmol⋅L^–1), NaHCO₃ (0.5, 3, 5, 10, and 20 mmol⋅L^–1), and MgCl₂ (0.5, 3, 5, 10, and 20 mmol⋅L^–1) at pH 8.0 and 25°C. The Michaelis constant K_m and the maximum reaction rate V_max were calculated according to the Lineweaver–Burk plot method, and the catalytic constant K_cat (K_cat = V_max/enzyme concentration) was calculated to measure the speed of an enzymatic reaction. The heat stability of the purified SaPEPCs was determined by measuring the enzyme activity at various temperatures (15–55°C). The pH stability was determined by an incubation with a 50 mmol⋅L^–1 Tris–HCl reaction buffer under the pH values from 7.0 to 10.0 at 25°C. The metal ion stability was determined using a reaction buffer containing 10 mmol⋅L^–1 EDTA and 10 mmol⋅L^–1 metal ions (Cu²⁺, Al³⁺, and Mn²⁺), respectively. The deionized water was used as the control, and the enzyme activity was measured at pH 8.0 and 25°C. The effect of metabolic effectors on enzyme activity was estimated in the presence of varying amounts of allosteric activators (0, 5, 10, 20, and 40 mmol⋅L^–1 glucose-6-phosphate or glycine) and inhibitors (0, 2, 5, 10, 15, 20, and 40 mmol⋅L^–1 L-malate) at pH 8.0 and 25°C.

For PEPC activity in S. aralocaspica, leaves (approximately 0.1 g) were homogenized on ice with 1.0 ml of an extraction buffer containing 100 mmol⋅L^–1 Tris-H₂SO₄ (pH 8.2), 7 mmol⋅L^–1 β-mercaptoethanol, 1 mmol⋅L^–1 EDTA, and 5% (v/v) glycerol. The homogenate was then centrifuged at 2,000 rpm, 4°C for 20 min. The supernatant was immediately used for the assay of PEPC activity according to the protocols described by Cao et al. (2015). For the enzymatic activity of recombinant proteins, the 10 ml samples from the different bacterial cultures were centrifuged at 12,000 g for 10 min, cell pellets were washed with a phosphate buffer, then sonicated for 10 times of 3 s of each with an interval of 10 s and centrifuged at 12,000 g, 4°C for 10 min. The supernatant was employed as a crude enzyme and kept on ice for immediate use. Enzyme activity was measured as described by Cheng et al. (2016). The absorbance of reaction mixtures was recorded by monitoring NADH oxidation at 340 nm on an UV-3010 spectrophotometer (Shimadzu, Kyoto, Japan). The total amount of proteins was determined at 595 nm (Bradford, 1976). One unit of PEPC enzyme activity was defined as an optical density value decrease of 0.01 per minute (Nomenclature Committee of the International Union Of Biochemistry (NC-IUB)., 1979).

Leaves (∼0.2 g) of S. aralocaspica were used for the extraction of soluble proteins according to the method described by Koteyeva et al. (2011). The supernatant was mixed with a loading buffer [250 mmol⋅L^–1 Tris–HCl, pH 6.8, 10% (w/v) SDS, 50% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, and 0.5% (w/v) bromphenol blue] as 4:1 in volume and boiled for 10 min after centrifugation at 10,000 g, 4°C for 10 min, the supernatant was subjected to the SDS-PAGE analysis. Protein concentration was determined using the Bradford Protein Assay Kit (Cat. PC0010, Solarbio, Beijing, China). The resolved protein samples (10 mg of each) were transferred to a PVDF membrane for an immunoblot analysis of the photosynthetic enzymes. All the primary antibodies used in this study were raised against the predicted optimal epitopic antigens of the conserved amino acid sequences of PEPC, pyruvate orthophosphate dikinase (PPDK), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from S. aralocaspica, the amino acid residues of the epitopic antigens, and the working dilution of these antibodies were as follows: anti-SaPEPC-C (EKLSSIDAQLR, common to PEPC) IgG (1:500), anti-SaPEPC-M {[EKLS(pS)IDAQLR], for the detection of the phosphorylation of the serine residue in this sequence of PEPC} IgG (1:500), anti-SaPPDK (KLATEKGRAAKPSL) IgG (1:200), and anti-SaRubisco large subunit (RBCL) (QARNEGRDLAREGN) IgG (1:500). The secondary antibody goat anti-rabbit IgG (conjugated horseradish peroxidase) (1:2000) was used for detection. Bound antibodies were visualized by enhanced chemiluminescence (Biosharp, Beijing, China), and the images were acquired by an luminescent image analyzer (FUJIFILM LAS-4000, Tokyo, Japan).

All data were plotted using GraphPad Prism Version 7.0 (GraphPad Software, San Diego, CA, United States) and analyzed using SPSS version 26.0 (SPSS Inc., Chicago, IL, United States). Univariate scatterplots displaying parametric data were present as mean and SD (Weissgerber et al., 2015). One-way ANOVA was used to test the significance of different treatments, and Tukey’s HSD test was performed for multiple comparisons to determine significant differences between the samples at 0.05, 0.01, 0.001, and 0.0001 significance levels. When the homogeneity of variance assumption was not met, differences were analyzed using Welch’s ANOVA and Games Howell post hoc test. Statistically significant differences between the groups at 0.05 significance level were determined by an unpaired Student’s t-test (homoscedastic) or unpaired Student’s t-test using Welch’s correction (heteroscedastic) of a two-tailed distribution (McDonald, 2014).

A total of three putative PEPC genes were identified by a local BLASTP search of S. aralocaspica genome, the deduced proteins were subjected to Pfam, SMART, and InterProScan databases to analyze the domains and active sites. Three non-redundant genes (GOSA_00009595-RA, GOSA_00006741-RA, and GOSA_00018957-RA) were confirmed as SaPEPCs, the first two were recognized as SaPEPC-1 (GenBank: KP985714.1) and SaPEPC-2 (GenBank: KX009562.1), respectively, which were identified in our previous work; the third one was similar to AtPPC4 in Arabidopsis and denominated as SaPEPC-4. Detailed information of SaPEPC family members is shown in Table 1.

To investigate the evolutionary relationship between SaPEPCs and PEPCs of other 26 representative eukaryotic species, including dicots, monocots, ferns, mosses, and algae, we constructed an unrooted neighbor-joining phylogenetic tree using 135 PEPC proteins (Figure 1). The results indicate that all PEPC family members can be categorized into two distinct clades: PTPCs and BTPCs, about 70% of the PEPCs were PTPCs. Similar distribution patterns of PTPCs and BTPCs were also found in different species (Supplementary Table 4). In the PTPC subfamily, compared with a dicot branch, monocots, mosses, and ferns were gathered together and formed another independent branch, which could further be divided into seven groups (PTPC I–PTPC VII). BTPC subfamily was distinctly classified into four groups (BTPC I–BTPC IV). Phylogenetic analysis also identified some closely related orthologous PEPCs among S. aralocaspica, A. thaliana, and C. quinoa (a C₃ plant, Geissler et al., 2015): SaPEPC-1, AtPPC2, and CqPPC1 were located on the same branch of PTPC IV; SaPEPC-2, AtPPC1/3, and CqPPC2 were assigned to PTPC II; whereas SaPEPC-4 was grouped into AtPPC4 and CqPPC4 cluster in BTPC III, suggesting that an ancestral set of PEPCs may exist prior to the divergence of S. aralocaspica, A. thaliana, and C. quinoa.

The amino acid sequence alignment among SaPEPCs, AtPPCs, CqPPCs, and ZmPEPCs showed that they shared typical conserved domains and functional sites in PTPC and BTPC genes. However, no N-terminal phosphorylation domain (SIDAQLR) was found in the polypeptide deduced from SaPEPC-4, CqPPC4, AtPPC4, and ZmPEPC3 genes, instead of harboring an RNTG tetrapeptide at the C-terminus, which was commonly found in BTPC (Figure 2A).

Different PEPC genes in the same species displayed great discrepancies in size. PEPC genes in PTPC II/IV groups contained 10 exons, whereas it was 20 in BTPC III (Figure 2B). By predicting the exon–intron structure in 27 plant species (including S. aralocaspica), we found that the length of PTPC genes was from about 4 to 13 kb, and that of BTPC genes ranged from 7.5 to 14 kb; whereas the length of PEPC exons was similar in the same branch, and the number of exons/introns was conserved (Supplementary Figure 2). The PTPC genes of dicots, monocots, and ferns contained 10–12 exons and 9–11 introns, whereas the BTPC genes contained 18–21 exons and 17–20 introns. The moss PTPC genes generally consisted of 11–14 exons and 10–13 introns, except for one member with only five exons and four introns; while the independent branches of moss BTPCs contained 32 exons and 31 introns. All algal PEPCs belonged to BTPC, generally containing 20–30 exons and 19–29 introns, but exceptionally, only one exon was found in that of Ostreococcus lucimarinus and Microcystis aeruginosa.

Although the exon number was largely different, the exon length of PTPC genes was similar to that of BTPC, especially exons 8, 9, and 10; whereas the intron length differed greatly, indicating that the size of PEPC genes largely depends on the introns. Similarly, in S. aralocaspica, although the length of coding region of SaPEPC-1 and SaPEPC-2 (both were 2,901 bp) and the number of exons/introns (10/9) were conserved, the size of SaPEPC-2 was 1,000 bp longer than that of SaPEPC-1, for the 3rd intron of the former was about 10 times longer than that of the latter (Figure 2B).

Multi-sequence alignment showed that PEPCs were highly conserved among S. aralocaspica, A. thaliana, and C. quinoa. The analysis of the top 10 conserved motifs of 135 PEPCs from 27 plant species revealed that all these motifs bore the prints (IPR021135) of PEPCase family, besides, motif-4 and motif-8 also contained the histidine (IPR033129) and lysine (IPR018129) active sites, respectively (Supplementary Figure 3 and Supplementary Table 5). SaPEPCs and other 121 PEPCs contained all these 10 motifs and were arranged in the same order, whereas the other five PTPCs and six BTPCs were lacking some of these motifs (Supplementary Table 6). It suggests that PEPCs in different species are generally conserved in gene structures, protein domains, and functional motifs but can also be genetically diverse.

The prediction of in silico subcellular localization showed that all three SaPEPC proteins were most probably localized in the cytoplasm, with an average possibility of 24.1% in the nucleus (Table 1). Our transient transformation assay in tobacco epidermal cells showed that SaPEPC-1 had a strong fluorescence signal in the cytoplasm, plasma membrane, and nucleus (Figure 3B) while the fluorescence signal of SaPEPC-2 was mainly observed in the nucleus (Figure 3C and Supplementary Figure 4), which is consistent with the prediction by the Plant-mPLoc and YLoc⁺ software.

The retrieved 2,500 bp sequences upstream of the start codon of SaPEPC genes were queried to the PlantCARE database for a cis-regulatory element prediction. A total of 98 cis-elements were detected from the three SaPEPC genes, in addition to the phytohormone and specific expression-related cis-elements, the other two cis-elements were involved in palisade mesophyll cell differentiation and four (ARE, WUN-motif, LTR, and MBS) in response to abiotic stresses. In particular, light-responsive cis-elements were up to 12 varieties (i.e., AE-box, AT1-motif, ATCT-motif, Box-4, chs-CMA1a, G-box, GA-motif, GT1-motif, I-box, MRE, TCCC-motif, and TCT-motif) (Figure 4A and Supplementary Table 7), suggesting that SaPEPCs may largely be involved in light regulation. Further analysis of these results might help in understanding the role of SaPEPC genes in development, photosynthesis, and response to stresses.

Two SaPEPC promoters driving GUS expression in vitro revealed that the GUS activity was gradually decreased with the progressive 5′-flanking deletion of the upstream sequence (F_7,24 = 46.26 and 42.69, p < 0.0001 for SaPEPC-1 and SaPEPC-2 under light, respectively; Welch’s F_7,10.03 = 263.99 and Welch’s F_7,10.14 = 32.85, p < 0.0001 for SaPEPC-1 and SaPEPC-2 under darkness, respectively) and significantly responded to light (e.g., t₆ = 6.748, p = 0.0005 for TSS1 and t₆ = 9.255, p < 0.0001 for P2-5, respectively) (Figures 4B,C). Interestingly, the upstream sequence starting from the TSS (named these segments as TSS1 and TSS2 for SaPEPC-1 and SaPEPC-2, respectively, which were lacking the 5′-UTR sequence compared with P1-6 and P2-6 fragments) significantly promoted GUS activity compared to that of P1-6 fragment of SaPEPC-1 (t₆ = 5.848, p = 0.0011 under light; t₆ = 3.343, p = 0.0155 under darkness) or P2-6 fragment of SaPEPC-2 (t₆ = 7.426, p = 0.0003 under light; t₆ = 5.310, p = 0.0018 under darkness), respectively, suggesting that a 5′-UTR region may apply a repressive effect on the activity of the SaPEPC promoter (Supplementary Figure 1). Further analysis revealed that multiple stem-loop structures might be formed at the RNA level in the 5′-UTR sequence of SaPEPCs, that of SaPEPC-2 presented a more complicated secondary structure with a folding free energy (ΔG) of −51.62 kcal mol^–1 (Supplementary Figure 5). These facts imply that the 5′-UTR sequence of SaPEPCs may play an important regulatory role in gene expression.

Based on the available RNA-Seq data (Wang et al., 2017, 2019), the temporal expression patterns of three SaPEPC genes in dry, imbibed, and germinated seeds were analyzed by using the dimorphic seeds. SaPEPC-1 accumulated more transcripts with the germination progression, and the brown seedlings responded quicker than the black ones; the transcripts of SaPEPC-2 were accumulated to remain relatively constant and abundant in dimorphic seeds and seedlings while SaPEPC-4 was expressed at lower levels in germination and both types of seeds (Figure 5). In our previous study, the transcriptional expression patterns of SaPEPC-1 and SaPEPC-2 were compared in terms of seed germination (0, 5, 10, and 15 days), different tissues (radicle, hypocotyl, and cotyledon), and salt stress (0, 100, 300, and 500 mmol⋅L^–1) in dimorphic seeds under normal conditions (Cheng et al., 2016). In this study, we strengthened these data by emphasizing the effect of light/dark on the expression of two SaPEPC genes without distinguishing dimorphic seeds, and a much longer developmental period (i.e., 0 h, 8 h, 12 h, 24 h, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, and 60 days), more different tissue types (radicles, hypocotyls, cotyledons, and roots, stems, leaves of 15- and 90-day plants), and more different types of abiotic stresses (NaCl, mannitol, ABA, and light intensity) were applied. In developing seedlings and adult plants from brown seeds, the transcript accumulation of two genes was increased gradually with seedling growth, and reached the highest value at 10 days after germination and 30 days after emergence, respectively (Figure 6). Light or darkness treatment in the germination stage revealed that SaPEPC-1 expression was more sensitive to light, and the transcript copies were approximately 20 times more than that in the dark on the 10th day of germination (Welch’s t_3.028 = 14.01, p = 0.0008) (Figures 6A,B). The results are consistent with our previous study (Cheng et al., 2016).

The spatial expression patterns of SaPEPC genes according to the available data showed that SaPEPC-2 was widely expressed in different tissues (root, stem, leaf, and fruit), SaPEPC-1 was preferentially expressed in leaves and fruits, with lower expression in roots while the expression of SaPEPC-4 was lower in all the tested tissues (Figure 5). With the help of qRT-PCR analysis, the expression patterns of SaPEPC-1 and SaPEPC-2 in different tissues were validated, partial results were consistent with the RNA-Seq data and our previous result (Cheng et al., 2016). In general, the accumulation of SaPEPC-1 transcripts was significantly higher than that of SaPEPC-2 in different tissues, especially in cotyledons (Welch’s t_3.089 = 15.03, p = 0.0005 under light and t₆ = 2.709, p = 0.0351 under darkness) and leaves (Welch’s t_3.005 = 14.40, p = 0.0007 for 15-day seedling and Welch’s t_3.000 = 13.74, p = 0.0008 for 90-day-adult plant). In comparison with the high level in leaves, SaPEPC-1 transcripts were hardly detected in developing roots and stems, whereas the SaPEPC-2 expressed relatively higher in roots compared to other tissue types (Figure 7). These results suggest that SaPEPC-1 and SaPEPC-2 may play diverse biological functions in plant developmental processes.

To investigate the responses of SaPEPCs to NaCl, mannitol, ABA, and different light intensities in developing seedlings, the transcriptional expression patterns of SaPEPC-1 and SaPEPC-2 were analyzed. The results showed that two SaPEPCs were significantly upregulated by increasing the light intensity (F_2,9 = 39.64, p < 0.0001 for SaPEPC-1 and F_2,9 = 28.99, p = 0.0001 for SaPEPC-2), especially under 300 (t₆ = 3.500, p = 0.0128) and 900 μmol⋅m^–2⋅s^–1 (t₆ = 4.586, p = 0.0037), the expression level of SaPEPC-1 was significantly higher than that of SaPEPC-2 (Figure 8D). Under various abiotic stresses, both SaPEPCs exhibited similarly positive responses to light or darkness while the transcript copies of SaPEPC-1 were significantly higher than that of SaPEPC-2 when exposed to 300 mmol⋅L^–1 NaCl (t₆ = 6.082, p = 0.0009), 600 mmol⋅L^–1 mannitol (t₆ = 9.432, p < 0.0001), and 10 μmol⋅L^–1 ABA (t₆ = 2.805, p = 0.0309) treatments under normal light conditions (Figures 8A–C).

Enzyme activity of SaPEPCs was measured in the leaves of different developmental stages and under different light intensities as well as abiotic stresses. As shown in Figures 9A–C, PEPC activity varied in consistency with the expression pattern of SaPEPC genes, which was significantly increased with the germination time extension (Welch’s F_3,5.152 = 101.5, p < 0.0001) and the stress enhancement (F_3,12 = 24.91, p < 0.0001 for salt stress and F_2,9 = 48.49, p < 0.0001 for drought stress). The diurnal variation in light intensity resulted in a gradual increase of PEPC activity in the morning, and reached the highest value (average 574.44 U⋅mg^–1 protein) at 12:00, then dramatically decreased thereafter until 22:00 (Welch’s F_7,9.908 = 75.16, p < 0.0001), appeared as a “unimodal” curve (Figure 9D). The protein accumulation of representative photosynthetic enzymes [total PEPC (PEPC-C), phosphorylated PEPC (PEPC-M), PPDK, and RBCL] was also analyzed by the varying light intensity from the morning to the evening (Figure 9E). Among them, the amount of RBCL appeared to be abundant and relatively constant; PEPC-C increased with time elongation and reached the highest level from 12:00 onward; PEPC-M and PPDK were remarkably accumulated from 12:00 to 18:00 (the period with the highest light intensity in a day), and decreased significantly from then on. Our results showed that these proteins could apparently be induced by increasing light intensity, especially PEPC phosphorylation, such an expression pattern was corresponding to the diurnal changes of PEPC enzyme activity, suggesting a close relationship between light intensity and PEPC phosphorylation, and the consequent PEPC activity.

Enzyme activity was further determined by the ectopic expression of SaPEPCs in E. coli under salt (NaCl), drought (PEG 6000), oxidation (MV), temperature, and acid/base (pH) stresses. SaPEPC-1 and SaPEPC-2 proteins were resolved by the SDS-PAGE and detected by an immunoblot analysis, which revealed a recombinant protein with the MW of 110 kDa in accordance with the theoretical values. SaPEPC-1 was expressed in the supernatant and inclusion bodies while SaPEPC-2 was mainly expressed in the inclusion bodies of the cells (Supplementary Figure 6). In our previous study, due to the lack of complete coding sequence of SaPEPC-2, only SaPEPC-1 recombinant strain was analyzed with the time course of growth and enzyme activity [under 400 mmol⋅L^–1 NaCl, 10% (w/v) PEG, 25°C, 75 μmol⋅L^–1 MV, and pH 5.0] (Cheng et al., 2016). In this study, we further supplemented the corresponding data of SaPEPC-2 recombinant strain, our results showed that the overexpression of SaPEPC-2 could also significantly enhance cell growth and enzyme activity under different stress conditions, except for the cases of growth at 25°C and the enzyme activity under 75 μmol⋅L^–1 MV (compared with SaPEPC-1 recombinant strain) (Supplementary Figures 7, 8). Based on the abovementioned analysis, the enzyme activity of both SaPEPC recombinant strains was determined simultaneously under the conditions of 400 mmol⋅L^–1 NaCl, 10% (w/v) PEG, 30°C, 25 μmol⋅L^–1 MV, and pH 5.0. With an increase in stress time (to a total of 12 h), the PEPC activity of two strains increased significantly and reached the maximum value at 3 h (Welch’s F_4,34.42 = 69.53, p < 0.0001 for SaPEPC-1 and Welch’s F_4,34.21 = 95.82, p < 0.0001 for SaPEPC-2), and then reduced but generally remained higher than that of the control (Figure 10). In general, the SaPEPC-2 activity was higher than that of SaPEPC-1.

Different conc. of PEP, Mg²⁺, and HCO₃^– were applied in the measurement of the recombinant SaPEPC activity to assess their effects. At pH 8.0 and 25°C, the highest K_m and V_max values of SaPEPC-1 or SaPEPC-2 were estimated to be approximately 0.237 or 0.231 mmol⋅L^–1 and 33.85 or 57.32 U⋅mg^–1 protein, respectively, with different conc. of PEP. On the contrary, the lowest K_m and V_max values were present under different conc. of HCO₃^–, the saturation conc. was quickly reached, and the catalytic efficiency (K_cat/K_m) was 39.33 × 10³ or 66.35 × 10³ (mmol⋅L^–1)^–1min^–1, respectively, which was two times higher than that with PEP and MgCl₂. Moreover, the catalytic efficiency of SaPEPC-2 with PEP or HCO₃^– was about two times as much as that of SaPEPC-1, indicating that SaPEPC-2 may possess a higher substrate affinity compared to SaPEPC-1 (Table 2 and Figure 11A).

The influence of various effectors on the stability of SaPEPC-1 and SaPEPC-2 activities was investigated. The enzymatic activity of two SaPEPCs remained relatively constant at a range of temperature from 15 to 40°C while declining rapidly from 40 to 55°C, and could almost not be detected at 55°C and above, between them, SaPEPC-2 was able to tolerate higher temperature than that of SaPEPC-1. The pH stability test showed that both SaPEPCs presented the highest activity at pH 8.3, whereas the activity decreased significantly when the pH value was less than 8.0 (Figure 11B). Al³⁺ ion showed a significant inhibition to the enzyme activity of two SaPEPCs, which was similar to the effect of EDTA (metal ion-chelating agent). When exposed to Cu²⁺ ion, only approximately 50% activity of both SaPEPCs was detected compared to the control. As the metal ion cofactors of PEPC, Mg²⁺, or Mn²⁺ ions displayed the highest activity and had no significant difference compared to the control (Figure 11C). The kinetics of the recombinant enzymes were examined in the presence of allosteric activators and inhibitors under standard conditions. As shown in Figure 11D, with an increase in the concentration of glucose-6-phosphate and glycine, the activity of both SaPEPCs showed a slight increase while L-malate significantly inhibited their activity.