Seed size is enhanced in NaCl-treated S. salsa
We examined fruit development in control vs. NaCl-treated S. salsa plants during the reproductive period. Black seeds matured earlier than brown seeds on the same branchlet. Both black and brown seeds from NaCl-treated plants were significantly larger than those from control plants (Fig. S1).
NaCl improves starch accumulation in S. salsa ovules
We measured starch accumulation in the embryo sacs of control and 200 mM NaCl-treated S. salsa plants (Fig. 1). Before fertilization (108 DAS), starch levels were significantly higher in the ovules of NaCl-treated plants than in control plants (Fig. 1a, b). After fertilization (110 DAS), the accumulated starch is used for embryo and endosperm development . Therefore, no starch accumulation was detected upon either control or 200 mM NaCl treatment after fertilization (Fig. 1c, d). However, the funiculi of the ovules of NaCl-treated plants accumulated more starch than those of the controls. The high levels of starch should provide enough raw materials for embryo development, perhaps explaining why seeds were larger in plants treated with 200 mM NaCl than in control plants.
NaCl treatment increases total soluble sugar levels in leaves, stems, and flowers during early reproductive growth
We measured total soluble sugar (TSS) contents in NaCl-treated and control S. salsa plants at the beginning of the reproductive growth phase (105 DAS). TSS contents were significantly higher in the leaves, stems, and flowers of 200 mM NaCl-treated plants compared to the controls, with levels 1.83-, 1.12-, and 1.59-fold those of the control, respectively. TSS levels were particularly elevated in the floral organs of plants treated with 200 mM NaCl, with levels 2.05- and 2.16-fold of those in leaves and stems, respectively (Fig. 2).
NaCl increases the net photosynthesis rate in S. salsa leaves
Starch and soluble sugars in plants are directly derived from photosynthesis. During the beginning of the reproductive growth period (103 DAS), the net photosynthetic rate was significantly (57.4%) higher in the leaves of S. salsa plants treated with NaCl compared to the control (Fig. 3a). However, we detected no significant difference in the transpiration rate or stomatal conductance between control and 200 mM NaCl-treated plants (Fig. 3b, c). By contrast, the intercellular CO2 concentration was 13.8% lower in NaCl-treated plants than in control plants (Fig. 3d).
NaCl treatment increases chlorophyll contents in S. salsa leaves
The efficiency of photosynthesis is related to chlorophyll contents in leaves. We therefore measured chlorophyll contents in the leaves of control and NaCl-treated S. salsa plants at 103 DAS. Chlorophyll contents were significantly higher in the leaves of NaCl-treated vs. control plants, including chlorophyll a, chlorophyll b, and total chlorophyll contents, with increases of 26.6%, 46.4%, and 31.5%, respectively (Fig. 4). A similar pattern was detected for chlorophyll contents in the flowers of S. salsa plants treated with NaCl (Fig. S2). During early flower development, the petals were green and contained chloroplasts (Fig. S3), indicating that S. salsa flowers can undergo photosynthesis during early development.
NaCl treatment enhances photochemical efficiency of PSII in S. salsa leaves
Both the potential photochemistry efficiency (Fv/Fm) and actual photochemistry efficiency (ΦPSII) of PSII were significantly higher in the leaves of NaCl-treated plants vs. the control (Fig. 5). However, there was no significant difference in Fv/Fm, an indicator of the maximum quantum yield of PSII in dark-adapted leaves, between the two treatment groups (Fig. 5a). By contrast, ΦPSII, which indicates the actual photochemistry efficiency under natural light conditions, was significantly (9.7%) higher in plants treated with NaCl than in the control plants at the beginning of the reproductive growth stage (Fig. 5b).
Sequencing Output And Assembly
To investigate the molecular mechanisms that improve flower and seed development in S. salsa plants grown in the presence of NaCl, we collected flowers from control and NaCl-treated (NaCl) plants and subjected them to RNA-sEq. After excluding low-quality sequences, we used the clean reads to generate a de novo transcriptome assembly using Trinity  and analyzed the differences in gene expression between control and NaCl-treated (NaCl) flowers after clustering by Corset . The transcripts and genes used in this analysis were described by Guo et al. .
Functional Annotation Of DEGs In Control Vs. NaCl-treated Flowers
We analyzed DEGs in the flowers of control vs. NaCl-treated plants using the RSEM software package . We performed functional annotation of DEGs between the two groups by BLAST analysis based on seven databases as described previously. To explore the biological functions of the DEGs, we mapped the DEGs to KEGG pathways. In total, 3640 DEGs were mapped to 124 KEGG pathways, such as “photosynthesis”, “starch and sucrose metabolism”, “fatty acid biosynthesis and elongation”, and “amino sugar and nucleotide sugar metabolism” .
DEGs related to antenna proteins and photosynthesis in S. salsa flowers
Light harvesting is the first step in photosynthesis. Light-harvesting complexes (LHCs) containing the photosynthetic pigments chlorophyll and carotenoid, the most important structures in this process, are located near PSI (photosystem I) and PSII [50, 51]. LHC proteins are encoded by a multi-gene family in the nucleus . Plants contain 14 types of LHC proteins (Lhca1–Lhca6 and Lhcb1–Lhcb8). Lhca-type proteins are located near the PSI reaction center, whereas Lhcb-type proteins are located near the PSII reaction center . In the present study, six DEGs were identified in S. salsa flowers that mapped to antenna proteins, of which four were upregulated and two were downregulated. Compared to the control, the four DEGs encoding Lhcb1 were upregulated in the flowers of NaCl-treated plants, including three that were highly expressed under NaCl treatment (Fig. 6). However, DEGs encoding Lhcb4 and Lhcb5 were downregulated in flowers in the NaCl-treated group (Fig. 6).
In addition to light harvesting, electron transfer is also a prerequisite for the completion of photosynthesis, especially under unfavorable environmental conditions. Salt stress significantly inhibits photosynthetic efficiency in plants [54, 55]. The photosynthetic electron transport chain in plants is composed of four protein complexes: PSII, the cytochrome (Cytb6f) complex, PSI, and ATP synthase. We identified 28 DEGs related to photosynthesis in the flowers of control vs. NaCl-treated plants, which may have led to functional changes in photosynthesis under high-salinity conditions (Fig. S4).
PSII consists of various types of chlorophyll binding components. This complex is responsible for light harvesting and electron transport during photosynthesis immediately after water oxidation . PSI, a chlorophyll-protein complex, catalyzes the transfer of electrons from plastocyanin/cytochrome c6 to ferredoxin/flavodoxin, a process driven by light . The PSI subunits PsaF and PsaN interact with ferredoxin or plastocyanin . In the flowers of NaCl-treated S. salsa plants, DEGs encoding PsaF were downregulated. By contrast, of the two DEGs related to PsaN, one was upregulated and one was downregulated (Fig. 6). Moreover, five DEGs related to the gamma chain of ATP synthase were identified, including one that was upregulated and four that were downregulated (Fig. 6).
DEGs related to carbon utilization and sucrose and starch metabolism in S. salsa flowers
In addition to DEGs related to light harvesting and electron transfer, we examined the expression of DEGs related to carbon utilization in flowers from the two groups of S. salsa plants. We identified 35 upregulated DEGs and 12 downregulated DEGs in the flowers of NaCl-treated plants compared to the control (Fig. S4). Of these, 16 genes were expressed at high levels specifically in flowers from NaCl-treated plants compared to the control. Detailed information is provided in Table S1.
Carboxylation is the first step in the CO2 assimilation process. Ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco, EC 126.96.36.199) is the key enzyme in this process, which links organic and inorganic matter in the biosphere . Two DEGs (Cluster-10319.94590 and Cluster-10319.106159) encoding Rubisco were upregulated in the flowers of NaCl-treated plants (Fig. 7). The increased CO2 fixation efficiency in S. salsa plants treated with NaCl might be due to the enhanced expression of genes encoding Rubisco. Similarly, in Dunaliella salina, photosynthesis-related genes are upregulated at the optimum NaCl concentration (1.7 M), which is associated with optimum growth . During the regeneration process of ribulose 1,5-diphosphate (RuBP), two DEGs (Cluster-10319.31330 and Cluster-10319.58613) encoding isomerases were upregulated in the flowers of NaCl-treated plants. Seven DEGs (Cluster-10319.110671, Cluster-10319.110672, Cluster-10319.92536, Cluster-10319. 110670, Cluster-10319.110668, Cluster-10319.92542, and Cluster-10319.9993) were upregulated in flowers from NaCl-treated plants (Fig. 7). These results indicate that the higher sugar contents in flowers and the higher starch contents in ovules are accompanied by the upregulated expression of the associated genes and high photosynthetic efficiency (Fig. 3a).
Starch is an essential source of carbon for plant development, especially in floral organs during reproductive growth. To investigate the reason for the different starch and sugar contents in S. salsa ovules and flowers in the two treatment groups, we examined the expression of DEGs involved in sugar and starch metabolism. We identified 133 DEGs related to sugar metabolism and sugar transport in S. salsa flowers (Fig. 8a). These DEGs might participate in sugar binding, sugar transport, and sugar phosphorylation, including 77 upregulated and 56 downregulated DEGs. We also identified 94 DEGs involved in starch metabolism in S. salsa flowers (Fig. 8b; detailed information is provided in Table S2). These genes, which may participate in starch binding, starch metabolic processes, and starch phosphorylation, included 55 upregulated and 39 downregulated DEGs. Finally, 30 and 27 DEGs involved in sugar and starch metabolism, respectively, were expressed at significantly higher levels in flowers from NaCl-treated vs. control plants; this upregulation likely increased the sugar and starch contents in these plants.
Sucrose synthetase (SS, EC: 188.8.131.52), sucrose phosphate synthase (SPS, EC: 184.108.40.206), fructokinase (EC: 220.127.116.11), and phosphoglucomutase (PGM1, EC: 18.104.22.168) are key enzymes in sucrose and starch metabolism. SS plays a vital role in the conversion of glucose and fructose to sucrose, as do SPS and PGM1. Based on our RNA-seq data, genes involved in this pathway were expressed at higher levels in S. salsa flowers from NaCl-treated vs. control plants, such as genes encoding SPS (two upregulated DEGs), SS (eight upregulated and two downregulated DEGs), and PGM1 (two upregulated DEGs). These findings are consistent with the high sugar content in the flowers of NaCl-treated vs. control plants (Fig. S5).
Verification Of RNA-seq Data Using qRT-PCR
To confirm the changes in gene expression detected by RNA-seq, we subjected ten randomly selected DEGs to quantitative qRT-PCR. The results from qRT-PCR are in close agreement with the RNA-seq data, with a correlation coefficient of R2 = 0.90 (Fig. 9), thus verifying the reliability of the RNA-seq results.