E. angustifolia plants flower and produce fruits in the saline habitat and flower but do not produce fruits in the non-saline habitat
We observed the flowering period and reproductive growth of E. angustifolia in two habitats. E. angustifolia flowers are bisexual, with 1–3 flower clusters per axil. The flowers are yellow on the inside, silvery white on the outside, and fragrant and are produced from early May to late June. In both habitats, flower buds formed in E. angustifolia, and the flowers opened (Fig. 1a, b). After flowering, fruits formed in E. angustifolia in the saline habitat (Fig. 1c), whereas plants in the non-saline habitat had no fruits, and all of the flowers fell off of the plants (Fig. 1d).
No significant difference in pollen viability or stigma receptivity between habitats
To determine whether the lack of fruit in E. angustifolia in the non-saline habitat was caused by poor pollen vitality, we dyed pollen collected from E. angustifolia in both habitats with Alexander’s solution. This stain dyes aborted pollen grains green and non-aborted pollen grains red. There was no difference in pollen vitality between the habitats: 80% of the pollen grains were viable (Fig. 2a, b). Pollen grains that germinated in vitro from plants grown in both non-saline and saline habitats were well developed, indicating they could germinate and form pollen tubes (Fig. 2c, d).
To determine whether insufficient pollen tube growth led to the lack of fertilization of E. angustifolia in the non-saline habitat, we determined the pollen germination rate and pollen tube length. As shown in Fig. S1, the pollen germination rate (Fig. S1 a) of E. angustifolia from both habitats was approximately 50%, and there was no significant difference in pollen tube length (Fig. S1 b) between habitats. These results indicate that the flowering and lack of fruit in E. angustifolia in non-saline habitats were not caused by pollen dysplasia.
To determine whether altered stigma receptivity prevents fruit formation in E. angustifolia in non-saline habitats, we measured peroxidase activity in stigmas, which represents stigma receptivity. As shown in Fig. 3b, E. angustifolia stigmas from the non-saline habitat were surrounded by blue staining and numerous bubbles, and there were no significant differences between these stigmas and stigmas from plants grown in the saline habitat (Fig. 3a). The stigmas of plants grown in both environments showed high receptivity, and the pollen vitality was high. These results indicate that poor pollen viability and stigma receptivity are not the reasons for the lack of fruit in E. angustifolia in the non-saline habitat.
Stigmas from the non-saline habitat lack pollen and pollen tubes under natural conditions but contain numerous pollen tubes after hand pollination
An aqueous solution of aniline blue is used as a dye that combines specifically with callose in the pollen tube wall. Under natural conditions, after self pollination, aniline blue staining of pistils revealed numerous pollen tubes in the styles and many pollen grains on the stigmas of plants grown in the saline habitat (Fig. 4a, d). By contrast, the styles of plants grown in the non-saline habitat had almost no pollen tubes in the styles and no pollen grains on stigmas (Fig. 4b, e). These results suggest that due to blocked anther dehiscence, no pollen grains reach the stigmas of plants in the non-saline habitat under natural conditions. At 4 h after hand pollination, many pollen tubes formed in the styles and many pollen grains were present on the stigmas of E. angustifolia grown in the non-saline habitat (Fig. 4c, f).
Blocked anther dehiscence occurs in the non-saline habitat
To further investigate whether blocked anther dehiscence led to the lack of fruit formation in the non-saline habitat, we observed flowers after anther dehiscence in plants from both habitats. As shown in Fig. 5a, the anthers were pale with almost no pollen grains in E. angustifolia in the saline habitat, as many pollen grains were released to the surfaces of the petals. However, in the non-saline habitat, the anthers were dark yellow and contained many pollen grains, and no pollen grains were present on the surfaces of the petals (Fig. 5b).
To determine if the color difference of the anthers from saline vs. non-saline habitats was due to the presence of pollen grains, we observed anthers after pollen released by DIC microscopy. The anthers of plants grown in the saline habitat contained almost no pollen grains and had undergone dehiscence, with only a single layer of cells (Fig. 5c). By contrast, the anthers of plants grown in the non-saline habitat contained numerous pollen grains after anther dehiscence, and no obvious stomium was observed in the anthers (Fig. 5d). The anther sections of E. angustifolia before dehiscence in two habitats were further prepared, no significant difference in pollen amount in anthers of the two habitats, obvious crack in anthers from saline habitat were observed, while not in anthers from non-saline habitat (Fig. S2). It is speculated that the anthers of the E. angustifolia in saline habitat can dehisce and loose pollens when the pollen grains are mature. However, the pollination in E. angustifolia flowers in non-saline habitat was inhibited.
Na+ content increases in leaves but not flowers in the saline habitat
Significantly (27.4%) higher Na+ contents were detected in the leaves of E. angustifolia grown in the saline vs. non-saline habitat, whereas no significant difference in Na+ content was observed in the flowers of these plants (Fig. 6a). Moreover, there was no significant difference in K+ content in the leaves or flowers of E. angustifolia plants grown in the saline vs. non-saline habitat (Fig. 6b). However, plant reproduction was not inhibited, but rather was improved, by the increased Na+ content in leaves under saline conditions.
Sequence assembly and quantitative real-time PCRvalidation
To investigate the molecular mechanism involved in the differences in anther development and dehiscence in E. angustifolia under saline vs. non-saline conditions, we collected anthers during early development and subjected them to RNA-seq analysis. The number of clean reads obtained from the both groups is shown in Table S3. Genes with differential expression in anthers between the two groups were filtered based on an adjusted P value <0.01 and |log2 (fold change)| > 1.5. The remaining genes were analyzed to identify the differentially expressed genes (DEGs) that might lead to failed dehiscence in E. angustifolia anthers in the non-saline habitat.
To validate the quality of the data obtained by RNA-seq, were subjected 20 DEGs involved in anther development to quantitative real-time PCR (qPCR). A high correlation (R2 = 0.88) between the results from RNA-seq and qPCR was obtained, indicating that the RNA-seq data were reliable (Fig. 7) and could be used for further analysis.
Analysis and annotation of DEGs
To evaluate the DEGs in E. angustifolia anthers under saline vs. non-saline conditions, we performed differential expression analysis using DESeq2 . In total, 8817 genes displayed significantly different expression levels between the saline and non-saline habitats. Among these, 5,063 were upregulated and 3,754 were downregulated in E. angustifolia anthers under saline vs. non-saline conditions (Fig. S3 a).
To identify the likely biological pathways involving the DEGs in anthers between the two habitats, we performed functional analysis of the DEGs using the Kyoto Encyclopedia Genes and Genomes (KEGG) database . In total, 1,919 DEGs were assigned to 116 KEGG pathways. These DEGs were mapped to many categories, such as “amino sugar and nucleotide sugar metabolism”, “biosynthesis of unsaturated fatty acids”, and “flavonoid biosynthesis” (Fig. S3 b). The differences in the pathways might be related to mechanisms used to ensure anther development in E. angustifolia in saline habitats. Furthermore, many other DEGs were involved in the categories “ABC transporters” and “starch and sucrose metabolism”, perhaps contributing to the anther dehiscence and pollination of flowers in E. angustifolia under saline conditions. During plant reproduction, plant hormone biosynthesis and signal transduction play crucial roles in development and plant responses to stress. Many of the DEGs were also assigned to the categories plant hormone biosynthesis and plant signal transduction.
DEGs involved in cell wall formation in E. angustifolia anthers
During cell and anther development, cell wall formation is fundamentally important for protecting the cells from adverse conditions, especially the formation of secondary cell walls (SCWs). In the present study, we identified DEGs related to cell wall modification in the anthers of E. angustifolia plants grown under saline vs. non-saline conditions. Thirteen DEGs associated with cell wall modification were upregulated in E. angustifolia anthers from the saline vs. non-saline habitat. In particular, three (Cluster-2772.54625, Cluster-2772.45882 and Cluster-2772.41972) genes were inducibly expressed under the saline habitat (Table S4). Actin and tubulin participate in anther development . Six DEGs encoding tubulin and one DEG encoding actin were upregulated and four of them (Cluster-2772.60317, Cluster-2772.61547, Cluster-2772.59057 and Cluster-2772.85525) were inducibly expressed under salinity (Table S4).
Lipoxygenase (LOX) and allene oxide cyclase (AOC) are involved in anther dehiscence . In E. angustifolia anthers, four DEGs encoding LOX and two DEGs encoding AOC were upregulated and DEGs (Cluster-2772.115415, Cluster-2772.82284 and Cluster-2772.46629) were inducibly expressed in the saline environment (Table 1). Lignin deposited in endothecium cells is essential prior to anther dehiscence . In E. angustifolia anther from the saline habitat, three DEGs encoding cinnamyl alcohol dehydrogenase (CAD) were upregulated and one (Cluster-2772.88336) was inducibly expressed in the saline environment (Table 1). In addition, two DEGs encoding E3 SUMO-protein ligase SIZ1 involved in the functional regulation of the endothecium in anthers [51, 52] were upregulated in E. angustifolia anthers under saline conditions. Notably, one DEG (Cluster-2772.16029) encoding receptor protein kinase (RPK), which plays a key role in anther dehiscence, was inducibly expressed in the saline environment [42, 43]. These results suggest that salinity induces or enhances the gene expression for cell wall formation in E. angustifolia anther to maintain anther dehiscence.
DEGs involved in hormone biosynthesis and signal transduction in E. angustifolia
Plant hormones play crucial roles in anther development. The expression levels of hormone-related genes correspond to higher concentrations of phytohormones or highly efficient signal transduction. To evaluate the possible reasons for the differences in anther development in E. angustifolia plants in non-saline vs. saline habitats, we identified three DEGs encoding the gibberellin receptor GID1, four DEGs encoding jasmonic acid-amino synthetase and two DEGs encoding auxin transporter-like protein, which were upregulated in plants grown in the saline environment. In particular, DEGs including Cluster-2772.3809, Cluster-25028.5, Cluster-2772.2343 and Cluster-2772.123259 were inducibly expressed in the saline environment (Table 2).
DEGs encoding transcription factors in E. angustifolia anthers
NAC, WRKY, and MYB TFs are involved in anther dehiscence in Arabidopsis [53-55]. In E. angustifolia anthers from the saline habitat, eleven DEGs encoding NAC TFs, nine encoding WRKY TFs and five DEGs encoding MYB TFs were upregulated compared to anthers from the non-saline habitat (Table 3). Moreover, NAC domain-containing protein 82, MYB 44, MYB6, MYB39 and WRKY40 were inducibly expressed in E. angustifolia anthers from the saline habitat.
DEGs involved in sugar transport and metabolism in E. angustifolia anthers
Sugar transport and metabolism play important roles in plant growth and development, especially during anther development. We detected enhanced and inducible expression of sugar transporter genes in E. angustifolia anthers from the saline habitat (Table S5). Three DEGs encoding sucrose synthase, one encoding sucrose phosphate synthase 1F and ten DEGs encoding sugar transporters or sugar transport proteins were upregulated in the anthers of plants of the saline habitat. In addition, eight of them (Cluster-2772.44272, Cluster-2772.44273, Cluster-2772.122621, Cluster-2772.17932, Cluster-2772.26904, Cluster-2772.115155, Cluster-2772.44021 and Cluster-2772.44963) were inducibly expressed in the saline habitat.
DEGs involved in ion content and ROS scavenging in E. angustifolia anthers
Altered levels of ions, especially Na+ and K+, are a critical indicator of plants grown in a saline environment. The transmembrane transport of ions occurs via transporters and channels located in the membrane, such as NHX (Na+/H+ antiporter), AKT (inward-rectifying K+ channel), and KEA (K+ efflux antiporter) proteins. In the anthers of plants from the saline habitat, four DEGs (Cluster-2772.37266, Cluster-2772.84379, Cluster-2772.125705, Cluster-2772.84388) encoding the Na+/H+ exchanger on the plasma membrane and tonoplast were upregulated, with at least a 2.34-fold (up to 11.97-fold) higher expression in anthers from the saline vs. non-saline habitat (Table 4). In addition, Cluster-5936.0 (encoding potassium channel AKT1) was upregulated 3.37-fold and Cluster-2772.33146 (encoding KEA) was upregulated 1.42-fold in the anthers of plants from the saline habitat. The expression levels of several ROS scavenging-related genes were also higher in E. angustifolia anthers from the saline habitat, including genes encoding catalase (CAT), peroxidase, and superoxide dismutase (SOD); the expression levels of these genes were at least 1.32-fold (up to 9.42-fold) higher in the anthers of plants from the saline vs. non-saline habitat. In particular, NHX6, four catalases, two PODs and one SOD were inducibly expressed the saline habitat (Table 4).