The Spartina alterniflora genome sequence provides insights into the salt-tolerance mechanisms of exo-recretohalophytes

Abstract

Spartina alterniflora is an exo-recretohalophyte Poaceae species that is able to grow well in seashore, but the genomic basis underlying its adaptation to salt tolerance remains unknown. Here, we report a high-quality, chromosome-level genome assembly of S. alterniflora constructed through PacBio HiFi sequencing, combined with high-throughput chromosome conformation capture (Hi-C) technology and Illumina-based transcriptomic analyses. The final 1.58 Gb genome assembly has a contig N50 size of 46.74 Mb. Phylogenetic analysis suggests that S. alterniflora diverged from Zoysia japonica approximately 21.72 million years ago (MYA). Moreover, whole-genome duplication (WGD) events in S. alterniflora appear to have expanded gene families and transcription factors relevant to salt tolerance and adaptation to saline environments. Comparative genomics analyses identified numerous species-specific genes, significantly expanded genes and positively selected genes that are enriched for 'ion transport' and 'response to salt stress'. RNA-seq analysis identified several ion transporter genes including the high-affinity K⁺ transporters (HKTs), SaHKT1;2, SaHKT1;3 and SaHKT1;8, and high copy number of Salt Overly Sensitive (SOS) up-regulated under high salt conditions, and the overexpression of SaHKT2;4 in Arabidopsis thaliana conferred salt tolerance to the plant, suggesting specialized roles for S. alterniflora to adapt to saline environments. Integrated metabolomics and transcriptomics analyses revealed that salt stress activate glutathione metabolism, with differential expressions of several genes such as γ-ECS, GSH-S, GPX, GST and PCS in the glutathione metabolism. This study suggests several adaptive mechanisms that could contribute our understanding of evolutional basis of the halophyte.

Keywords: Spartina alterniflora; evolution; genome; glutathione metabolism; salinity adaptation.

Figure 1

Genome features of S. alterniflora. From the outermost to the innermost circles are (a) karyotyping result, (b) GC content, (c) gene density, (d) transposable element proportion and (e) intra‐genome colinear blocks connected by curved lines. The colinear blocks represents segmental duplications.

Figure 2

Comparative analysis of gene families and evolution of the S. alterniflora. (a) Phylogenetic tree of 15 species constructed based on the single‐copy genes, with Physcomitrella patens as the outgroup. Gene family expansions and contractions in each plant species are shown in the right of tree. Divergence times on the tree were estimated using the MCMCtree and TimeTree. (b) Comparative analysis of salt stress‐responsive potassium ion channel genes in S. alterniflora and other investigated plant species. (c) Distribution of synonymous substitution rates (Ks) among collinear paralogs in S. alterniflora, Paspalum vaginatum and Zoysia japonica.

Figure 3

Comparative genomics analysis of S. alterniflora and other plant species. (a) Comparative GO analysis of the significantly expanded gene families in S. alterniflora and the other 13 species. (b) Phylogenetic tree of the EXPB genes in S. alterniflora and the other 13 species. The colour shadows represent the expansion clade members in S. alterniflora. (c) Sequence alignment of TTG1 proteins in diverse species (highlighted in red font). The black line indicates the protein sequence alignment position (located within the WD40 domain) where the mutation occurred. (d) GO enriched terms of the PSGs in S. alterniflora and other halophytes. (e) Common and species‐specific gene families in S. alterniflora and the other 13 species. (f) GO analysis of the 1614 species‐specific gene families in S. alterniflora.

Figure 4

Transcriptomic analysis of the S. alterniflora in response to salt treatment. (a) and (b) Up‐ and down‐regulated DEGs and GO enrichment of S. alterniflora in response to salt stress. (c) KEGG enrichment analysis of the up‐regulation DEGs in S. alterniflora. (d) The probable ABA‐dependent signalling pathway and the expression patterns of ABA‐responsive genes of S. alterniflora in response to salt stress. (e) The distribution of ABRE element on the 2‐kb upstream regions of bZIP, JAZ and AP2 genes. The regulatory element was identified in the PlantCARE database. (f) The differentially expressed salt‐tolerance‐related genes under salt stress. The green, purple, blue represent the gene expression with no changed, up‐regulated and down‐regulated under salt stress, respectively. The number represents the number of genes corresponding to the change. (g) Phylogenetic relationship of HKT gene family in S. alterniflora, maize, rice and Arabidopsis. The light green and light yellow represents the Types I and II of HKT genes.

Figure 5

Localization of SaHKT2;4 in the plasma membrane and impact of salt stress on SaHKT2;4 transgenic Arabidopsis and wild‐type plants. (a) Subcellular localization of SaHKT2;4 protein. The pAN580‐GFP‐SaHKT2;4 vector was transformed into rice seedling protoplasts, with the pAN580‐GFP vector serving as a control. Bar = 10 μm. (b) Phenotypic comparison of SaHKT2;4 overexpression lines and Col‐0 under 0 and 150 mm NaCl for 10 days. (c, d) Detection of O₂ ⁻ and H₂O₂ through histochemical staining using DAB (c) and NBT (d). (e–g) Quantification of Na⁺ (e) and K⁺ (f) contents in rosette leaves of WT and transgenic Arabidopsis upon treatment with 0 and 150 mm NaCl, with Na⁺/K⁺ ratios calculated (g). (h–l) Antioxidant responses and osmoregulation in WT and transgenic Arabidopsis under salt stress, measured through chlorophyll content (h), peroxidase (POD) activity (i), catalase (CAT) activity (j), proline levels (k) and malondialdehyde (MDA) content (l) in rosette leaves upon treatment with 100 and 200 mm NaCl. The error bars represent standard deviations, and statistically significant differences are indicated as follows: *P < 0.05; **P < 0.01 (Student's t‐test).

Figure 6

Salt stress promotes glutathione metabolism in S. alterniflora. (a) Number of the class I metabolites detected from the treatment samples. (b) Veen analysis of the differentially abundant metabolites (DAMs) under 300 and 600 mm NaCl treatments. There are 142 DAMs identified among the two treatments. (c) KEGG enrichment results of the DAMs in response to salt stress. (d) Proposed metabolism pathway for glutathione in S. alterniflora. Also indicated are expression patterns of corresponding key metabolism genes in response to salt stress. γ‐ECS, γ‐glutamylcysteine synthetase; γ‐EC, γ‐glutamylcysteine; GSH‐S, glutathione synthetase; GSH, glutathione; GSSG, glutathione disulfide; GR, glutathione reductase; GPX, glutathione peroxidase; GST, glutathione S‐transferase; PCS, phytochelatin synthase; NO, nitric oxide; GSNO, S‐nitrosoglutathione; P‐SNO, S‐nitrosylated protein; X, xenobiotic substrates; P‐SH, protein harbouring reduced SH groups; P‐SG, S‐glutathionylated protein. (e) Summary of cis‐acting elements in the promoter regions of genes related to the glutathione metabolic pathway. (f) qRT‐PCR analysis of the expression patterns of upstream TF genes. 0, 300 and 600 mm represent the concentrations of NaCl. The error bars represent standard deviations, and statistically significant differences are indicated as follows: *P < 0.05; **P < 0.01 (Student's t‐test).