Physiological Characteristics and Biomass Of Rice Seedlings Alleviated By Alginate Oligosaccharides Under Salt Stress
Compared with NaCl treatment, AOS +NaCl treatment reduces membrane lipid peroxidation levels, increases the biomass of two rice varieties, and increases the antioxidant indexes of two rice varieties (Figure 1). After AOS treatment, GSH content in FL478 and IR29 increases significantly (12.9% and 7.5%) (Figure. 1E).
Compared with the control, the ASA activities of FL478 and IR29 under NaCl treatment significantly increase (11.6% and 14.5%) (Figure 1F). In contrast, the CAT activities significantly decreased (10.4% and 18.1%) (Figure 1B). SOD and APX activities of the IR29 cultivar are significantly increased (23.1% and 49.7%) (Figure 1A, C), and the GSH of the FL478 cultivar is significantly increased (13.7%) (Figure 1E). Compared with NaCl treatment, AOS + NaCl treatment significantly increases SOD and CAT activities (9.6% and 5.4%) and GSH and AsA contents (4.1% and 11.2%) of rice FL478, respectively (Figure 1A, B, E, F). IR29 significantly increases SOD and APX activities (increase by 19.3% and 35.1%) and GSH and AsA contents (increase by 15.2% and 14.5%) (Figure 1A, C, E, F).
NaCl stress increases the MDA content in the leaves of both rice seedlings, and IR29 rice samples increased significantly (28.3%). Compared with NaCl treatment, AOS + NaCl treatment reduces the MDA content of both varieties, with rice IR29 content reaching a significant level (13.25%).
After AOS treatment, the two rice varieties' aboveground and underground dry weights increase but do not reach a significant level. Salt stress had no significant effect on the aboveground and underground dry weights of FL478, but significantly decreased the aboveground and underground dry weights of rice IR29 (by 13.4% and 23.8%). Compared with NaCl treatment, the aboveground and underground dry weights of IR29 after AOS + NaCl treatment increased significantly (8.3% and 14.0%) but did not reach the control levels (Figure 1G, H).
The results showed that AOS could reduce the effect of salt stress on rice by increasing SOD activity and AsA and GSH contents. At the same time, the impact of AOS on the antioxidant system of the two rice varieties was different. Exogenous AOS application could increase the CAT content of FL478 to offset the effects of salt stress. AOS can improve salt tolerance by increasing the APX content, reducing membrane lipid peroxidation damage and increasing the biomass of IR29 rice.
Transcriptome Responses Of Rice Seedlings To Salt Stress And Sodium Alginate Oligosaccharide Treatment
RNA sequences from 18 samples were analyzed (Table S1). Each sample produced an average of approximately 6.68GB of bases. The average Q30 is 93.47%, the sequencing quality and library construction of reaction samples were good, and the data accuracy was high. Each sample was clean read against a reference genome sequence. The mapping ratio is 89.91-91.17%, and the unique mapping ratio is 87.55%-88.87%. These data met the requirements of subsequent analysis, and the correlation coefficient of three replicates in each treatment is above 0.9 (Figure S2).
The number of differentially expressed genes was analyzed by transcriptome sequencing. The p <0.05 and Log2FC≥1 were used to screen differentially expressed genes. Compared with CK, there were 732 differentially expressed genes (483 up-regulated and 249 down-regulated) and 305 (169 down-regulated and 136 down-regulated) in NaCl and AOS + NaCl, respectively. There were 662 differentially expressed IR29 genes (272 up-regulated, 390 down-regulated) and 1030 (307 up-regulated, 723 down-regulated), respectively. There were 1015 differentially expressed genes in FL478 and IR29 varieties treated with NaCl+AOS (567 up-regulated and 448 down-regulated) and 225 (41 up-regulated and 184 down-regulated), respectively, compared with NaCl treatment (Table 1). RT-qPCR was used to detect the expression levels of eight identified transcriptome (RNA-ref) differential genes to verify the RNA-ref data (Figure S2). The results of RT-qPCR are positively correlated with those of RNA-ref.
Table 1 Statistics of the number of differentially expressed genes in rice seedlings
Combinations
|
Up-regulation
|
Down-regulation
|
All DEGs
|
FLCK-vs-FLNaCl
|
483
|
249
|
732
|
FLCK-vs-FLNaCl+AOS
|
169
|
136
|
305
|
FLNaCl-vs-FLNaCl+AOS
|
567
|
448
|
1015
|
IRCK-vs-IRNaCl
|
272
|
390
|
662
|
IRCK-vs-IRNaCl+AOS
|
307
|
723
|
1030
|
IRNaCl-vs-IRNaCl+AOS
|
41
|
184
|
225
|
Table 1 and Figure 2 show the number of down-regulated genes in rice seedlings under different treatments, revealing the expression patterns of FL478 and IR29 in response to salt, alginate oligosaccharides, alginate oligosaccharides and salt treatments. There are more differentially expressed genes of CK vs NaCl in FL478 than for CK vs AOS + NaCl, with more up-regulated genes than down-regulated genes in FL478 treated with NaCl vs AOS + NaCl. In IR29, more differentially expressed genes were treated with AOS + NaCl than with NaCl alone, with more down-regulated genes than up-regulated genes treated with NaCl vs AOS + NaCl. These results indicated that the two rice varieties had different regulatory pathways when developing salt tolerance and their responses to alginate oligosaccharides.
Cluster analysis reveals that alginate oligosaccharides and salts significantly change rice differential gene expression (Figure 2A, D). Further analysis using Venn diagrams showed that CK vs NaCl and NaCl vs AOS + NaCl shared fewer genes in the FL478 and IR29 varieties (Figure 2). These results indicated that rice plants responded to salt stress and alginate oligosaccharides with different genes.
Alginate Oligosaccharides Regulate Functional Genes To Alleviate Salt Stress In Rice
NaCl and AOS +NaCl treatments significantly affected gene expression in both rice varieties. In FL478/IR29 rice seedlings, the specific differential genes up-regulated and down-regulated by CK vs AOS +NaCl were 146/240 and 105/489, respectively. The significant effect of exogenous alginate oligosaccharides on the gene expression of rice plants under salt conditions is demonstrated schematically in Figure 2.
Through GO enrichment of differential genes, the most common types of up-regulated and down-regulated genes in FL478 rice seedlings are involved as integral components of membranes (GO:0016021), plasma membranes (GO:0005886), ATP binding (GO:0005524), Cytoplasm (GO:0005737) and Nucleus components (GO:0005634) (Figure 3A). Among the up-regulated and down-regulated genes in IR rice seedlings, the primary common categories were: integral components of membranes (GO:0016021), plasma membranes (GO:0005886), nucleus (GO:0005634), ATP binding (GO:0005524), and metal ion binding targets (GO:0046872) (Figure 3C).
Among 410 up-regulated genes in FL478 rice seedlings, ADP binding (GO:0043531) and plant-type hypersensitive genes were the most enriched responses (GO:0009626), with additional categories of microtubule (GO:0005874), Golgi membrane (GO:0000139), Golgi apparatus (GO:0005794), ubiquitin-protein transferase activity (GO:0004842), and cell wall organization (GO:0071555). Among the 490 down-regulated genes, the GO-rich types were cell wall (GO:0005618), Peroxisome (GO:0005777), chitinase activity (GO:0004568), chitin binding (GO:0008061), DNA binding (GO:0003677), chitin catabolic process (GO:0006032), and polysaccharide catabolic process (GO:0000272) (Figure 3B).
The 139 up-regulated genes in IR29 rice seedlings included the most enriched category of GO:0043531 (ADP binding), GO:0009626 (plant-type hypersensitive response), GO:0004386 (helicase) activity), GO:0006281 (DNA repair), and GO:0009870 (defense response signaling pathway, resistance gene-dependent). Among the down-regulated genes, dominant GO categories include GO:0009535 (chloroplast thylakoid membrane), GO:0009941 (chloroplast envelope), GO:0006979 (response to oxidative stress), GO:0009579 (thylakoid), GO:0009536 (plastid), GO:0061630 (ubiquitin protein ligase) activity), and GO:0042744(peroxide catabolic process) as shown in Figure 3D.
Gene Expression In Rice Seedlings Under Salt Stress Induced By Alginate Oligosaccharides
This study elucidates that compared with the control treatment, some genes were differentially expressed only under the alginate oligosaccharides + salt treatment. In FL478, 13 photosynthetic, 5 Serine/threonine kinases, 9 cell wall, 6 carbohydrate and 12 plant hormone-related genes were significantly different to the control data. The genes up-regulated IR29 expression levels under salt stress under sodium alginate oligosaccharide treatment correlated with 22 photosynthetic, 7 carbohydrates, 11 serine/threonine kinase-related genes, 15 plant hormones, 18 salt response and 24 transcription factors (Table S3). Among them, OsCAB1, OsLhcb2.1, OsLhcb6, OsLHCB4 and OsLhcp participated in the regulation of photosystem II, OsPMEI28 participated in the regulation of monosaccharide and lignin content in stems, OsGA2ox6 participated in the regulation of gibberellin, and OsRLCK106 participated in response to rice salt stress. These outcomes indicate that sodium alginate oligosaccharides play an active role in regulating salt tolerance in rice through multiple pathways. The photosynthetic components (OsPORA, OsDIMT2) are related to some identical genes in the two varieties. In contrast, other specific differentially expressed genes were different in the two varieties, indicating that alginate oligosaccharides may have different transcriptional regulation modes for FL478 and IR29.
Changes Of Metabolites In Rice Seedlings Treated With Alginate Oligosaccharides Under Salt Stress
To evaluate the effects of exogenous alginate oligosaccharides treatment on the metabolic variations between FL478 and IR29 rice seedlings under salt stress. Differential metabolites were screened using the filters VIP≥1, Fold-Change≥1.2 or≤0.83, and p-value<0.05. FL478 and IR29 samples treated with CK, S and A+S were compared. Two principal components (PC1=50.17%, PC2=23.19%) in positive ion mode and two principal components (PC1=65.12%, PC2=15.73%) in negative ion mode were obtained using PCA analysis, with differences between the two cultivars in both modes (see Figure 4A, D). A total of 851 metabolites were identified in all samples under both modes. Among them, 549 differential metabolites (VIP≥1, Fold-Change≥1.2 or ≤0.83, p-value<0.05) were significantly abundant in at least one group (Table 2).
Table 2 Breakdown of differential metabolites for both rice species.
Group
|
Total
|
Up
|
Down
|
FLCK-vs-FLNaCl
|
115
|
44
|
71
|
FLCK-vs-FLNaCl+AOS
|
60
|
13
|
47
|
FLNaCl-vs-FLNaCl+AOS
|
88
|
55
|
33
|
IRCK-vs-IRNaCl
|
90
|
39
|
51
|
IRCK-vs-IRNaCl+AOS
|
80
|
26
|
54
|
IRNaCl-vs-IRNaCl+AOS
|
116
|
65
|
51
|
Forty-four metabolites increased, and 71 decreased in salt-treated FL478 rice seedlings compared to the control seedlings. The abundance of 39 and 51 metabolites in IR29 rice seedlings increased and decreased, respectively. After alginate oligosaccharides + salt treatment, the metabolite abundance of FL478 rice seedlings increased by 13 and decreased by 47 compared with the control samples. In IR29 rice seedlings, the metabolite abundance increased and decreased by 26 and 54 species, respectively, compared with the control data (Table 2). The differential metabolites of rice after treatment were analyzed using Venn diagrams. In the treatment with alginate oligosaccharides + salt, the abundance of 4 and 17 differential metabolites increased in the FL478 and IR29 rice seedlings, while 18 and 33 differential metabolites decreased, respectively (Figure 4B, C, E, F). Therefore, the metabolite expression differences induced by alginate oligosaccharides in the two rice varieties changed significantly under salt stress. In FL478, the differential metabolites Flavonoids (Vicenin II, Wedelolactone), Indole and derivatives (6-Hydroxymelatonin), and Gamma butyrolactones (Dehydroascorbic acid) abundance all increased significantly. The abundance of differential metabolites such as Carbohydrates(D-(+)-Glucosamine)、Terpenoids(2,6-Di-tert-butyl-1,4-benzoquinone)、Fatty acyls(10-Nitrolinoleate) was significantly reduced. Amino acids (L-Histidine、Aspartame、L-Norleucine、N-Acetyl-L-methionine), Terpenoids (Rehmannioside C, Andrographolide) and other differential metabolites are significantly increased in abundance in IR29. The abundance of differential metabolites such as Rehmannioside C and Andrographolide increased significantly in IR29. Flavonoids (Neomangiferin 5,7-dihydroxychromone, Vitexin, and Wedelolactone), Polyketides [PK] (Dodecyl sulfate, 6-Gingerol) and other differential metabolite levels were significantly reduced in IR29 (Table S4). Among them, 6-Hydroxymelatonin, Wedelolactone and L-Histidine play an active role in the antioxidant process.
Table 3 Differential metabolites with significant up-regulation or down-regulation in FL478 only under alginate oligosaccharides + salt treatment and in the control samples.
Metabolite ID
|
Metabolite Name
|
P-value
|
VIP
|
Log2FC
|
15.16_486.26264
|
Andrastin A
|
0.00
|
1.53
|
-0.33
|
0.844_174.01649
|
Dehydroascorbic acid
|
0.00
|
1.87
|
0.64
|
17.37_350.16205
|
7-(2,3-Dimethylphenyl)-2-methoxy-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione
|
0.00
|
1.69
|
-0.57
|
1.642_297.10731
|
N(6)-oh-me-adenosine
|
0.00
|
1.75
|
-0.54
|
15.159_220.14657
|
2,6-Di-tert-butyl-1,4-benzoquinone
|
0.01
|
1.64
|
-0.36
|
9.905_314.04186
|
Wedelolactone
|
0.01
|
1.25
|
0.35
|
17.766_354.19332
|
2-(3,4-Dimethoxyphenyl)-N-(2-piperidinophenyl)acetamide
|
0.01
|
1.84
|
-0.82
|
11.85_478.25486
|
Diflucortolone pivalate
|
0.01
|
2.43
|
-0.75
|
9.363_307.21470
|
10-Nitrolinoleate
|
0.01
|
2.76
|
-2.95
|
2.302_248.11946
|
6-Hydroxymelatonin
|
0.01
|
1.87
|
0.86
|
1.073_179.07961
|
D-(+)-Glucosamine
|
0.02
|
1.50
|
-0.50
|
7.915_467.22892
|
1-{[(1s,4s,6s)-6-isopropyl-3-methyl-4-{[5-(4-pyridinyl)-1,3,4-oxadiazol-2-yl]methyl}-2-cyclohexen-1-yl]methyl}-3-phenylurea
|
0.02
|
2.43
|
-2.77
|
7.196_397.14212
|
Methyl 2,7,7-trimethyl-5-oxo-4-[3-(2-thienyl)-1H-pyrazol-4-yl]-1,4,5,6,7,8-hexahydro-3-quinoline carboxylate
|
0.02
|
1.43
|
-0.36
|
5.726_594.15930
|
Vicenin II
|
0.02
|
1.22
|
0.38
|
11.851_330.18063
|
(1r,4as)-7-(2-hydroxypropan-2-yl)-1,4a-dimethyl-9-oxo-3,4,10,10a-tetrahydro-2h-phenanthrene-1-carboxylic acid
|
0.03
|
1.59
|
-1.24
|
8.181_467.22889
|
N-(3,4-dimethoxybenzyl)-2-[(3r,4s)-3-{[5-(4-fluorophenyl)-1,2-oxazol-3-yl]methyl}-4-piperidinyl]acetamide
|
0.03
|
1.56
|
-1.27
|
10.031_490.27684
|
N-({(2R,4S,5R)-5-[3-(3,4-Dimethoxyphenyl)-1-methyl-1H-pyrazol-5-yl]-1-azabicyclo[2.2.2]oct-2-yl}methyl)-2-ethylbutanamide
|
0.04
|
1.14
|
-0.45
|
13.967_334.21199
|
(3E)-3-(Hydroxymethyl)-2-oxo-5-[(1S,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-3-pentanoic acid
|
0.04
|
1.02
|
-0.40
|
16.124_382.27154
|
1a,1b-dihomo prostaglandin f2О±
|
0.04
|
1.17
|
-0.93
|
10.011_472.26648
|
4-cyano-n-({(1s,4s,6s)-6-isopropyl-3-methyl-4-[2-(4-methyl-1-piperazinyl)-2-oxoethyl]-2-cyclohexen-1-yl}methyl)benzamide
|
0.04
|
2.00
|
-0.71
|
8.793_376.20933
|
3-[4-(tert-Butyl)anilino]-1-[4-(tert-butyl)phenyl]-2,5-dihydro-1H-pyrrole-2,5-dione
|
0.05
|
1.12
|
-0.98
|
7.725_528.18623
|
1,4:3,6-Dianhydro-2-[(benzylsulfonyl)amino]-5-{[4-(4-biphenylyl)-2-pyrimidinyl]amino}-2,5-dideoxy-L-iditol
|
0.05
|
1.62
|
-2.80
|
A KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis of the metabolites was undertaken to investigate the primary metabolic pathways modified by alginate oligosaccharide treatment of FL478 and IR29 rice seedlings under salt stress. FL478 and IR29 were annotated into 4 and 16 KEGG pathways for samples treated with alginate oligosaccharides + salt. The co-enriched FL and IR pathways include Ascorbate and aldarate metabolism (ko00053) and Glutathione metabolism (osa00480). FL's unique metabolic enrichment pathways include amino sugar and nucleotide sugar metabolism (ko00520) and tryptophan metabolism (ko00380). The unique metabolic pathways for IR include histidine metabolism (ko00340), beta-alanine metabolism(ko00410), stilbenoid, diarylheptanoid and gingerol biosynthesis (ko00945), flavonoid biosynthesis (ko00941), aminoacyl-tRNA biosynthesis (ko00970), and biosynthesis of secondary metabolites (ko01110) (Table S5).
Combined Transcriptome And Metabolome Analysis
To explore the effects of alginate oligosaccharides on genes and metabolites of rice under salt stress, differential genes and metabolites of control and salt and control and alginate oligosaccharides + salt treatment groups were simultaneously enriched into KEGG pathways. Twenty and eight KEGG pathways were identified due to salt exposure in FL478 and alginate oligosaccharides + salt treatments, while 15 and nine KEGG pathways were identified for salt exposure and alginate oligosaccharides + salt treatments in IR29 (Figure 5). Glutathione metabolism (ko00480), Ascorbate and aldarate metabolism (ko00053) in FL478 are in these pathways. For IR29, the pathways for beta-Alanine metabolism (ko00410), Glutathione metabolism (ko00480), Ascorbate and aldarate metabolism (ko00053), and Aminoacyl-tRNA biosynthesis (ko00230) are significantly enriched only under the alginate oligosaccharides + salt treatment. Among them, glutathione metabolism (ko00480), ascorbic acid and hemorakine metabolism (ko00053) are significantly enriched in FL478 and IR29 (Figure 6). The differential metabolite dehydroascorbic acid is simultaneously enriched in both pathways. Histidine abundance increases significantly during sodium alginate oligosaccharide treatment under salt stress. The related gene GAD3 is also significantly up-regulated (Figure 7). Alginate oligosaccharides may induce the GAD3 gene to induce important histidine metabolism modulation.
Table 4 Co-enrichment pathways of transcriptome and metabolome in rice seedlings treated with alginate oligosaccharides + salt
Varieties
|
Pathway
|
Metabolite ID
|
Metabolite Name
|
logFC
|
FL478
|
Glutathione metabolism(ko00480)
|
0.844_174.01649
|
Dehydroascorbic acid
|
0.64
|
|
Ascorbate and aldarate metabolism(ko00053)
|
0.844_174.01649
|
Dehydroascorbic acid
|
0.64
|
IR29
|
beta-Alanine metabolism(ko00410)
|
0.771_155.06943
|
L-Histidine
|
0.35
|
|
Glutathione metabolism(ko00480)
|
0.844_174.01649
|
Dehydroascorbic acid
|
-0.41
|
|
Ascorbate and aldarate metabolism(ko00053)
|
0.844_174.01649
|
Dehydroascorbic acid
|
-0.41
|
|
Aminoacyl-tRNA biosynthesis(ko00970)
|
0.771_155.06943
|
L-Histidine
|
0.35
|