Transcriptome analysis of Tamarix ramosissima leaves in response to NaCl stress

Abstract

Halophyte Tamarix ramosissima. Lcdcb (T. ramosissima) are known as the representative of Tamarix plants that are widely planted in salinized soil. However, molecular mechanisms towards salt tolerance and adaptation are largely rare. In this study, we carried out RNA-sequence and transcriptome analysis of T. ramosissima in response to NaCl stress, screened differentially expressed genes (DEGs) and further verified by qRT-PCR. Results showed that 105702 unigenes were spliced from the raw data of transcriptome sequencing, where 54238 unigenes were retrieved from KEGG, KOG, NR, and SwissProt. After 48 hours of NaCl treatment, the expression levels of 6374 genes were increased, and 5380 genes were decreased in leaves. After 168 hours, the expression levels of 3837 genes were up-regulated and 7808 genes were down-regulated. In particular, 8 transcription factors annotated to the KEGG Pathway were obtained, involving the WRKY and bZIP transcription family. In addition, KEGG pathway annotation showed that expression of 39 genes involved in ROS scavenging mechanisms were significantly changed, in which 21 genes were up-regulated and 18 genes were down-regulated after 48 hours as well as 15 genes were up-regulated and 24 genes were down-regulated after 168h. Simultaneously, the enzyme activities of SOD and POD were significantly enhanced under NaCl treatment, but the enzyme activity of CAT was not significantly enhanced. Moreover, WRKY, MYB and bZIP may participate in the process of salt resistance in T. ramosissima. This study provides gene resources and a theoretical basis for further molecular mechanisms of salt tolerance in T. ramosissima.

1. Introduction

Salinized soil has high salt content and poor soil physical and chemical properties, which seriously hindered the growth and development of plants [1]. Salinized soil contains a lot of Na⁺ type salt which can destroy the stability of protein and membrane, and produces osmotic stress and ion poisoning to initiate reactive oxygen ROS (reactive oxygen species) signals in the cell, make dysfunction of the cell, affect the growth of plants, and causes plant death in severe cases [2]. In the past decades, the area of salinized soil has continued to expand due to global human activities and climate changes. Favorably, it is becoming a hotspot to carry out afforestation, restore salinized soil and improve the ecological environment in salinized soil areas.

In recent years, RNA-Seq technology has been widely used to study molecular mechanisms of plant resistance to adverse stresses, including salt stress [3, 4]. Previous studies showed that reactive oxygen species (ROS) would be produced in multiple cell compartments, including chloroplasts [5], mitochondria [6], and peroxisomes [7] under salt stress conditions. A relatively low concentration of ROS is known as an important signal molecule to regulate the normal plant growth and responses to abiotic stresses [8, 9]. However, excessive accumulation of ROS can adversely cause cell oxidative damage [10]. To adapt to ROS damage, higher plants have evolved corresponding regulatory mechanisms to maintain the stability of life activities. Notably, ROS scavenging enzymatic systems, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX), are prone to play important roles to adapt to undesired abiotic stresses [11]. In addition, plants can also take advantage of the ascorbic acid- reduced glutathione (AsA-GSH) cycle, which contains APX, glutathione reductase (GR) and AsA, and GSH, and so on, to eliminate the damage of ROS [12].

In particular, transcription factors are the most important regulators for plants to respond to various abiotic stresses [13]. In details, reports are focused on the involvement of transcription factors of WRKY [14], bHLH [15], bZIP [16] NAC [17], MYB [18], AP2/ERF [19] under salt stress. In cotton, the WRKY transcription factor gene GhWRKY34 was induced by salt stress in transgenic Arabidopsis [20]. In Paspalum notatum, the WRKY transcription factor genes are involved in regulating the expression of SOD and related oxidoreductase genes to adapt to salt stress [21]. Notably, Tamarix plants have evolved a complex regulatory network for a long time to adapt to the adverse abiotic stresses [22]. T. hispida is often used as a biological material to explore the molecular mechanisms towards salt stress tolerance. Overexpression of the T. hispida ThCOL2 gene can regulate the activity of protective enzymes and reduce the accumulation of O²⁻ and H₂O₂ that enhanced the ROS scavenging ability and improved the adaptability of transgenic T. hispida under salt stress [23]. Overexpression of ThbZIP1 enhanced the activity of POD and SOD, increased the content of soluble sugar and soluble protein that further improved salt tolerance in transgenic T. hispida [24]. T. ramosissima usually grows in arid and semi-arid regions with high salt content [25]. Moreover, low concentration (<100mM) NaCl promoted while high concentration (≥200mM) NaCl stress inhibited the growth of T. Ramosissima [26]. Liu and her colleagues found that T. ramosissima exhibited the strongest salt tolerance among 3 Tamarix species, including Tamarix gansuensis H.Z.Zhang, Tamarix leptostachys Bunge, and Tamarix ramosissima. Lcdcb, and T. ramosissima were chosen as the representative species of Tamarix plants for further mechanisms studies.

In this study, we performed high-throughput transcriptome sequencing in T. ramosissima under NaCl stress and screened and verified DEGS at the transcriptional level. This study lays a theoretical basis to reveal molecular mechanisms towards salt tolerance in Tamarix plants, and provides gene resources for further variety breeding of salt-tolerant Tamarix plants.

2. Materials and methods

2.1. Plant materials

T. ramosissima seedlings were provided by the Dongying Experimental Station of Shandong Academy of Forestry Sciences. Experiments were completed at the Key Laboratory of Forest Tree Genetic Breeding and Biotechnology of the Ministry of Education of Nanjing Forestry University from October 2019 to March 2021. 5-month-old T. ramosissima seedlings with uniform growth were transferred to a 24-well hydroponic box (size: 40cm*30cm*16cm), supplemented with 1/2 Hoagland nutrient solution, and then placed in a greenhouse that was maintained at 26 ± 2°C (day) whose relative humidity stays between 40% and 55% for 1 month after training before treatment. The culture solution was changed every 3^rd day.

2.2 NaCl treatment

In the control group (CK), seedlings were suffered with 1/2 Hoagland nutrient solution. In the treatment group, seedlings were cultured in 1/2 Hoagland nutrient solution, supplemented with 200 mM NaCl. 8 plants were used in each group, and the experiments were repeated 3 times. The culture solution was changed every 3^rd day. The leaf samples were collected at 0h, 48h, and 168h, respectively, and immediately frozen in liquid nitrogen, and then moved to a -80°C refrigerator for storage.

2.3 Phenotype and antioxidative enzyme activity analysis in T. ramosissima leaves

Leaves of T. ramosissima were collected after 0h, 48h and 168h of NaCl treatment, respectively, and the distribution of salt secretion on the leaf surface was observed using a JSZ6S stereo microscope (Jiangnan, China). The activities of SOD [27], POD [28] and CAT [29] were determined and analyzed, according to the description of the commercial Extraction Kits (Jiancheng Limit Co., Nanjing, China).

2.4 Transcriptome sequencing

The frozen leaf samples were used for 3-generation high-throughput transcriptome sequencing, using Illumina HiSeq^™4000, in Guangzhou GENE Denovo Company. The purified PCR products were analyzed by pair-end sequencing (PE150) on the platform according to standard operations, and then fastp was used for quality control [30]. The original data was first filtered to obtain clean reads, then assembled [31]. These assembled fragments without N terminal obtained by reading overlap are used as the assembled Unigene, and then use Blast2 GO [32] and KOBAS [33] to obtain Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. The Illumina raw sequencing data were submitted to the National Center for Biotechnology Information (NCBI) Short Reads Archive (SRA) database under accession number SRP356215.

2.5 Screening methods for differentially expressed genes

DESeq2 software was used to analyze the reads count data to obtain the final correct FDR value (FDR value means BH corrected p value) [34]. A corrected p value of <0.05 is considered to be significantly enriched. Based on the results of the difference analysis, genes with FDR value <0.05 and |log2FC| > 1 are considered to be significantly different genes.

2.6 Quantitative Real-Time PCR (qRT-PCR) validation of DEGs

Eight putative genes (Unigene0104732, Unigene0028215, Unigene0083695, Unigene0069097, Unigene0090596, Unigene0024962, Unigene0007135 and Unigene0088781) were randomly selected to verify the accuracy of the RNA-Seq results by the qRT-PCR technique. The total RNA was extracted with Omega RNA Extraction Kit (Shanghai, China), and then reverse-transcribed the 1-strand cDNA by using the PrimerScript^™ RT Master Mix Kit (TaKaRa, Dalian, China). Specific primers of DEGs are designed via the Primer-BLAST server (S1 Table). qRT-PCR samples were labelled with PowerUp^™ SYBR Green Master mix reagent (Thermo Fisher, China) and then performed on ABI ViiA^™ 7 Real-time PCR system (ABI, USA). A total of 3 biological repeats were performed, each with 4 technical repetitions. Actin was used as the internal reference gene, and the relative expression level was calculated by the 2^−ΔΔCt method.

3 Results

3.1 Phenotype and antioxidative enzyme activity analysis in T. ramosissima leaves

In the CK group, there was no salt secretion in the leaves of T. ramosissima at 0h, 48h and 168h. However, leaves began to excrete a small amount of salt at 48h under 200 mM NaCl treatment, and the salt secretion reached the maximum amount at 168h under NaCl treatment (Fig 1). These findings showed that the amount of salt secretion in leaves increased along with the prolongation of NaCl treatment time.

Fig 1. Salt secretion from T. ramosissima leaves under NaCl stress.

[Leaf salt secretion are checked in the leaves of T. ramosissima at 0h, 48h and 168h under 200mM NaCl treatment (The red labels indicate the salt secretion products)].

The activities of SOD, POD and CAT in the leaves of T. ramosissima showed an increasing trend under 200mM NaCl for 48h and 168h, compared with the control group. In particular, the activities of SOD and POD were significantly higher than those of the control group after either 48h or 168h. The CAT activity increased slightly but had no significant change, compared with the control group (Fig 2). These results showed that SOD and POD activities under salt stress in the leaves of T. ramosissima.

Fig 2. Changes of SOD, POD and CAT enzyme activities in leaves of T. ramosissima under salt stress.

(Note: The different letters indicate significant differences at the p value < 0.05. The graph illustrates the changes of enzyme activities in SOD, POD and CAT compared with CK group at 48h and 168h after 200mM NaCl treatment).

3.2 Sequencing quality analysis

Using IlluminaHiSeq^™4000, obtained multiple high-quality bases at 0h, 48h and 168h were obtained in T. ramosissima leaves under 200mM NaCl stress, and Q20 reached more than 95%, and Q30 reached more than 90% and the GC content is above 44% (Table 1), indicating that the quality of the transcriptome sequencing is relatively high, which is reliable for further analysis.

Table 1. Filtered reads quality statistics.

Sample	Raw data (bp)	Clean data (bp)	Q20 (%)	Q30 (%)	GC (%)
CK1-0h	6341519400	6017997220	97.34%	92.65%	45.15%
CK2-0h	6216526200	6057604903	97.54%	93.11%	45.12%
CK3-0h	6627399900	6507140412	97.77%	93.55%	45.24%
NaCl1-48h	6654895800	6541177895	98.78%	95.93%	45.04%
NaCl2-48h	6888168900	6782061623	98.72%	95.71%	44.94%
NaCl3-48h	6720560700	6605897734	98.79%	95.94%	44.90%
NaCl1-168h	6691086000	6551036697	98.85%	96.17%	45.42%
NaCl2-168h	6181114500	6032346218	98.83%	96.16%	45.44%
NaCl3-168h	6396633600	6256461214	98.93%	96.43%	45.35%

3.3 Unigene basic notes

Results showed that a total of 105,702 Unigenes were spliced in this study. There are 53,385, 46062, 31587, and 36087 Unigenes with gene annotations in the Nr, KEGG, KOG, and SwissProt databases, respectively, and a total of 27,670 Unigenes were simultaneously screened with annotations in the four major databases (Fig 3).

Fig 3. Annotation diagrams obtained form 4 major databases.

(Distribution map of 4 major databases annotated to genes).

3.4 Quantitative expression analysis of DEGs

Taking the CK group as the control, the transcription data of T. ramosissima under NaCl stress treatment at 0h, 48h and 168h were compared and analyzed, respectively. DEGs were screened with the standard of FDR value <0.05, p value <0.05 and |log2FC| > 1 after correction. In the CK-0h v.s. NaCl-48h comparison group, a total of 11,754 gene expression levels were detected, in which 6374 genes were up-regulated and 5380 genes were down-regulated under NaCl treatment. In the CK-0h v.s. NaCl-168h comparison group, a total of 7768 gene expression changes were detected, in which 2542 genes were induced and 5226 genes were reduced under NaCl treatment (Fig 4).

Fig 4. Analysis of DEGs.

(The differentially expressed genes were up-regulated and down-regulated in the comparison groups of CK-0h v.s. NaCl-48h and CK-0h v.s. NaCl-168h).

3.5 GO analysis of DEGs

Through GO annotation analysis, the above-mentioned DEGs can be divided into 3 categories: biological processes, cellular components, and molecular functions, and a total of 51 different classification groups were observed (Fig 5). In detail, in the CK-0h v.s. NaCl-48h comparison group, the up-regulated genes were slightly more than the down-regulated genes, while in the CK-0h v.s. NaCl-168h comparison group, the down-regulated genes were significantly more than the up-regulated genes. In the broad category of biological process, DEGs are mainly concentrated in cellular process, metabolic process, single-organism process and response to stimulus (Table 2). In the molecular function category, DEGs are mainly concentrated in catalytic activity, binding, transporter activity and structural molecule activity (Table 3). Among the major categories of cell components, DEGs are mainly enriched in cell, cell part, organelle and membrane (Table 4). In addition, the number of up-regulated DEGs was significantly lower and the overall number of DEGs decreased, along with the time of NaCl treatment. We speculate that T. ramosissima leaves may respond to high NaCl stress by affecting the expression level of related DEGs at the transcription level.

Fig 5. GO enrichment analysis of DEGs.

(The first and outer circle: the top 20 GO terms are enriched, outside the circle is the scale of the number of genes. Different colors represent different Ontologies. The second circle: the number of the GO term in the background gene and the Q value. The more genes, the better the bars. Long, the smaller the Q value, the darker the color. The third circle: the bar graph of the ratio of up-regulated genes, the dark color represents the proportion of up-regulated genes, and the light color represents the proportion of down-regulated genes. The specific value is displayed below. The fourth and inner circle: the ratio of each GO term Rich Factor value (number of differential genes in this GO term divided by all numbers), background grid lines, each grid represents 0.1).

Table 2. GO enrichment analysis of the biological process of DEGs.

GO ID	GO Term	CK-0hv.s.NaCl-48h (DEGs)		CK-0hv.s.NaCl-168h (DEGs)
		Up	Down	Up	Down
GO:0023052	signaling	168	268	98	262
GO:0065007	biological regulation	659	563	269	728
GO:0050789	regulation of biological process	584	514	240	655
GO:0002376	immune system process	73	124	29	130
GO:0032501	multicellular organismal process	398	276	150	394
GO:0050896	response to stimulus	796	875	411	962
GO:0051704	multi-organism process	178	242	95	261
GO:0032502	developmental process	519	373	201	511
GO:0044699	single-organism process	1269	1222	592	1400
GO:0048511	rhythmic process	18	15	15	13
GO:0048519	negative regulation of biological process	90	37	22	70
GO:0048518	positive regulation of biological process	48	61	23	75
GO:0000003	reproduction	222	156	93	226
GO:0040011	locomotion	1	4	1	0
GO:0022414	reproductive process	220	152	91	223
GO:0098754	detoxification	4	0	1	2
GO:0051179	localization	457	436	197	498
GO:0001906	cell killing	0	1	0	4
GO:0040007	growth	83	68	46	102
GO:0022610	biological adhesion	2	4	4	10
GO:0009987	cellular process	1395	1456	659	1638
GO:0008152	metabolic process	1420	1479	659	1630
GO:0071840	cellular component organization or biogenesis	499	417	217	495

Table 3. GO enrichment analysis of molecular function of DEGs.

GO ID	GO Term	CK-0hv.s.NaCl-48h (DEGs)		CK-0hv.s.NaCl-168h (DEGs)
		Up	Down	Up	Down
GO:0001071	nucleic acid binding transcription factor activity	79	63	33	102
GO:0004871	signal transducer activity	11	26	3	30
GO:0005215	transporter activity	146	117	57	152
GO:0016209	antioxidant activity	12	23	6	18
GO:0009055	electron carrier activity	6	9	5	7
GO:0060089	molecular transducer activity	14	19	4	18
GO:0005198	structural molecule activity	119	165	52	130
GO:0003824	catalytic activity	999	1062	458	1212
GO:0098772	molecular function regulator	16	8	8	17
GO:0000988	transcription factor activity, protein binding	1	1	1	2
GO:0005488	binding	939	967	415	1104
GO:0045182	translation regulator activity	0	0	0	0

Table 4. GO enrichment analysis of cellular component of DEGs.

GO ID	GO Term	CK-0hv.s.NaCl-48h (DEGs)		CK-0hv.s.NaCl-168h (DEGs)
		Up	Down	Up	Down
GO:0044425	membrane part	311	296	146	333
GO:0016020	membrane	686	732	342	789
GO:0031012	extracellular matrix	1	2	0	2
GO:0044421	extracellular region part	3	3	8	2
GO:0044420	extracellular matrix component	0	1	0	0
GO:0005576	extracellular region	76	97	49	112
GO:0009295	nucleoid	2	2	0	1
GO:0030054	cell junction	193	226	93	248
GO:0099512	supramolecular fiber	2	0	0	5
GO:0019012	virion	0	3	0	2
GO:0044423	virion part	0	3	0	2
GO:0031974	membrane-enclosed lumen	6	12	8	9
GO:0044422	organelle part	502	506	232	497
GO:0032991	macromolecular complex	327	311	119	298
GO:0043226	organelle	1167	1077	595	1180
GO:0005623	cell	1345	1290	667	1430
GO:0044464	cell part	1340	1284	664	1428

3.6 KEGG pathway analysis of DEGs in T. ramosissima leaves under NaCl stress KEGG pathway analysis showed that 1762 and 1366 DEGs were annotated in the comparison group of CK-0h v.s. NaCl-48h and CK-0h v.s. NaCl-168h, respectively (Fig 6), which directly reflected the changes of gene expression in leaves of T. ramosissima under NaCl stress. Among the top 10 KEGG pathways from the CK-0h v.s. NaCl-48h comparison group, Ribosome (ko03010) annotated to 435 DEGs, accounting for 24.69%, followed by biosynthesis of secondary metabolites (ko01110), phytopathogen interaction (ko04626), phenylpropane biosynthesis (ko00940), plant hormone signal transduction (ko04075) and plant MAPK signaling pathway (ko04016), respectively, annotated to 416 (23.61%), 88 (4.99%), 69 (3.92%), 69 (3.92%) and 59 3.35% DEGs. Among the top 20 pathways from the CK-0h v.s. NaCl-168h comparison group, the metabolic pathway (ko1100) was annotated to 614 DEGs, accounting for 44.95%, followed by the Biosynthesis of secondary metabolites (ko01110), Ribosomes (ko03010), Oxidative phosphorylation (ko00190), Plant hormone signal transduction (ko04075) and Plant pathogen interaction (ko04626), respectively, annotated to 351 (25.70%), 296 (21.67%), 104(7.61%), 72 (5.27%) and 63 (4.61%) DEGs. In summary, DEGs are significantly enriched on the KEGG pathway, such as metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction, in T. ramosissima under NaCl treatment.

Fig 6. Top 10 pathway analysis.

(Distribution of differentially expressed genes in the top 10 KEGG pathway in the CK-0hv.s.NaCl-48h and CK-0hv.s.NaCl-168h comparison groups).

3.6 Analysis of antioxidant DEGs in T. ramosissima leaves under NaCl stress

Results showed that the expression of 39 ROS scavenging-related DEGs in the leaves of T. ramosissima were changed under salt treatment (Table 5). Notably, there were 21 up-regulated genes and 18 down-regulated genes from the CK-0h v.s. NaCl-48h comparison group. The largest number of up-regulated genes is GST (7), followed by POD (4), CAT (4), APX (3), GR (2) and SOD (1). The largest number of down-regulated genes is GST (8), followed by POD (4), SOD (3), APX (2) and GPX (1). In the CK-0h v.s. NaCl-168h comparison group, there were 15 up-regulated genes and 24 down-regulated genes. In particular, the largest number of up-regulated genes are CAT(3), APX(3) and GST(3), followed by SOD(2), POD(2), and GR(2). The largest number of down-regulated genes is GST(12), followed by POD(6), SOD(2), APX(2), CAT(1) and GPX(1) (Fig 7). These results showed implying that the antioxidant mechanism might be initially enhanced in T. ramosissima leaves accompanied with corresponding physiological responses to resist NaCl stress during the first 48 hours. However, the antioxidant mechanism might be initially inhibited in T. ramosissima leaves under a long time (168 h) of NaCl treatment, just to adapt to salt stress.

Table 5. Antioxidant DEGs annotated to the KEGG pathway.

Pathway	Gene ID	Description	Log2FoldChange
			CK-0hv.s.NaCl-48h	CK-0hv.s.NaCl-168h
SOD
ko04146	Unigene0033269	SOD4 protein, partial	-1.01	-0.17
	Unigene0049419	superoxide dismutase [Mn]	7.82	9.54
	Unigene0050462	superoxide dismutase	-1.51	-0.60
	Unigene0082550	superoxide dismutase	-0.57	0.26
POD
ko01100;ko01110;ko00940	Unigene0009260	peroxidase 20	-0.46	-0.59
	Unigene0013825	peroxidase	1.79	-1.76
	Unigene0013827	peroxidase	1.17	-0.61
	Unigene0014843	peroxidase	-2.95	-1.80
	Unigene0029752	peroxidase 17	1.51	-0.44
	Unigene0049353	peroxidase 5	-4.98	0.70
	Unigene0086491	peroxidase 52	0.85	-0.74
	Unigene0094375	peroxidase 31	-0.32	1.59
CAT
ko01100;ko01110;ko01200;ko00630;ko04146;ko04016;ko00380	Unigene0046159	catalase isozyme 1	0.58	0.73
	Unigene0046160	catalase, partial	0.77	1.93
	Unigene0087092	leaf catalase	0.07	-0.48
	Unigene0103080	catalase isozyme 1	5.74	12.41
APX
ko01100;ko00480	Unigene0008032	L-ascorbate peroxidase 3	-0.45	-0.24
	Unigene0008033	L-ascorbate peroxidase 3	0.49	0.60
	Unigene0008513	peroxidase domain-containing	0.59	-0.51
	Unigene0048033	cytosolic ascorbate peroxidase	-0.02	0.08
	Unigene0105664	thylakoid ascorbate peroxidase precursor, partial	0.90	1.55
GPX
ko01100;ko0048; ko00590	Unigene0035407	glutathione peroxidase	-0.18	-0.65
GST
ko01100;ko00480	Unigene0001041	glutathione S-transferase	-3.17	-11.56
	Unigene0004890	glutathione S-transferase T1-like	-1.88	-0.68
	Unigene0007072	glutathione S-transferase U17-like	-0.08	0.49
	Unigene0012650	glutathione S-transferase Mu 1-like	13.69	7.20
	Unigene0015109	glutathione S-transferase U8-like	0.11	-0.42
	Unigene0020552	glutathione S-transferase	-0.14	-0.07
	Unigene0041633	microsomal glutathione S-transferase 3-like	0.08	-0.34
	Unigene0048538	glutathione S-transferase U10-like	-3.33	-2.73
	Unigene0056773	glutathione S-transferase	-0.70	0.14
	Unigene0064942	glutathione S-transferase L3	0.28	-0.10
	Unigene0069058	glutathione-S-transferase	0.33	0.92
	Unigene0069060	glutathione S-transferase L3-like	-0.03	-2.16
	Unigene0081745	glutathione S-transferase U10-like	0.05	-0.14
	Unigene0082147	glutathione S-transferase F11-like	2.47	-0.45
	Unigene0098941	glutathione S-transferase U9	-0.17	-5.91
GR
ko01100;ko00480	Unigene0075696	glutathione reductase	0.47	0.12
	Unigene0098587	glutathione reductase-like	8.98	10.52

Fig 7. Antioxidant mechanism related genes under NaCl treatment in T. ramosissima.

(Notes: GR, glutathione reductase; GST, glutathione S-transferases; APX, ascorbate peroxidase; GPX, glutathione peroxidase; CAT, Catalases; POD, peroxidase; SOD, Superoxide dismutase. The number of up-regulated and down-regulated activities of each enzyme in the reactive oxygen species scavenging mechanism shown in the figure in the comparison of CK V.S. NaCl-48h and CK V.S. NaCl-168h).

3.7 Analysis of transcription factor DEGs in T. ramosissima leaves under NaCl stress

Transcription factors play a major role in regulating plant growth and adaptation to adverse environments, including salt stress. In this study, According to the transcriptome sequencing of T. ramosissima leaves, many transcription factors were discovered. In this study, we did select 8 statically significant DEGs, which were specifically observed and annotated in the KEGG database. In particular, 5 WRKY (ko04626 and ko04016) annotated genes were responsive to NaCl treatment. In particular, the expression levels of Unigene0010090, Unigene0077293 and Unigene0079542 exhibited a downward trend at 0h, 48h and then an upward trend at 168h. The expression level of Unigene0014406 and Unigene0024962 continuously increased until 168h. In addition, 3 bZIP transcription factors were annotated to the KEGG pathway (ko04016, ko01100, ko01110, ko01200, ko01212, ko04146, ko00071, ko00640, ko00410, ko01040, ko00592 and ko04075). The expression level of Unigene0026888 and Unigene0008868 exhibited a downward trend at 48h and then an upward trend at 168h, while Unigene0010561 showed a downward trend at 48h and then an upward trend at 168h (Table 6).

Table 6. Gene annotation of transcription factors.

Gene ID	Description	Pathway	Log2 fold change
			CK-0hv.s.NaCl-48h	CK-0hv.s.NaCl-168h
Unigene0010090	Transcription factor WRKY33	ko04626;ko04016	-0.75	-0.40
Unigene0014406	WRKY DNA-binding protein 27	ko04626;ko04016	0.46	0.79
Unigene0024962	WRKY transcription factor 1	ko04626	0.64	0.69
Unigene0077293	WRKY transcription factor	ko04626;ko04016	-1.96	-2.04
Unigene0079542	WRKY transcription factor 11	ko04626	-0.20.	0.18
Unigene0026888	bZIP4	ko04016	0.67	0.36
Unigene0008868	bZIP2	ko01100;ko01110;ko01200;ko01212;ko04146;ko00071;ko00640;ko00410;ko01040;ko00592	0.6439	-0.011
Unigene0010561	bZIP10	ko04075	-0.8186	-0.38502

3.8 Quantitative Real-Time PCR (qRT-PCR) validation of differential expression

We further randomly selected 8 DEGs involved in salt stress for qRT-PCR verification (S2 Table). Results showed that the expression level of Unigene0104732, Unigene0083695 and Unigene0069097 were induced at 48h but reduced at 168h, while genes of Unigene0090596, Unigene0007135 and Unigene0088781 were decreased at 48h but increased at 168h. Notably, Unigene0024962 was continuously increased while Unigene0028215 was continuously decreased under NaCl stress. These qRT-PCR verification results are completely consistent with the expression trends observed from the transcriptome sequencing analysis (S1 Fig). Nonetheless, the transcriptome data obtained in this study is accurate and reliable.

4. Discussion

Tamarix plants have evolved a series of complex mechanisms to resist and adapt to salt stress in the long term. In particular, Tamarix plants have typical multicellular salt glands, which can reduce plant damage from ion poisoning, which is one of the important morphological characteristics of Tamarix plants to adapt to the saline environment [25]. In this study, electron microscopy analysis showed that leaf salt secretion increased with the prolongation of NaCl treatment, suggesting that T. tamariska may alleviate and respond to the toxicity of salt stress via the salt secretion pathway, thereby adapting to the adverse long-term salt stresses.

The response of plants under salt stress is quite complex that needs multiple gene families and regulatory mechanisms involved in many biological pathways, including metabolism, signal transduction, energy production and transportation, ion penetration and transportation [35]. A comprehensive transcriptome analysis of T. ramosissima plants helps to reveal the molecular mechanisms of Tamarix plants in response to salt stress.

4.1 Enhanced active oxygen scavenging capacity in T. ramosissima

Plants are usually affected by various unfavorable environmental factors, including salt stress, that results in a large amount of ROS accumulation. In this present study, 39 ROS scavenging-related DEGs were significantly regulated under NaCl stress, implying that ROS scavenging mechanisms were indispensable for T. ramosissima plants under NaCl stress.

SOD, POD and CAT enzymes are involved in ROS scavenging in plant cells. Under abiotic stresses, expression of SOD, POD and CAT related genes are prone to be up-regulated [36]. Notably, the expression level of the TaSOD1.7 gene in leaves increased significantly, and the salt tolerance of transgenic wheat was enhanced [37]. In this study, 1 SOD, 4 POD and 4 CAT related genes were induced, while 3 SOD and 4 POD related genes are down-regulated at 48 h of NaCl treatment. Even168 hours after finishing NaCl treatment, there are still 2 SOD, 2 POD and 3 CAT related genes were up-regulated and 2 SOD, 6 POD and 1 CAT related genes were down-regulated. Together, these genes mentioned above may contribute to the increased enzyme activities of SOD and POD in T. ramosissima leaves under NaCl treatment.

Both APX and GPX are the key enzymes for enzymatically removing ROS, protecting cells by catalyzing H₂O₂, and playing an important role in plant adversity stress. APX family genes are involved in plant tolerance to drought, heat, salt, oxidation and biological stresses [38]. In this study, the expression of 3 APX related genes was up-regulated and 2 genes were down-regulated under NaCl treatment. However, only one GPX related gene was down-regulated. We guess that APX and GPX genes in T. ramosissima leaves may respond to salt stress in different ways.

Glutathione S-transferase (GST) is involved in detoxification and antioxidant defence, protecting plants from different abiotic stresses and adversities, and playing multiple roles in plant growth and development [39–42]. In particular, GST was involved in salt stress and GST-related genes are non-sensitive to low and medium NaCl (≤100 mM) concentration but are sensitive to high NaCl (≥200 mM) stress [39, 46]. In this study, 3 GST related genes were up-regulated and 8 genes were down-regulated in the leaves at 48 h, and 3 genes were up-regulated and 12 genes were down-regulated at 168 h, implying that there may exist both positive and negative regulation of GST in T. ramosissima under NaCl stress, and the negative regulation may be the dominant.

GR is one of the plant antioxidant enzymes and a flavoprotein oxidoreductase, which exists in eukaryotes and prokaryotes, and plays an important role in the elimination of ROS in the process of plant oxidative stress [43]. The expression of Oryza sativa GR3 [44] and Jatropha JcGR [45] are all up-regulated under salt stress. Similar to the previous studies, two GR related genes were up-regulated in T. ramosissima leaves under NaCl stress. We speculate that GR might be involved in the adaptation of T. ramosissima to NaCl stress in a positive direction.

4.2 Responsive transcription factors under NaCl treatment ins T. ramosissima

Transcription factors are indispensable for plants to respond to abiotic stresses [13]. WRKY is one of the most important transcription factor families, which has been verified to participate in a variety of metabolic processes and plays an important role in the regulation of transcriptional reprogramming related to plant biological and abiotic stress responses [46–48]. In this study, Unigene0024962, which encodes a WRKY transcription factor, was up-regulated by NaCl treatment, while Unigene0079542 was decreased at 48 h but increased at 168 h, which is consistent with the previous reports in WRKY family genes in soybean [49] and Reaumuria trigyna [50]. The expression of CaWRKY27 in pepper was down-regulated by NaCl stress [51], which is in contrast to our findings. Notably, Unigene0014406 was continuously induced by NaCl treatment, suggesting that this gene may be inevitably involved in the response of T. ramosissima to salt stress. In addition, bZIP transcription factors play an important regulatory role in plant growth and environmental stress response [52, 53]. The expression of Unigene0008868 was significantly up-regulated by NaCl treatment, which is consistent with that of Huang’s findings in ramie, implying that the bZIP transcription factor plays an important regulatory role in response to salt stress [54]. Moreover, MYB is one of the most diversified transcription factor families in plants in terms of quantity and function, which plays important roles under abiotic stresses [55, 56]. In this study, the expression level of Unigene0088781 decreased in the first 48 hours and then increased in a long time under NaCl treatment, suggesting that this gene may be active, especially during a specific period under salt stress. Similar findings were also observed in Medicago sativa seedlings [57] and Rosa rugosa petals [58].

5. Conclusions

The raw data of transcriptome sequencing of T. ramosissima leaves in response to NaCl stress was spliced into 105702 Unigenes, and 54238 annotated Unigenes were retrieved in the 4 major functional databases of KEGG, KOG, NR and SwissProt. The expression profiles of DEGs are slightly different between short time (48h) and long time (168) treatments. In particular, ROS scavenging genes and transcription factor encoding genes (including WRKY, MYB and bZIP family) are sensitive to NaCl treatment with distinct regulatory statuses. This study provides the theoretical basis and gene resource for further molecular mechanisms towards salt tolerance in T. ramosissima.

Supporting information

S1 Fig. Verification of DEGs by qRT-PCR.

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S1 Data

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S1 Table. Sequences of specific primers.

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S2 Table. Randomly select 8 differentially expressed genes.

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Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This work was jointly supported by the following grants: the Agricultural Variety Improvement Project of Shandong Province (2019LZGC009), China’s National Science Foundation through grants (32071612), and the China Scholarship Council (202108320311). Agricultural Variety Improvement Project of Shandong Province provided experimental materials (Hongxia Zhang). China’s National Science Foundation through grants (Jiang Jiang)and the China Scholarship Council provide test sites and data collection(Yahui Chen). Agricultural Variety Improvement Project of Shandong Province, China’s National Science Foundation through grants and the China Scholarship Council provided great help in research design, data collection and analysis.