Transcriptome analysis of Tamarix ramosissima leaves in response to NaCl stress.

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 3rd 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 3rd 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.

Verification of DEGs by qRT-PCR.

(PDF)

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(XLSX)

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Sequences of specific primers.

(PDF)

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Randomly select 8 differentially expressed genes.

(PDF)

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25 Jan 2022

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Reviewer #1: Abstract

1. L26-34: reorganize and summarize the core contents.

Method

2.1 and 2.2：Whether the seedlings, Hoagland nutrient solution, and treatment conditions are sterile? If not, the nutrient medium is easy to be polluted, resulting in bacterial treatment of seedlings.

L96 format error: temperature description

Result

1. Why is the treatment group designed for 2d (48h) and 7d (168h)? The response at the transcriptome level is usually relatively rapid. Did such a long time of NaCl treatment produce phenotypic differences of T. ramosissima? IF it is, pictures should be provided,

2. 3.3 The title is inappropriate.

3. 3.4 What is the significance of the analysis of GO notes？Figure2 and Table 2,3,4…Go enrichment analysis can be performed instead of GO annotation analysis statistics…..And what led the authors to this conclusion about “T. ramosissima leaves may respond to high NaCl stress by inhibiting the expression level of related DEGs at the transcription level”?

4. 3.6 According to what the 39 genes were concerned and screened from all DEGs? the GO annotation or KEGG or others

5. What led the authors to this conclusion about “antioxidant mechanism might be initially inhibited in T. ramosissima leaves under long time (168 h) of NaCl treatment, just to adapt to salt stress”? The results are not enough to support the conclusion….. The two diagrams in Figure 5 can be combined.

6. 3.7 The title is inappropriate. According to what the 8 genes were concerned and screened from all DEGs? Only these eight? Are there other TFs involved in stress regulation.

7. 3.8 Unigenes mentioned in 3.6 and 3.7 should be selected and analyzed by qRT-PCR.

8 The authors should detect the related enzyme activities such as POD, SOD, CAT etc. from control and treated T. ramosissima to support the conclusion.

Discussion

1. The author should condense the discussion part.

2. 4.1 L284: The results are not enough to support the conclusion.

L290: “Overexpression of SbGST in transgenic tobacco plays a crucial role in salt stress tolerance” What is the role?

4.2

L310-313:What do the authors want to say?

L322-323: What led the authors to this conclusion about “that this gene needs to be active especially in a specific period under salt stress”? The results are not enough to support the conclusion

Others

1. Where's the legends of the figures？

2. Writing problems in L149, 152, 160, 210,223,225,255,264,271

Reviewer #2: A transcriptome analysis of T. ramosissima in response to NaCl stress was performed in this study, and some differentially expressed genes (DEGs) were found. However, the methods, results and conclusions should be considered more appropriately and rigorously.

Here are the details:

Introduction Section:

Lines 65-66: GhWRKY34 is not a wheat gene, please correct it after carefully reading the reference 20.

Materials and Methods Section:

1. In terms of the materials, why choose leaves not roots to study molecular mechanisms towards salt stress?

2. Lines 109-110: The filtering parameters of Fastp should be added. The original data was first filtered to obtain clean reads, then assembled! Not “the original data was filtered and assembled to obtain clean reads.” Please correct it, and add the method as well as software used in assembly.

Results section:

Table 1 : Please use scientific notation for the data.

Line 170: “We speculate that T. ramosissima leaves may respond to high NaCl stress by inhibiting the expression level of related DEGs at the transcription level.” What exactly does these down-regulated “DEGs” related to?

Line 201-202: “Results showed the expression of 39 ROS scavenging-related DEGs in the leaves of T.ramosissima were changed under salt treatment.” Please revise it.

Lines 223-224: How many transcription factors in DEGs? Why only WRKY and bZIP were mentioned? What’s the P value and log2FC of these 8 genes?

Line 237-238: What kind of genes are Unigene0104732, Unigene0083695 and Unigene0069097? Why randomly choose these three genes instead of choosing genes mentioned above, like WKRY, bZIP?

One of the main conclusion,“T. ramosissima may adapt to salt stress by enhancing the ROS scavenging mechanism, including SOD, POD, CAT, APX, GPX, GST, and GR.” I don’t think this conclusion is reliable. As the result showed there were some SOD, POD and CAT down-regulated whether after 48 h or 168 h salt treatment. The enzyme activity of SOD, POD, CAT should be measured to see whether ROS scavenging mechanism is enhanced.

Besides, the article needs careful revision; there are many typos and format error in it.

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Submitted filename: Plos One.pdf

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2 Feb 2022

Journal: PLOS ONE

Title: Transcriptome analysis of Tamarix ramosissima leaves in response to NaCl stress

Manuscript ID: PONE-D-21-38993

Dear Dr. Guangxiao Yang,

Thank you for giving us the opportunity to resubmit our revised paper for publication in Plos One. We greatly appreciate the efforts of you, the associate editor and all reviewers for the constructive comments that have helped shape this manuscript into a better form. We have thoroughly revised the article and addressed all the concerns by either editing the manuscript or clarifying the details in the revised manuscript. Please see the detailed point-by-point response (marked in blue) added below. A 'point-by-point response' to each of the reviewer comments and a marked-up version of the manuscript with the changes highlighted in blue were also uploaded with the clean version of the manuscript files.

Sincerely,

Ning Zhang, Ph.D.

Faculty of Forestry, The University of British Columbia,

Vancouver, British Columbia, Canada

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Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

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Data Availability Statement

All data that support the findings of this study are available. The Illumina raw sequencing data were submitted to the National Center for Biotechnology Information (NCBI) Short Reads Archive (SRA) database under accession number SRP356215.

Response to Reviewer 1

Dear Reviewer:

Many thanks for all your valuable comments and suggestions. We carefully revised the manuscript and tried our best to improve it to be a better state to meet the journal’s requirements. The revised manuscript has been submitted to the journal, and looking forward to your further consideration.

Abstract

1. Lines:26-34: reorganize and summarize the core contents.

Response: According to your suggestion, we reorganized the abstract and improved greatly in the revised manuscript. Again, thank you for your suggestions.

Method：

2. 2.1 and 2.2：Whether the seedlings, Hoagland nutrient solution, and treatment conditions are sterile? If not, the nutrient medium is easy to be polluted, resulting in bacterial treatment of seedlings.

Response: Thanks a lot for your valuable comments. To be honest, the Hoagland nutrient solution used in the study was sterilized before treatment. Before NaCl treatment, the cuttings seedlings were rinsed 3-5 times with sterilized distilled water, and then transferred to the incubator, which was disinfected with 75% alcohol before treatment. Notably, the nutrient solution was changed every third day to avoid media pollution, especially under 26 ℃ in the short-term treatment.

3.Line96 format error: temperature description.line 96 format error: temperature description： “laced in……at 26 (± 2) ℃ .”

Response: Sorry for the incorrect format. It has been revised in the manuscript.

Results:

1. Why is the treatment group designed for 2d (48h) and 7d (168h)? The response at the transcriptome level is usually relatively rapid. Did such a long time of NaCl treatment produce phenotypic differences of T. ramosissima? IF it is, pictures should be provided.

Response: The purpose of selecting 2d (48h) and 7d (168h) is to observe the changes of gene expression under salt treatment in both the short and the long term. According to previous reports (Song and Su, 2013, Weed Sci; Song et al., 2015, Bio Plantaraum; Song et al., 2016, Sci Horticulturae; Liang et al., 2020 Biomed Res Int; Chen et al., 2021, Int J Genomics), 2d (48h) after stress treatment is a typical time point to monitor the expression levels of responsive genes. While under 7d (168h) treatment, plants exhibited significant physiological phenotype and it is better to discover which DEGs are responsive under salt stress.

2. 3.3 DEGs quantitative analysis

Response: Thank you for your useful suggestions. In the revised manuscript, it was changed into ‘Quantitative expression analysis of DEGs’.

3. 3.4 What is the significance of the analysis of GO notes？Figure2 and Table 2,3,4…Go enrichment analysis can be performed instead of GO annotation analysis statistics…..And what led the authors to this conclusion about “T. ramosissima leaves may respond to high NaCl stress by inhibiting the expression level of related DEGs at the transcription level”?

Response: Appreciate your valuable suggestions, and definitely, Go enrichment analysis was performed instead of GO annotation analysis statistics in the revised submission. GO analysis can clearly understand the expression levels of DEGs in three categories: biological processes, cellular components, and molecular functions. According to the distribution of the subclasses of DEGs in each subclass under each major class, it is easy to find which subclasses of DEGs’ expression levels were sensitive to salt stress.

4. 3.6 According to what the 39 genes were concerned and screened from all DEGs? the GO annotation or KEGG or others.

Response: Among all these DEGs, only 39 ROS-related DEGs were specifically found and annotated in the KEGG database.

5. What led the authors to this conclusion about “antioxidant mechanism might be initially inhibited in T. ramosissima leaves under long time (168 h) of NaCl treatment, just to adapt to salt stress”? The results are not enough to support the conclusion….. The two diagrams in Figure 5 can be combined.

Response: Frankly speaking, we did analyze the enzymatic activities of SOD, POD and CAT in our previous studies, which were increased at 7d, 15d and 30d. under NaCl stress, compared with the control (Y Chen et al. 2021, 49(15): 142-146, Jiangsu Agricultural Science, in Chinese), Here, we supplied the enzymatic activities of SOD, POD and CAT in our previous studies, which were increased at 2d (48h) and 7d (168h) in the newly revised manuscript (as shown in Fig. 1).

Figure 1

(Note: The different letters indicate significant differences at the P < 0.05 level

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.)

6. 3.7 The title is inappropriate. According to what the 8 genes were concerned and screened from all DEGs? Only these eight? Are there other TFs involved in stress regulation.

Response: 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.

7. 3.8 Unigenes mentioned in 3.6 and 3.7 should be selected and analyzed by qRT-PCR.

Response: Thank you for your valuable comments. In order to verify the reliability of the transcriptome sequencing data, we randomly selected 8 DEGs (including some of the transcription factors screened in this article), according to the description of verification methods in previous publications (He et al., Forests. 2020, 11(8); Lu et al., Biotechnology Bulletin (in Chinese).2020, 36(12):42-53).

.

8 The authors should detect the related enzyme activities such as POD, SOD, CAT etc. from control and treated T. ramosissima to support the conclusion.

Response: Thank you for your kind suggestions. Definitely, we carried out such experiments previously as mentioned above (Chen et al. 2021, 49(15): 142-146, Jiangsu Agricultural Science, in Chinese).

Discussion：

1. The author should condense the discussion part.

Response: We tried our best to rewrite the discussion part. Please see the newly submitted manuscript.

2. Line 284: 4.1 The results are not enough to support the conclusion.

Response: Thanks for the kind reminder. After second thoughts, we revised the content as follows: Enhanced active oxygen scavenging capacity in T. ramosissima.

3. Line 290: “Overexpression of SbGST in transgenic tobacco plays a crucial role in salt stress tolerance” What is the role?

Response: These findings in tobacco showed that the expression of SbGST gene was up-regulated under the stress conditions of salt, cold, drought and salicylic acid. Among them, overexpression of SbGST gene in transgenic tobacco promoted seed germination and growth under salt stress. These results confirmed that the expression of SbGST genes was up-regulated under different stresses, and the overexpression of tua class SbGST genes in transgenic tobacco played a crucial role in abiotic stress tolerance. We revised the manuscript in the revised manuscript.

4. Lines 310-313 :4.2What do the authors want to say?

Response: In the 4.2 section of the Discussion part, we want to discuss the differentially expressed responsive transcription factor genes and their possible role under NaCl treatment ins T. ramosissima.

5. Lines 322-323 : What led the authors to this conclusion about “that this gene needs to be active especially in a specific period under salt stress”? The results are not enough to support the conclusion.

Response: Many thanks for your valuable comments. After second thought, we have already improved this part in the newly submitted manuscript.

Others：

1. Where's the legends of the figures？

Response: Sorry for the careless mistakes. The legends of the figures are supplied in the revised manuscript. Thanks a lot.

2. Writing problems in L149, 152, 160, 210,223,225,255,264,271.

Response: Sorry for our spelling mistakes. All problems have been revised in the newly submitted manuscript.

To Reviewer 2

Reviewer #2: A transcriptome analysis of T. ramosissima in response to NaCl stress was performed in this study, and some differentially expressed genes (DEGs) were found. However, the methods, results and conclusions should be considered more appropriately and rigorously.

Response:

Response: Appreciate your valuable comments and suggestions. We carefully revised the manuscript and tried our best to improve it to be a better state to meet the journal’s requirements. The revised manuscript has been submitted to the journal, and looking forward to your further consideration

Introduction Section:

1. Lines 65-66: GhWRKY34 is not a wheat gene, please correct it after carefully reading the reference 20.

Response: Thank you so much, and sorry for our typo. GhWRKY34 is a gene belonging to cotton, which has been revised in the new submission.

Materials and Methods Section:

1. In terms of the materials, why choose leaves not roots to study molecular mechanisms towards salt stress?

Response：Thank you for your valuable comments. Frankly, the root growth of T. ramosissima is quite slow and rare, especially under salt stress, and the phenotype of roots is not easily altered under the short term of NaCl treatment. While the leaves are the dominant fresh biomass in T. ramosissima, and exhibited salt secretion even under the short term of salt stress, which was not observed in roots (Fig. 2). Therefore, we chose the leaves of T. ramosissima as the research object to explore responsive DEGs under salt stress.

2. Lines 109-110: The filtering parameters of Fastp should be added. The original data was first filtered to obtain clean reads, then assembled! Not “the original data was filtered and assembled to obtain clean reads.” Please correct it, and add the method as well as software used in assembly.

Response: Thanks a lot for your valuable suggestion. The filtering parameters of Fastp were set in accordance with Chen et al. (2018, Bioinformatics, 34(17):i884-i890), which has been revised in the manuscript.

Results section:

Table 1: Please use scientific notation for the data.

Response: According to your valuable suggestion, it has been revised throughout the manuscript.

2. Line 170: “We speculate that T. ramosissima leaves may respond to high NaCl stress by inhibiting the expression level of related DEGs at the transcription level.” What exactly does these down-regulated “DEGs” related to?

Response：Thanks for the useful comment. After second thought, we delete this sentence to avoid misunderstanding.

3. Lines 201-202: “Results showed the expression of 39 ROS scavenging-related DEGs in the leaves of T.ramosissima were changed under salt treatment.” Please revise it.

Response: According to your suggestion, this sentence was changed into: ‘Results showed that the 39 ROS scavenging-related DEGs were responsive to salt treatment in the leaves of T. ramosissima’.

4. Lines 223-224: How many transcription factors in DEGs? Why only WRKY and bZIP were mentioned? What’s the P value and log2FC of these 8 genes?

Response: In total, more than 300 differentially expressed transcription factors genes were screened in this study partially annotated in GO and KEGG databases. In order to verify the reliability of the transcriptome sequencing data, we randomly selected 8 DEGs, which were specifically observed and annotated in the KEGG database, in this study. In addition, the P values of these 8 differentially expressed transcription factor genes were all 0 among the comparisons of CK-0h v.s. NaCl-48h, NaCl-48h vs NaCl-168h and CK-0h v.s. NaCl-168h, indicating a significant high Log2 Fold Change of 8 differentially expressed transcription factors. All these details mentioned here were listed in the new Table 1. Appreciate your valuable comments.

Table 1

Gene ID Description Pathway Log2 Fold Change

CK-0h v.s. NaCl-48h NaCl-48h v.s. NaCl-168h CK-0h v.s. NaCl-168h

Unigene0010090 Transcription factor WRKY33 ko04626;ko04016 -0.75 0.35 -0.40

Unigene0014406 WRKY DNA-binding protein 27 ko04626;ko04016 0.46 0.33 0.79

Unigene0024962 WRKY transcription factor 1 ko04626 0.64 0.05 0.69

Unigene0077293 WRKY transcription factor ko04626;ko04016 -1.96 -0.08 -2.04

Unigene0079542 WRKY transcription factor 11 ko04626 -0.20. 0.38 0.18

Unigene0026888 bZIP4 ko04016 0.67 -0.31 0.36

Unigene0008868 bZIP2 ko01100;ko01110;ko01200;ko01212;ko04146;ko00071;ko00640;ko00410;ko01040;ko00592 0.64 -0.65 -0.01

Unigene0010561 bZIP10 ko04075 -0.82 0.43 -0.39

5. Lines 237-238: What kind of genes are Unigene0104732, Unigene0083695 and Unigene0069097? Why randomly choose these three genes instead of choosing genes mentioned above, like WKRY, bZIP?

Response: More than 300 differentially expressed transcription factors genes were screened in this study partially annotated in GO and KEGG databases. In order to verify the reliability of the transcriptome sequencing data, we randomly selected 8 DEGs, which were specifically observed and annotated in the KEGG database, according to the description of verification methods in previous publications (He et al., Forests. 2020, 11(8); Lu et al., Biotechnology Bulletin (in Chinese).2020, 36(12):42-53).

6. One of the main conclusion, “T. ramosissima may adapt to salt stress by enhancing the ROS scavenging mechanism, including SOD, POD, CAT, APX, GPX, GST, and GR.” I don’t think this conclusion is reliable. As the result showed there were some SOD, POD and CAT down-regulated whether after 48 h or 168 h salt treatment. The enzyme activity of SOD, POD, CAT should be measured to see whether ROS scavenging mechanism is enhanced.

Response: Frankly speaking, we did analyze the enzymatic activities of SOD, POD and CAT in our previous studies, which were increased at 7d, 15d and 30d. under NaCl stress, compared with the control (Y Chen et al. 2021, 49(15): 142-146, Jiangsu Agricultural Science, in Chinese), Here, we supplied the enzymatic activities of SOD, POD and CAT in our previous studies, which were increased at 2d (48h) and 7d (168h) in the newly revised manuscript (as shown in Fig. 1).

Figure 1

(Note: The different letters indicate significant differences at the P < 0.05 level

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.)

7. Besides, the article needs careful revision; there are many typos and format error in it.

Response: We would like to thank you again for these valuable comments. We carefully revised the manuscript and tried our best to improve the English level. Hopefully, this revision work will meet your requirement! We need your further help and consideration. Many thanks again！

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

28 Feb 2022

3 Mar 2022

Journal: PLOS ONE

Title: Transcriptome analysis of Tamarix ramosissima leaves in response to NaCl stress

Manuscript ID: PONE-D-21-38993

Dear Edit,

Thank you for giving us the opportunity to resubmit our revised paper for publication in Plos One. We greatly appreciate the efforts of you, the associate editor and all reviewers for the constructive comments that have helped shape this manuscript into a better form. We have thoroughly revised the article and addressed all the concerns by either editing the manuscript or clarifying the details in the revised manuscript. Please see the detailed point-by-point response (marked in blue) added below. A 'point-by-point response' to each of the reviewer comments and a marked-up version of the manuscript with the changes highlighted in blue were also uploaded with the clean version of the manuscript files.

Sincerely,

Ning Zhang, Ph.D.

Faculty of Forestry, The University of British Columbia,

Vancouver, British Columbia, Canada

Response to Editor

1. Please upload a Response to Reviewers letter which should include a point by point response to each of the points made by the Editor and / or Reviewers. (This should be uploaded as a 'Response to Reviewers' file type.)

Response: Thank you for your valuable comments. We have finished editing

2. Please remove your figures/ from within your manuscript file, leaving only the individual TIFF/EPS image files. These will be automatically included in the reviewer’s PDF.

Response: According to your suggestion, We have removed all pictures in the manuscript from the manuscript, and each picture is packaged separately as a PDF file for upload.

Response to Reviewer 1

Dear Reviewer:

Many thanks for all your valuable comments and suggestions. We carefully revised the manuscript and tried our best to improve it to be a better state to meet the journal’s requirements. The revised manuscript has been submitted to the journal, and looking forward to your further consideration.

Abstract

1. Lines 31-32, Lines 37-38. Please restate the relevant contents of transcription factors

Response: According to your suggestion, we restated all these relevant contents of transcription factors in the revised manuscript. Thank you so much for valuable comments.

2. Line 36 “the enzyme activities of SOD, POD, and CAT were significantly enhanced under NaCl treatment”. The CAT activity had no significant change in Figure2.

Response: Thank you for your useful suggestions. We improved the description of this sentence in the revised manuscript that as following: ‘The enzyme activities of SOD and POD were significantly enhanced under NaCl treatment, but the enzyme activity of CAT was not significantly enhanced’.

3. Line 88 “NaCl stress and screened and verified EGs at the transcriptional level.” Please rewrite this sentence

Response: Sorry for our mistake. In the revised manuscript, it was revised as: ‘NaCl stress and screened and verified DEGs at the transcriptional level’.

Method：

1. Line 99 “maintained at 26 ± 2 ℃ (day)() with a relative humidity of 40” Please rewrite this sentence.

Response: Sorry for our mistake. This sentence was revised in the newly submitted manuscript that followed as: ‘maintained at 26 ± 2 ℃ (day) whose relative humidity stays between 40% and 55%’.

2. Line 125 “2.5 Differential gene screening” Please rewrite this little title

Response: According to your valuable suggestion, it has been revised throughout the manuscript as follows: ‘2.5 Screening methods for differentially expressed genes’.

Results:

1. Line 127: maybe “corrected P value” and “FDR” is the same parameters?

Response: Many thanks for valuable comments. FDR value means BH corrected p value. This has been revised in the manuscript.

2. Lines 209-210: “We speculate that T. ramosissima leaves may respond to high NaCl stress by reducing the expression level of related DEGs at the transcription level.” This conclusion is inappropriate.

Response: Thank you for your valuable comments. It has been revised in the manuscript that followed as: ‘We speculate that T. ramosissima leaves may respond to high NaCl stress by affecting the expression level of related DEGs at the transcription level’.

3. Line 228 “3.6 KEGG pathway analysis of DEGs”. Line 275 “3.8 KEGG pathway analysis of key DEGs”. Please rewrite these little titles.

Reply: We rectified these little titles in the newly submitted manuscript as follows: ‘KEGG pathway analysis of DEGs in T. ramosissima leaves under NaCl stress’ and ‘3.8 Analysis of transcription factor DEGs in T. ramosissima leaves under NaCl stress’.

Others：

1. The reason why or how the author chose to pay attention to the eight transcription factors should be explained clearly in the manuscript.

Response: Many thanks for your valuable comments. It has been revised in the manuscript.

2. And the language of the manuscript needs to be carefully revised. Please revise the manuscript carefully.

Response: We would like to thank you again for these valuable comments. We carefully revised the manuscript and tried our best to improve the English level. Hopefully, this revision work will meet your requirement! We need your further help and consideration. Many thanks again!

To Reviewer 2

Dear Reviewer:

Response: Appreciate your valuable comments and suggestions. We carefully revised the manuscript and tried our best to improve it to be a better state to meet the journal’s requirements. The revised manuscript has been submitted to the journal, and looking forward to your further consideration

Introduction Section:

1. Lines 70-71: “In cotton, the WRKY transcription factor gene GhWRKY34 was induced by salt stress in transgenic Arabidopsis and wheat.” This sentence is still inaccurate, please correct it after carefully reading the reference.

Response: Sorry for our mistakes. It has been revised in the manuscript that followed as: ‘In cotton, the WRKY transcription factor gene GhWRKY34 was induced by salt stress in transgenic Arabidopsis’.

Results:

1. Figure 1：The lower left corner of the Figure 1 was CK-48h? not NaCl-0h? Besides, please add scale bar.

Response: Sorry for our mistake. It has been revised in the manuscript.

2. Figure 2: Please make sure that the data of Figure 2 is unpublished in your previous studies.

Response: We indeed confirm that the data for Figure 2 is not published previously.

3. Table 1: "0.00%" ? Besides, please use scientific notation for the data.

Response: Sorry for our mistake. It has been deleted in the manuscript.

4. Lines 263-265: “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.” How do you explain the fact that the antioxidant mechanism was significantly enhanced after 168 h of NaCl treatment (Figure 2).

Response: Sorry for the inaccurate description appeared in the original submission. Under prolonged (168 hours) NaCl treatment, the antioxidant machinery of T. ramosissima leaves may play a key role in up-regulating genes to adapt to salt stress.

5. As many DEGs were mentioned in 3.7 and 3.8, and some conclusions had been drawn, at least 2-3 genes of the these DEGs should be selected and analyzed by qRT-PCR to support your results and conclusion.

Response: Thank you for your valuable comments. In order to verify the reliability of the transcriptome sequencing data, we randomly selected 8 DEGs (including some of the transcription factors screened in this article), according to the description of verification methods in previous publications [He et al., Forests. 2020, 11(8); Lu et al., Biotechnology Bulletin (in Chinese).2020, 36(12):42-53]. In addition, Unigene0024962, as a verified gene, is also a transcription factor annotated to the KEGG Pathway mentioned above.

6. P in “P value” should be italic. All the Figure legends and Table titles should be corrected to meet PLOS ONE's requirements.

Response：According to your valuable suggestion, “p value” was written in italic, and all Figure legends and Table titles was corrected in the newly submitted manuscript.

We would like to thank you again for these valuable comments. We carefully revised the manuscript and tried our best to improve the English level. Hopefully, this revision work will meet your requirement! We need your further help and consideration.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

7 Mar 2022

Transcriptome analysis of Tamarix ramosissima leaves in response to NaCl stress

PONE-D-21-38993R2

Dear Dr. Zhang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Guangxiao Yang, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

21 Mar 2022

PONE-D-21-38993R2

Transcriptome analysis of Tamarix ramosissima leaves in response to NaCl stress

Dear Dr. Zhang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr Guangxiao Yang

Academic Editor

PLOS ONE