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Physiological and transcriptomic analysis of the effect of overexpression of the NTPIP2;4 gene on drought tolerance in tobacco

BMC Plant Biology volume 25, Article number: 63 (2025) Cite this article

Abstract

Aquaporins are widely present in the plant kingdom and play important roles in plant response to abiotic adversity stresses such as water and temperature extremes. In this study, we investigated the regulatory role of NTPIP2;4 on drought tolerance in tobacco at physiological and transcriptional levels. In this experiment, we constructed an NtPIP2;4 overexpression vector and genetically transformed tobacco variety 'K326' to investigate the mechanism of NtPIP2;4 gene in regulating drought tolerance in tobacco at physiological and transcriptomic levels. Physiological analyses showed that overexpression plants showed low wilting under drought conditions compared to wild-type (WT), and NtPIP2;4 overexpression tobacco plants showed enhanced superoxide dismutase (SOD) and catalase (CAT) activities, lower levels of superoxide anion (O2-), malondialdehyde (MDA), and hydrogen peroxide (H2O2) than the control, and significantly higher proline (Pro) content than the control. The leaves of NtPIP2;4 overexpressing plants and wild-type controls after drought were subjected to transcriptome sequencing, and RNA-seq analysis showed that a total of 1752 differentially expressed genes (DEGs) were obtained under drought conditions, with 1005 DEGs of up-regulated and 747 DEGs of down-regulated differentially expressed genes. The DEGs were enriched mainly in the plant MAPK signaling pathway, the plant hormone signal transduction pathway, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism and plant-pathogen interaction pathways. We also investigated the drought pathway of MAPK pathway and the auxin pathway mechanism of plant hormone signal transduction pathway, and found that the transcript levels of the genes of the relevant pathways changed, and hypothesized that NtPIP2;4 might regulate the drought resistance of plants through the expression of the relevant genes induced by auxin. This study demonstrates that overexpression of NtPIP2;4 gene can enhance the drought resistance of tobacco plants, which will provide a basis for the research on the function of tobacco NtPIP2;4 gene and the creation of new germplasm resources.

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Introduction

The normal transportation of water molecules is essential to maintain cell function and normal life activities, and the movement of water in and out of cells is a process mainly controlled by water channel proteins [1]. A central process in maintaining intracellular water homeostasis is the movement of water from the soil through xylem conduits, a process that involves three different mechanisms of water movement, namely, symplast, apoplast, and transmembrane transport [2]. Among them, transmembrane transport of water is realized through specific water channel proteins in biological membranes. Aquaporins (AQPs) are widely found in most of the organisms in nature, e.g., plants, animals and bacteria [3]. AQPs can regulate the efficient, rapid and reversible transmembrane transport of water, and also transport a small amount of ions and small molecule solutes, thus regulating the balance of osmotic potential in the plant body, and have a variety of functions such as regulating plant seed germination, growth and development, and responding to a variety of adversity stresses [4,5,6]. In recent years, the roles of plant water channel proteins in different plant physiologies have attracted much attention from scholars, especially with regard to abiotic stresses. Heterologous expression of the Pearl Millet Aquaporin Gene PgPIP2;6 gene into tobacco improved abiotic stress tolerance by enhancing transpiration efficiency [7]. Under abiotic stress, Arabidopsis plants overexpressing EsPIP1;4 exhibited increased antioxidant enzyme activities and increased K+/Na+ ratios compared with control plants, improving abiotic stress tolerance [8].

The HvPIP2;5 gene was obtained from barley, and overexpression lines were obtained after successful introgression, and it was found that the overexpression lines had an increased biosynthesis capacity of osmotic regulating substances as well as antioxidant enzyme activities under drought stress, which resulted in an enhanced drought-resistant capacity [9]. Numerous studies have shown that PIPs directly or indirectly regulate water uptake and retention capacity of plants and thus play a role in resisting drought stress, and PIPs in citrus roots showed down-regulated expression after drought treatment [10, 11]. Maize ZmPIP2;5 overexpressing plants have higher water conductivity in root endodermal cells, faster leaf growth rate and higher survival under mild salt stress [12]. Bamboo PePIP2;7 overexpression was up-regulated in Arabidopsis under abiotic stresses such as strong light, drought, and NaCl, and resulted in higher seed germination, longer taproot length, higher SOD activity, and lower MDA content [13] suggests that PePIP2;7 is leaf-specific and involved in the stress response. After jojoba ScPIP1 was heterologously expressed in Arabidopsis thaliana, overexpression lines showed increased proline content and significantly decreased ion leakage and malondialdehyde content under drought conditions, thus enhancing drought tolerance [14]. Thus, it can be seen that AQPs play an important role in the plant's defense against adversity stress. Althouth they are well-studied in the model crop Arabidopsis thaliana [15, 16], relatively few studies have been conducted in tobacco.

When the plant is under drought stress, the auxin content of the plant will change to adapt to the drought environment [17]. In tobacco, it was found that drought stress regulated the formation of lateral root (LR) in tobacco seedlings by regulating auxin synthesis and polar transport [18]. A study of the relationship between drought-related gene expression and phytohormones in Arabidopsis revealed that 641 hormone-responsive genes were involved in the drought response, of which 95 genes were overlapping genes in the drought and IAA response [19]. Studies have shown that in Populus euphratica, PeFUS3 can activate the genes PIN2, PIN6a and AUX1, which are closely related to auxin transport, thereby regulating the development of lateral roots ( LR ) under drought conditions [20].

In this study, we constructed an NtPIP2;4 overexpression vector and genetically transformed it in tobacco K326 to investigate the function of tobacco NtPIP2;4 gene under drought conditions, and combined with transcriptome technology to preliminarily investigate the signaling mechanism in drought stress, so as to provide a technical basis for the enhancement of drought resistance and molecular genetic improvement of tobacco.

Materials and methods

Experimental materials

Nicotiana tabacum L. (K326) seeds, provided by the Key Laboratory for Tobacco Quality Research Guizhou Province. In this experiment, tobacco K326 was used as the wild-type (WT) control, and all overexpression lines were generated in the K326 background.

Vector construction and genetic transformation

According to the CDS sequence of NTPIP2;4 gene, tobacco K326 genomic DNA was used as template to clone NTPIP2;4 gene. The recombinant plasmid containing the target gene was constructed using E. coli receptor cells, and the successfully constructed recombinant plasmid was transformed into Agrobacterium tumefaciens strain GV3101 (Fig. 1). Tobacco K326 was transformed by Agrobacterium-mediated method, and the transgenic plants were identified by GUS and PCR techniques. The primers used for transgenic plant detection are shown in Table 1.

Fig. 1
figure 1

Schematic illustration of the pBWA(V)HS-PIP2;4-GUS expression vector

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Table 1 Primers used in the study
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Drought treatment

The overexpressed T1 generation seeds and wild-type WT seeds were sown on a special substrate for tobacco, placed in an artificial climate chamber with 65% relative humidity, 16 h/28 ℃ during the day and 8 h/20 ℃ at night, and floated until the seedling stage, and the tobacco plants with the same growth and at the stage of seedling were selected and transplanted into 7 × 7.3 × 5 cm seedling squares to slow down, and then the treatment was started after the tobacco plants had finished the slowing down process. The treatment groups were watered thoroughly before the start of the treatment and were not watered throughout the subsequent period. Leaves of each strain were collected at 0, 7 and 14 days of drought and 12 h after re-watering for subsequent experiments. Three biological replicates were set up for each treatment, and the light and temperature conditions during the treatment period were 16 h/28 ℃ light and 8 h/20 ℃ dark. The contents of SOD, CAT, MDA, Pro, H2O2, and O2− were determined using commercial assay kits according to the manufacturer's instructions. Referring to YC/T142-2010 “Investigating and measuring methods of agronomical character of tobacco”, 15 plants were randomly sampled from three lines (P4, P5, and WT) at time of seedling desired to plant, rosette stage and fast growing period, and the related data, such as plant height, stem circumference, leaf length, leaf width, and leaf area, were measured.

RNA sequencing and transcriptomics analysis

Total RNA of the samples was extracted using the Vazyme kit FastPure Universal Plant Total RNA Isolation Kit, and excess DNA impurities were removed using the RNA Purification Kit. The quality and concentration of RNA were determined using a KAIAO Ultra-Micro Spectrophotometer. Higher purity RNA samples (OD260/280 = 1.85 ~ 2.20, concentration ≥ 30 ng/μL) were finally used. Based on the Illumina platform, the Illumina NovaSeq Reagent Kit method was used for library construction. The target library was obtained after mRNA isolation and enrichment, reverse transcription of the enriched mRNA into cDNA after randomly breaking it into short fragments of about 300 bp, connect the adapter, purify and screen the products after connecting the adapter, and obtain the target library, and finally sequenced on the Illumina NovaSeq X Plus platform [21]. The raw sequencing data were filtered by fastp (https://github.com/OpenGene/fastp) software [22]. The reads after quality control were compared with the whole tobacco genome obtained from NCBI using HiSat2 software (http://ccb.jhu.edu/software/hisat2/index.shtml) to obtain mapped reads for subsequent transcript assembly and data analysis. Genes and transcripts were aligned with the NR, Swiss-Prot and EggNOG databases [23], and differential genes were analyzed for KEGG and GO enrichment, and the data obtained were analyzed on the Majorbio cloud platform (https://cloud.majorbio.com/) for analysis and manipulation.

RNA sequencing samples: T1 generation plants and control WT were sampled after 7 d of natural drought, respectively, and after determining PIP2;4 gene expression, three plants each were selected for transcriptome analysis based on the analysis results.

qRT-PCR validation

Total RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit, and then reverse transcribed to obtain cDNA using the RT mix with DNase All-in-One kit. qRT-PCR was performed using the SYBR Prime qPCR Set Reagent. The qRT-PCR reaction system used was SYBR Prime qPCR Set reagent. The internal reference gene for qRT-PCR of tobacco gene was L25 [24], and WT was the control for drought study of tobacco overexpression plants. Three biological replicates and three qRT-PCR replicates were designed.

Data statistics and analysis

Excel 2016, Origin 2021 and IBM SPSS Statistics 26 software were utilized to process and graph all the data of this experiment.

Results and analysis

NtPIP2;4 transgenic plants showed elevated NtPIP2;4 expression levels

To evaluate the function of NtPIP2;4, NtPIP2;4 overexpression vector was transformed into tobacco K326 for expression. A total of seven transgenic plants showed a significant increase in NtPIP2;4 expression by GUS and qRT-PCR (Fig. 2). The highest expression was found in P4 and P5, which were 5.8-fold and 12.6-fold higher than that of WT, and were utilized to investigate the effect of NtPIP2;4 on drought tolerance in tobacco (Fig. 3).

Fig. 2
figure 2

GUS staining of leaves of T0 generation plants and positive PCR results of NtPIP2;4 overexpressed plants. A The white leaves on the left No.1 are WT, and the blue leaves on the back are NtPIP2;4 overexpression plants P1-P7 in turn. B M is DL2000 DNA marker; NO. 1 represents a positive (plasmid) control; NO. 2 represents negative control (water); NO. 3 represents wild type; NO. 4–10 represents NtPIP2;4 overexpressed positive plants

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Fig. 3
figure 3

Relative expression of NtPIP2;4 overexpression positive plants

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Overexpression of NtPIP2;4 improved tobacco drought resistance

The results are shown in Fig. 4, the degree of WT wilting of wild-type plants was significantly higher than that of overexpression plants after 7 d of treatment, with the best performance of P4 plants and the second best performance of P5 plants, meanwhile, the growth of all the plants was recovered after 12 h of rewatering, but it could be clearly observed that the degree of yellowing of the foot leaves and the degree of wilting of the wild-type control was higher, and the same overexpression of the NtPIP2;4 gene was found in the P4 and P5 lines of the plants showed a better degree of recovery.

Fig. 4
figure 4

Phenotypic records of natural drought treatment for 14 d and rehydration for 12 h

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To further investigate the effect of overexpression of NTPIP2;4 on the physiology and biochemistry of mutant tobacco, superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), proline (Pro), hydrogen peroxide (H2O2), and superoxide anion (O2−) contents in leaves of overexpressed and wild-type tobaccos were determined under drought treatment. The results were shown in Fig. 5. Before drought stress, there was no significant difference in H2O2 and O2−content among the strains. After 7 and 14 days of drought stress, the H2O2 content and O2−content of WT were significantly higher than those of P4 and P5. Therefore, NTPIP2;4 overexpression plants had lower ROS compared with WT. Drought stress significantly increased the antioxidant enzyme activities of the plants. After 7 d and 14 d of drought stress, SOD and CAT activities were significantly lower in WT than in P4 and P5 tobacco. MDA content reflects the degree of cell membrane damage in plant cells, and Pro content can be used as a biochemical indicator of plant resistance. There was no significant difference in the MDA content of each strain before drought stress, and MDA contents of P4 and P5 overexpression plants were significantly lower than those of WT after 7 d and 14 d of drought treatment. Prior to drought stress, there was no significant difference in proline content between WT and transgenic plants. Under drought stress treatment, the proline content of P4 and P5 plants was significantly higher than that of WT. Therefore, the levels of H2O2, O2− and MDA content in the overexpression lines after drought stress treatment were significantly lower than that of WT (Fig. 5), whereas the activities of the related antioxidant enzymes (SOD, CAT) and the content of proline were enhanced and higher than that of the WT plants, reaching significant levels. Therefore, we hypothesized that the improved drought tolerance of transgenic plants might be related to reduced ROS accumulation, high antioxidant enzyme activities, low degree of membrane damage, fast osmoregulation, and high solute accumulation.

Fig. 5
figure 5

Measurements in activity of various physiological indicators under natural drought treatment. Within 14 days of natural drought treatment, the Changes in physiological activity or content of various substances between NtPIP2;4 overexpressed plants and wild-type plants. White columns represent wild-type WT, gray represents NtPIP2;4 overexpressing strain P4, and black represents NtPIP2;4 overexpressing strain P5. (A) The activity of catalase(CAT); (B) Hydrogen peroxide (H2O2) content; (C) Malondialdehyde (MDA) content; (D) Superoxide anion (O2−) content; (E) Proline (Pro) content; (F) The activity of superoxide dismutase(SOD)

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In order to investigate the function of NtPIP2;4 overexpression in tobacco growth and development, two T1 generation strains (P4 and P5) were selected and planted with wild-type seedlings at the appropriate time in the field, and agronomic traits such as plant height, stem girth, leaf length, leaf width and leaf area were measured and analyzed in the time of seedling desired to plant, rosette stage and fast growing period of the three strains, respectively. The results are shown in Table 2. We found that there were no significant differences among the three strains in the early reproductive stages (time of seedling desired to plant and rosette stage), but the two overexpression strains P4 and P5 showed significant differences in leaf size and stem circumference, which were the two agronomic traits that were dominant when the tobacco grew to the fast growing period.

Table 2 Correlation analysis of agronomic traits of three strains (P4, P5, WT)
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Transcriptome analysis

Sequencing and sample quality analysis

In order to further understand how NtPIP2;4 regulates drought tolerance in plants, we conducted a whole gene expression study on WT and P5 plants using RNA-seq. As shown in the Table 3, a total of six cDNA libraries were constructed in this study, and as shown in Table 3, this transcriptome averaged a total of 6.7 Gb per cDNA library; 41,118,658–46,862,614 filtered Clean reads were obtained for each cDNA library, and 6,148,403,094–7,013,854,259 bp of filtered Clean bases, with the percentage of Q20 bases exceeding 98% and the percentage of Q30 bases all exceeding 94.9%. Sequence comparison of the Clean reads of the six libraries with the tobacco reference genome showed a comparison rate of more than 95%, indicating that the sequencing data can be used for subsequent transcriptome analysis.

Table 3 Analysis of sequence assembly and alignment of tobacco transcriptome under drought stress
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Transcriptome sequence annotation and PCA analysis

The total number of genes annotated to databases obtained from this transcriptome was 54,408, and the number of genes annotated to six databases, including GO, KEGG, COG, NR, Swiss-Prot, and Pfam, was 46,024, 22,388, 48,828, 54,360, 42,378, and 41,554, respectively, and the number of genes annotated accounted for 94.4% (Fig. 6). As can be seen from Fig. 7, the three treatments of WT control were close to each other, with good reproducibility among the biological replicates of each treatment; however, two of the three overexpression plants were more clustered, and P5-25 was more far away, which suggests that overexpression of NtPIP2;4 had a greater impact in the expression of other genes, thus distinguishing it from the wild type. In addition, it can be seen that the contribution of X-axis principal component 1 (PC1) to distinguish the samples was 50.84%, and the contribution of Y-axis principal component 2 (PC2) was 16.63%. The above results indicate that the transcriptome sequencing is of high quality and meets the requirements for subsequent analysis.

Fig. 6
figure 6

Number of GO annotation differential genes obtained from 6 databases

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Fig. 7
figure 7

Principal component analysis (PCA) between samples. Orange indicates the control group WT, and blue indicates that the test group overexpressed P5 plants

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Comparative analysis of differential expression of all genes

In this study, the software DESeq2 was used to analyze all the data between groups, as a way to screen to obtain the differential genes (DEGs) that were specifically expressed between groups. As shown, a total of 1752 DEGs were obtained, with 1005 DEGs for up-regulated differential genes and 747 DEGs for down-regulated ones (Fig. 8). In addition by analyzing the heatmap of differential gene enrichment, it was found that highly expressed DEGs were more clustered in this strain P5-25 (Fig. 9).

Fig. 8
figure 8

Comparative analysis of expression levels of differentially expressed genes. Red indicates up-regulated DEGs, blue indicates down-regulated DEGs, and gray indicates genes with no significant difference. The larger the value of -log10, the more significant it is

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Fig. 9
figure 9

Enrichment heatmap of differentially expressed genes. Red indicates that the expression level of a differential gene is up-regulated, the darker the color, the higher the expression level; blue indicates that the expression level of a differential gene is down-regulated, and the darker the color, the lower the expression level

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GO analysis of differential genes under drought stress

To understand the effect of overexpression of the NtPIP2;4 gene on tobacco, we performed GO analysis to investigate the differences between WT and P5 strains. The results showed (Fig. 10) that there were a total of 1,752 GO category species enriched for differential genes between overexpressed P5 and WT. Through GO analysis, we found that DEGs were mainly clustered in three major categories: molecular function, cellular component and biological process. The main biological process categories were “metabolic process” and “cellular process”, and the DEGs in the molecular function category were related to “catalytic activity” and “binding”. Finally, “protein-containing complex” and “cellular anatomical entity” are the main categories of cellular component in leaves.

Fig. 10
figure 10

GO annotation results of differentially expressed genes. Green represents biological process, red represents cellular component, and blue represents molecular function

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By comparing each aggregation pathway, we screened the top 20 enrichment pathways enriched with DEGs, and the results showed that the leaves could be enriched with genes involved in bioregulatory metabolic process, cellular process, response to stimulus, transcription regulator activity, binding, catalytic activity and other related genes, and the number of DEGs aggregated in three pathways, namely, cellular anatomical entity, binding and catalytic activity, was more prominent. The number of DEGs in these three pathways is more prominent.

KEGG analysis of differential genes under drought stress

KEGG enrichment analysis was performed on the differentially expressed genes (DEGs), and the results are shown in Fig. 11. In the WT vs P5 group, the DEGs were primarily enriched in five main categories: metabolism (10 pathways), environmental information processing (2 pathways), genetic information processing (5 pathways), cellular processes (1 pathway), and organismal systems (1 pathway). The most significantly enriched pathway in metabolism was carbohydrate metabolism; in genetic information processing, it was folding, sorting and degradation; in environmental information processing, it was signal transduction; in cellular processes, it was transport and catabolism; and in organismal systems, it was environmental adaptation.

Fig. 11
figure 11

KEGG annotation results of differential genes. Red represents metabolism, light blue represents genetic information processing, green represents environmental information processing, dark blue represents cellular processes, and orange represents organismal systems

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There were 394 genes enriched to KEGG pathway. The top 10 enriched pathways enriched in DEGs were screened, and the second category of pathways of genes in tobacco leaves after drought stress were able to be enriched in the MAPK signaling pathway-plant, the plant-pathogen interaction pathway, the plant hormone signal transduction pathway, the amino sugar and nucleotide sugar metabolism pathway, and the protein processing in endoplasmic reticulum pathway (Table 4).

Table 4 Enrichment analysis of KEGG pathways (Top 10) of different genes in tobacco under drought stress
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Analysis of key genes of signal transduction pathways in tobacco transcriptome under drought stress

Drought had a significant effect on signaling pathways in tobacco leaves, and functional enrichment in the second category of GO-enriched pathways gained the top 20 pathways (P < 0.05), among which the aminoglycan metabolic process, cell recognition, recognition of pollen, and chitin metabolic process were enriched in significant abundance (Fig. 12). In addition, GO enrichment involved the highest number of genes in two pathways, the plasma membrane and the defense response pathway, suggesting that the NtPIP2;4 gene can regulate other genes at the plasma membrane and plays a key role in signaling processes that assist in responding to abiotic stresses in plants.

Fig. 12
figure 12

Annotation results of GO function enrichment. The length of the orange column indicates the significance of differential gene enrichment, and the blue broken line indicates the number of differential gene enrichment

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In the process of coping with drought stress, a series of metabolic adjustments and changes will accompany in plants to adapt to this arid environment. These changes can ensure that plants can maintain normal physiological functions and growth status under water deficit conditions, and show good environmental adaptability [25]. KEGG functional enrichment pathways were selected from the top 20 pathways with significant P value, and the enrichment abundance in the pathways of phenylpropanoid biosynthesis, plant hormone signal transduction, pentose phosphate pathway, sesquiterpenoid and triterpenoid biosynthesis, porphyrin metabolism, tryptophan metabolism, and glutathione metabolism were significantly enriched (Fig. 13), and the number of differential genes enriched in three pathways, namely, plant hormone signal transduction, MAPK signaling pathway-plant, and plant-pathogen interaction, was high, and previous studies have also shown that the above pathways have important roles in resisting abiotic stresses [26].

Fig. 13
figure 13

Annotation results of KEGG function enrichment. The redder the bubble color, the more significant the enrichment degree, and the larger the bubble diameter, the more the number of differential genes

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Screening and functional analysis of NtPIP2;4 downstream differential genes

To screen the candidate differential genes of NtPIP2;4 under drought stress, we selected the differential genes contained in the top five pathways with high significance (P < 0.05) among KEGG-enriched pathways (Table 5), which were MAPK signaling pathway-plant, plant-pathogen interaction pathway, plant hormone signal transduction pathway, amino sugar and nucleotide sugar metabolism pathway, starch and sucrose metabolism pathway. Then after screening the genes without nomenclature, genes without annotation of their functions and genes with expression levels below 2.0, we obtained 24 candidate target genes.

Table 5 Functional annotations of differentiated genes GO and KEGG after selecting
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Among these 24 genes, 22 differential gene expressions were up-regulated, mainly annotated to UDP glucose-associated dehydratase, LRR (Leucine Rich Repeat) receptor-like serine/threonine-protein kinase, reductase, pathogenesis-related protein, chitinase, RPM1-interacting protein; two genes were down-regulated, mainly annotated to auxin-responsive protein and LRR receptor-like serine/threonine-protein kinase protein functions. This suggests that NtPIP2;4 may regulate the corresponding drought stress in plants at room temperature through these up-regulated differential genes.

The qRT-PCR validation of differentially expressed genes

A total of 1752 differentially expressed genes were detected in transcriptome sequencing, from which the relevant genes with high variability, a total of 8 DEGs, were selected for fluorescence quantitative PCR validation. The results are shown in Fig. 14. The expression trend of all genes detected by qRT-PCR was similar to the trend of the results in transcriptome sequencing, and the expression levels of the eight genes were up-regulated to different degrees under drought-induced conditions, among which the relative expression of gene-LOC107790992 was the highest, which further proves the availability and accuracy of the results of transcriptome sequencing (Fig. 14).

Fig. 14
figure 14

The qRT-PCR verification of differential genes

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Discussion

Drought stressors greatly limit plant distribution, alter plant growth and development, and reduce crop yields. The adaptive regulatory responses including morphology, physiology, and gene expression made by plants in response to drought stress lead to optimized choices [25, 27, 28]. Some previous studies claimed that OsPIP2;6 overexpression obtained in rice found that OsPIP2;6 overexpressing lines had better growth under drought stress, which laterally proved that OsPIP2;6 helps to improve drought tolerance in rice [29]; the expression of OsPIP2;4 in leaves and roots of rice Giza178 cultivar overexpressing PIP2;4 was significantly higher than that of the control in control condition [30]; the differential expression of two genes, SiPIP3;1 and SiSIP1;1, in Italian dogwood in response to high temperature, drought and salt stress, and overexpression in a heterologous yeast system also indicated that the transgenic cells were able to tolerate dehydration and salt stress, suggesting that these proteins were involved in the tolerance mechanism [31]. To understand the relationship between NtPIP2;4 and drought tolerance in tobacco, we constructed an overexpression vector for NtPIP2;4 to genetically transform tobacco and investigated its role in drought tolerance. The expression of NtPIP2;4 in overexpression plants was significantly greater than that in WT plants.

Under normal growth conditions, reactive oxygen species (ROS) production and scavenging are in a dynamic equilibrium [32]. In order to protect the integrity of cell membranes, proteins and metabolic enzyme activities, it stimulates the activation of the antioxidant defense system, which generates a series of scavenging reactive oxygen species-related enzymes and non-enzymatic substances that work together to defend against the attack of reactive oxygen species [33]. Crops under drought stress produce reactive oxygen species (ROS) as a secondary stress causing cell membrane lipid peroxidation [34]. Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are scavenging enzymes in plants that mainly target reactive oxygen radicals and are able to essentially maintain the relative homeostasis of hydrogen peroxide in plants [35]. The degree of crop tolerance to abiotic stresses is correlated with the ability to scavenge ROS, and under severe drought conditions, the content of CAT and APX in wheat leaves were about 1.5-fold and 2.9-fold, respectively [36]. Under drought conditions, strains with TaAQP7 overexpression maintained higher germination rate, root length, relative water content, SOD vigor and CAT vigor [37]. In this study, SOD and CAT activities of NtPIP2;4 overexpressing tobacco plants were enhanced under drought stress, and the contents of superoxide anion, malondialdehyde, and hydrogen peroxide were lower than those of the control, while the proline content was significantly higher than that of the control. Proline content was significantly increased, indicating that overexpression of the NtPIP2;4 gene could improve the ability of tobacco to resist drought stress.

Auxin regulates the effect of drought on plants through content changes [17]. GH3 gene is an early rapid auxin response gene [38]. In apple, under long-term drought stress, MdGH3 RNAi plants performed better than wild-type plants, and the concentration of IAA increased, which enhanced the drought resistance of plants [39]. The Arabidopsis thaliana plants overexpressing GH3-5 gene showed auxin response deficiency and high drought resistance phenotype [40]. Activation of the TLD1/Os GH3.13 gene in the functionally acquired mutant tld1-D in rice led to a reduction in IAA concentration and a significant change in plant shape, which resulted in increased survival by reducing plant water loss [41]. MAPK phosphorylates a variety of substrates, including transcription factors, protein kinases, and cytoskeleton-associated proteins, etc., and plays an important role in regulating plant response to adversity (salinity, drought, temperature extremes, heavy metals, etc.). H2O2 is a key plant signaling molecule and induces a MAPK cascade in response to adversity stress. ROS produced by plants stimulated by abiotic factors such as salt, low temperature, and drought lead to the activation of MEKK1-MKK4/5-MPK3/6 in response to adversity stress [26, 42, 43]. GP1pro is a selective plant inducer whose target protein is NbPIP2;4. GP1pro binds to the target protein NbPIP2;4 and transports apoplast-to-cytoplasm H2O2 to regulate plant immunity [44]. In addition, overexpression of SiPIP2;4 gene in tobacco enhanced drought tolerance under drought conditions [45]. To further understand the drought resistance mechanism of NtPIP2;4, we characterized the effect of NtPIP2;4 expression on gene expression under drought 7 d conditions using RNA-seq. Differential genes were mainly enriched in the MAPK signaling pathway-palnt, plant hormone signal transduction, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, and the plant-pathogen interaction pathway. These pathways are accompanied by the responses of the corresponding functional genes of serine/threonine-protein kinase. However, the expression of two differential genes was down-regulated, one of which was the auxin response protein gene. Auxin regulates the effect of drought on plants through changes in its content, so it is speculated that this is the down-regulation of some genes caused by drought stress in plants, but at the same time more genes that regulate threonine kinase are up-regulated to be expressed, so we hypothesize that NtPIP2;4 may regulate the drought tolerance of plants through the expression of auxin-dependent related genes and then regulate drought tolerance.

Conclusion

In this study, we constructed an overexpression vector for the NTPIP2;4 gene and transformed it into the baked tobacco variety K326. the expression of NTPIP2;4 was relatively high in leaves and roots. Under drought stress, the osmoregulatory capacity of tobacco was improved by NtPIP2;4 overexpression to reduce membrane damage, thus improving drought resistance of tobacco.

Using transcriptome analysis, we found that under drought stress conditions, NtPIP2;4 overexpression resulted in a significant enrichment of differential genes in the MAPK signaling pathway-plant, plant-pathogen interaction, plant hormone signal transduction, amino sugar and nucleotide sugar metabolism and starch and sucrose metabolism pathways. Further exploring the pathway mechanism between MAPK signaling and plant hormone signal transduction in drought response in depth, we hypothesized that NtPIP2;4 may regulate drought resistance in plants through auxin-induced expression of related genes. Subsequently, preliminary validation of the downstream regulated genes was performed by quantitative PCR with the transcriptome, and the differential genes were significantly up-regulated in NtPIP2;4 overexpressing plants.

Data availability

The protein sequence and nucleotide sequence of the NtPIP2;4 gene (Gene ID: 107763243) were obtained through the NCBI website (https://www.ncbi.nlm.nih.gov/) and the website is open to all researchers. Nicotiana tabacum L. (K326) seeds, provided by the Key Laboratory for Tobacco Quality Research Guizhou Province, were used as the experimental material. The datasets supporting the conclusions of this article are included in the article.

Abbreviations

CDS:

Coding sequence

SPSS:

Statistical Product and Service Solutions

qRT-PCR:

Quantitative Real-time PCR

DEG:

Differentially expressed gene

KEGG:

Kyoto Encyclopedia of Genes and Genomes

GO:

Gene Ontology

SOD:

Superoxide dismutase

CAT:

Catalase

PCA:

Principal component analysis

Pro:

Proline

MDA:

Malondialdehyde

ROS:

Reactive oxygen species

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Acknowledgements

Thanks to all who participated in this study.

Funding

This study was financially supported by the Science and Technology Project of Guizhou Tobacco Company (2021XM04). We declare that we have no financial with Guizhou Tobacco Company that can inappropriately influence our work.

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Authors and Affiliations

  1. College of Tobacco Science, Guizhou University, Guiyang, 550025, China

    Xu Luo, Yuanshuai Shi, Jie Tan, Tao Long, Juntao Song & Yang Liu

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  1. Xu Luo
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  2. Yuanshuai Shi
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  3. Jie Tan
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  4. Tao Long
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  5. Juntao Song
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  6. Yang Liu
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Contributions

X.L. and Y.L. planned and designed the research. X.L., T.L. and J.S. did the main experiment work. X.L., Y.S. and J.T. analyzed the data. All of the authors carried out the field experiment. X.L. wrote the manuscript. Y.L. revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Yang Liu.

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Luo, X., Shi, Y., Tan, J. et al. Physiological and transcriptomic analysis of the effect of overexpression of the NTPIP2;4 gene on drought tolerance in tobacco. BMC Plant Biol 25, 63 (2025). https://doi.org/10.1186/s12870-024-05896-7

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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