https://orcid.org/0009-0003-0451-472X
ABSTRACT
The plant-specific stress response protein NRP (asparagine-rich protein) is characterized by an asparagine-rich domain at its N-terminus and a conserved development and cell death (DCD) domain at its C-terminus. Previous transcriptional studies and phenotypic analyses have demonstrated the involvement of NRP in response to severe stress conditions, such as high salt and ER Endoplasmic reticulum-stress. We have recently identified distinct roles for NRP in biotic- and abiotic-stress signaling pathways, in which NRP interacts with different signaling proteins to change their subcellular localizations and stability. Here, to further explore the function of NRP, a transcriptome analysis was carried out on nrp1nrp2 knock-out lines at different life stages or under different growing conditions. The most significant changes in the transcriptome at both stages and conditions turned out to be the induction of the synthesis of secondary metabolites (SMs). Such an observation implicates that NRP is a general stress-responsive protein involved in various challenges faced by plants during their life cycle, which might involve a broad alteration in the distribution of SMs.
1. Introduction
As sessile organisms, plants have evolved various mechanisms in order to enable normal growth and flexible functions under fluctuating conditions, especially those unfavorable environments.Citation1 These resistant responses to stress in plants rely on the expression of some essential genes,Citation2 and asparagine-rich protein (NRP) is one of them. NRP was initially identified by the differential display of mRNAs in the hypersensitivity of soybean after infecting with Pseudomonas syringae pv. glycinea.Citation3 It consists of an asparagine-rich N terminal (~25%) and a Development and Cell Death domain (DCD domain), which is highly conserved and has been found in many other different plants, such as Arabidopsis, rice, and pine.Citation3,Citation4
There are two homologous NRPs in Arabidopsis thaliana, NRP1 (AT5G42050, 349aa) and NRP2 (AT3G27090, 296aa), both with DCD domains that are associated with several different stress responses. Notably, NRP2 is constitutively expressed, while NRP1 can be induced in plants. It is reported that a single gene mutation of either NRP1 or NRP2 has no effect on the normal growth of Arabidopsis. Knockout of both NRP1 and NRP2 can promote senescence significantly, and the plant exhibits a highly sensitive phenotype in unfavorable conditions.Citation5
According to earlier research, expression of NRP can increase in the early phase of stress response while the plants suffer from abiotic pressure, including NaCl-induced salt stress, oxidative stress, and mechanical perturbation.Citation6 Unfolded protein response (UPR) is usually regarded as a critical way for cells to survive in ER stress, but cells will enter the apoptosis procedure if the stress continues constantly.Citation7 bZIP60 is an important transcription factor in the UPR that activates the expression of many ER stress-related genes.Citation8 Interestingly, NRP1 can be activated and acts as a pro-survival factor to inhibit cell death during ER stress through the combination between the UPRE-1 element in its promoter and bZIP60.Citation5
NRP can be upregulated significantly to participate in the program cell death (PCD) process after the treatment of cycloheximide or the infection of pathogenic bacteria in plants,Citation3 and this pathway is not really the same as the typical PCD response mediated by salicylic acid (SA).Citation9,Citation10 In our previous study, it has been found that NRP can regulate plant flowering by interaction with CRY2 during fungal infection.Citation11 NRP can recognize the fungal effector protein PevD1, reduce the turn-over rate of flowering-accelerating crypto-chrome CRY2, and promote an early flowering response in Arabidopsis during Verticillium Dahliae infection.Citation11–16 NRP can also amplify the abscisic acid (ABA) signaling by increasing the degradation of FyPP3,Citation17 a dephosphorylation factor of transcription factor ABI5, leading to the activation of ABA-responsive element recognizing transcription factor ABI5 and the alteration of the germination rate of transgenic seeds.Citation4,Citation6,Citation18–21
We are interested in the special contribution of NRP in various stress conditions. In this study, in order to get a more global view of the anti-stress process of NRP involved in and reveal the signaling pathways in Arabidopsis associated with the NRP, a transcriptome analysis was conducted based on nrps knockout Arabidopsis. Notably, the most significant changes in the transcriptome turned out to be the induction of the synthesis of secondary metabolites (SMs) at both different life stages and different growing environment.
1.1. Materials and methods
1.1.1. Plant materials and growth conditions
Genotypes of A. thaliana: Wildtype (WT); nrp1(SALK_041306 KO); nrp2(GK_520C04 KO); nrp1nrp2; and NRP-OE (nrp1nrp2 complemented with Pro35S: NRP-GFP).
Arabidopsis seeds were surface-sterilized with 75% ethanol and rinsed with distilled water, then stratified at 4°C for 2 d. Seeds after stratification were plated on ½ Murashige & Skoog medium containing 0.8% (w/v) agar and 1% (w/v) sucrose, pH 5.7, and grown in a 16 h:8 h, light:dark 22°C :18°C photoperiod with a photosynthetic photon flux density of 100 mmol m−2s−1 for 7 d. Then, the plants were used in relevant experiments or transplanted into soil for 5 weeks in the same condition.
1.2. Construction of double mutant line
The double mutants (nrp1nrp2) were generated by crossing nrp1 with nrp2 and were then isolated from the F3 population by PCR and confirmed by reverse transcription (RT)-PCR.
1.3. Stress treatments
The 7-day-old plants are soaked in a liquid culture medium with 100 µM ABA for 1 h before collecting for RNA sequencing.
1.4. Total RNA extraction
The samples for RNA sequencing were collected at two time points, one from 7-day-old seedlings and the other from 5-week-old plants, and both were compared with wide-type Arabidopsis planted side-by-side. The samples were frozen immediately in liquid nitrogen and stored at −80°C for further RNA isolation.Citation22 The total RNA sequencing on the Illumina HiSeq platform was performed (Genewiz, China).
1.5. RNA seq and statistical analysis
The clean data of each sample is about 6GB, and the reads have an average quality score greater than 30 (Q 30%). To estimate gene expression levels, the fragments per kilobase of exon per million fragments mapped (FPKM) value was used and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment was obtained and visualized.Citation22–26
2. Results
2.1. Loss-of-function of NRP1 and NRP2 shows dwarf phenotype in normal growing environment
We observed the phenotype of nrp1nrp2 after obtaining the double homozygous mutant by crossing nrp1 with nrp2. As can be seen in , nrp1nrp2 have shorter roots and smaller cotyledons than WT, whereas single-gene-knockout plants, nrp1 or nrp2, are comparable to the wild plants (). Interestingly, expressing Pro35S: NRP1 in nrp1nrp2 could rescue its dwarf phenotype and root lengths. The complemented seedlings show no significant difference compared to WT ().
Figure 1. Phenotype observation in normal growth condition.
2.2. Transcriptome analyses of WT and nrp1nrp2 with or without ABA reveal the role of NRPs in the development and growth of plants
First, we performed a statistical analysis on the number of genes differentially expressed in WT and nrp1nrp2. Genes with more than twofold change in expression and Q-value ≤0.05 are screened. Loss of NRPs causes differential expression of 306 genes in normal growing conditions with 128 genes upregulated and 178 genes downregulated ().
Figure 2. Volcanic diagram of the expression difference in Arabidopsis.
NRP is a plant-specific stress responsive protein, ABA can induce a large upregulation of NRP expression, and NRP has a positive regulatory effect on ABA-mediated seed germination and gene expression.17 Due to the essential role of ABA in stress response and development of plants, we treated WT and nrp1nrp2 with ABA at a concentration of 100 µMCitation27 here and then analyzed the number of genes differentially expressed in both two types of plants again. The results showed that the number of genes differentially expressed in both plants after ABA treatment amounted to 618, including 49 upregulated genes and 569 downregulated genes (). Combined with phenotypes, nrp1nrp2 are more sensitive to ABA than the WT plants, in which the differential expression of many genes is involved.
2.3. KEGG enrichment analysis shows that phenylpropanoid biosynthesis is remarkably influenced in nrps knockout plants
To better clarify the specific contribution of these differentially expressed genes, we enriched all metabolic pathways in KEGG using hypergeometric tests and selected the 30 most significant pathways to display. In the two groups (WT-VS-KO12, i.e., WT versus nrp1nrp2; WT-ABA-VS-KO12-ABA, i.e., WT versus nrp1nrp2 with ABA treatment), 27.78% of the differentially expressed genes were annotated in these 30 metabolic pathways when the two types of plants grow in normal condition (). Moreover, there are also 19.58% of the differentially expressed genes in total pathways that are annotated when plants are treated with ABA (100 µM) (). As can be seen from the results, expressions of genes associated with phenylpropanoid biosynthesis have the most significant change.
Figure 3. KEGG enrichment of genes expressed at two growth conditions.
It is reported that various SMs are critical in anti-stress response in plants.Citation28 Many studies have documented that plants can synthesize and release SMs to protect themselves and inhibit the invasion of pathogens.Citation29–33 Phenylpropanoid biosynthesis is a major pathway for many SMs production,Citation34 including SA, lignin, suberin, or condensed tannins, and can be upregulated by a variety of biotic and abiotic stimuli. All these phenylpropanoid-based polymers contribute substantially to the inhibition of pathogens and the protection from environmental damage, such as wounding, mechanical damage, salt stress, and drought.Citation34,Citation35
Several phytohormones including SA are produced via phenylpropanoid biosynthesis pathway. SA can amplify pathogen infection signals and activate the plant defense program upon infection of various pathogens.Citation36 Therefore, the perturbation of phenylpropanoid may influence the biosynthesis of SA and alter the defense levels in plants. Lignins are the main components of certain plant cell walls and serve as the first barrier against pathogens.Citation37 Furthermore, flavonoids, which include the synthesis of anthocyanin, are also produced via the phenylpropanoid pathway.Citation35 Based on these observations, we believe that the phenylpropanoid biosynthesis plays critical roles in the defense process of plants and that NRPs are indispensable components of this process.
2.4. Genes associated with SMs also have differential expression at different life stages in WT and nrp1nrp2 in normal growth conditions
Previous studies have shown that NRP is expressed in high amounts in seedling cotyledons, sepals, and stem-borne leaves of inflorescences and moderately in all other detected tissues. The expression level of NRP in rosette leaves increases with the continuation of aging.Citation6 In Arabidopsis, wilting of rosette leaves and ripening of pods begin around fifth week under normal growth condition. According to the KEGG enrichment analysis, we also carried out a transcriptome analysis of WT and nrp1nrp2 mutants in 7-day-old seedlings and 5-week-old plants without any stress treatments. There are 1173 genes expressed differentially between WT and nrp1nrp2 mutants in 7-day-old seedlings and 735 genes expressed differentially in 5-week-old plants. About 6.5% genes appeared simultaneously in the differentially expressed genes at these two stages (). Less differential genes were detected in 5-week-old plants than in 7-day-old seedlings, which might be due to cellular senescence, slowing or stagnating metabolic activities in the mature plants.
Figure 4. The presentation of Venn diagram expressed at two-time points.
Interestingly, the synthesis of several groups of SMs was altered in nrps knockout plants compared to WT at both two time points (), in which phenylpropanoid biosynthesis pathway is influenced significantly. Differences in the differential expression of genes associated with SMs at different life stages may lead to changes in cellular activity and changes in plant interactions with the external environment. nrp1nrp2 mutants may be less resistant to adverse external environments due to changes in the expression of secondary metabolism-related genes. Compared with their roles in 5-week-old mature plants, nrps mutation in seedlings has a more profound effect on gene expression, which may be due to the higher sensitivity of seedlings to environmental stresses.
Figure 5. KEGG enrichment of genes differentially expressed at two time points in WT and nrp1nrp2 mutants.
3. Discussion
Plants have evolved various mechanisms for living in changeable environments, especially those unfavorable conditions.Citation38–40 Among these mechanisms, the production of SMs (SMs) is one of the most important parts. SMs are those low-molecular weight organic compounds that are not directly required for plant growth, including phenolics, terpenes, and nitrogen/sulfur-containing compounds that can mediate the interaction between plants and environments.Citation41 The accumulation of such metabolites often occurs when plants suffer from stresses,Citation33,Citation42,Citation43 which is always regarded as an adaptive capacity acting through various signaling pathways. Lack of callose defense responses in Arabidopsis mutants defective in glucosinolate (GLS) biosynthesisCitation44 or in Benzoxazinoid-deficient bx1 maize mutants,Citation45 for example, leads to unfavorable growth for plants in the environment where aphids are present. GLS and phenolic compounds are associated with water uptake and transport under abiotic stress conditions.Citation46,Citation47 Additionally, SMs can be produced in vitro, and some of them are valuable for human health,Citation48 or used as industrial biochemicals.Citation49
Recent reports indicate that there are extensive crosstalk and communications between stress response pathway and plant SMs, which involves many molecules, such as ABA, salicylic, and calcium.Citation50,Citation51 However, the specific mechanism is still ambiguous due to the variety of these SMs and difficulties in analytical limitations.
In our previous work, we found NRP plays a vital role in response to both biotic and abiotic stress.Citation11,Citation17,Citation52 In this study, according to the phenotype observation, nrps knockout plants are quite different from WT and more sensitive to salt and osmotic stress.Citation6 To further explore the specific functions of NRP, we carried out transcriptome analyses in WT and nrp1nrp2 mutants treated with or without ABA (ABA can increase the expression of NRP and induce plant stress responses), and then we found that phenylpropanoid biosynthesis is tremendously affected by the lack of NRP in either distinct growth condition or different life stages. It is reported that phenylpropane biosynthesis is the crucial link in the synthesis of SMs, and phenylpropanoid serves as a rich source of metabolites in plants.Citation53 In early land plants, SMs such as flavonoids and lignin were produced through the phenylpropanoid pathway, contributing to plant colonization.Citation54 Under abiotic stress conditions, phenylpropanoid biosynthetic pathway is activated, resulting in the accumulation of various phenolic compounds that have the potential to scavenge harmful reactive oxygen species.Citation55
Considering these results, we hypothesize that NRP may exert its anti-stress function by regulating the synthesis of downstream SMs such as phenylpropanoid, which may influence plant immune response globally. Plants may use NRP to protect themselves during pathogen infection or environmental damage.
Acknowledgments
We are grateful to Dr Qingqiu Gong (Shanghai Jiao Tong University) for support in the analysis of RNA-seq data.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Additional information
Funding
References
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