Drought stress induces a biphasic NO accumulation in Arabidopsis thaliana

ABSTRACT

We have recently reported the proteomic signature of the early (≤30 min) drought stress responses in Arabidopsis thaliana suspension cells challenged with PEG. We found an over-representation in the gene ontology categories “Ribosome” and “Oxidative stress along with an increased abundance of late embryogenesis abundant (LEA) and early response to dehydration (ERD) proteins. Since nitric oxide (NO) plays a pivotal role in plant responses to drought stress and induces LEA and DREB proteins, here we monitored the levels of NO in Arabidopsis cell suspensions and leaf disks challenged with PEG, and performed comparative analyses of the proteomics and transcriptomics data in public domain to search for a common set of early drought and NO responsive proteins.

We show that under drought-stress, NO shows a biphasic time course, much like in response to ozone stress and that among the early drought and NO responsive proteins, the categories “DNA binding”, “Nucleotide binding” and “Transcription regulator activity” are enriched. Taken together, present study suggests that in Arabidopsis the changing NO levels may play a critical role in early drought responsive processes and notably in the transcriptional and translational reprograming observed under drought stress.

In many environments, plants face drought stress, a stress that can be exacerbated by concomitantly high solar irradiation and high air temperature. These conditions inhibit photosynthesis cause the production of reactive oxygen species (ROS) and oxidative stress that negatively impacts yields of crop plants.¹ Nitric oxide (NO), a reactive nitrogen species (RNS), is an important signaling molecule involved in many biological processes including stomatal closure.² A large body of evidence indicates the involvement of NO in environmental responses to abiotic stress such as salt, heat shock, drought, UV irradiance and ozone.^3,4 Microarray analysis of NO-responsive transcripts in Arabidopsis (e.g. a 3-h treatment)⁵ identified, e.g. late embryogenesis abundant (LEA) proteins and transcription factors, the dehydration responsive element-binding proteins DREB1 and DREB2.

In a recent study,⁶ we investigated the early response (10 and 30 min) of the Arabidopsis thaliana proteome to severe drought stress imposed through polyethylene glycol 6000 (PEG 6000) treatment. PEG 6000 is a high molecular weight solute that does neither penetrate cell wall pores nor does it enter the apoplastic space, rather it causes cytorrhysis (i.e. protoplasts and cell walls contraction) due to the water potential loss resulting from severe water loss.⁷ For this reason, PEG is widely regarded as a convenient compound to mimic drought stress effects induced by air-drying in planta. The study by Alqurashi and colleagues⁶ reported that 310 proteins were differentially expressed (at least ±2.0 fold change) after PEG treatment, and many of them have a role in endocytic processes. The study also highlights that three proteins recently annotated as Cwf18 pre-mRNA splicing factor (AT3G05070), an uveal autoantigen with coiled-coil/ankyrin (AT4G15790) and a transcription factor with DUF 662 domain (AT5G03660) dramatically increase in abundance. Interestingly, AT5G03660 is annotated as salt, drought, cold and abscisic acid (ABA) responsive and contains a cAMP- and cGMP-dependent protein kinase phosphorylation site.

Since drought stress has been associated with changing levels of NO,⁸ we have been interested to test the NO response in our experimental system and to investigate if a part of the observed PEG-induced dehydration response could conceivably have been mediated by NO. To this end we challenged Arabidopsis cells with either 10% or 40% of PEG 6000 and monitored the intracellular induction of NO production with 4-amino-5-methylamino-2ʹ,7ʹ-difluororescein diacetate (DAF-FM) DA fluorescent probe (Figure 1). Compared with the control cells, the levels of NO started to increase after 5 min of PEG treatment to peak after 15 min and remain stable up to 30 min. Since this assay measures DAF-FM accumulation, it does not reflect the accurate NO concentration overtime in PEG-treated cells. Rather, it indicates, that compared to control cells, the 10% PEG induces a strong and rapid increase of fluorescence within 15 min (inset of Figure 1). This NO accumulation inside the cells observed in response to 10% PEG is most probably transient and the rate of NO production likely falls to zero between 25 and 30 min after PEG addition (Figure 1). The rate of survival and morphology of PEG-treated cells were similar to the control cells as shown in Alqurashi and colleagues.⁶ In a subsequent experiment, we monitored the release of NO post-PEG 6000 induced stress. To this end, Arabidopsis leaf disks were challenged with either 10% or 40% of PEG 6000 for 30 min, then PEG was washed-out and the DAF-2 probe, which is able to penetrate the cell membrane and detect the release of NO, was added. We sampled NO released in the incubation buffer after 15, 30, 45 and 60 min, corresponding to 45, 60, 75 and 90 min after PEG addition, respectively. We first noted that NO released from leaf disks challenged with 40% PEG 6000 was significantly higher compared to 10%, suggesting that the NO release is dose-dependent (Figure 2). The NO increase became significant after 45 min of treatment with 40% PEG and after 60 min for leaf disks treated with 10% PEG (Figure 2). Overall, our results indicate that PEG-induced drought stress causes a biphasic NO accumulation, with the highest levels reached after 15 min of PEG-treatment and after 45 and 60 min of recovery for 40% and 10% PEG, respectively. This is in-line with our previous report showing a biphasic NO signature in O₃-treated tomato plants.¹⁰ In addition, in Arabidopsis a rapid accumulation of NO is essential for a proper O₃ response and this rapid response likely depends on the NO-ROS balance.¹¹ Crosstalk between NO and hydrogen peroxide (H₂O₂) seems also to occur in Scrophularia striata where, as a consequence of the 72 h PEG exposure of the cultured cells, a positive correlation between H₂O₂ and NO contents has been reported.¹² It has been postulated that accumulation of NO as ROS could stimulate mechanism of tolerance in salt stress plant. NO production strongly increased after PEG treatment in suspension cultures of the stress tolerant ecotype dune reed of Phragmites communis Trin., whereas no significant differences were observed in those of the drought sensitive swamp reed ecotype.¹³ In addition, in rapeseed NO pre-treatment alleviated oxidative damage and thereby conferred an increased tolerance to osmotic stress caused by PEG 6000 treatment.¹⁴

Figure 1.

Time course of NO production in Arabidopsis suspension cells.

The suspensions were incubated with 15 microM of the probe 4-amino-5-methylamino-2ʹ,7ʹ-difluorofluorescein diacetate (DAF-FM DA) at 25°C for 30 min. To remove the superfluous DAF-FM DA that did not enter into the cells, the probe-loaded cells were rinsed three times with sodium phosphate buffer (pH 7.4). Next, 10% (w/v) PEG 6000 was added to the suspension cells. The osmotic potential of 10% PEG 6000 (100 gL⁻¹) was −1.48 bar according to Michael and Kaufmann (1973).⁹ NO production was visualized at 0, 5, 15, 25 and 30 min using a fluorescence microscope. Excitation at 488 nm and emission at 510 nm was used. (A–D): NO visualization inside the cells after 5, 15 and 30 min from PEG addition and in control cells without PEG. Magnification is identical for all images, bar 100 μm. The control samples were without PEG. (E): quantification of NO fluorescence was performed using ImageJ analysis software. Each value represents the mean ±SE of 5 independent experiments (over 800 cells were visualized in each experiment). The values with different letters indicate significant differences at p ≤ 0.01 (Two-way ANOVA, Duncan’s test). Inset: transient NO increase in PEG-treated cells. Values were calculated as difference between the value of DAF-2T fluorescence detected at each time point and the previous one.

Figure 2.

NO released from Arabidopsis leaf disks treated with PEG.

Six leaf disks (7 mm diameter) from 4-week-old Arabidopsis plants were incubated in an Eppendorf tube containing 2.5 mL of incubation buffer (10 mM Tris-HCl, 10 mM KCl, pH 7.5) supplemented or not with 10% and 40% (w/v) PEG 6000. The osmotic potential of 10% PEG 6000 (100 gL⁻¹) was −1.48 bar and 40% PEG 6000/40 gL⁻¹) −17.57 bar, according to Michael and Kaufmann (1973).⁹ Samples were kept in darkness at 25°C under continue agitation. In the control samples the buffer was without PEG. After 30 min leaf disks were washed to remove PEG with fresh incubation buffer and then 10 microM of 4,5-diaminofluorescein (DAF-2) probe dissolved in the incubation buffer was added. At different time points (15, 30, 45 and 60 min), aliquots of the incubation medium were sampled, and the highly fluorescent compound triazolofluorescein (DAF-2T) formed from DAF-2 and NO reaction, was measured using a fluorescence spectrophotometer (BioSpectrometer, Eppendorf), with excitation at 470 nm and emission at 520 nm. Blank contained the incubation buffer and DAF-2 without leaf disks. Each value represents the mean ± SE of three independent experiments (four replicates in each experiment). The values with different letters indicate significant differences at p ≤ 0.01 (Two-way ANOVA, Duncan’s test).

The role of NO in the response to drought is also associated with its ability to reduce stomatal aperture as well as controlling other physiological processes such as photosynthesis, proline accumulation and developmental processes.¹⁵ Moreover, NO has also been shown to modulate plant stress responses by regulating gene-encoding proteins involved in signal transduction such as kinases and phosphatase proteins¹⁶ and directly modify target proteins by posttranslational modification including metal nitrosylation, tyrosine nitration and S-nitrosylation.¹⁷ Given the biphasic accumulation of NO, we speculate that NO may play a dual role in the response to drought stress. Firstly, the early transient NO elevation may trigger physiological responses including posttranslational protein modification and hence control protein activity and stability. It remains to be seen if some of the modified protein are RNA-binding proteins¹⁸ and/or transcription factors that will then affect of the transcriptional program. Experimental evidence for an early NO accumulation associated with posttranslational modification of proteins by S-nitrosylation (cysteine residues) and nitration (tyrosine residues) via peroxynitrite generated by a reaction between NO and oxygen under salt stress has been presented.¹⁹ These modifications can alter enzyme activity, block the sites of regulatory proteins or induce posttranslational modifications, thereby influencing signaling pathways, including those of ABA (nitrosylation of OST1 protein and SnRK2),^20,21 auxin (nitrosylation of TIR1 receptor)²² and cytokinin (nitrosylation of type-A response regulators).²³

Secondly, the later and more persistence elevation of higher levels of NO (that in our experimental model occurs 30–45 min after stress application) might be of importance in inducing and sustaining an adaptive change to the stress by inducing and controlling transcription and protein biosynthesis of stress responsive genes.

We have also undertaken a comparison of differentially expressed proteins after PEG-induced dehydration stress (10 and 30 min)⁶ and NO-responsive genes from previous studies.^5,11 We found 29 common proteins including BAK1-interacting receptor-like kinase 1, basic-leucine zipper (bZIP) transcription factor, G-box binding factor 3, glutathione peroxidase 2 and 6, glutathione S-transferase phi 7, ERD11 as well as vacuolar protein sorting-associated protein 60.1 (Table 1). In addition, gene ontology (GO) analysis (performed with “AgriGo”, http://bioinfo.cau.edu.cn/agriGO/(Nov. 2018)) on the proteins in Table 1 showed 18 out of the 29 proteins (62%) significantly enriched in DNA binding (GO:0003677, p-value 2.3e⁻⁰⁸, FDR 3.4e⁻⁰⁷), Transcription regulator activity (GO:0030528, p-value 3.6e⁻⁰⁶, FDR 2.6e⁻⁰⁵) and Nucleotide binding (GO:0003676, p-value 8.8e⁻⁰⁶, FDR 4.3e⁻⁰⁵) as well as enriched localization to the nucleus (GO: 0005634, p-value 1.5e⁻⁰⁵, FDR 2.5e⁻⁰⁴; Figure 3). Such molecular function and cellular component enrichment concur with previous study on NO regulation.²⁴ Given that GO enrichments can infer biological function(s),²⁵ we propose that drought-induced NO generation triggers NO-dependent transcription and protein synthesis that directly affect transcriptional activities and conceivably participate in the change of the transcriptional program required for adaptive responses. It will therefore be interesting to further dissect the roles of NO and the in particular the transcriptional and translational responses that are linked to the biphasic NO signature.

Table 1.

List of proteins common between PEG drought stress and NO-responsive proteins.

Accession number	Protein annotation	GO category enrichment
AT3G53990*	Adenine nucleotide α-hydrolases-like
AT2G44730	Alcohol dehydrogenase transcription factor Myb/SANT-like	A, B, C
AT3G48090	α/β-Hydrolase
AT5G48380	BAK1-interacting receptor-like kinase 1
AT5G11260	Basic-leucine zipper (bZIP) transcription factor	A, B, C, D
AT5G44080	Basic-leucine zipper (bZIP) transcription factor	A, B, C, D
AT3G55120	Chalcone-flavanone isomerase	D
AT3G05380	Always early 2	A, B, C
AT1G44810*	DNA-binding storekeeper protein-related transcriptional regulator	B
AT4G00238	DNA-binding storekeeper protein-related transcriptional regulator	D
AT1G02930	Early response to dehydration (ERD11)
AT2G46270	G-box binding factor 3	A, B, C
AT5G40370	Glutaredoxin
AT2G31570	Glutathione peroxidase 2
AT4G11600	Glutathione peroxidase 6
AT1G02920	Glutathione S-transferase phi 7	D
AT2G35370	Glycine decarboxylase complex H
AT5G23420	High-mobility group box 6	A, B, C, D
AT5G61890	Integrase-type DNA-binding protein	A, B, C, D
AT1G15340	Methyl-CPG-binding domain 10	A, C
AT3G15790	Methyl-CPG-binding domain 11	A, C
AT3G46580	Methyl-CPG-binding domain 5	A, C
AT3G22060	Receptor-like protein kinase-related protein
AT5G13330	Ethylene-responsive transcription factor 113	A, B, C, D
AT3G57040	Two-component response regulator 9	B, D
AT4G00990	Transcription factor jumonji domain-containing protein	A, B, C
AT4G34131	UDP-glucosyl transferase 73B3
AT3G10640	Vacuolar protein sorting-associated protein (VPS60.1)
AT2G30620	Winged-helix DNA-binding transcription factor	A, C, D

All proteins were significant (p-value ≤ 0.05) with * indicating a >twofold change. A, DNA binding, B, Transcription regulator activity, C, Nucleotide binding, D, Nucleus.

Figure 3.

Venn diagram of enriched GO terms in proteins common between PEG drought stress and NO-responsive proteins.

The Venn diagram shows 18 enriched of the 29 proteins common between PEG drought stress study and NO-response study. The enriched terms of the GO analysis include DNA binding, Transcription regulator activity and Nucleotide binding as well as enriched localization to the nucleus.