Rice WNK1 is regulated by abiotic stress and involved in internal circadian rhythm

Abstract

In Mammalian system the WNK (with no lysine kinase) serine-threonine protein kinase gene family is suggested to be involved in regulating ion homeostasis and other pathophysiological processes including cancer, hypertension and renal ion transport. In plant system the information about WNK genes is very poor. However, WNK-like genes have also been identified in plants, including ten in Arabidopsis, designated AtWNK1-AtWNK10. Here we report the cloning and characterization of a homologue of AtWNK1 gene from Oryza sativa indica cultivar Pusa Basmati-1 rice and designated as OsWNK1. The specific feature of this gene is lysine residue in kinase subdomain II, which is essential for the coordination of ATP in the active center and conserved among all other kinases, is absent. OsWNK1 was found to respond differentially under various abiotic stresses like cold, heat, salt, drought. OsWNK1 gene showed rhythmic expression profile under diurnal and circadian conditions at the transcription level. Our data indicates that OsWNK1 in rice might play a role in abiotic stress tolerance and that it is involved in internal rhythm.

Protein kinases are essential regulators of numerous cellular processes ranging from cell cycle to differentiation. One of the members of protein kinases found in higher organisms is WNK (With No lysine (K)) kinases, a subfamily of serine/threonine protein kinases related to the STE20/PAK-like family.¹ A lysine residue in kinase subdomain II, which is essential for the coordination of ATP in the active center and conserved among all other kinases, is missing in this subfamily.^1,2 It was shown to be replaced by a lysine residue in subdomain I that is characteristic for members of the WNK family.³ WNK kinases have been found in numerous eukaryotes, but are not found in yeast and it has been proposed that they are restricted to multicellular organisms.⁴

Ten members of the WNK family have been reported from the model plant Arabidopsis but only a couple of them have so far been characterized.^5,6 Arabidopsis WNK gene family suggested to regulate flowering time by modulating the photoperiod pathway.⁶ AtWNK1 phosphorylate the putative circadian clock component APRR3 in vitro and might be involved in a signal transduction cascade regulating its biological acitivity.⁷ A member of Arabidopsis WNK family (AtWNK8) interacts with subunit C of the vacuolar H⁺-ATPase (V-ATP-ase) via a short C-terminal domain. AtWNK8 is shown to autophosphorylate intermolecularaly and phosphorylate Arabidopsis subunit C (AtVHA-C) at multiple sites as determined by MALDI-TOF MS analysis.⁸ Wang et al.⁶ reported the involvement of members of WNKs in regulating flowering time. T-DNA knock out mutations in AtWNK2, AtWNK5 and AtWNK8 resulted in early flowering and a T-DNA knockout mutant of wnk1 had much delayed flowering time.

The 24 hour periodicity of circadian rhythms enables organisms to coordinate their activities with the external light dark cycles by anticipating the onset of dawn or dusk. Circadian rhythms in plants include movement of organs, such as leaves, petals and stomata opening.⁹ Plants have adapted their growth and development to use the diurnal cycling of light and dark. This is manifested at both, physiological and molecular level with expression of some genes occurring only at certain time of the day. A number of circadian-regulated genes have been identified through extensive analysis with DNA microarrays of Arabidopsis.^10,11 The day/night cycling of gene expression is called diurnal rhythm and is achieved primarily by two mechanisms: first by light called diurnal and second by a free running internal circadian clock.¹² Eleven percent of the genes, encompassing genes expressed at both high and low levels, showed a diurnal expression pattern and 2% cycled with a circadian rhythm.¹¹ APPR3 was found to be substrate of WNK1 in Arabidopsis and both these gene showed same rhythmic transcriptome expression. This provided molecular link between the putative clock component, APRR3 and WNK1 which is implicated as a signal transducer. Nakamachi et al.⁵ found that transcript of Arabdiopsis WNK1 and three other members (WNK2, WNK4 and WNK6) are under the control of circadian rhythms.

We, in the present report, identified and partially characterized a novel type of protein kinase at the transcript level from rice (Oryza sativa indica cultivar Pusa Basmati-1) namely OsWNK1. Primary amino acid sequence of its catalytic kinase domain significantly showed differences from MAPKK (in fact, it has been designated as a MEK1 in rice database). However, close inspection revealed that it is more similar to a set of Arabidopsis and mammalian WNKs. The expression pattern of OsWNK1 under different abiotic stress condition as well in different tissues were analyzed. The role of the gene in circadian regulation was also investigated.

Reverse transcriptase polymerase chain reaction using gene specific primers designed against the OsMEK1 sequences available in database from japonica rice was used to clone MEK1 from the leaves of Oryza sativa indica-cultivar group var. Pusa Basmati-1. Primers used for cloning full length OsWNK1 gene and partial OsWNK1 was designed from the sequence available in database for OsMEK1 (AF080436) (forward primer-5′-GAG TAC CAG GGG ATG GAG GT, reverse primer-5′-TGA CCA TCT GGA CTG CTC TG). After cloning in pGEM-T Easy vector the full length gene was sequenced. The nucleotide sequence consisting of 2,037 base pair was submitted to the GenBank database and an accession number DQ837532 was obtained. Close inspection of the amino acid sequence revealed that lysine in subdomain II, a very important feature of subdomain II as a nucleotide (or ATP) recognition site is missing in this gene sequence. This lysine (K) is replaced by asparagine (N) residue (Fig. 1A) at position 55 in subdomain II. We therefore named this gene as OsWNK1. Asparagine in place of lysine was also found in OsWNK2, OsWNK3, OsWNK5, OsWNK7 and OsWNK8, while it was serine (S) in OsWNK4 and OsWNK9 and glycine (G) in OsWNK6. The consensus sequence of S/T-X₅-S/T between VII and VIII domain, a prerequisite to be a member of MAP kinase kinase was also missing. The amino acid sequence of this gene and other WNKs from Arabidopsis, human and rat were aligning with each other and showed common ancestry (Fig. 1B).

Figure 1.

Phylogenetic analysis of OsWNK1. (A) N-terminal region of OsMKKK1 (AAM47616.1), OsMKK1 (DQ917757), OsMAPK1 (AF216315) aligned with WNKs of rice, OsWNK1* (DQ837532), OsWNK2 (AAU44135); OsWNK3 (XP_476903); OsWNK4 (BAD27820); OsWNK5 (XP_478910); OsWNK6 (AAX95458); OsWNK7 (AK072172); OsWNK8 (NP_00106598.1); OsWNK9 (NP_001066222.1) showing change of amino acid (boxed area) lysine to asparagine in OsWNK1 (at 55^th position from start), OsWNK2, OsWNK3, OsWNK5, OsWNK7, OsWNK8; lysine to serine in OsWNK4, OsWNK9 and lysine to glycine in OsWNK6. (B) Phylogenetic relationship between OsWNK1 from indica rice cultivar with all other known WNKs from Arabidopsis, Japonica rice, mouse and humans. The sequences used for generating phylogenetic tree are OsWNK1* (DQ837532); OsWNK1 (XP_478792); OsWNK2 (AAU44135); OsWNK3 (XP_476903); OsWNK4 (BAD27820); OsWNK5 (XP_478910); OsWNK6 (AAX95458); OsWNK7 (AK072172); OsWNK8 (NP_00106598.1); OsWNK9 (NP_001066222.1); AtWNK1 (NP_001030637.1); AtWNK2 (NP_9743541.1); AtWNK3 (NP_680105.1); AtWNK4 (NP_200643.1); AtWNK5 (NP_566954.2); AtWNK6 (NP_001030723.1); AtWNK7 (NP_849787.1); AtWNK8 (NP_568599.1); AtWNK9 (NP_001031960.1); MmWNK1 (AAQ77243.1); MmWNK2 (Q3UH66); MmWNK3 (AAH60731.1); MmWNK4 (AA021955.1); HsWNK1 (AAH21121.1); HsWNK2 (CAI4449.2); HsWNK3 (CAI43129.2); HsWNK4 (NP_115763.2). Os, Oryza sativa; At, Arabidopsis thaliana; Hs, Homo sapiens; Mm, Mus musculus.

To have an insight in the regulation of the gene under different abiotic stress, the expression patterns of OsWNK1 gene was analyzed under cold, heat, salinity and drought stress. Northern blot analysis revealed that OsWNK1 gene was differentially regulated in reponse to these stress stimuli (Fig. 2A). The transcript of OsWNK1 was induced in drought and cold stress. In case of salinity stress the transcript was downregulated with time. With cold treatment, the induction of transcript was observed up to 3 h followed by repression of the same at 6 and 12 h. With heat treatment, the transcript showed no change upto 2 h followed by repression after 3 h. Under salinity stress the repression in the transcript level was observed within an hour of stress treatment (Fig. 2A). Analysis of the translational product of OsWNK1 gene using antibody generated against the poorly conserved peptide (polyclonal antibody against OsWNK1 was prepared from the peptide, DNLNGERRMKSSLNC, designed from non-conserved region of amino acid sequence and the antibody was raised in rabbit), revealed accumulation of protein under drought, heat and cold stress (Fig. 2B). As observed in the transcript regulation, the OsWNK1 protein also exhibited repression with time under salinity stress (Fig. 2B). The specificity of the antiserum was tested against OsWNK1 expressed as GST fusion (OsWNK1: GST) in bacterial system (data not shown).

Figure 2.

Regulation of OsWNK1: (A) northern blot analysis (B) immuno blot analysis, of OsWNK1 during abiotic stresses (d-days, h- hours). (C) qRT-PCR analysis of tissue specific expression of OsWNK1 in rice (n = 3). (D) Transcript analysis of OsWNK1 gene by northern blot for a 24 h period showed diurnal rhythm. Rice plants were grown under 16 h light and 8 h dark in growth chamber with constant temperature of 28°C and sampled at regular intervals. (E) The quantitative data from (D) are shown schematically as the relative amounts of transcripts. (F) Transcript analysis of OsWNK1 gene by northern blot for 72 h period in continuous dark revealed circadian rhythm. Rice plants were grown for 20 days under 16 h light and 8 h dark at 28°C, before switching off the light for next 72 h. RNA samples were prepared from the leaves of plants growing in continuous darkness at 3 hour interval for next 72 h. (G) The quantitative data from (F) are shown schematically as the relative amounts of transcripts. Lower parts in (B), (D) and (F) shows methylene blue staining of ribosomal RNA for equal loading. The maximum level of transcript is taken as 10 arbitrarily value in order to clarify the profile in (E) and (G).

The tissue specific accumulation of transcript of OsWNK1 studied in a mature rice plant by quantitative real time PCR (qRT-PCR) revealed that the gene is expressed at higher level in panicle followed by shoots, roots and leaves (Fig. 2C). Gene specific primers were designed for OsWNK1 gene, OsWNK1-F, 5′-AAC CAG GGG GAG GTC AAG AT-3′ and OsWNK1-R, 5′-GGT GCG TGC ACT CGC TAT AC-3′ and their specificities were confirmed by running the RT-PCR product in agarose gel, before they were used in real time PCR analysis.

Expression of WNK1 gene in Arabidopsis has been reported to be under the control of circadian rhythms,⁵ implying a circadian associated protein kinase function to the member of WNK family. To analyze whether OsWNK1 is also controlled by circadian clock, the expression of the gene in 24 h period was tested in two week old rice plants growing in 16/8 h light/dark cycle. Northern blot analysis showed that the transcript level of OsWNK1 started accumulating with the onset of light and reached to maximum level at 7 h after the lights were switched on. The transcript level declined gradually in light and reached to its minimum at 19 h before showing an upward trend with the onset of darkness (Fig. 2D and E) in a rhythmic pattern. Further, to implicate the role of OsWNK1 in circadian regulation, the rhythmic expression of WNK1 transcript was tested under constant external cues. The plants growing at 16/8 light/dark cycles were moved to continuous darkness for 3 days and sampled at an interval of every 3 h from day one. Northern blot analysis revealed that an initial increase in the transcript level with the extended darkness that peaks at 10 h on day one before exhibiting a decline in the accumulation of transcripts with the onset of dark on day one. This trend was repeated with slight change in peak expression time points and followed cyclic pattern in day 2 and 3 (Fig. 2F and G). Though accumulation of the transcripts decreased substantially at the end of day three, i.e., after 61 h onwards the rhythm observed for OsWNK1 indicated that the expression of OsWNK1 gene is under the control of circadian clock. However, analyzing the quantitative data represented in Figure 2G by FFT-NLS software did not indicated a very strong cycling. To implicate the role of OsWNK1 in circadian clock regulation, we analyzed the expression of OsPRR1, a member of OsPRR quintet involved in circadian regulation from rice^13,14 under similar continuous dark conditions for 72 h by semi quantitative RT PCR. The data revealed that OsPRR1 also showed a persistent rhythm with variable amplitude very much similar to the rhythm of OsWNK1 (Data not shown).

With no lysine kinases (WNK) have received considerable attention since human WNKs were genetically linked to the regulation of blood pressure.² By annotation Arabidopsis has been reported to have the largest WNK family consist of ten members, whereas the larger rice genome encodes only nine members, though Hermesdorf et al.⁸ reported seven. A lysine residue in kinase subdomain II, which is essential for the coordination of ATP in the active center and conserved among all other kinases including MAP kinase kinase,¹⁵ is missing in this subfamily.^1,2 It was shown to be replaced by a lysine residue in subdomain I that is characteristic for members of the WNK family.³ WNKs therefore represent an ancient group of protein kinases and it seems possible that the characteristic placement of the catalytic lysine residue in subdomain I found in all WNKs is older than the placement in subdomain II found in other protein kinases.⁸ Here we identified OsWNK1 as a novel protein kinase that has not previously been characterized from Oryza sativa. Although the inferred OsWNK1 protein is classified as a member of the MAPK family in Oryza sativa database, it should be considered as a novel type of Ser/Thr kinase homologs that have recently been found in mammals and Arabidopsis. Interestingly, the WNKs from plants shows higher similarity among themselves compared to those from humans and mouse (Fig. 1B). The regulation and expression of WNKs by osmolarity and changes in salt intake has been reported in human and rat.^16–18 In Arabidopsis V-ATPases are potential targets of WNK kinases and their associated signalling pathways.⁸ There is no report of expression analysis of OsWNK1 in rice under any stress so far. We, therefore, analyzed the expression of OsWNK1 with various abiotic stress treatments like salt, cold, heat and drought. Our results suggest that OsWNK1 gene has differential expression in various abiotic stresses. The expression of the gene was induced in drought and transient upregulation was observed during cold and heat stress, while salinity stress resulted in downregulation of the OsWNK1 transcripts as well as protein. Tissue specific expression of OsWNK1 by quantitative real time PCR revealed that the expression is relatively higher in panicle followed by shoots in mature plants. MPSS has been used previously for genome level expression analysis in several systems including Arabidopsis.^19,20 We found high variability in the abundance of different WNK specific mRNA tags (measured as transcript per million, TPM) in different tissue and stress specific libraries (data not shown). Among the members analyzed OsWNK5 has shown higher TPM in cold induced libraries. Unfortunately, OsWNK1 expression analyses were not performed as this gene information was not represented in MPSS database.

In higher plants, clock-controlled circadian rhythms are a longstanding issue in physiology, and a newly emerging paradigm of molecular biology. Studies on such plant clocks and various genes assosciated with clocks have been rapidly progressing in Arabidopsis^4,21,22 and rice.²³ It has been reported that APRR3, a component of the clock-associated APRR1/TOC1 quintet is a substrate of the novel WNK1 protein kinase in Arabidopsis,⁷ and that the genes for WNK1 and APRR3 showed a similar rhythmic transcription profile. Our analysis with the expression of OsWNK1 under diurnal conditions resulted in a distinct rhythmic expression at transcript level.

To be under circadian clock control, a particular rhythm should comply with the following three criteria: persist under constant conditions with a period of about 24 h; be entrained to 24 h by environmental cues (such as day and night shifts or cycles of varying temperatures); and display the same period.¹² Most studies of circadian functions in plants have been performed under continuous conditions. Rice plants entrained under 16 h light/8 h dark conditions when transferred to continuous dark the rhythmic expression continued up to 72 h. This was very similar with the rhythmic expression of OsPRR1, a known marker gene involved in circadian regulation.^13,14 The consistency of the rhythm in continuous dark suggests that rhythmic expression of OsWNK1 was endogenously generated and may be under the regulation of circadian clock. The findings of this study provided us with insight into the mechanism underlying the circadian and diurnal rhythm in Oryza sativa, suggesting a possible molecular link of this rhythm with OsWNK1. Rice is one of the most important crops and the model plant for monocot species. This study is a first step toward systematic functional characterization of member of WNK family in monocot rice.