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NTHK1 transcripts were first accumulated in the palisade parenchyma cells and ..... Acknowledgements. We thank Dr H. J. Klee (University of Florida), Dr J. C. Lagarias ... the National High Tech Project (2001AA222131). References. Abeles ...
The Plant Journal (2003) 33, 385–393

Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco Can Xiey, Jin-Song Zhangy,ô, Hua-Lin Zhouy, Jian Li, Zhi-Gang Zhang, Dao-Wen Wang and Shou-Yi Chen Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, Peoples Republic of China Received 20 August 2002; revised 16 October 2002; accepted 25 October 2002.  Correspondence (fax þ86 10 64873428; e-mail [email protected], ô[email protected]). y Contributed equally to this work.

Summary A histidine kinase-based signaling system has been proposed to function in ethylene signal transduction pathway of plants and one ethylene receptor has been found to possess His kinase activity. Here we demonstrate that a His kinase-like ethylene receptor homologue NTHK1 from tobacco has serine/threonine (Ser/Thr) kinase activity, but no His kinase activity. Evidence obtained by analyzing acid/base stability, phosphoamino acid and substrate specificity of the phosphorylated kinase domain, supports this conclusion. In addition, mutation of the presumptive phosphorylation site His (H378) to Gln did not affect the kinase activity whereas deletion of the ATP-binding domain eliminated it, indicating that the conserved His (H378) is not required for the kinase activity and this activity is intrinsic to the NTHK1-KD. Moreover, confocal analysis of NTHK1 expression in insect cells and plant cells suggested the plasma membrane localization of the NTHK1 protein. Thus, NTHK1 may represent a distinct Ser/Thr kinase-type ethylene receptor and function in an alternative mechanism for ethylene signal transduction. Keywords: two-component kinase, Ser/ Thr kinase, ethylene receptor, NTHK1, plasma membrane localization, tobacco.

Introduction Protein kinases play key roles in cellular regulation and signal transduction. These can be classified into three categories according to the amino acid residues these phosphorylated, namely serine/threonine (Ser/Thr) protein kinases (STKs), tyrosine protein kinases and histidine protein kinases (HKs). The Ser/Thr/Tyr protein kinases catalyze the formation of a phosphoester linkage between a phosphate group and the specific Ser/Thr/Tyr residues in protein substrate, whereas in HKs, His phosphorylation occurs on nitrogen, producing a phosphoramidate bond (Robinson et al., 2000). Although HKs are a distinct protein kinase family from Ser/Thr/Tyr kinases according to the difference in chemical bond formation during phosphorylation, these are probably still structurally related. It has been found that the phytochromes in prokaryotes are HKs, whereas the phytochromes in higher plants were demonstrated to possess STK activity (Yeh and Lagarias, 1998; Yeh et al., 1997). Also, the mitochondrial protein kinase family and the antisigma factor SpoIIAB from Bacillus subtilis phosphorylate the substrates on Ser residues although these exhibit ß 2003 Blackwell Publishing Ltd

sequence similarity to HK (Min et al., 1993; Elich and Chory, 1997). Moreover, a HK-like DokA from Dictyostelium can be phosphorylated in vivo on a Ser residue upon osmotic stress (Oehme and Schuster, 2001). HK-based signaling systems (so-called two-component systems) are prevalent in prokaryotes and mediate many adaptive responses (Parkinson and Kofoid, 1992; Robinson et al., 2000). A few similar systems have been found in some eukaryotes such as slime mold, fungi, yeast and higher plants (Alex et al., 1996; Chang and Stewart, 1998; Maeda et al., 1994; Wang et al., 1996). In plants, a HK-based signaling mechanism has been demonstrated to function in ethylene-signaling pathway. Ethylene is a gaseous hormone that plays important roles in plant growth and development such as seed germination, seedling growth, flowering, fruit ripening, organ abscission and senescence. It is also involved in plant responses to stress and pathogen infection (Abeles et al., 1992). Genetic and mutational analysis of Arabidopsis has led to the identification of five ethylene receptors and all of these receptors showed 385

386 Can Xie et al. structural similarity to HK (Bleecker and Kende, 2000; Wang et al., 2002). The ethylene receptor family can be divided into two subfamilies. The first subfamily (subfamily I) is represented by ETR1 and ERS1, and possesses three transmembrane segments at the N-terminus plus a conserved HK domain. A conserved receiver domain is present at the C-terminus of ETR1, but not ERS1 (Chang et al., 1993; Hua et al., 1995). The second subfamily (subfamily II) is exemplified by ETR2, EIN4 and ERS2. These all have an additional hydrophobic region at the N-terminus, followed by three transmembrane segments and a more diverged HK domain. A receiver domain is also occurred at the C-terminus of ETR2 and EIN4, but not ERS2 (Hua et al., 1998; Sakai et al., 1998). Homologues of ethylene receptor have been isolated from tomato (Tieman and Klee, 1999; Wilkinson et al., 1995; Zhou et al., 1996), tobacco (Knoester et al., 1997; Terajima et al., 2001; Zhang et al., 1999; 2001a,b) and other plants (Sato-Nara et al., 1999; Vriezen et al., 1997). Previously, we have isolated an ethylene-receptor homolog gene NTHK1 from tobacco and found that its encoded protein belonged to the second subfamily of the ethylene receptors. NTHK1 transcripts accumulated during flower organ formation and embryo development and in response to wounding, salt and drought stresses. In response to wounding (cutting), NTHK1 transcripts were first accumulated in the palisade parenchyma cells and then spread to the spongy parenchyma cells (Zhang et al., 2001a). The NTHK1 proteins in these cells followed similar expression patterns (Xie et al., 2002). HK activity has been demonstrated in the ethylene receptor ETR1 in the first subfamily (Gamble et al., 1998) and ETR1 was reported to be localized to endoplasmic reticulum (Chen et al., 2002). However, whether the members in the second subfamily have any HK activity remains unclear. In addition, where these are located remains to be investigated. Here we examined the kinase activity of NTHK1 by expressing it in yeast and found that it possesses STK activitity but not HK activity. Confocal microscopy of its expression in insect cells and plant cells demonstrated its plasma membrane localization. The biological significance of the STK activity in NTHK1 was also discussed.

Results Purification of the NTHK1 domains NTHK1 contains a putative signal peptide, three transmembrane segments, a putative HK domain and a receiver domain (Figure 1a). The putative signal peptide was defined according to the prediction method of Nielsen et al. (1997) and the possible cleavage site was between position 21 and 22. Other domains were recognized using the SMART program (Schultz et al., 1998) and by comparison with ETR1 (Chang et al., 1993; Gamble et al., 1998). The putative

Figure 1. Purification of different domains of NTHK1 as GST fusions. (a) Schematic representation of NTHK1 and the four versions used for yeast expression. For NTHK1, the first box indicates the putative signal peptide, the next three boxes represent the transmembrane segments. The rectangular box represents the putative His kinase domain. The oval box represents the putative receiver domain. (b) Coomassie blue staining of SDS–PAGE gel containing protein markers and purified GST fusion proteins. (c) Western blot analysis of the purified GST fusion proteins using an antiGST monoclonal antibody.

phosphorylation site and the phosphate receiver site were identified at H378 and D689, respectively (Zhang et al., 2001a). To address the biochemical property of NTHK1, we expressed four truncated proteins corresponding to the kinase domain (NTHK1-KD, amino acids 145–636), the kinase domain with a mutation of the conserved His (H378) to Gln [NTHK1-KD(H378Q)], the truncated kinase domain without the ATP-binding motif [NTHK1-KD(DATP), amino acids 145–445] and the receiver domain (NTHK1-RD, amino acids 624–762) as GST fusions, respectively, in yeast (SPQ01) (Figure 1a,b). Yeast was used because initial experiments expressing the kinase domain as His6 or GST fusion proteins in E. coli resulted in a very small amount of proteins and thus not enough for further analysis (data not shown). The yeast-expressed proteins were purified by GST affinity resin. Fusion proteins displayed an apparent molecular mass of 80, 80, 60 and 41 kDa for NTHK1-KD, ß Blackwell Publishing Ltd, The Plant Journal, (2003), 33, 385–393

Ser/Thr kinase activity in the ethylene receptor

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NTHK1-KD(H378Q), NTHK1-KD(DATP) and NTHK1-RD, respectively, when analyzed by SDS-PAGE, consistent with the predicted molecular mass for the GST fusion proteins (Figure 1b). The GST protein itself was also expressed, purified and used as a control in later analysis. The expressed GST fusions were confirmed on Western blots using the mouse anti-GST monoclonal antibody (Figure 1c). Kinase activity of the NTHK1 kinase domain Because the ethylene receptor ETR1 has been demonstrated to have HK activity, we then examined whether the purified NTHK1-KD fusion had any kinase activity. As indicated in Figure 2, when the NTHK1-KD fusion protein was incubated with [g-32P] ATP, 32P was incorporated into the fusion protein in the presence of Mn2þ and the molar percent incorporation was estimated to be 2%. When Mn2þ was removed, no 32P was incorporated in the presence of Ca2þ or Mg2þ. The presence of Mn2þ plus Mg2þ or Ca2þ together did not result in more phosphorylation than Mn2þ alone, indicating that these cations do not function synergistically. This cation specificity is similar to that of ETR1 (Gamble et al., 1998). Sodium pyrophosphate inhibited the kinase activity of the NTHK1-KD fusion. GST itself doesn’t exhibit any 32P-incorporation (Figure 2).

Figure 2. NTHK1-KD has protein kinase activity in the presence of Mn2þ in in vitro phosphorylation assay. GST fusion protein NTHK1-KD (0.5 mg) was incubated with [g-32P]-ATP for 60 min in the presence of 5 mM Mg2þ, 5 mM Ca2þ, 5 mM Mn2þ, 5 mM Mg2þ and 5 mM Mn2þ, 5 mM Ca2þ and 5 mM Mn2þ, 5 mM Mn2þ and 2 mM pyrophosphate (PPi), or absence of them. GST was used as a control. The phosphorylated proteins were then separated on SDS–PAGE, transferred onto PVDF membrane and autoradiographed (top) or Coomassie blue stained (bottom).

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 33, 385–393

Figure 3. NTHK1-KD autophosphorylates on its Ser/Thr residues. (a) Hydrolytic stability of autophosphorylated NTHK1-KD before (top) and after (middle) treatment with water, HCl or NaOH. After autoradiograph, the treated protein blots were then subject to Coomassie blue staining (bottom). (b) Phosphoamino acid analysis of the phosphorylated NTHK1-KD. The positions of the phosphoamino acids were identified by spraying with ninhydrin (left) and the labeled residues were revealed by autoradiography (right).

To examine which classes of residues are phosphorylated, the phosphorylated NTHK1-KD protein was transferred onto polyvinylidene difluoride (PVDF) membrane and treated with acid or base to test its hydrolytic stability. We found that the phosphorylated residues were stable under acidic conditions but sensitive to basic conditions (Figure 3a), and the residual level left after NaOH treatment disappeared after longer treatment (data not shown), implying that the phosphorylated NTHK1-KD contained phosphoserine, phosphothreonine or phosphotyrosine, but not phosphohistidine. We further conducted the phosphoamino acid analysis. The phosphorylated NTHK1-KD was hydrolyzed with concentrated HCl and the products, together with the phosphoamino acid standards, were analyzed by two-dimensional thin-layer chromatography electrophoresis (Figure 3b). The labeled spots corresponded to the positions of phosphoserine and phosphothreonine, indicating that NTHK1-KD contained phosphoserine and phosphotheronine. We then investigated whether the phosphorylation of NTHK1-KD was resulted from any co-purified proteins from yeast. Because NTHK1 does not have the two conserved glycines in the G1 box for Gly mutation (Zhang et al., 2001a), we deleted the putative ATP-binding motif from NTHK1-KD (Figure 1), which corresponded to the regions of N, G1, F and G2 boxes from ETR1. The results in Figure 4 showed that elimination of the ATP-binding motif in NTHK1-KD abolished the phosphorylation in NTHK1KD(DATP), indicating that the kinase activity was intrinsic to NTHK1-KD, but not from other co-purified proteins. We further mutated the conserved His (H378) to Gln to test its

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Figure 4. Phosphorylation assay of different versions of NTHK1-KD. NTHK1-KD or other versions (0.5 mg) was incubated under phosphorylating conditions with no substrate or with MBP (5 mg), NTHK1-RD (2 mg) or GST (5 mg). GST, MBP or their combination was also incubated under phosphorylating conditions without NTHK1-KD as controls. The phosphorylated proteins were resolved on SDS–PAGE gel, transferred to PVDF membranes and subjected to autoradiography (top) or Coomassie blue staining (bottom).

effect on phosphorylation (Figures 1 and 4). The same site in ETR1 (H353) has been proven to be essential for the phosphorylation (Gamble et al., 1998). The results (Figure 4) showed that the mutation form [NTHK1-KD(H378Q)] did not affect the phosphorylation, suggesting that H378 is not an intermediate phosphorylation site in NTHK1-KD. We examined whether NTHK1-KD can phosphorylate the receiver domain NTHK1-RD and in vitro substrate myelin basic protein (MBP). MBP has been used as substrate to identify new protein kinases from mammals and plants (Cicirelli et al., 1988; Hardin and Wolniak, 1998). It is shown that the NTHK1-KD fusion phosphorylated MBP, but not NTHK1-RD or the GST control (Figure 4). NTHK1-KD(H378Q) also phosphorylated MBP. At the same time, the NTHK1-KD was autophosphorylated (Figure 4). MBP, GST and their combination did not show any 32P-incorporation in the absence of NTHK1-KD. Subcellular localization of NTHK1 We further studied the possible function of the putative signal peptide and the three transmembrane segments. Three constructs, namely Bac1-(gene fragments plus) GFP, Bac2-GFP and Bac3-GFP were made by introducing the corresponding NTHK1 GFP open reading frame into plasmid pFASTBAC1 (Figure 5a). The GFP fusions were

expressed in insect cells (sf21). Under the confocal microscope, we observed that the expressed full-length NTHK1 protein for Bac1-GFP, as revealed by GFP, was localized on the plasma membrane, indicating that NTHK1 was a transmembrane protein (Figure 5b, left). The truncated NTHK1 without the putative signal peptide (for Bac2-GFP) was also present on plasma membrane (Figure 5b, middle). This phenomenon may imply that deletion of the putative signal peptide does not affect the localization of the truncated protein. The reason for this remains unknown. When the signal peptide and the three transmembrane segments were all removed, the truncated protein (for Bac3-GFP) was mainly distributed in cytoplasm (Figure 5b, right). This result demonstrated that the three transmembrane segments were essential for plasma membrane localization of the NTHK1 protein in cells. To investigate the localization of NTHK1 in plant cells, we made three more constructs Puc1-GFP, Puc2-GFP and Puc3GFP. Each construct contained the fusion gene encoding the NTHK1 protein (or its deletions) plus a GFP protein (Figure 6a). The genes were controlled by 35S promoter. These constructs were transformed into tobacco protoplasts by electroporation and the expressions of the GFP fusions were examined under a confocal microscope for green fluorescence. The result in Figure 6(b) showed that, whereas the GFP protein control was present in the cytoplasm, the full-length NTHK1 protein and the NTHK1 without the putative signal peptide were all localized on the plasma membrane (Figure 6b(i–iii)). For the truncated protein without the putative signal peptide and the three transmembrane segments, its expression was concentrated in the cytoplasm (Figure 6b(iv)). This result was consistent with the expression pattern of NTHK1 in insect cells, implying the plasma membrane localization of NTHK1 in plant cells and the function of the three transmembrane segments in NTHK1 localization. Discussion From comparison of amino acid sequences, NTHK1 showed 35% identity to ETR1. The G1, F, and G2 box in the putative HK domain diverged from that of ETR1, and these boxes have been demonstrated to be necessary for the HK activity (Gamble et al., 1998; Zhang et al., 2001a). The divergence implies some difference between NTHK1 and ETR1. In the present study, we characterized NTHK1 by expressing its putative kinase domain in yeast and found that NTHK1 in vitro possessed STK activity but not HK activity. Several lines of evidence support this conclusion. First, acid/base stability analysis of the phosphorylated protein showed that NTHK1-KD was stable under acidic condition, but unstable under basic condition. This phenomenon is characteristic of phosphoserine, phosphothreonine and phosphotyrosine, but not ß Blackwell Publishing Ltd, The Plant Journal, (2003), 33, 385–393

Ser/Thr kinase activity in the ethylene receptor

Figure 5. NTHK1 is localized on the plasma membrane of insect cells. Bars represent 10 mm. (a) Schematic representation of constructs made for NTHK1 expression in insect cells. All constructs have a GFP gene fused to the 30 -end of the corresponding NTHK1 gene. (b) Expression of the three NTHK1–GFP fusion proteins in insect cells. Confocal images are shown. Green fluorescence for GFP was associated with plasma membrane in Bac1-GFP and Bac2-GFP expression, whereas the fluorescence was mainly localized in the cytoplasm in Bac3-GFP expression. Cells were counter-stained with a PI solution to reveal the nuclei.

phosphohistidine because phosphohistidine is base stable and acid labile. Second, phosphoamino acid analysis of the hydrolyzed NTHK1-KD clearly indicated that the phosphorylated amino acids were Ser and Thr. Third, mutation of the conserved His (H378) to Gln did not affect the phosphorylation of NTHK1-KD. Fourth, NTHK1-KD can phosphorylate MBP, which is commonly used as a substrate for a number of Ser/Thr/Tyr kinases (Cicirelli et al., 1988; Hardin and Wolniak, 1998). Moreover, deletion of the ATP-binding motif resulted in the loss of the phosphorylation in NTHK1-KD(DATP), implying that the kinase activity was intrinsic to NTHK1-KD. Although NTHK1 has STK activity, it exhibited no obvious sequence similarity to the known STK. Thus, it may represent a distinct class of receptor STK. However, where the phosphorylated Ser/Thr residues are located remains to be identified and more work needs to be done to further exclude the possibility that the phosphorylated site is on the GST portion although such a case is less likely. Other possibilities might also exist that deletion of the ATP-binding motif eliminate the co-purification of a contaminating Ser/Thr kinase. Mutagenesis of the essential residues in the catalytic domain, if identified as in the case of ETR1 (Gamble et al., 1998), would have improved the analysis. In addition, another His residue in NTHK1, which is seven residues away from the H378 and more conserved in the ethylene receptor homologs, might serve as a His phospho-intermediate. Mutation of this site would make a better understanding of its function. ß Blackwell Publishing Ltd, The Plant Journal, (2003), 33, 385–393

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Figure 6. NTHK1 is localized on the plasma membrane of plant cells. (a) Schematic representation of versions made for NTHK1 expression in tobacco protoplasts. All constructs have a GFP gene fused to the 30 -end of the corresponding NTHK1 gene. (b) Expression of GFP control and the NTHK1–GFP fusions in tobacco protoplasts. Confocal images of the green fluorescence for GFP are shown. (i) Confocal images from protoplasts expressing Puc-GFP (control). (ii) Confocal images from protoplasts expressing Puc1-GFP. (iii) Confocal images from protoplasts expressing Puc2-GFP. (iv) Confocal images from protoplasts expressing Puc3-GFP. Bars represent 2.5 mm.

NTHK1 cannot phosphorylate its receiver domain NTHK1-RD in the present in vitro studies. Therefore, the receiver domain may play roles in aspects other than accepting a phosphate, probably in regulation or interaction, etc. ETR1 cannot phosphorylate its receiver domain in vitro either (Gamble et al., 1998). This fact indicates that both ETR1 and NTHK1 may work in a more or less different way from their bacterial counterparts. However, other possibilities may also be present since no in vivo study has been performed in plants. The STK activity of NTHK1 showed dependence for Mn2þ. This cation specificity was similar to that of ETR1. However, NTHK1 showed almost no kinase activity with Mg2þ. This is in contrast to ETR1, which still exhibited significant activity in the presence of Mg2þ (Gamble et al., 1998). The reason for this difference is not known. It is probably due to the unique structural feature occurred in NTHK1 for its STK activity. The presence of STK activity in NTHK1 indicates an alternative mechanism for ethylene signal transduction in addition to the pathway mediated by HK-type ethylene receptor ETR1. In this alternative pathway, the phosphor-

390 Can Xie et al. ylation state of NTHK1 may activate the downstream components or cascades. It is also possible that the STK activity of NTHK1 will directly phosphorylate the downstream components and the related cascades. A CTR1 protein belonging to the mitogen-activated protein kinase cascade has been reported to interact with ETR1 (Clark et al., 1998). However, the downstream events for NTHK1 are still not known although the possibility exists that similar mitogenactivated protein kinase cascade be used. The possibility also exists that the STK activity of NTHK1 in vitro may be indirectly involved in the in vivo ethylene signal transduction. The co-occurrence of STK and HK in the ethylene receptor family may reflect their multiple roles played in plant growth, development and responses to biotic and abiotic stresses. Because different tissues or organs at various stages have different sensitivity to ethylene, two types of kinase activity and the corresponding signaling pathways may meet the needs of plants to bind ethylene, and then respond and adapt to the changes in internal and environmental factors. Also, these may facilitate the cross-talk and integration among different signaling pathways in order for the plants to generate complicated and elaborate responses to survive and to complete life cycle smoothly and quickly (Chory and Wu, 2001; McCarty and Chory, 2000). Recently, Gamble et al. (2002) reported that the enzymatic activity was probably not required for the ethylene insensitivity conferred by the mutant etr1-1 receptor. However, the ETR1 kinase activity may be still involved in responses other than ethylene triple responses. In fact, NTHK1 expression has been found to go up in response to wounding, salt and drought stresses (Xie et al., 2002; Zhang et al., 2001a). Another divergent ethylene receptor homologue NTHK2 was induced upon wounding, drought stress and heat shock (Zhang et al., 2001b). Some other ethylene receptors have been reported to involve in defense response (Ciardi et al., 2000; 2001; Knoester et al., 1998; Lund et al., 1998) and most of them were also highly expressed in reproductive organs (Hua et al., 1998; Sakai et al., 1998; Tieman and Klee, 1999; Zhang et al., 2001a,b). By expressing NTHK1 in insect cells and tobacco protoplasts, we found that NTHK1 was localized on the plasma membrane. The three transmembrane segments are essential for the plasma membrane localization. However, presence or absence of the putative signal peptide doesn’t affect this conclusion. This fact implies that the information needed for the trafficking and targeting of NTHK1 are within the protein but not in the putative signal peptide. Alternatively, the putative signal peptide may simply be another transmembrane segment that redirects the amino terminus to the cytoplasm. Although the present work indicated the plasma membrane localization of NTHK1, other approaches should be adopted to investigate its in vivo situa-

tion in plant. Recently, Chen et al. (2002) reported the localization of ETR1 to the endoplasmic reticulum of Arabidopsis, which is different from the localization of the present NTHK1. This difference probably resulted from the divergence in the putative signal peptide, the hydrophobic transmembrane regions or other unknown sequences between subfamily I and II members of the ethylene receptor family. The different localization of ETR1 and NTHK1 may indicate their roles in sensing ethylene from different places, i.e. from inside of the cell or from outside of the cell. Additionally, the differences in localization and kinase activity between NTHK1 and ETR1 revealed the complexity of the functions of these ethylene receptors in regulation of plant growth, development and stress responses. By over-expression of NTHK1 in tobacco and Arabidopsis, we have observed reduced ethylene sensitivity through the examination of the triple responses in transgenic plants (Xie et al., 2002; Zhang JS et al. unpublished results). These results are consistent with previous studies (Hua and Meyerowitz, 1998; Tieman et al., 2000), indicating that the NTHK1 is a functional ethylene receptor in plant. Further research should focus on which Ser/Thr residues are phosphorylated and what are the downstream components for NTHK1 and how they interact during the signal transduction process. These studies will give a full picture as to how NTHK1 works in plants.

Experimental procedures Recombinant expression of truncated NTHK1 proteins To express different truncated version of NTHK1 as fusions to glutathione S-transferase (GST) in yeast, DNA fragments corresponding to the putative kinase domain (NTHK1-KD, amino acids 145–636), the kinase domain without the ATP-binding motif [NTHK1-KD(DATP), amino acids 145–445] and the putative receiver domain NTHK1-RD (amino acids 624–762) were amplified from the original NTHK1 plasmid. For NTHK1-KD, the sense primer is 50 AGGGGATCCATGCTGAAAAA GAAAACTTGG-3 and the antisense primer is 50 -AGAACGCTAGCCCCCTGG AGGAGTGAGTG-30 . For NTHK1-KD(DATP), the sense primer is the same as that for NTHK1-KD, and the antisense primer is 50 -GCAGCTAGCATGTAGCTGAAAATGCCT CAT-30 . The primers for NTHK1-RD are 50 TATGGATCCTCTTCTGATCACT CGCATC-30 and 50 -GCAGCTAGCACATCATCACGTGATTATG-30 . A mutant version [NTHK1-KD(H378Q)] of the kinase domain, which has a Gln (CAG) at position 378 instead of the original conserved phosphorylation site His (CAT), was also generated by using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The mutation was confirmed by sequencing. The PCR products for NTHK1-KD, NTHK1-KD(H378Q), NTHK1-KD(DATP) and NTHK1-RD were digested with BamHI and NheI, cloned into the yeast expression vector pESP-2 (Stratagene) and confirmed by sequencing. The recombinant plasmids were transformed into Schizosaccharomyces pombe SP-Q01 yeast strain (leu 1–32 h, Stratagene) and the positive colonies were identified by their ability to grow on Edinburgh minimal medium ß Blackwell Publishing Ltd, The Plant Journal, (2003), 33, 385–393

Ser/Thr kinase activity in the ethylene receptor supplemented with thiamine. Growing the cells in Edinburgh minimal medium without thiamine induced expression of GST fusion protein. After induction, the yeast cells were broken and the supernatant was loaded onto the GST affinity resin in the column. The resin was washed extensively with washing buffer [50 mM Tris–HCl, pH 7.3, 100 mM NaCl, 0.1% (v/v) Tween-20, 10% (v/v) glycerol] and finally eluted for GST fusion proteins with elution buffer (10 mM reduced glutathione, 50 mM Tris–HCl, pH 8.0). The free glutathione was removed on a centriprep concentrator and the buffer was changed to storage buffer (50 mM Tris–HCl, pH 7.6, 50 mM KCl, 2 mM DTT, 10% (v/v) glycerol). The expression and purification of the GST fusion proteins were confirmed by Western blotting using a mouse anti-GST monoclonal antibody (Amersham).

In vitro phosphorylation assay The purified GST fusion proteins NTHK1-KD, NTHK1-KD(H378Q) and NTHK1-KD(DATP) were examined for their kinase activity and GST is used as a control. Unless specified otherwise, phosphorylation was performed in a 25-ml assay buffer [50 mM Tris–HCl, pH 7.6, 50 mM KCl, 2 mM DTT, 10% (v/v) glycerol] containing 0.5– 1 mg GST fusion proteins in the presence of 5 mM MnCl2. The phosphorylation was initiated by adding 25 mCi of [g-32P] ATP (30 Ci mmol1), incubated at 228C for 60 min and terminated by the addition of EDTA to a final concentration of 10 mM. The phosphorylated proteins were subjected to 10% SDS–PAGE and transferred onto PVDF membranes (Amersham). The incorporated phosphate was visualized by autoradiography. Coomassie blue staining of the same membrane was also performed to verify the protein loading. The stability of the incorporated phosphate was determined by treating the membranes with water, 1 M HCl or 3 M NaOH for 2 h at room temperature. The treated membranes were then subjected to autoradiography.

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NTHK1 expression in insect cells Three constructs (Bac1-GFP, Bac2-GFP and Bac3-GFP) were made for NTHK1 expression in insect cells using the Bac-to-BacTM baculovirus expression system (Gibco BRL). Each constructs contained the NTHK1 coding sequence (or its deletions) plus a GFP gene. For Bac1-GFP, two primers BacNTHKF1: 50 -TTGGGATCCATGTTAAGGACATTAGCATTAG-30 and BacNTHKR1: 50 -ACAGTCGACCGTGATTATGCTTGCCTG-30 were used to amplify the NTHK1 encoding the full-length protein (amino acids 1–762) from the original NTHK1 plasmid. For Bac2-GFP, primers BacNTHKF2: 50 -TTAGGATCCATGGATAATGGTTTCCCTCGTTG-30 and BacNTHK1R1 were used to amplify the gene fragment encoding the truncated NTHK1 protein without the putative signal peptide (amino acids 22–762). For Bac3-GFP, primers BacNTHKF3: 50 AGGGGATCCATGCTGAAAA AGAAAACTTGG-30 and BacNTHKR1 were used to amplify the gene fragment encoding the truncated NTHK1 protein without the putative signal peptide and the three transmembrane segments (amino acids 145–762). The PCR products were digested with BamHI and SalI, cloned into pFASTBAC1 under the polyhedrin promoter and confirmed by sequencing. The coding region of GFP protein was amplified from a p35S-GFP plasmid and fused to the 30 -end of the above pFASTBAC1 plasmids after digestion with SalI and XbaI. The three recombinant pFASTBAC1 plasmids (Bac1-GFP, Bac2-GFP and Bac3-GFP) were transformed into DH10BAC competent cells where the expression cassette of pFASTBAC was transposed to a baculovirus shuttle vector by site-specific transposition. The resulted recombinant bacmids were then introduced into insect cells (sf21) and the expression of the proteins as revealed by GFP was observed under fluorescence microscopy or confocal microscopy. In confocal microscopy, the selected cells were optically sectioned at a 2-mm interval and an average of 15 sections was made for each cell.

NTHK1 expression in tobacco protoplasts Phosphoamino acid analysis After electrophoresis, the phosphorylated NTHK1-KD was transferred onto PVDF membrane and autoradiographed. The area on the membrane corresponding to the labeled NTHK1-KD was excised and hydrolyzed in 5.7N HCl at 1108C for 60 min. The supernatant was lyophilized and dissolved in distilled water. The samples together with the phosphoamino acid standards (Sigma) were spotted onto 0.1 mm cellulose TLC plates (Merck) and subjected to two-dimensional thin-layer chromatography electrophoresis as described (Kamps and Sefton, 1989). The positions of the three phosphoamino acid standards were visualized by spraying the plates with 0.25% ninhydrin in acetone followed by incubation in oven at 658C until the purple spots of the standards occurred. The plate was then autoradiographed to identify the labeled phosphoamino acids.

Substrate phosphorylation For substrate phosphorylation, NTHK1-KD or NTHK1-KD(H378Q) (0.5 mg) was incubated under phosphorylating conditions with no substrate or with myelin basic protein (MBP) (5 mg), NTHK1-RD (2 mg) or GST (5 mg). GST, MBP or their combination was also incubated under phosphorylating conditions without NTHK1-KD and used as controls. The phosphorylated proteins were resolved on SDS–PAGE gel, transferred to PVDF membranes and subjected to autoradiography or Coomassie blue staining. ß Blackwell Publishing Ltd, The Plant Journal, (2003), 33, 385–393

Three constructs (Puc1-GFP, Puc2-GFP and Puc3-GFP) were made for NTHK1 expression in tobacco protoplasts. Each construct contained the NTHK1 coding sequence (or its deletions) plus a GFP gene and the fusion gene was controlled by a 35S promoter. For Puc1-GFP, two primers BacNTHKF1 (the same as in insect expression) and GFP-R (50 -GCTGGTACCTTATTTGTATAGTTCATCCATG-30 ) were used to amplify the DNA fragment corresponding to the NTHK1 protein (amino acids 1–762) plus a GFP from the Bac1GFP plasmid used in the insect expression experiment. For Puc2GFP, two primers BacNTHKF2 and GFP-R were used to amplify the DNA fragment corresponding to the truncated NTHK1 protein (amino acids 22–762, without the putative signal peptide) plus a GFP from the Bac2-GFP plasmid. For Puc3-GFP, two primers BacNTHKF3 and GFP-R were used to amplify the DNA fragment corresponding to the truncated protein (amino acids 145–762, without the putative signal peptide and the three transmembrane segments) plus a GFP from the Bac3-GFP plasmid. The PCR products were digested with BamHI and KpnI, and cloned into a plasmid pUC-35S-GA5-NOS that has been cut by BamHI and KpnI to remove its insert. The resulted plasmids were confirmed by sequencing. These plasmids were transformed into tobacco protoplasts for transient expression. The pUC-35S-GFP-NOS plasmid (Puc-GFP) was used as a control. Protoplasts from the very young leaves of tobacco (SR1) were prepared and transformed by electroporation as described (Watanabe et al., 1987). Protoplasts were transformed with 15 mg of each plasmid, resuspended in 3 ml of medium and cultured in 30-mm petri dishes in dark at 288C for 30 h

392 Can Xie et al. before observation under a confocal microscope. Optical sections were made according to the instructions.

Acknowledgements We thank Dr H. J. Klee (University of Florida), Dr J. C. Lagarias (University of California at Davis) and Dr M. D. Gale (John Innes Centre) for critical reading of the manuscript. This research was supported by the National Natural Science Foundation of China (39900009), the National Transgenic Research Projects (J00-A-008– 02), the National Key Basic Research Project (G19990117003) and the National High Tech Project (2001AA222131).

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