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Characterization of a wheat histidine-containing phosphotransfer protein (HP) that is regulated by cytokinin-mediated inhibition of leaf senescence
Plant Science 168 (2005) 1507–1514 www.elsevier.com/locate/plantsci
Characterization of a wheat histidine-containing phosphotransfer protein (HP) that is regulated by cytokinin-mediated inhibition of leaf senescence Qing-Hu Ma a,*, Bin Tian a,b a
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxin Cun, Xiangshan, Beijing 100093, China b Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
Received 27 February 2004; received in revised form 4 February 2005; accepted 7 February 2005 Available online 20 March 2005
Abstract A wheat histidine-containing phosphotransfer (HP) cDNA (TaHP1) was isolated from a wheat seedling cDNA library using a RT-PCR product as a probe. The predicted amino acid sequence of TaHP1 is homologous to the histidine-containing phosphotransfer module of the multistep His-Asp phosphorelay from maize and Arabidopsis. The occurrence of HP in wheat was extensively inspected, two homologues were retrieved from wheat EST database and were named as TaHP2 and TaHP3, respectively. TaHP1, 2 and 3 are high homology and form as TaHP1 gene family. Phylogenetic analysis suggests that two classes of HP existed in plants. The secondary and three-dimensional structure analysis by molecular modeling revealed that the basic structure of plant HP proteins is similar with that of YDP1, a yeast HP protein. However, a slight difference in helix arrangement was found between TaHP1 and AHP1, the representative of class I and class II HP proteins, respectively. TaHP1 is present as a single copy gene in the wheat genome as demonstrated by DNA gel blot analysis. RNA gel blot hybridization demonstrated that TaHP1 family was actively expressed in seedlings, but could not be detected in root tissues. Treatment of leaf segments with BA induced TaHP1 family transcript accumulation in a dose-dependent manner, while treatment with trans-zeatin did not. These effects paralleled BA-mediated retardation leaf senescence. These results suggest that HP, like response regulator (RR), is also involved in the resetting or fine-tuning of cytokinin signal transduction and participation in cytokinin-mediated signaling during specific physiological processes, such as leaf senescence. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Histidine-containing phosphotransfer; Structural analysis; Cytokinin signal transduction; Leaf senescence; Triticum aestivum L.
1. Introduction Protein phosphorylation is one of the most commonly occurring signal transduction systems in the regulation of biological processes. In eukaryotes, phosphorylation and dephosphorylation of tyrosine or serine/threonine residues and phosphoprotein phosphatases have been well studied. The ‘‘two-component regulatory system’’ or ‘‘multistep His-Asp phosphorelay’’, was once thought to be unique to prokaryotes [1], but recently has been found in eukaryotes * Corresponding author. Tel.: +86 10 62836236; fax: +86 10 62590833. E-mail address: [email protected] (Q.-H. Ma).
[2]. In higher plants, genetic and biochemical studies have demonstrated that ethylene receptors in Arabidopsis (ETR1, ETR2, ERS and EIN4) belong to this system [3]. Recent work also demonstrated that this system was also involved in cytokinin signal transduction [4,5]. Cytokinins constitute a major class of plant growth regulators that have been shown to be involved in a wide range of physiological processes, including the onset of senescence, promotion of cell division, directional flow of assimilates and alterations of source/sink relationship, and bud differentiation [6]. During the past few decades, intensive efforts have been made to identify the cytokinin receptors that play essential roles in cytokinin-mediated
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.02.022
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signal transduction. Recently a receptor-like histidinekinase (CKI1) that may be involved in cytokinin signaling was identified in Arabidopsis [7]. In addition, three genes encoding sensory His-protein kinases that function as cytokinin receptors (AHK4, AHK2 and AHK3) have been identified [8,9]. It has been demonstrated that AHK4 is capable of functioning as a cytokinin sensor in E. coli and fission yeast [10]. Further evidence has proved that AHK4 is the primary receptor that can directly bind cytokinins and transduce cytokinin signals across the plasma membrane in Arabidopsis [11]. The multistep His-Asp phosphorelay typically consists of three functional modules: a sensory His-protein kinase, a His-containing phosphotransfer protein (HP), and a response regulator (RR). In these systems, signals are transmitted by phosphoryl group between histidine and aspartate residues. Such regulatory modules have been recently isolated from Arabidopsis and maize. In Arabidopsis, ARR1 to ARR7, IBC6 and IBC76, and AtRR1 to AtRR4 have been found to encode the response regulator, and 22 genes for RR were identified in the Arabidopsis genome (Arabidopsis Genome Initiative, 2000). On the basis of structural features, these RRs were divided into two types, type-A and type-B RRs [12]. Six HPs, AHP1 to AHP6, have been isolated from Arabidopsis [13,14]. Six type-A RRs (ZmRR1, ZmRR2, ZmRR4–ZmRR7), 3 type-B RRs (ZmRR8–ZmRR10), and 3 HPs (ZmHP1–ZmHP3) were found in maize [15–17]. It was demonstrated that the expression of type-A RRs was cytokinin-responsive and was also induced by nitrate in Arabidopsi [18]. All type-A RR genes expressed in maize leaves were up-regulated by exogenous cytokinin [17]. Supplying inorganic nitrogen to the whole plant induced the accumulation of ZmRR1 and ZmRR2 transcripts, suggesting that ZmRR1 and ZmRR2 are possibly involved in nitrogen signal transduction mediated by cytokinins [15]. Unlike sensor and response regulator modules, HPs do not exhibit any catalytic function of their own, rather, they appear to be phosphodonors to response regulators that function as substrates for RR in the phosphorelay reaction. In this case, HP may be a reservoir for the signal in the multistep His-Asp phosphorelay system. In vitro experiments demonstrated that putative signaling factors can transfer the phosphoryl group from His80 of ZmHP2 to Asp90 of ZmRRs [15]. However, the steady-state transcript levels of AHPs and ZmHPs were not affected by cytokinins and their regulations and physiological functions are still largely unclear. Compared with RR, much remains to be elucidated about the roles of HP modules in cytokinin signal transduction. (i) Are there any subtypes for HPs as are found within the RRs? (ii) Is HP induced by any signal or in response to any specific physiological processes? To answer these questions, a cDNA clone encoding an HP homologue was isolated from wheat and its expression pattern in relation to cytokinins and leaf senescence was determined. Furthermore, phylogenetic and structural
analysis of HP proteins from plants provides evidence for the subclassification of HP proteins.
2. Materials and methods 2.1. Plant materials and nucleotide acid isolation Wheat plants (Triticum aestivum L. cv H4564) were grown in a naturally lit glasshouse at 25 8C with normal irrigation and fertilization. Leaf segments (5 cm long, 10 per treatment) were cut from 10-day-old seedlings of wheat and placed with abaxial side down in a Petri dish (diameter 9 cm) contained one circle of Whatman No. 1 paper wetted with cytokinin solutions (trans-zeatin or 6-benzylaminopurine). Leaf segments incubated on filter paper wetted with H2O were used for control. The dishes were wrapped in foil and left in the dark at 25 8C. The leaf samples were then collected, quick-frozen in liquid nitrogen and kept at 80 8C before further analysis. Total RNA was isolated from wheat tissues by TRI reagent (Molecular Research Center, Inc, Cincinnati, USA) according to the manufacturer’s instructions. Poly(A)+RNAwas isolated using PolyAT tract1 mRNA Isolation Kit (Promega). Genomic DNA was purified from young wheat leaf tissues according to the protocol described by Dellaporta et al. [19]. 2.2. Isolation of histidine-containing phosphotransfer sequence for use as a probe cDNA synthesis was based on the rapid amplification of cDNA ends method [20] using the oligonucleotide primer 50 -GACTCGAGTCGACATCGA(T)17-30 . PCR was conducted using the forward primer: 50 -GT(T/C/G/ A)GA(T/C)TT(T/C)GA(T/C)(A/C)(G/A)(T/C/G/A)GT(T/ C/G/A)GA(T/C)GC-30 and reverse primer: 50 -GACTCGAGTCGACATCG-30 . The forward primer was synthesized according to the highly conserved amino acid sequences identified in histidine-containing phosphotransfer modules (VDFDS(N/K/R/H/Q)VD). The PCR products were resolved on a 1.0% agarose gel and purified using a GlassMAX1 DNA Isolation Kit (Gibco). The purified fragments were cloned into pGEM-T Easy vector (Promega). After sequencing, positive clones which shown a high sequence similarity to histidine-containing phosphotransfer proteins were used as a probe for screening the wheat seedling cDNA library. 2.3. Construction and screening of a wheat seedling cDNA library A wheat seedling cDNA library was constructed following the manufacturer’s instructions (Stratagene). cDNA was prepared using the ZAP-cDNA synthesis Kit in conjunction with the Uni-ZAP unidirectional vector. This phage vector was multiplied in E. coli XL1-Blue cells. About 2 105 recombinant phages were screened by lifting
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onto Hybond-N+ membrane (Amersham) and hybridizing with a 32P-labelled, PCR-generated probe at 42 8C according to standard procedures [21]. Positive isolates were purified by three rounds of plating and hybridization. cDNA inserts were sequenced using an ABI 377 automated DNA sequencing machine following established protocols. Sequence similarities were analyzed using the SIMAlignment Tool [22] and data from GenBank database. Evolutionary relationships were determined using the Clustal W method with PAM 250 residue weight table [23]. The similar sequences were retrieved by an extensive computer-aided similarity search in the TIGR database for wheat ESTs (http://www.tigr.org/tdb/tgi/tagi). The contig EST sequences were deposited into GenBank as the Third Party Annotation (TPA). 2.4. Secondary and three-dimensional structure prediction Secondary and three-dimensional structures were predicted using SWISS-MODEL [24] by submitting the sequence of HP proteins to the prediction server PHD at Columbia University (http://cubic.bioc.columbia.edu/predictprotein), and the three-dimensional figure was prepared with the program MOLMOL [25] (http://www.mol.biol.ethz.ch/wuthrich/software/molmol). 2.5. Leaf senescence analysis The leaf segments were treated with trans-zeatin (Z) or 6benzylaminopurine (BA) as described in Section 2.1. The amount of chlorophyll extracted from leaf segments was used as an indicator of leaf senescence. Chlorophyll content was determined by spectroscopy. Leaf samples were ground in 3 ml of ice-cold AA solution (80% acetone, 1% ascorbic acid). The homogenate was centrifuged at 3000 g for 10 min. The pellet was resuspended in 3 ml AA solution and centrifuged again. Both supernatants were combined and adjusted to 10 ml. The chlorophyll content was determined from the absorption coefficients by the protocol of Porra et al. [26].
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3. Results 3.1. Analysis of HP cDNA from wheat A cDNA fragment of 502 bp was obtained by RT-PCR using wheat leaf poly(A)+RNA as a template. Sequence analysis indicated this fragment showed high identity with a histidine-containing phosphotransfer (HP) from maize (more than 70% identity in amino acid level). Using this cDNA fragment as a probe, six positively hybridizing plaques were detected after high-stringency hybridization of a wheat seedling cDNA library. Restriction analysis showed these isolates had the same restriction fragment pattern. Complete DNA sequence analysis of three positive clones showed that they belong to the same cDNA, designated as TaHP1. The isolated TaHP1 clone (GenBank accession no. AY342358) was found to be 775 bp long with a 35 nucleotide 50 -untranslated region, a 295 nucleotide 30 untranslated region and a poly(A)+ tail. An open reading frame (ORF) predicted to encode a 148 amino acid polypeptide was located between nucleotide positions 36 and 479. The predicted protein has a calculated molecular mass of 16,200 and an isoelectric point of 4.8. The deduced protein sequence of TaHP1 showed high identity (more than 50% identity at the amino acid level) with HPs from maize and Arabidopsis. 3.2. Compilation of HP genes in wheat After extensively searching the wheat EST database in TIGR (http://www.tigr.org/tdb/tgi/tagi), 13 clones were identified as possible HP homologues. Only two EST sequences showed high homology with TaHP1 (95% identity in amino acid level). These two ESTs were annotated as TaHP2 and TaHP3 and submitted into GenBank as a Third Party Annotation (accession numbers as BK005644 and BK005645, respectively). The alignment of TaHP1, TaHP2 and TaHP3 was showed in Fig. 1. The other 11 clones have low homology with TaHP1 (The identity at the amino acid
2.6. DNA and RNA gel blot analysis Genomic DNA from wheat leaf tissues was digested with appropriate restriction enzymes and resolved on a 1.0% agarose gel. An amount of 10 mg of total RNA was electrophoresed on 1.4% (W/V) formaldehyde agarose gels. DNA and RNA were blotted onto Hybond-N+ membrane (Amersham) using established protocols [21]. The blots were hybridized at 42 8C in 6 SSC, 5 Denhardt, 0.5% SDS, 100 mg ml1 salmon sperm DNA with 50% formamide and washed with 0.1 SSC plus 0.1% SDS at 65 8C. Probes were 32P-labelled using a Ready-to-Go DNA Labeling Kit (Amersham). Northern blots were quantified using Phosphor Image and mRNA levels were normalized by comparison to a soybean 18S rRNA.
Fig. 1. Alignment of the predicted amino acid sequence of TaHP1 with TaHP2 and TaHP3. Gaps, indicated by dots, were inserted to maximize homology.
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level was found to range from 25 to 65%). Their functions await further investigation. After searching maize EST sequences in TIGR, four possible HP homologues were identified. However, three of them are identical to ZmHP1, ZmHP2 and ZmHP3, which were previously reported [17]. The forth maize clone shared only a 20% identity to HP, and may not represent an authentic HP protein.
3.3. Phylogenetic and structural analyses of HP proteins Phylogenetic reconstruction with 11 plant HP proteins revealed two main classes (Fig. 2). A yeast HP protein, YDP1 [27], was used as outgroup. The HP proteins from wheat and maize make up class I, while class II includes HP proteins from Arabidopsis and Catharanthus roseus [28]. Several conserved motifs were found in the amino acid sequences of these plant HP proteins, including motif 1: G**D*QF*QLQ, motif 2: FV*EV**LF, motif 3: VHQLKGSS, and motif 4: GA*KVK**C (*designates positions with unconserved amino acids). Motifs 1 and 2 are located in the N-terminus of the HP proteins, whereas motif 4 is located in the C-terminus. The motif 3, which contain the active histidine residue, was also conserved in YDP1. Wheat HP proteins showed closer with ZmHP2, a Hiscontaining phosphotransfer protein that was reported to be involved in the nitrogen signal transduction in maize [16].
The possible secondary and three-dimensional structures of TaHP1 and AHP1, representatives of class I and class II HP proteins, respectively, were predicted by SWISSMODEL software using the yeast HP protein (YDP1) as a template. The comparison spans the N-terminal 100 amino acids only. The predicted secondary structure of plant HP proteins consisted primarily of alpha helices (79–83%) scattered in random coil and beta-sheet. The basic structure of TaHP1 and AHP1 is very similar with that of YDP1 (Fig. 3), which is composed of four alpha-helices. These helices are labeled A, B, C and D, according to their assignment within the YDP1 structure. The structures of helices B, C and D are well conserved in TaHP1, AHP1 and YDP1, although some variations were found in helix A. TaHP1 has a longer helix A compared to AHP1 (13 amino acids in TaHP1 versus 5 amino acids in AHP1). This makes the angle between helix B and C greater in TaHP1 than in AHP1. The conserved motif 1 and 4, as suggested by primary sequence analysis, were located in the beginning of helix A and D, respectively. The motif 2 and 3 were located in the center of helix B and C, respectively. 3.4. Genomic complexity of TaHP1 To determine the copy number of TaHP1 gene, DNA gel blot analysis was performed using wheat genomic DNA digested individually with the restriction enzymes EcoRI, BamHI, HindIII, and XbaI, which are predicted not to cut (EcoRI, BamHI and XbaI) or cut once (HindIII) within the TaHP1 cDNA. The blot was probed with TaHP1 cDNA under high-stringency conditions. As shown in Fig. 4, two strong bands were detected in BamHI and XbaI digestions, while three bands were detected in EcoRI and HindIII digestions. These results, when interpreted in consideration of the hexaploid nature of wheat, suggest that TaHP1 exists as a single-copy gene in the wheat genome. TaHP1 shares a high degree of homology with TaHP2 and TaHP3 (97% and 95% identical at the nucleotide and amino acid levels, respectively), which suggests that these three cDNAs belong to the same gene. Wheat is an allopolyploid that is made up of three closely related genomes. Therefore, these three cDNAs are likely from the different genomes. 3.5. Tissue specific expression of the TaHP1 gene family
Fig. 2. Dendrogram showing phylogenetic relationships between HP proteins from various plants. The tree was constructed using the Clustal W method with PAM 250 residue weight table, bootstrap values are shown in each branch. AHP1 (Accession number AF370265), AHP2 (BAA36336), AHP3 (BAA36337), and AHP4 (AB023046) from Arabidopsis thaliana; CrHPt1 (AF346308) from Catharanthus roseus; ZmHP1 (AB024293), ZmHP2 (AB024292), and ZmHP3 (AB089191) from Zea mays; TaHP1 (AY342358), TaHP2 (BK005644), and TaHP3 (BK005645) from Triticum aestivum; YDP1 (S67799) from Saccharomyces cerevisiae.
RNA gel blot analysis was performed to examine the gene expression pattern of TaHP1 in 10-day-old wheat seedlings. As shown in Fig. 5, TaHP1 mRNA was detected in seedlings, but not in roots. The analysis through the numbers of EST and their origin of library indicated that the expression of TaHP2 and TaHP3 genes was detected in most vegetative tissues, such as leaves, roots and seedlings. Since TaHP1 shares a high similarity to TaHP2 and TaHP3 and all three genes are expressed in leaf tissues, the signal from our Northern hybridization may represent a composite from the expression of all three genes. For convenience, in the text we
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Fig. 3. The predicted three-dimensional structures of TaHP1 and AHP1. N: N-terminus, H: active histidine residue, A, B, C, D: different helices named according to YPD1 [27].
will refer to the signal as TaHP1 family. In maize, ZmHP2 transcript was detected in all organs, including leaves, stems, husks, young corns with silk, tassels and roots [16]. The AHP2 gene from Arabidopsis was constitutively expressed in most tissues, while the expression of AHP1 and AHP3 genes exhibited remarkable organ specificity [13]. A HP EST clone from rice was also found to have the high number in the cDNA library of drought-stressed leaf tissues (data not shown). It is speculated from the tissue specific expression patterns that various HP proteins are likely to be involved in the regulation of different physiological processes in plants. 3.6. Cytokinin regulation on TaHP1 family expression levels in relation to leaf senescence To explore the possible functions of HP in cytokinin signal transduction, the regulation of TaHP1 family mRNA levels by exogenous cytokinins was checked during the senescence process in detached wheat leaves. It has been
Fig. 4. DNA gel blot analysis of genomic DNA isolated from wheat leaf tissues (10 mg loaded in each lane) probed with TaHP1 cDNA. The restriction enzymes used are listed at the top of the figure and molecular weight markers are indicated.
Fig. 5. RNA gel blot analysis of TaHP1 gene family expression in wheat tissues. RNA was hybridized with a TaHP1 cDNA fragment. S: seedling; R: root. Hybridization with a soybean 18S rDNA probe has been included to confirm that the RNA preparations are undegraded and to serve as an internal control for variations in gel loading and blotting.
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Table 1 Chlorophyll content (mg g1 fresh weight) of wheat leaf segments in response to cytokinins Concentration (M)
BA
Z
0 1 108 1 107 1 106 1 105 1 104
0.76 0.11 0.81 0.11 0.96 0.13 1.18 0.08 1.38 0.14 1.52 0.13
0.76 0.11 0.79 0.09 0.84 0.08 0.87 0.12 0.92 0.18 0.97 0.16
Leaf segments were detached from 10-day-old seedlings of wheat and treated with benzyladenine (BA) or trans-zeatin (Z) at different concentrations. Values represent the mean of three independent replicates S.D.
known that natural and synthetic cytokinins have markedly different activities in the leaf senescence retardation assay, particularly within the Gramineae [29]. The effects of BA and trans-zeatin on leaf senescence retardation were also measured in wheat. As shown in Table 1, BA was markedly more effective that trans-zeatin on the retardation of leaf senescence. The pronounced concentration of BA was 1 106 M. This is in agreement with the measurement in oat [29]. TaHP1 family mRNA levels were determined for each leaf treatment by Northern hybridization and normalized relative to the 18S rRNA signal (Fig. 6). The accumulation of TaHP1 family transcripts in leaf segments was induced after treatment with BA, when the concentration of BA was greater than 1 106 M. In comparison, trans-zeatin treatment, which did not have much effect on the retardation of leaf senescence, had little influence on TaHP1 family mRNA levels at concentration up to 1 104 M. The time-course of BA induction on TaHP1 family expression was checked by Northern blot hybridization. The results indicated that BA stimulated maximal TaHP1 family mRNA accumulation at 12 h after treatment (Fig. 7). These results were confirmed after the signals were normalized relative to the 18S rRNA signal (data not shown).
4. Discussion In this report, we isolated a gene encoding HP from wheat. Two similar clones were retrieved from wheat EST database. These three clones (named as TaHP1, TaHP2 and
Fig. 7. RNA gel blot analysis of TaHP1 gene family expression in wheat leaf segments after treatment with BA (100 mM) for different times as indicated. Hybridization with a soybean 18S rDNA probe has been included to confirm that the RNA preparations are undegraded and to serve as an internal control for variations in gel loading and blotting.
TaHP3, respectively) showed high identity (more than 50% identity at the amino acid level) with HPs from maize and Arabidopsis. Since previous works have indicated the function of HP proteins in maize and Arabidopsis [13– 17], the high homology of TaHP1, TaHP2 and TaHP3 proteins suggested that they might also be involved in cytokinin signal transduction. According to their phylogenetic origins, 11 plant HP proteins that have reported in literatures can be clayed into two classes (Fig. 2). Class I contains HP proteins from wheat and maize. Class II contains HP proteins from Arabidopsis and Catharanthus roseus. Four conserved motifs were found in HP proteins and these motifs showed little difference between two groups of HP proteins. Therefore, the most differences in primary sequence between Class I and II HP proteins arise from their N- and C-terminal sequence. Furthermore, since there is a limited number of plants for which HP protein sequence data are available, it is difficult to ascribe different classes of HP proteins to different groups of plants, for example, assigning class I HP proteins to monocotyledon plants and class II to dicotyledon plants. As more HP sequence data is released, HP proteins with class II characteristics may be identified in monocotyledon plants. However, the phylogenetic analysis does indicate that different types of HP proteins are present in plants, as is the case for the response regulator (RR). The secondary and three-dimensional structure analysis by molecular modeling revealed that the basic structure of plant HP proteins is composed of four alpha-helices, which
Fig. 6. RNA gel blot analysis of TaHP1 gene family expression in wheat leaf segments after treatment with BA or trans-zeatin for 24 h with various concentrations as indicated. Hybridization with a soybean 18S rDNA probe has been included to confirm that the RNA preparations are undegraded and to serve as an internal control for variations in gel loading and blotting.
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is very similar with that of YDP1, a yeast HP protein (Fig. 4), although YDP1 and plant HP proteins share only 16–21% amino acid sequence identity. The active histidine residue is located in helix C, which forms a binding surface with helix D for the response regulator. This indicates that there is a high degree of conservation in three-dimensional structure between yeast and plant HP proteins. A slight difference in helix arrangement was found between TaHP1 and AHP1, representatives of class I and class II HP proteins, respectively. AHP1 has a much shorter helix A than TaHP1. These differences coupled with differences in amino acid side-chains in these helices may be to affect binding to the response regulator. Consequently, different HP proteins may interact with specific response regulators in various signaling pathways. Since many isoforms of HP and RR are found in the same plant species, as in wheat, maize and Arabidopsis, the specific molecular recognition and discrimination between different HPs and RRs is expected to be critically important in order to avoid cross-talk among these isoforms. The expression profile also indicated the diversity among the different HP genes. Some HP genes are constitutively expressed in plant tissues, such as ZmHP1, ZmHP2 and ZmHP3 from maize, AHP2 from Arabidopsis, and TaHP2 and TaHP3 from wheat. The expression of other HP genes, however, is confined to specific tissues. For example, AHP1 is expressed predominantly in roots, and barely detected in leaves and stems. Furthermore, some HP genes are specifically expressed in response to a particular physiological process such as a HP clone in drought-stressed rice. Furthermore, the constitutively expressed HP genes ZmHP1, ZmHP2 and ZmHP3 showed preferential interactions with certain ZmRRs in the yeast two hybrid assay [17]. These data further support the idea that different forms of HP proteins are likely to play specific roles in different plant tissues or physiological processes. In agreement with the above analyses, the expression of TaHP1 family exhibited some degree of cytokinin-inducibility. This induction is evident after BA treatment, but not after trans-zeatin treatment, which demonstrates the close relationship of BA and retardation of leaf senescence. Two-component elements could potentially be regulated by cytokinin signals and also by other signals. This allows resetting or fine-tuning in cytokinin signal transduction, and also provides a cross-talk mechanism in plant signaling networks. This has been demonstrated for response regulators. For example, the expression of ZmRR1 from maize was regulated by nitrogen signals [15], while the expression of some ARRs was regulated by stress [30] and nitrogen signals [18]. Cytokinin-dependent induction of ZmRR1 and ZmRR2 was repressed by co-treatment with ABA in a dose-dependent manner in maize [31]. The responsiveness of HP to cytokinin and other stimuli, however, has not been well characterized. The expression data from TaHP1 family suggest that HP, like RR, is not only involved in the regulation of cytokinin signal transduction
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by alternation of gene expression, but also participates in cytokinin-mediated signaling in special physiological processes, such as leaf senescence and drought stress.
Acknowledgements We wish to sincerely thank Dr. Bettina Deavours (Plant Biology Division, The Samuel Roberts Noble Foundation, USA) and Dr. Charles H. Hocart (Australian National University, Australia) for critical reading of the manuscript. This work was supported by the State Key Basic Research and development Plan of China (2003CB114300), the National Natural Science Foundation of China (No. 30400222), the Chinese National Special Foundation for Transgenic Plant Research and Commercialization (J2002B007), and the Natural Science Foundation of Beijing (No. 503211).
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Characterization of a wheat histidine-containing phosphotransfer protein (HP) that is regulated by cytokinin-mediated inhibition of leaf senescence Qing-Hu Ma a,*, Bin Tian a,b a
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxin Cun, Xiangshan, Beijing 100093, China b Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
Received 27 February 2004; received in revised form 4 February 2005; accepted 7 February 2005 Available online 20 March 2005
Abstract A wheat histidine-containing phosphotransfer (HP) cDNA (TaHP1) was isolated from a wheat seedling cDNA library using a RT-PCR product as a probe. The predicted amino acid sequence of TaHP1 is homologous to the histidine-containing phosphotransfer module of the multistep His-Asp phosphorelay from maize and Arabidopsis. The occurrence of HP in wheat was extensively inspected, two homologues were retrieved from wheat EST database and were named as TaHP2 and TaHP3, respectively. TaHP1, 2 and 3 are high homology and form as TaHP1 gene family. Phylogenetic analysis suggests that two classes of HP existed in plants. The secondary and three-dimensional structure analysis by molecular modeling revealed that the basic structure of plant HP proteins is similar with that of YDP1, a yeast HP protein. However, a slight difference in helix arrangement was found between TaHP1 and AHP1, the representative of class I and class II HP proteins, respectively. TaHP1 is present as a single copy gene in the wheat genome as demonstrated by DNA gel blot analysis. RNA gel blot hybridization demonstrated that TaHP1 family was actively expressed in seedlings, but could not be detected in root tissues. Treatment of leaf segments with BA induced TaHP1 family transcript accumulation in a dose-dependent manner, while treatment with trans-zeatin did not. These effects paralleled BA-mediated retardation leaf senescence. These results suggest that HP, like response regulator (RR), is also involved in the resetting or fine-tuning of cytokinin signal transduction and participation in cytokinin-mediated signaling during specific physiological processes, such as leaf senescence. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Histidine-containing phosphotransfer; Structural analysis; Cytokinin signal transduction; Leaf senescence; Triticum aestivum L.
1. Introduction Protein phosphorylation is one of the most commonly occurring signal transduction systems in the regulation of biological processes. In eukaryotes, phosphorylation and dephosphorylation of tyrosine or serine/threonine residues and phosphoprotein phosphatases have been well studied. The ‘‘two-component regulatory system’’ or ‘‘multistep His-Asp phosphorelay’’, was once thought to be unique to prokaryotes [1], but recently has been found in eukaryotes * Corresponding author. Tel.: +86 10 62836236; fax: +86 10 62590833. E-mail address: [email protected] (Q.-H. Ma).
[2]. In higher plants, genetic and biochemical studies have demonstrated that ethylene receptors in Arabidopsis (ETR1, ETR2, ERS and EIN4) belong to this system [3]. Recent work also demonstrated that this system was also involved in cytokinin signal transduction [4,5]. Cytokinins constitute a major class of plant growth regulators that have been shown to be involved in a wide range of physiological processes, including the onset of senescence, promotion of cell division, directional flow of assimilates and alterations of source/sink relationship, and bud differentiation [6]. During the past few decades, intensive efforts have been made to identify the cytokinin receptors that play essential roles in cytokinin-mediated
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.02.022
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signal transduction. Recently a receptor-like histidinekinase (CKI1) that may be involved in cytokinin signaling was identified in Arabidopsis [7]. In addition, three genes encoding sensory His-protein kinases that function as cytokinin receptors (AHK4, AHK2 and AHK3) have been identified [8,9]. It has been demonstrated that AHK4 is capable of functioning as a cytokinin sensor in E. coli and fission yeast [10]. Further evidence has proved that AHK4 is the primary receptor that can directly bind cytokinins and transduce cytokinin signals across the plasma membrane in Arabidopsis [11]. The multistep His-Asp phosphorelay typically consists of three functional modules: a sensory His-protein kinase, a His-containing phosphotransfer protein (HP), and a response regulator (RR). In these systems, signals are transmitted by phosphoryl group between histidine and aspartate residues. Such regulatory modules have been recently isolated from Arabidopsis and maize. In Arabidopsis, ARR1 to ARR7, IBC6 and IBC76, and AtRR1 to AtRR4 have been found to encode the response regulator, and 22 genes for RR were identified in the Arabidopsis genome (Arabidopsis Genome Initiative, 2000). On the basis of structural features, these RRs were divided into two types, type-A and type-B RRs [12]. Six HPs, AHP1 to AHP6, have been isolated from Arabidopsis [13,14]. Six type-A RRs (ZmRR1, ZmRR2, ZmRR4–ZmRR7), 3 type-B RRs (ZmRR8–ZmRR10), and 3 HPs (ZmHP1–ZmHP3) were found in maize [15–17]. It was demonstrated that the expression of type-A RRs was cytokinin-responsive and was also induced by nitrate in Arabidopsi [18]. All type-A RR genes expressed in maize leaves were up-regulated by exogenous cytokinin [17]. Supplying inorganic nitrogen to the whole plant induced the accumulation of ZmRR1 and ZmRR2 transcripts, suggesting that ZmRR1 and ZmRR2 are possibly involved in nitrogen signal transduction mediated by cytokinins [15]. Unlike sensor and response regulator modules, HPs do not exhibit any catalytic function of their own, rather, they appear to be phosphodonors to response regulators that function as substrates for RR in the phosphorelay reaction. In this case, HP may be a reservoir for the signal in the multistep His-Asp phosphorelay system. In vitro experiments demonstrated that putative signaling factors can transfer the phosphoryl group from His80 of ZmHP2 to Asp90 of ZmRRs [15]. However, the steady-state transcript levels of AHPs and ZmHPs were not affected by cytokinins and their regulations and physiological functions are still largely unclear. Compared with RR, much remains to be elucidated about the roles of HP modules in cytokinin signal transduction. (i) Are there any subtypes for HPs as are found within the RRs? (ii) Is HP induced by any signal or in response to any specific physiological processes? To answer these questions, a cDNA clone encoding an HP homologue was isolated from wheat and its expression pattern in relation to cytokinins and leaf senescence was determined. Furthermore, phylogenetic and structural
analysis of HP proteins from plants provides evidence for the subclassification of HP proteins.
2. Materials and methods 2.1. Plant materials and nucleotide acid isolation Wheat plants (Triticum aestivum L. cv H4564) were grown in a naturally lit glasshouse at 25 8C with normal irrigation and fertilization. Leaf segments (5 cm long, 10 per treatment) were cut from 10-day-old seedlings of wheat and placed with abaxial side down in a Petri dish (diameter 9 cm) contained one circle of Whatman No. 1 paper wetted with cytokinin solutions (trans-zeatin or 6-benzylaminopurine). Leaf segments incubated on filter paper wetted with H2O were used for control. The dishes were wrapped in foil and left in the dark at 25 8C. The leaf samples were then collected, quick-frozen in liquid nitrogen and kept at 80 8C before further analysis. Total RNA was isolated from wheat tissues by TRI reagent (Molecular Research Center, Inc, Cincinnati, USA) according to the manufacturer’s instructions. Poly(A)+RNAwas isolated using PolyAT tract1 mRNA Isolation Kit (Promega). Genomic DNA was purified from young wheat leaf tissues according to the protocol described by Dellaporta et al. [19]. 2.2. Isolation of histidine-containing phosphotransfer sequence for use as a probe cDNA synthesis was based on the rapid amplification of cDNA ends method [20] using the oligonucleotide primer 50 -GACTCGAGTCGACATCGA(T)17-30 . PCR was conducted using the forward primer: 50 -GT(T/C/G/ A)GA(T/C)TT(T/C)GA(T/C)(A/C)(G/A)(T/C/G/A)GT(T/ C/G/A)GA(T/C)GC-30 and reverse primer: 50 -GACTCGAGTCGACATCG-30 . The forward primer was synthesized according to the highly conserved amino acid sequences identified in histidine-containing phosphotransfer modules (VDFDS(N/K/R/H/Q)VD). The PCR products were resolved on a 1.0% agarose gel and purified using a GlassMAX1 DNA Isolation Kit (Gibco). The purified fragments were cloned into pGEM-T Easy vector (Promega). After sequencing, positive clones which shown a high sequence similarity to histidine-containing phosphotransfer proteins were used as a probe for screening the wheat seedling cDNA library. 2.3. Construction and screening of a wheat seedling cDNA library A wheat seedling cDNA library was constructed following the manufacturer’s instructions (Stratagene). cDNA was prepared using the ZAP-cDNA synthesis Kit in conjunction with the Uni-ZAP unidirectional vector. This phage vector was multiplied in E. coli XL1-Blue cells. About 2 105 recombinant phages were screened by lifting
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onto Hybond-N+ membrane (Amersham) and hybridizing with a 32P-labelled, PCR-generated probe at 42 8C according to standard procedures [21]. Positive isolates were purified by three rounds of plating and hybridization. cDNA inserts were sequenced using an ABI 377 automated DNA sequencing machine following established protocols. Sequence similarities were analyzed using the SIMAlignment Tool [22] and data from GenBank database. Evolutionary relationships were determined using the Clustal W method with PAM 250 residue weight table [23]. The similar sequences were retrieved by an extensive computer-aided similarity search in the TIGR database for wheat ESTs (http://www.tigr.org/tdb/tgi/tagi). The contig EST sequences were deposited into GenBank as the Third Party Annotation (TPA). 2.4. Secondary and three-dimensional structure prediction Secondary and three-dimensional structures were predicted using SWISS-MODEL [24] by submitting the sequence of HP proteins to the prediction server PHD at Columbia University (http://cubic.bioc.columbia.edu/predictprotein), and the three-dimensional figure was prepared with the program MOLMOL [25] (http://www.mol.biol.ethz.ch/wuthrich/software/molmol). 2.5. Leaf senescence analysis The leaf segments were treated with trans-zeatin (Z) or 6benzylaminopurine (BA) as described in Section 2.1. The amount of chlorophyll extracted from leaf segments was used as an indicator of leaf senescence. Chlorophyll content was determined by spectroscopy. Leaf samples were ground in 3 ml of ice-cold AA solution (80% acetone, 1% ascorbic acid). The homogenate was centrifuged at 3000 g for 10 min. The pellet was resuspended in 3 ml AA solution and centrifuged again. Both supernatants were combined and adjusted to 10 ml. The chlorophyll content was determined from the absorption coefficients by the protocol of Porra et al. [26].
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3. Results 3.1. Analysis of HP cDNA from wheat A cDNA fragment of 502 bp was obtained by RT-PCR using wheat leaf poly(A)+RNA as a template. Sequence analysis indicated this fragment showed high identity with a histidine-containing phosphotransfer (HP) from maize (more than 70% identity in amino acid level). Using this cDNA fragment as a probe, six positively hybridizing plaques were detected after high-stringency hybridization of a wheat seedling cDNA library. Restriction analysis showed these isolates had the same restriction fragment pattern. Complete DNA sequence analysis of three positive clones showed that they belong to the same cDNA, designated as TaHP1. The isolated TaHP1 clone (GenBank accession no. AY342358) was found to be 775 bp long with a 35 nucleotide 50 -untranslated region, a 295 nucleotide 30 untranslated region and a poly(A)+ tail. An open reading frame (ORF) predicted to encode a 148 amino acid polypeptide was located between nucleotide positions 36 and 479. The predicted protein has a calculated molecular mass of 16,200 and an isoelectric point of 4.8. The deduced protein sequence of TaHP1 showed high identity (more than 50% identity at the amino acid level) with HPs from maize and Arabidopsis. 3.2. Compilation of HP genes in wheat After extensively searching the wheat EST database in TIGR (http://www.tigr.org/tdb/tgi/tagi), 13 clones were identified as possible HP homologues. Only two EST sequences showed high homology with TaHP1 (95% identity in amino acid level). These two ESTs were annotated as TaHP2 and TaHP3 and submitted into GenBank as a Third Party Annotation (accession numbers as BK005644 and BK005645, respectively). The alignment of TaHP1, TaHP2 and TaHP3 was showed in Fig. 1. The other 11 clones have low homology with TaHP1 (The identity at the amino acid
2.6. DNA and RNA gel blot analysis Genomic DNA from wheat leaf tissues was digested with appropriate restriction enzymes and resolved on a 1.0% agarose gel. An amount of 10 mg of total RNA was electrophoresed on 1.4% (W/V) formaldehyde agarose gels. DNA and RNA were blotted onto Hybond-N+ membrane (Amersham) using established protocols [21]. The blots were hybridized at 42 8C in 6 SSC, 5 Denhardt, 0.5% SDS, 100 mg ml1 salmon sperm DNA with 50% formamide and washed with 0.1 SSC plus 0.1% SDS at 65 8C. Probes were 32P-labelled using a Ready-to-Go DNA Labeling Kit (Amersham). Northern blots were quantified using Phosphor Image and mRNA levels were normalized by comparison to a soybean 18S rRNA.
Fig. 1. Alignment of the predicted amino acid sequence of TaHP1 with TaHP2 and TaHP3. Gaps, indicated by dots, were inserted to maximize homology.
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level was found to range from 25 to 65%). Their functions await further investigation. After searching maize EST sequences in TIGR, four possible HP homologues were identified. However, three of them are identical to ZmHP1, ZmHP2 and ZmHP3, which were previously reported [17]. The forth maize clone shared only a 20% identity to HP, and may not represent an authentic HP protein.
3.3. Phylogenetic and structural analyses of HP proteins Phylogenetic reconstruction with 11 plant HP proteins revealed two main classes (Fig. 2). A yeast HP protein, YDP1 [27], was used as outgroup. The HP proteins from wheat and maize make up class I, while class II includes HP proteins from Arabidopsis and Catharanthus roseus [28]. Several conserved motifs were found in the amino acid sequences of these plant HP proteins, including motif 1: G**D*QF*QLQ, motif 2: FV*EV**LF, motif 3: VHQLKGSS, and motif 4: GA*KVK**C (*designates positions with unconserved amino acids). Motifs 1 and 2 are located in the N-terminus of the HP proteins, whereas motif 4 is located in the C-terminus. The motif 3, which contain the active histidine residue, was also conserved in YDP1. Wheat HP proteins showed closer with ZmHP2, a Hiscontaining phosphotransfer protein that was reported to be involved in the nitrogen signal transduction in maize [16].
The possible secondary and three-dimensional structures of TaHP1 and AHP1, representatives of class I and class II HP proteins, respectively, were predicted by SWISSMODEL software using the yeast HP protein (YDP1) as a template. The comparison spans the N-terminal 100 amino acids only. The predicted secondary structure of plant HP proteins consisted primarily of alpha helices (79–83%) scattered in random coil and beta-sheet. The basic structure of TaHP1 and AHP1 is very similar with that of YDP1 (Fig. 3), which is composed of four alpha-helices. These helices are labeled A, B, C and D, according to their assignment within the YDP1 structure. The structures of helices B, C and D are well conserved in TaHP1, AHP1 and YDP1, although some variations were found in helix A. TaHP1 has a longer helix A compared to AHP1 (13 amino acids in TaHP1 versus 5 amino acids in AHP1). This makes the angle between helix B and C greater in TaHP1 than in AHP1. The conserved motif 1 and 4, as suggested by primary sequence analysis, were located in the beginning of helix A and D, respectively. The motif 2 and 3 were located in the center of helix B and C, respectively. 3.4. Genomic complexity of TaHP1 To determine the copy number of TaHP1 gene, DNA gel blot analysis was performed using wheat genomic DNA digested individually with the restriction enzymes EcoRI, BamHI, HindIII, and XbaI, which are predicted not to cut (EcoRI, BamHI and XbaI) or cut once (HindIII) within the TaHP1 cDNA. The blot was probed with TaHP1 cDNA under high-stringency conditions. As shown in Fig. 4, two strong bands were detected in BamHI and XbaI digestions, while three bands were detected in EcoRI and HindIII digestions. These results, when interpreted in consideration of the hexaploid nature of wheat, suggest that TaHP1 exists as a single-copy gene in the wheat genome. TaHP1 shares a high degree of homology with TaHP2 and TaHP3 (97% and 95% identical at the nucleotide and amino acid levels, respectively), which suggests that these three cDNAs belong to the same gene. Wheat is an allopolyploid that is made up of three closely related genomes. Therefore, these three cDNAs are likely from the different genomes. 3.5. Tissue specific expression of the TaHP1 gene family
Fig. 2. Dendrogram showing phylogenetic relationships between HP proteins from various plants. The tree was constructed using the Clustal W method with PAM 250 residue weight table, bootstrap values are shown in each branch. AHP1 (Accession number AF370265), AHP2 (BAA36336), AHP3 (BAA36337), and AHP4 (AB023046) from Arabidopsis thaliana; CrHPt1 (AF346308) from Catharanthus roseus; ZmHP1 (AB024293), ZmHP2 (AB024292), and ZmHP3 (AB089191) from Zea mays; TaHP1 (AY342358), TaHP2 (BK005644), and TaHP3 (BK005645) from Triticum aestivum; YDP1 (S67799) from Saccharomyces cerevisiae.
RNA gel blot analysis was performed to examine the gene expression pattern of TaHP1 in 10-day-old wheat seedlings. As shown in Fig. 5, TaHP1 mRNA was detected in seedlings, but not in roots. The analysis through the numbers of EST and their origin of library indicated that the expression of TaHP2 and TaHP3 genes was detected in most vegetative tissues, such as leaves, roots and seedlings. Since TaHP1 shares a high similarity to TaHP2 and TaHP3 and all three genes are expressed in leaf tissues, the signal from our Northern hybridization may represent a composite from the expression of all three genes. For convenience, in the text we
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Fig. 3. The predicted three-dimensional structures of TaHP1 and AHP1. N: N-terminus, H: active histidine residue, A, B, C, D: different helices named according to YPD1 [27].
will refer to the signal as TaHP1 family. In maize, ZmHP2 transcript was detected in all organs, including leaves, stems, husks, young corns with silk, tassels and roots [16]. The AHP2 gene from Arabidopsis was constitutively expressed in most tissues, while the expression of AHP1 and AHP3 genes exhibited remarkable organ specificity [13]. A HP EST clone from rice was also found to have the high number in the cDNA library of drought-stressed leaf tissues (data not shown). It is speculated from the tissue specific expression patterns that various HP proteins are likely to be involved in the regulation of different physiological processes in plants. 3.6. Cytokinin regulation on TaHP1 family expression levels in relation to leaf senescence To explore the possible functions of HP in cytokinin signal transduction, the regulation of TaHP1 family mRNA levels by exogenous cytokinins was checked during the senescence process in detached wheat leaves. It has been
Fig. 4. DNA gel blot analysis of genomic DNA isolated from wheat leaf tissues (10 mg loaded in each lane) probed with TaHP1 cDNA. The restriction enzymes used are listed at the top of the figure and molecular weight markers are indicated.
Fig. 5. RNA gel blot analysis of TaHP1 gene family expression in wheat tissues. RNA was hybridized with a TaHP1 cDNA fragment. S: seedling; R: root. Hybridization with a soybean 18S rDNA probe has been included to confirm that the RNA preparations are undegraded and to serve as an internal control for variations in gel loading and blotting.
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Table 1 Chlorophyll content (mg g1 fresh weight) of wheat leaf segments in response to cytokinins Concentration (M)
BA
Z
0 1 108 1 107 1 106 1 105 1 104
0.76 0.11 0.81 0.11 0.96 0.13 1.18 0.08 1.38 0.14 1.52 0.13
0.76 0.11 0.79 0.09 0.84 0.08 0.87 0.12 0.92 0.18 0.97 0.16
Leaf segments were detached from 10-day-old seedlings of wheat and treated with benzyladenine (BA) or trans-zeatin (Z) at different concentrations. Values represent the mean of three independent replicates S.D.
known that natural and synthetic cytokinins have markedly different activities in the leaf senescence retardation assay, particularly within the Gramineae [29]. The effects of BA and trans-zeatin on leaf senescence retardation were also measured in wheat. As shown in Table 1, BA was markedly more effective that trans-zeatin on the retardation of leaf senescence. The pronounced concentration of BA was 1 106 M. This is in agreement with the measurement in oat [29]. TaHP1 family mRNA levels were determined for each leaf treatment by Northern hybridization and normalized relative to the 18S rRNA signal (Fig. 6). The accumulation of TaHP1 family transcripts in leaf segments was induced after treatment with BA, when the concentration of BA was greater than 1 106 M. In comparison, trans-zeatin treatment, which did not have much effect on the retardation of leaf senescence, had little influence on TaHP1 family mRNA levels at concentration up to 1 104 M. The time-course of BA induction on TaHP1 family expression was checked by Northern blot hybridization. The results indicated that BA stimulated maximal TaHP1 family mRNA accumulation at 12 h after treatment (Fig. 7). These results were confirmed after the signals were normalized relative to the 18S rRNA signal (data not shown).
4. Discussion In this report, we isolated a gene encoding HP from wheat. Two similar clones were retrieved from wheat EST database. These three clones (named as TaHP1, TaHP2 and
Fig. 7. RNA gel blot analysis of TaHP1 gene family expression in wheat leaf segments after treatment with BA (100 mM) for different times as indicated. Hybridization with a soybean 18S rDNA probe has been included to confirm that the RNA preparations are undegraded and to serve as an internal control for variations in gel loading and blotting.
TaHP3, respectively) showed high identity (more than 50% identity at the amino acid level) with HPs from maize and Arabidopsis. Since previous works have indicated the function of HP proteins in maize and Arabidopsis [13– 17], the high homology of TaHP1, TaHP2 and TaHP3 proteins suggested that they might also be involved in cytokinin signal transduction. According to their phylogenetic origins, 11 plant HP proteins that have reported in literatures can be clayed into two classes (Fig. 2). Class I contains HP proteins from wheat and maize. Class II contains HP proteins from Arabidopsis and Catharanthus roseus. Four conserved motifs were found in HP proteins and these motifs showed little difference between two groups of HP proteins. Therefore, the most differences in primary sequence between Class I and II HP proteins arise from their N- and C-terminal sequence. Furthermore, since there is a limited number of plants for which HP protein sequence data are available, it is difficult to ascribe different classes of HP proteins to different groups of plants, for example, assigning class I HP proteins to monocotyledon plants and class II to dicotyledon plants. As more HP sequence data is released, HP proteins with class II characteristics may be identified in monocotyledon plants. However, the phylogenetic analysis does indicate that different types of HP proteins are present in plants, as is the case for the response regulator (RR). The secondary and three-dimensional structure analysis by molecular modeling revealed that the basic structure of plant HP proteins is composed of four alpha-helices, which
Fig. 6. RNA gel blot analysis of TaHP1 gene family expression in wheat leaf segments after treatment with BA or trans-zeatin for 24 h with various concentrations as indicated. Hybridization with a soybean 18S rDNA probe has been included to confirm that the RNA preparations are undegraded and to serve as an internal control for variations in gel loading and blotting.
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is very similar with that of YDP1, a yeast HP protein (Fig. 4), although YDP1 and plant HP proteins share only 16–21% amino acid sequence identity. The active histidine residue is located in helix C, which forms a binding surface with helix D for the response regulator. This indicates that there is a high degree of conservation in three-dimensional structure between yeast and plant HP proteins. A slight difference in helix arrangement was found between TaHP1 and AHP1, representatives of class I and class II HP proteins, respectively. AHP1 has a much shorter helix A than TaHP1. These differences coupled with differences in amino acid side-chains in these helices may be to affect binding to the response regulator. Consequently, different HP proteins may interact with specific response regulators in various signaling pathways. Since many isoforms of HP and RR are found in the same plant species, as in wheat, maize and Arabidopsis, the specific molecular recognition and discrimination between different HPs and RRs is expected to be critically important in order to avoid cross-talk among these isoforms. The expression profile also indicated the diversity among the different HP genes. Some HP genes are constitutively expressed in plant tissues, such as ZmHP1, ZmHP2 and ZmHP3 from maize, AHP2 from Arabidopsis, and TaHP2 and TaHP3 from wheat. The expression of other HP genes, however, is confined to specific tissues. For example, AHP1 is expressed predominantly in roots, and barely detected in leaves and stems. Furthermore, some HP genes are specifically expressed in response to a particular physiological process such as a HP clone in drought-stressed rice. Furthermore, the constitutively expressed HP genes ZmHP1, ZmHP2 and ZmHP3 showed preferential interactions with certain ZmRRs in the yeast two hybrid assay [17]. These data further support the idea that different forms of HP proteins are likely to play specific roles in different plant tissues or physiological processes. In agreement with the above analyses, the expression of TaHP1 family exhibited some degree of cytokinin-inducibility. This induction is evident after BA treatment, but not after trans-zeatin treatment, which demonstrates the close relationship of BA and retardation of leaf senescence. Two-component elements could potentially be regulated by cytokinin signals and also by other signals. This allows resetting or fine-tuning in cytokinin signal transduction, and also provides a cross-talk mechanism in plant signaling networks. This has been demonstrated for response regulators. For example, the expression of ZmRR1 from maize was regulated by nitrogen signals [15], while the expression of some ARRs was regulated by stress [30] and nitrogen signals [18]. Cytokinin-dependent induction of ZmRR1 and ZmRR2 was repressed by co-treatment with ABA in a dose-dependent manner in maize [31]. The responsiveness of HP to cytokinin and other stimuli, however, has not been well characterized. The expression data from TaHP1 family suggest that HP, like RR, is not only involved in the regulation of cytokinin signal transduction
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by alternation of gene expression, but also participates in cytokinin-mediated signaling in special physiological processes, such as leaf senescence and drought stress.
Acknowledgements We wish to sincerely thank Dr. Bettina Deavours (Plant Biology Division, The Samuel Roberts Noble Foundation, USA) and Dr. Charles H. Hocart (Australian National University, Australia) for critical reading of the manuscript. This work was supported by the State Key Basic Research and development Plan of China (2003CB114300), the National Natural Science Foundation of China (No. 30400222), the Chinese National Special Foundation for Transgenic Plant Research and Commercialization (J2002B007), and the Natural Science Foundation of Beijing (No. 503211).
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