Mol Genet Genomics DOI 10.1007/s00438-014-0821-9

Original Paper

Expression and functional analysis of genes encoding cytokinin receptor‑like histidine kinase in maize (Zea mays L.) Bo Wang · Yanhong Chen · Baojian Guo · Muhammad Rezaul Kabir · Yingyin Yao · Huiru Peng · Chaojie Xie · Yirong Zhang · Qixin Sun · Zhongfu Ni 

Received: 15 June 2013 / Accepted: 28 January 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Cytokinin signaling is vital for plant growth and development which function via the two-component system (TCS). As one of the key component of TCS, transmembrane histidine kinases (HK) are encoded by a small gene family in plants. In this study, we focused on expression and functional analysis of cytokinin receptorlike HK genes (ZmHK) in maize. Firstly, bioinformatics analysis revealed that seven cloned ZmHK genes have different expression patterns during maize development. Secondly, ectopic expression by CaMV35S promoter in Arabidopsis further revealed that functional differentiation exists among these seven members. Among them, the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX was obviously reduced as compared to wild type. Additionally,

ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type. Although the causal link between ZmHK genes and cytokinin signaling pathway is still an area to be further elucidated, these findings reflected that the diversification of ZmHK genes expression patterns and functions occurred in the course of maize evolution, indicating that some ZmHK genes might play different roles during maize development.

Communicated by S. Hohmann.

Introduction

B. Wang and Y. Chen contributed equally to this work. Electronic supplementary material  The online version of this article (doi:10.1007/s00438-014-0821-9) contains supplementary material, which is available to authorized users. B. Wang · Y. Chen · B. Guo · M. R. Kabir · Y. Yao · H. Peng · C. Xie · Y. Zhang · Q. Sun · Z. Ni (*)  State Key Laboratory for Agrobiotechnology, Key Laboratory of Crop Heterosis Utilization (MOE), Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing 100193, China e-mail: [email protected] B. Wang · Y. Chen · B. Guo · M. R. Kabir · Y. Yao · H. Peng · C. Xie · Y. Zhang · Q. Sun · Z. Ni  National Plant Gene Research Centre (Beijing), Beijing 100193, China

Keywords  Maize · Cytokinin · Histidine kinase · Gene expression · Functional analysis

Cytokinin signaling plays a vital role in many aspects of plant growth and development, including root and shoot growth, photomorphogenesis, flowering timing, senescence, seed development and the circadian clock (D’Aloia et al. 2011; Mok and Mok 2001; Zheng et al. 2006). At the molecular level, these processes are controlled by a two-component system (TCS) (Hwang and Sheen 2001; Heyl 2003; Kakimoto 2003; Ferreira and Kieber 2005; Ren et al. 2009), which mainly consists of a transmembrane histidine kinase receptor (HK), a nuclear response regulator (RR) and an intermediate histidine phosphotransfer protein (HP) that transfers the signal from the transmembrane receptor to the nucleus. Among these, histidine kinase protein typically contains

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a conserved histidine kinase domain and a C-terminal receiver domain. The transmitter domain includes characteristic sequence motifs and a conserved His residue that is the site of autophosphorylation (West and Stock 2001). A domain in the predicted extra cytoplasmic region, designated cyclase/histidine kinase-associated sensory extracellular (CHASE) possesses hormone binding activity (Yamada et al. 2001; Anantharaman and Aravind 2001; Vyroubalová et al. 2009; Hothorn et al. 2011; Lomin et al. 2012). Genome-wide analysis revealed that cytokinin receptor-like histidine kinases are encoded by a small gene family in Arabidopsis and rice, with the member of 3 and 5, respectively (Riefler et al. 2006; Ito and Kurata 2006). Expression analysis suggested that these genes are, in general, transcribed in a wide variety of tissues and organs (Nishimura et al. 2004; Ito and Kurata 2006). Genetic studies revealed that there is functional redundancy among HK genes. For example, single mutants of each AHK in Arabidopsis have no obvious phenotype, but in the ahk2/ahk3 double mutant and the ahk2/ahk3/ahk4 triple mutant, a severe phenotype was observed, including abolished sensitivity to cytokinins, reduced meristematic activity, severely inhibited root growth, as well as impaired reproductive development with reduced fertility. On the other hand, it has been reported that a loss-of-function mutation in AHK3, but not in the other cytokinin receptor genes, conferred a reduced sensitivity to cytokinin in cytokinin-dependent delay of leaf senescence, which revealed that there is functional differentiation among these genes (Kim et al. 2006). In maize, genome-wide analysis exhibited that there was eleven ZmHKs with highly conserved CHASE domain (Chu et al. 2011) and six ZmHK genes (ZmHK1, ZmHK1b1, ZmHK1b2, ZmHK1a2, ZmHK2 and ZmHK3) have been cloned (Muñiz et al. 2010). Although functional analysis of ZmHK1, ZmHK2, and ZmHK3a by expressing in E. coli and the cytokinin binding study have been reported (Yonekura-Sakakibara et al. 2004; Lomin et al. 2011), the expression pattern and function of the maize cytokinin receptor-like histidine kinase gene family still need a further investigation. In this article, we provide a detailed information on the expression patterns of seven maize genes encoding cytokinin receptor-like histidine kinase. Moreover, comparative analysis was further performed by the generation and characterization of transgenic Arabidopsis plants overexpressing individual ZmHK genes. We found that these seven ZmHK genes showed distinctive expression patterns and functions in plant growth and development, which will pave the way for elucidating the precise role of ZmHK genes in plant growth and development in the future.

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Mol Genet Genomics

Materials and methods Plant materials and treatments Maize (Zea mays L.) inbred line Zong31 was used in this study. Plants were grown in the greenhouse in a 16-h light/8-h dark cycle, maintaining a temperature of 29 and 25 °C for the light and dark cycles, respectively. Fourteen different tissues from Zong31 were harvested and frozen immediately in liquid nitrogen then stored at −80 °C for further use. For 6-benzylaminopurine (6-BA) treatments, seedlings of Zong31 were incubated in 6-BA with various concentrations for 24 h, and roots of the treated seedlings were collected for quantitative real-time PCR analysis. The Columbia (Col-0) ecotype of Arabidopsis thaliana was used as the wild type. Plants were grown in the greenhouse in soil at 20 °C under long-day conditions (16 h light/8 h dark). For the in vitro seedling assays, seeds were surface sterilized and cold treated at 4 °C for 3 days in the dark and then exposed to white light. Seedlings were grown at 20 °C on horizontal or vertical plates containing MS medium, 3 % sucrose, and 0.9 % agarose (Merck) unless otherwise specified. Maize HK gene clone and motif detection Based on the previous study reported by Chu et al. (2011), specific primer pairs of seven genes encoding cytokinin receptor-like histidine kinase were designed (supplementary Table S1), and seedling root cDNA of maize inbred line Zong31 was used to amplify ZmHK genes. Putative motifs of ZmHK protein were predicted by Simple Modular Architecture Research Tool (SMART) and TMHMM Server 2.0 software. Promoter motifs were predicted in PlantCARE (http://sphinx.rug.ac.be:8080/PlantCARE/). Expression analysis of ZmHK genes A comprehensive atlas of global transcription profiles across developmental stages and plant organ of inbred line B73 was established (Sekhon et al. 2011), and pictorial representations of expression of ZmHK1, ZmHK1a2, ZmHK1b1, ZmHK2, ZmHK2a2 and ZmHK3b can be found at http://www.maizegdb.org. In addition, the expression data of ZmHK1b2 gene are available at http://www.plexdb. org, and electronic fluorescent pictograph of this gene was generated by using the eFP Browser software (Winter et al. 2007). Isolation of total RNA and reverse transcription Total RNA was extracted using High-purity Total RNA Rapid Extraction Kit (Spin-column) (Bioteke). The

Mol Genet Genomics

amount and quality of the total RNA were checked through electrophoresis in 1 % agarose gel. The concentration of RNA was measured with spectrophotometer NanoDrop, ND1000 (Nano Drop Technologies). 2 μg total RNA was reverse transcribed to cDNA in 20 μl reactions using the M-MLV reverse transcriptase (Promega Corporation, USA). Reverse transcription was performed for 60 min at 42 °C with a final denaturation step at 70 °C for 15 min. Aliquots of 2 μl of the obtained cDNA were subjected to RT-PCR analysis. Quantitative real‑time PCR Specific primers of seven ZmHK genes, three AtPINs (AtPIN1, AtPIN3 and AtPIN7), three AHKs genes (AHK2, AHK3 and AHK4) and ARR5 (supplementary Table S2) were used for SYBR green-based quantitative real-time PCR analysis, and ZmActin and AtActin8 were amplified as endogenous control. cDNAs of Arabidopsis seedling roots at 16 days after germination and cDNAs of Zong31 seedling roots after 6-BA treatments were used for analysis. The reaction conditions performed were as follow: 94 °C for 4 min, followed by 40 cycles: 94 °C for 30 s, 66/58 °C for 30 s, 72 °C for 30 s, and then 72 °C for 5 min. Quantification results were expressed in terms of the cycle threshold (CT) value determined according to the manually adjusted baseline. Relative gene expression in different samples was determined using a previously described method (Livak and Schmittgen 2001). Briefly, differences between the CT values of target gene and endogenous control were calculated as ΔCT  =  CT target − CT endogenous control, and expression levels of target genes relative to endogenous control were determined as 2−CT. For each sample, PCR was repeated three times, and the average values of 2−CTwere used to determine difference in gene expression. Statistical analysis was performed using t test. Plasmid construction and generation of transgenic Arabidopsis lines Total RNA extracted from roots of Zong31 was used as template to synthesize the first strand of cDNA using oligodT as a primer. The reaction was used to PCR-amplify cDNA sequences corresponding to the coding region of ZmHK genes using the Reverse Transcriptase Super Script III (Invitrogen), and the resulting PCR fragments were cloned into the Gateway entry vector pDNOR221. After confirmation by DNA sequencing, the ZmHK fragments were recombined into the pB2GW7 destination vector which can express under control of the CaMV35S promoter. The resulting constructs were transformed into Agrobacterium tumefaciens strain GV3101, which was

then used for the transformation of plants (Col-0) by the floral dip method (Zhang et al. 2006). Seed assay Only seed batches that had been harvested and stored at the same time and under the same conditions were used for the seed germination assay. Seeds were sterilized and plated on Murashige and Skoog (MS) medium containing 0.9 % agar, but no sucrose. After 3 days of stratification at 4 °C in the dark, plates were incubated at 20 °C in the darkness. The number of germinated seeds at 48 h after sowing was calculated. Experiments were done in triplicate with 100 seeds for each experiment and genotype. Toluidine blue O staining of mature seeds at 8 days after pollination (DAP) was performed as reported by Debeaujon et al. (2000). Observations and photographs were taken with a Nikon Stereo Microscope (Nikon, Tokyo, Japan). Chlorophyll retention assay Seedlings were grown in soil for 24 days, the third and fourth leaves were then detached and incubated in distilled water in small Petri dishes for 4 days in darkness. Three samples were measured for each genotype, each sample consisting of eight leaves. Chlorophyll was extracted with 95 % ethanol for 24 h in darkness. Light absorption at 647 and 664 nm was determined with a spectrophotometer, normalized to fresh weight, and the chlorophyll content was calculated as described (Chen and Gallie 2006). Leaf morphometric measurements At day 24 after germination, the rosette diameter and total number of leaves of six randomly chosen plants per genotype were measured and then harvested individually. The leaf blades of each rosette were dissected, and total leaf blade area was measured with a calibrated leaf area meter (LI-3000A; LI-COR). To check the accuracy of the area measurement of small leaves, digital pictures of a tworeplicate series of Col-0 leaves (n  = 10) were taken and total leaf area was measured with a public domain image analysis software (ImageJ version 1.32; http://rsb.info.nih. gov/ij/). The seventh leaf was cut from 24 DAG seedlings and immersed in boiling water in a beaker for 10 min to kill the cells, then immersed in 95 % ethyl alcohol, until decolorized. When the process was complete, the leaf was transferred to hot (96 °C) 85 % lactic acid for 8 min. Finally, the leaf was transferred to cold acid (room temperature) for microscopic inspection by Nikon Stereo Microscope.

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Mol Genet Genomics

Results Cloning of cytokinin receptor‑like histidine kinase genes in maize According to previous study reported by Chu et al. (2011), the cDNAs of seven genes encoding cytokinin receptorlike histidine kinase were amplified from seedling root of Zong31 with primer pairs (supplementary Table S1) to validate the full-length cDNAs and fragments of about 3,000 bp were obtained (data not shown). The PCR products were cloned into pDNOR221 and three clones of each gene were sequenced. Sequence analysis revealed that the ORF sequences of these seven cytokinin receptor-like histidine kinase genes ranged from 2,925 (ZmHK1) to 3,150 bp (ZmHK3b) in length, with the sizes of deduced proteins from 974 to 1,049 amino acids (supplementary Table S3). Consistent with the results reported by Chu et al. (2011), all of the ZmHK proteins contain three highly conserved regions of about 65, 184 and 133 amino acid residues, respectively, which correspond to the histidine kinase domain, receiver-like domain and receiver domain (Fig. S1). Moreover, all of them contained a CHASE domain related to cytokinin receptor at the N-terminus, suggesting functional similarities among them. It should to be noted that six of seven ZmHK proteins contained two hydrophobic membrane-spanning domains, whereas ZmHK1 only contained one. In addition, CHASE domain of ZmHK3b is shorter than others due to alternative splicing in mRNA processing (Chu et al. 2011) (Fig. S1). Promoter motif prediction and cytokinin inducibility of ZmHK genes Putative promoter sequences (2,000 bp upstream the 5′UTR region) of ZmHK genes were obtained from the draft maize genome sequence and database search of plant promoters (PlantCARE) detected one or more hormone and stressresponse elements (Lescot et al. 2002). Notably, ABA- and MeJA-responsive elements were detected in all ZmHK promoter regions; however, the number of these elements differs in different members (Fig. S2). For example, four ABA- and four MeJA-responsive elements were identified in ZmHK1a2 promoter, whereas only two MeJA-responsive elements were identified in ZmHK3b promoter (Fig. S2). It is worth noting that pair genes ZmHK1b1 and ZmHK1b2 have the same response element in their promoter region, whereas the others (ZmHK1 and ZmHK1a2; ZmHK2 and ZmHK2a2) are more divergent (Fig. S2). For example, total number of responsive element in ZmHK1 is less than those of ZmHK1a2, and heat stress-responsive element only existed in ZmHK1a2. In addition, auxin-responsive element was found in ZmHK2 but not in its sister pair ZmHK2a2

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Fig. 1  Relative mRNA abundances of ZmHK genes in Zong31 seedling root after exogenous cytokinin treatment. Seeds of Zong31 were imbibed at 28 °C for 48 h, then were transferred to hydroponic culture. After 3 days, the seedlings were transferred to other hydroponic cultures containing 0 M (black bars), 5 × 10−6  M (open bars) and 5 × 10−5 M (gray bars) 6-BA for 24 h, and the treated seedling roots were collected for quantitative real-time PCR analysis. Asterisks and double asterisks refer to significance at p