Elsevier

Gene

Volume 651, 20 April 2018, Pages 106-117
Gene

Research paper
Identification and expression analysis of the IPT and CKX gene families during axillary bud outgrowth in apple (Malus domestica Borkh.)

https://doi.org/10.1016/j.gene.2018.01.101Get rights and content

Highlights

  • A total of 12 MdIPTs and 12 MdCKXs were identified in the apple genome and their systematic phylogenetic, structural, and synteny analyses were performed.

  • Analyses of the response of MdIPT, MdCKX and MdPIN1 genes to 6-BA treatment indicated that the initial up-regulation of MdIPTs may not be induced directly by 6-BA but rather due to the export of auxin from the axillary bud.

  • All of the detected MdIPTs were significantly up-regulated initially after decapitation, suggesting that apple buds may also be a primary source for the synthesis of CKs during the activation of bud outgrowth.

  • Results of the expression patterns of MdIPT, MdCKX and MdPIN1 genes after decapitation and Lovastatin treatments indicated that the absence of CK biosynthesis is not sufficient for the activation of bud break and the bud auxin export in apple tree with intact apical dominance or decapitated apple tree.

  • MdCKX8 and MdCKX10 exhibited little response to decapitation, but was significantly up-regulated by 6-BA and Lovastatin, and their function in axillary bud outgrowth is worth further investigation

Abstract

Cytokinins (CKs) play a crucial role in promoting axillary bud outgrowth and targeting the control of CK metabolism can be used to enhance branching in plants. CK levels are maintained mainly by CK biosynthesis (isopentenyl transferase, IPT) and degradation (dehydrogenase, CKX) genes in plants. A systematic study of the IPT and CKX gene families in apple, however, has not been conducted. In the present study, 12 MdIPTs and 12 MdCKXs were identified in the apple genome. Systematic phylogenetic, structural, and synteny analyses were performed. Expression analysis of these genes in different tissues was also assessed. MdIPT and MdCKX genes exhibit distinct expression patterns in different tissues. The response of MdIPT, MdCKX, and MdPIN1 genes to various treatments (6-BA, decapitation and Lovastatin, an inhibitor of CKs synthesis) that impact branching were also investigated. Results indicated that most of the MdIPT and MdCKX, and MdPIN1 genes were upregulated by 6-BA and decapitation treatment, but inhibited by Lovastatin, a compound that effectively suppresses axillary bud outgrowth induced by decapitation. These findings suggest that cytokinin biosynthesis is required for the activation of bud break and the export of auxin from buds in apple tree with intact primary shoot apex or decapitated apple tree. MdCKX8 and MdCKX10, however, exhibited little response to decapitation, but were significantly up-regulated by 6-BA and Lovastatin, a finding that warrants further investigation in order to understand their function in bud-outgrowth.

Introduction

Branching patterns and density in apple (Malus domestica) are important parameters in commercial plantings of the apples, as tree architecture greatly affects fruit yields and tree management options. The appropriate number and size of branches can foster the development of robust saplings, induce early flowering, high biomass, and can be critical to achieving the beneficial tree structures in modern apple orchards (Quinlan, 1981; Wertheim et al., 2001; Wertheim et al., 2003; Gudarowska and Szewczuk, 2004; Kviklys, 2006; Bostan, 2010; Elfving, 2010; Wang et al., 2017). The scion/interstem/rootstock combination of ‘Nagafu No. 2’ (a ‘Fuji’ cultivar)/T337/M. robusta Rehd. is one of the most widely used apple trees in commercial orchards in China. This genetic combination, however, exhibits problems with natural shoot branching. Therefore, understanding the molecular mechanism responsible for apple shoot branching would be greatly beneficial. Lateral apple shoot branches generally arise from the outgrowth of axillary buds, which is controlled by a complex interaction of phytohormones and environmental conditions.

Earlier studies have demonstrated that lateral bud outgrowth is inhibited by basipetal transport of auxin from terminal buds (Thimann and Skoog, 1933; Morris, 1977). Auxin, however, does not move directly into the lateral buds (Prasad et al., 1993; Booker, 2003) and direct auxin application onto lateral buds does not inhibit outgrowth (Brown et al., 1979). This suggests that auxin is indirectly responsible for hindering bud outgrowth, which supported the hypothesis that other hormones are involved as second messengers of auxin in buds during bud break (Sachs and Thimann, 1967; Bangerth, 1994; Li et al., 1995). It was found that CK functions as the principle second messenger during bud outgrowth (Snow, 1937; Bangerth, 1994; Leyser, 2003) and blocks the auxin signal from entering into the axillary bud (Chatfield et al., 2000) and thus directly promoted bud outgrowth. Auxin, however, can directly inhibit the biosynthesis of CKs through an AXR1-dependent auxin signaling pathway (Nordstrom et al., 2004), and thus suppress bud outgrowth (Bangerth, 1994; Tanaka et al., 2006).

There is evidence that the bud outgrowth is correlated with the expression of cytokinin biosynthetic genes (Ferguson and Beveridge, 2009). CKs can be synthesized throughout the plant (Nordstrom et al., 2004). The first and rate-limiting step of CK biosynthesis is catalyzed by adenosine phosphate-isopentenyl transferases (IPTs) (Hirose et al., 2008), which can be expressed throughout the plant (Miyawaki et al., 2004) and exist as adenosine phosphate-IPTs (ATP/ADP IPTs) and tRNA-IPTs. Previous studies have indicated that ATP/ADP IPTs control the biosynthesis of isopentenyladenine (iP)- and trans-zeatin (tZ)-type CKs, whereas tRNA IPTs are mainly used for the synthesis of cis-zeatin (cZ)-type CKs (Miyawaki et al., 2004; Sakakibara, 2006). The degradation of CKs is catalyzed by CK oxidase/dehydrogenases (CKXs). All of the IPT protein sequences contain one or two conserved IPPT-binding domains, and FAD- and CK-binding domains exist in every CKX protein sequence.

Recent studies have explored the functions of IPT and CKX genes in various plant species. In Arabidopsis, ipt single or multiple mutants exhibited significant reductions in branching (Muller et al., 2015). The mutation of multiple IPT genes produced cytokinin-deficient plants with a smaller Shoot Apical Meristem (SAM) size that produces fewer organ primordia, relative to wild-type plants (Miyawaki et al., 2006). In pea (Pisum sativum L.), PsIPT1 and PsIPT2 genes were significantly induced in nodal stem tissues after decapitation, and increased CK levels were present in the nodal tissue and axillary buds (Tanaka et al., 2006). Three-year-old transgenic Asakura-sanshoo lines, containing a CaMV35S-IPT construct, exhibited morphological characteristics typically brought about by overproduction of CK, including reduced stem elongation, decreased leaf surface area, increased branching, and delayed leaf senescence (Zeng and Zhao, 2016). LEACO10.821kb-ipt transgenic chrysanthemum lines displayed an enhanced flowering and branching phenotype (Khodakovskaya et al., 2009). A knockdown of LjIPT3 (LjIPT3i) by RNAi in Lotus japonicus reduced the levels of endogenous cytokinins and influenced nodule development (Chen et al., 2014). Other experiments have shown that ectopic expression of AtSTM induces the expression of IPT7, thereby enhancing the levels of CKs, while the application of exogenous CK partly restored the phenotype of a stm mutant (Yanai et al., 2005). The atmyb2 transgenic Arabidopsis plants exhibit upregulated expression of AtIPT genes, contain higher levels of CKs, and produce a more branching phenotype (Guo and Gan, 2011). In apple, however, MdIPT5b (MDP0000232324) was found to have a crucial role in inducing the dwarfing properties characteristic of ‘M.9’ rootstock (Feng et al., 2017). The overexpression of MdIPT3a (MDP0000013380) gene in tobacco plants resulted in the inhibition of root development, enhanced outgrowth of axillary buds, and a delay in flowering (Zhu et al., 2012). The native MhIPT3 identified in Malus hupehensis Rehd. was the most similar to AtIPT3 and its expression was strongly induced by the application of nitrate to root or leaves of the nitrogen-deprived seedlings (Peng et al., 2008).

In Arabidopsis, 35S:CKX7 overexpression plants results in lower levels of cytokinin metabolites, particularly cis‑zeatin (cZ) and N‑glucoside cytokinins (Kollmer et al., 2014). Holst et al. (2011) produced Arabidopsis plants whose shoot primordia were CK-deficient by expressing a CKX gene under the control of the AINTEGUMENTA (ANT) promoter. The resulting transgenic plants had a reduced ability of both the vegetative and the reproductive shoot apical meristems to initiate new leaves and flowers. The overexpression of AtCKX1 in an atmyb2 mutant of Arabidopsis reduced endogenous CK levels and restored the bushy phenotype of the atmyb2 mutant to the wild-type (Guo and Gan, 2011). Overexpression of AtCKX genes in tobacco induced cytokinin deficiency and profoundly influenced root and shoot development (Werner et al., 2001). Additionally, enhancing the expression of six different AtCKX genes reduced the activity of vegetative and floral shoot apical meristems and leaf primordia (Werner et al., 2003). In barley, compact CK levels were established by the down-regulation of CKXs and reinforced the de novo biosynthesis of CKs (Mrizova et al., 2013), while RNAi silencing of HvCKX1 resulted in plant with a higher number of seeds and spikes per plant and thus an overall higher yield (Zalewski et al., 2014). Rice osckx2 mutants that exhibit specific suppression of OsCKX2 expression through shRNA-mediated gene silencing, display better growth and productivity by increasing tiller number and grain weight. Insertional activation of OsCKX2 consistently resulted in increased expression of CKX2 and reduced tiller number and growth (Yeh et al., 2015).

Although the roles of IPT and CKX genes in influencing the levels of CKs and branching have been extensively studied in Arabidopsis and other species, little information is available regarding the expression of these genes in response to axillary bud outgrowth in apple. Therefore, a genome-wide identification of MdIPT and MdCKX gene family members was performed in apple. A systematic analysis of the gene characterization, gene structures, gene phylogeny and synteny was also conducted. The expression levels of MdIPT and MdCKX genes in different tissues (axillary buds, roots, stems, leaves, stem tips, flowers, and fruits), and in response to treatments (6-BA, decapitation and Lovastatin) that impact branching, were also examined. Changes in gene expression in response to these branching-related treatments provided evidence of their potential roles in apple bud outgrowth. Our results provide a foundation for further analysis of the functional role of these genes in apple shoot branching.

Section snippets

Identification and chromosome location of MdIPT and MdCKX genes in apple

IPT and CKX proteins sequences in Arabidopsis, downloaded from the Arabidopsis genome (TAIR; http://www.Arabidopsis.org/), were used as query sequences to search the apple genome sequence database located on the website of the Genome Database for Rosaceae (GDR; https://www.rosaceae.org/). A BLASTp search was performed and sequences were selected for further analysis when their E value was less than −5. All of the obtained putative apple IPT and CKX gene sequences were submitted to the Pfam

Identification and genetic analysis of MdIPT and MdCKX genes

Arabidopsis AtIPT and AtCKX proteins were used as query sequences (Takei et al., 2001; Schmulling et al., 2003) to identify the MdIPT and MdCKX genes in the apple genome. After manual filtering, a total of 12 MdIPT genes and 12 MdCKX genes with confirmed conserved domains were identified and annotated (Table 1, Table 2). The identified MdIPT and MdCKX genes were named based on chromosome order. The 12 MdIPT genes were distributed on 6 chromosomes (chr3, 6, 10, 11, 13 and 16) (Fig. S1a). The CDS

Discussion

Gene family analysis has become a useful approach for better understanding gene structure, function, and evolution. IPT and CKX genes are CK biosynthesis and CK degradation genes that play an important function in fine-turning cytokinin levels in plants.

The function and evolution of IPT and CKX gene families have been analyzed in a variety of plants, including Arabidopsis (9 AtIPT and 7 AtCKX genes) (Takei et al., 2001; Schmulling et al., 2003), rice (10 OsIPT and 11 OsCKX genes) (Ashikari et

Acknowledgments

This work was supported by the National Apple Industry Technology System of Ministry of Agriculture of the People's Republic of China (CARS-28), Science and Technology Innovative Engineering Project in Shaanxi province of China (2015NY114), Yangling Subsidiary Center Project of National Apple Improvement Center and Collaborative Innovation of Center Shaanxi Fruit Industry Development. Innovation project of science and technology plan projects of Shaanxi province (2016TZC-N-11-6).

Author contributions

Ming Tan, Guofang Li, Juanjuan Ma, Dong Zhang and Mingyu Han participated in the experimental design and data analysis. Ming Tan, Guofang Li, Siyan Qi and Xiaojie Liu. Xilong Chen performed material sampling, field measurements and the laboratory data measurement. Ming Tan, Guofang Li and Mingyu Han participated in the paper writing and manuscript amend. All authors participated in revising the work and approved the manuscript.

Conflicts of interest

The authors declare no conflict of interest.

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