The genes for gibberellin biosynthesis in wheat - Springer Link

Funct Integr Genomics (2012) 12:199–206 DOI 10.1007/s10142-011-0243-2

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The genes for gibberellin biosynthesis in wheat Yuanyuan Huang & Wenlong Yang & Zhong Pei & Xiaoli Guo & Dongcheng Liu & Jiazhu Sun & Aimin Zhang

Received: 18 May 2011 / Revised: 11 July 2011 / Accepted: 17 July 2011 / Published online: 19 August 2011 # Springer-Verlag 2011

Abstract The gibberellin biosynthesis pathway is well defined in Arabidopsis and features seven key enzymes including ent-copalyl diphosphate synthase (CPS), entkaurene synthase (KS), ent-kaurene oxidase (KO), entkaurenoic acid oxidase (KAO), GA 20-oxidase, GA 3-oxidase, and GA 2-oxidase. The Arabidopsis genes were used to identify their counterparts in wheat and the TaCPS, TaKS, TaKO, and TaKAO genes were cloned from Chinese Spring wheat. In order to determine their chromosome locations, expression patterns and feedback regulations, three TaCPS genes, three TaKS genes, three TaKO genes, and three TaKAO genes were cloned from Chinese Spring wheat. They are mainly located on chromosomes 7A, 7B, 7D and 2A, 2B and 2D. The expression patterns of TaCPS, TaKS, TaKO, and TaKAO genes in wheat leaves, young spikes, peduncles, the third and forth internodes were

investigated using quantitative PCR. The results showed that all the genes were constitutively expressed in wheat, but their relative expression levels varied in different tissues. They were mainly transcribed in stems, secondly in leaves and spikes, and the least in peduncles. Feedback regulation of the TaCPS, TaKS, TaKO, and TaKAO genes was not evident. These results indicate that all the genes and their homologs may play important roles in the developmental processes of wheat, but each of the homologs may function differently in different tissues or during different developmental stages. Keywords Wheat . Gibberellin biosynthesis enzymes . Gene cloning . Chromosome location . Expression regulation

Introduction Authors Yuanyuan Huang and Wenlong Yang contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s10142-011-0243-2) contains supplementary material, which is available to authorized users. Y. Huang : W. Yang : D. Liu : J. Sun : A. Zhang (*) The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China e-mail: [email protected] Z. Pei : X. Guo College of Biological Sciences, China Agricultural University, Beijing 100193, China Y. Huang Graduate University of Chinese Academy of Sciences, Beijing 100049, China

Gibberellin is an important hormone in plants. It participates in the regulation of many developmental processes, such as seed germination, stem elongation, leaf stretching, flower induction, and seed development (Hedden and Kamiya 1997; Olszewski et al. 2002; Hedden 2003; Salamini 2003; Biemelt et al. 2004; Grennan 2006; Yamaguchi 2008). The biosynthesis of gibberellin is well elucidated in model plants and mainly involves seven kinds of enzymes, including ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 20-oxidase, GA 3-oxidase, and GA 2-oxidase (Yamaguchi 2006). These enzymes are divided into three groups according to their subcellular localization and catalytic properties (Yamaguchi et al. 2001). The terpene cyclases, including CPS and KS, are localized in the proplastids and catalyze the original

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substrate geranylgeranyl diphosphate (GGDP) to entkaurene. The cytochrome P450 monooxygenases, including KO and KAO, are localized to the endoplasmic reticulum (ER) and catalyze ent-kaurene to GA12 aldehyde through a series of oxidation reactions. The soluble 2-oxoglutaratedependent dioxygenases (2ODD), including GA20ox and GA3ox, believed to be soluble and cytoplasmic, catalyze a hydroxylation reaction to generate the bioactive gibberellins from GA12 aldehyde, such as GA1, GA4, and GA7. Also, another 2-oxoglutarate-dependent dioxygenase, GA2ox, inactivates GAs from gibberellins and their precursors (Schomburg et al. 2003; Zhu et al. 2006; Lo et al. 2008; Rieu et al. 2008; Yamaguchi 2008). All of these enzyme genes have been isolated from different plants (Sun and Kamiya 1994; Helliwell et al. 1998; Yamaguchi et al. 1998; Otomo and Kenmoku 2004; Davidson et al. 2005), and their functions were determined with their related mutants (Bensen et al. 1995; Davidson et al. 2003; Davidson and Smith 2004; Grennan 2006). CPS, KS, KO, and KAO are single-copy genes in most plants. Their mutants display severe dwarfism and loss of fertility, which could be recovered after spraying with exogenous active gibberellins (Silverstone et al. 1997; Helliwell and Poole 1999; Helliwell et al. 2001; Olszewski et al. 2002; Prisic and Xu 2004; Xu et al. 2007). At present, only a few reports have been published on genes involved in GA synthesis in wheat, such as TaCPS, TaKAO, TaGA20ox, and TaGA3ox (Spielmeyer et al. 2004; Appleford et al. 2006; Zhang et al. 2007; Toyomasu 2008; Khlestkina et al. 2010). Three TaKAO genes are localized to chromosomes 7AS, 4AL, and 7DS using partial genome sequences and the ITMI mapping population (Khlestkina et al. 2010). Three TaGA20ox1 genes, located on chromosomes 5BL, 5DL, and 4AL, are expressed in the nodes and ears of elongating stems, and also in developing and germinating embryos (Appleford et al. 2006). Three TaGA3ox2 genes are expressed in the internodes, nodes, and ears of elongating stems and germinating embryos (Appleford et al. 2006). Only one TaCPS gene, TaCPS3, was confirmed to participate in GA synthesis using mRNA expression patterns (Toyomasu 2008). To elucidate the GA biosynthesis process in wheat, we focused on cloning and functional analysis of the upstream genes of the GA biosynthesis pathway in the wheat cultivar Chinese Spring.

Funct Integr Genomics (2012) 12:199–206

were sampled and quickly frozen in liquid nitrogen and stored at −80°C until RNA was isolated for polymerase chain reaction (PCR)-based cloning. The Chinese Spring nulli-tetrasomic lines, grown in an experimental field, were sampled for DNA preparation. Paclobutrazol and gibberellin treatments Plants were grown in a greenhouse using liquid culture (Hoagland and Arnon 1950), and 2-week-old seedlings were treated with 30 μM Paclobutrazol (PAC, an inhibitor of GA synthesis) or 30 μM of gibberellic acid (GA3). Chinese Spring seedlings cultured with water were used as a control. After 2 weeks of treatment, the seedlings were collected and frozen in liquid nitrogen, and stored at −80°C for RNA isolation. Cloning and sequencing of the TaCPS, TaKS, TaKO, and TaKAO genes Total RNAs of wheat samples were extracted using TRIzol reagent (Invitrogen) and purified by RNase-free DNaseI treatment (Promega). The concentration of RNA was determined using a UV spectrometer. The first strand of cDNA was synthesized by MMLV (Promega) using the oligo-dT primer according to the manufacturer’s instructions. The quality of the cDNA was checked by PCR with the tubulin gene in wheat. Genomic DNA was extracted using a CTAB extraction buffer. The concentration and quality of the DNA were checked using a UV spectrometer and agarose gel electrophoresis. The wheat EST database was blasted using the nucleotide sequences of the HvCPS, HvKS, HvKO, and HvKAO genes (GenBank accession numbers: AY551435, AY551436, AY551434, and AF326277). The ESTs with high similarity to the HvCPS, HvKS, HvKO, and HvKAO genes were selected and aligned using the DNASTAR software. Specific primers for wheat CPS, KS, KO, and KAO were designed and the full-length sequences of wheat CPS, KS, KO, and KAO were determined by PCR-based cloning. The PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced by a commercial sequence company SinoGenoMax Co., Ltd. Chromosomal localization of TaCPS, TaKS, TaKO, and TaKAO

Materials and methods Plant materials The wheat cultivar Chinese Spring was grown in a greenhouse until the heading stage. The stems and leaves

To assign the homologs of the TaCPS, TaKS, TaKO, and TaKAO genes to the wheat genome, the Chinese Spring nulli-tetrasomic line was used for chromosomal localization. First, specific primers for all of the genes were designed, and PCR was performed using the DNA of the


Chinese Spring nullisomic–tetrosomic line as a template. Then the homologs of the TaCPS, TaKS, TaKO, and TaKAO genes were assigned to their respective wheat chromosome. Quantitative real-time PCR analysis Total RNA was extracted from various organs of the Chinese Spring cultivar as mentioned above, and residual genomic DNA was removed by digestion with DNase I (Promega) prior to reverse transcription (RT). Equivalent amounts of total RNA (2 μg) from each tissue sample were reverse-transcribed into cDNA (Promega) according to the manufacturer’s instructions. The quantitative real-time PCR experiment (qPCR) was performed on a Roche LightCycler 480 system with the LightCycler 480 SYBR Green 1 Master Kit. Primer efficiencies were estimated by analyzing the amplification curves with the Roche LightCycler 480 Basic Software. To normalize the qPCR data, 12 wheat reference genes were tested using different wheat samples, and their average expression stability values were assessed using the geNorm software. Ta4050 (TC number in http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi) was finally chosen as the reference gene for its high stability among all of the analyzed samples. qPCR was performed using a 10-μl reaction containing 5-μl SYBR green mix, 2 μl 15-fold-diluted cDNA template, and 3 μl 1 μM forward and reverse primers. Three biological replicates of each sample, together with two technical replicates, were performed for all of the homologs of TaCPS, TaKS, TaKO, and TaKAO.

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and 2,496-bp coding regions, respectively. The three TaKS genes are 2,682, 2,721, and 2,770-bp long with 2,559, 2,541, and 2,541-bp coding regions, respectively. The three TaKO genes are 1,765, 1,785, and 1,747-bp long and all have 1,536-bp coding regions. The three TaKAO genes are 1,677, 1,609, and 1,781-bp long with 1,479, 1,476, and 1,476-bp coding regions, respectively. The sequences of each homolog of the above genes showed very high similarity to each other in their coding regions, and the differences among these homologous genes were mainly in the 5′ and 3′ untranslated regions (UTRs) (Fig. 1 for TaCPS and Additional file 1 a, b, c for TaKS, TaKO, and TaKAO, respectively). Chromosomal localization of TaCPS, TaKS, TaKO, and TaKAO The Chinese Spring nulli-tetrasomic lines were used as templates to determine the chromosomal localization of each homologous gene, and they were designated according to their chromosome localizations. The three TaCPS genes, localized on chromosomes 7A, 7B, and 7D, were named TaCPS-A, TaCPS-B, and TaCPS-D, respectively; the three TaKS genes, localized on chromosomes 2A, 2B, and 2D, were named TaKS-A, TaKS-B, and TaKSD, respectively; the three TaKO genes, localized on chromosomes 7A, 7B, and 7D, were named TaKO-A, TaKO-B, and TaKO-D, respectively; the three TaKAO genes, located on chromosomes 4A, 7A, and 7D, were named TaKAO-A2, TaKAO-A1, and TaKAO-D, respectively (Additional file 2). Expression patterns of TaCPS, TaKS, TaKO, and TaKAO

Results Isolation and sequence analysis of TaCPS, TaKS, TaKO, and TaKAO Through blasting the wheat EST database with the nucleotide sequences of HvCPS, HvKS, HvKO, and HvKAO, the ESTs with high similarity to HvCPS (TC367000, TC320899, TC335688, CD898505), HvKS (TC153894, BE419989), HvKO (AK334773), and HvKAO (AK334174, BE413961) were selected and aligned using the DNASTAR software. Specific primers were then designed to isolate the full-length wheat CPS, KS, KO, and KAO genes. Three TaCPS genes (GenBank accession numbers: GU980886–GU980888), three TaKS genes (FR719731, GU980889, and GU980890), three TaKO genes (GU980893–GU980895), and three TaKAO genes (GU980891, GU980892, and GU143912) were cloned from the Chinese Spring cultivar. The three TaCPS genes are 2,570, 2,599, and 2,706 bp in length with 2,496, 2,499,

To understand the transcription levels of TaCPS, TaKS, TaKO, and TaKAO, qPCR was performed to assess the expression patterns of these genes in leaves, young spikes, peduncles, and the third and fourth internodes. The results showed that all the genes were constitutively expressed in wheat, but their relative expression levels varied in different tissues. They were mainly transcribed in the stems, then in the leaves and spikes, and the least in peduncles. The relative expression levels of the genes from different genomes also differed. TaCPS-B had higher expression levels than TaCPS-A and TaCPS-D; TaKS-D was more highly expressed than TaKS-A and TaKS-B, TaKO-A, and TaKO-D were more highly expressed than TaKO-B, and TaKAO-A2 was more highly expressed than TaKAO-A1 and TaKAO-D (Fig. 2). These results indicate that all of the genes and their homologs may play important roles in wheat developmental processes, but each of the homologs may function differently in tissues or during developmental stages.

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Fig. 1 Nucleotide sequence alignments of three homologs of TaCPS



Fig. 1 (continued)

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Fig. 2 The expression patterns of TaCPS, TaKS, TaKO, and TaKAO in wheat leaves (L), young spikes (YS), peduncles (P), and the third (3IN) and fourth internodes (4IN). a TaCPS, b TaKS, c TaKO, d TaKAO

Feedback regulation of TaCPS, TaKS, TaKO, and TaKAO Some studies revealed that the active types of gibberellins could feedback regulate the expression levels of the GA20ox and GA3ox genes, which are the downstream genes in the GA synthesis pathway. TaCPS, TaKS, TaKO, and TaKAO are the upstream genes in the GA biosynthesis pathway. Whether their expression is regulated by their products is unknown. So the feedback regulation of TaCPS, TaKS, TaKO, and TaKAO was estimated in Chinese Spring seedlings by treatment with 30 μM GA3 or 30 μM PAC (an inhibitor of GA synthesis) solution. The results revealed that the expression levels of TaCPS, TaKS, TaKO, and TaKAO were not obviously altered by GA3 or PAC, except for TaCPS-B and TaCPS-D, which were upregulated by PAC and downregulated by GA3, and TaKO-D and TaKAO-D, which were upregulated by PAC (Additional file 3). These results further confirmed that all of the genes and their homologs are necessary in the developmental process.

Discussion Gibberellin is an important phytohormone that plays a part in many aspects of growth and development in plants. The synthesis and metabolism of gibberellins exist in fungi and all flowering plants (Hedden et al. 2001; Bottini et al. 2004; Tudzynski 2005). Common wheat is a hexaploid plant with designated A, B, and D genomes, but the number of genes involved in GA synthesis and the roles that they play in wheat are still unknown. In the present study, we cloned the CPS, KS, KO, and KAO genes from bread wheat, i.e., the

Chinese Spring cultivar, and the chromosomal localization of each homolog of TaCPS, TaKS, TaKO, and TaKAO were determined by PCR using Chinese Spring nulli-tetrasomic lines. The results were consistent with those of Spielmeyer et al. (2004) using DNA gel plot analysis, i.e., the homologs of TaCPS, TaKS, TaKO, and TaKAO were mainly located on chromosomes 7A, 7B, 7D and 2A, 2B and 2D. Of which, the TaKAO-A2 located on chromosomes 4A might be resulted from the translocation of chromosomes 4A and 7B in the evolution of hexaploid wheat (Devos et al. 1995). Meanwhile, the expression analysis of TaCPS, TaKS, TaKO, and TaKAO genes were carried out, and the results showed that TaCPS, TaKS, TaKO, and TaKAO are constitutively expressed in the leaves, young spikes, peduncles, and stems of wheat, but different expression patterns exist among the TaCPS, TaKS, TaKO, and TaKAO genes derived from the A, B, and D genomes. The expression levels of TaCPS and TaKS were lower than those of TaKO and TaKAO, which is consistent with the CPS, KS, KO, and KAO genes in Arabidopsis. GGDP is the precursor of gibberellins and other terpenes, CPS and KS catalyze GGDP to ent-kaurene, and CPS is a rate-limiting enzyme that determines the level of GGDP converted into gibberellins (Yamaguchi 2006). Overexpression of AtCPS in Arabidopsis results in high levels of ent-kaurene, but overexpression of AtKS in Arabidopsis does not increase ent-kaurene content, indicating that they have different mechanisms of regulation (Fleet and Yamaguchi 2003). Arabidopsis has two copies of the AtKAO gene, AtKAO1 and AtKAO2, and if AtKAO1 loses its function, AtKAO2 will replace it (Helliwell et al. 2001). Wheat contains three copies each of TaCPS, TaKS, TaKO, and TaKAO and they


may function redundantly, like AtKAO; thus, rare mutants of TaCPS, TaKS, TaKO, and TaKAO can be found in wheat. Many studies showed that GAs downregulate GA20ox and GA3ox, but do not alter the expression of CPS, KS, KO, and KAO (Itoh et al. 2001; Spielmeyer et al. 2002; Yamaguchi 2008; Jia and Zhang 2009). In the present study, feedback regulation by GA3 or PAC (an inhibitor of GA biosynthesis) on the expression of TaCPS, TaKS, TaKO, and TaKAO was not found, although different copies of TaCPS, TaKS, TaKO, and TaKAO showed different expression levels. This indicates that TaCPS, TaKS, TaKO, and TaKAO may all be necessary in wheat. Acknowledgments This work was supported by the National Natural Science Foundation of China (90717118 and 30521001).

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