MOLECULAR ANALYSIS OF A CANDIDATE GENE TO MANIPULATE PLANT HEIGHT, GIBBERELLIN 2-OXIDASE1 FROM...

In: Plant Breeding ISBN: 978-1-60741-624-1 Editor: Niko Huttunen and Taavi Sinisalo © 2009 Nova Science Publishers, Inc.

Chapter 9

MOLECULAR ANALYSIS OF A CANDIDATE GENE TO MANIPULATE PLANT HEIGHT, GIBBERELLIN 2-

OXIDASE1 FROM SUNFLOWER (HELIANTHUS ANNUUS)

Vania Michelotti1, Filippo G. Carzoli1, Marco Fambrini1, Mariangela Salvini1,2 and Claudio Pugliesi1,*

1Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica, Università di Pisa, Via Matteotti 1B, I-56124 Pisa, Italy

2Scuola Normale Superiore, Università di Pisa, Piazza dei Cavalieri 7, I-56124 Pisa, Italy

ABSTRACT

Gibberellin (GA) signalling pathways are prime targets for producing dwarf high-yielding crop varieties. The GA 2-oxidases catalyze the 2β-hydroxylation of several biologically active GAs, leading to the homeostatic regulation of their endogenous level. A gene, HaGA2ox1, and its corresponding cDNA that contains all the features and exhibits homology to GA 2-oxidases of several plant species has been isolated from vegetative shoots of sunflower (Helianthus annuus). Sequence analysis revealed that HaGA2ox1 consists of three exons and two introns. The predicted amino acid sequence of HaGA2ox1 shares the highest similarities (89% of identity) with the GA 2-oxidase of Lactuca sativa (LsGA2ox1). The phylogenetic analysis divides the large GA 2-oxidase family into three separate classes. The amino acid sequence of HaGA2ox1, included in the class I, predicts that it would be able to catabolize C19-GAs, including bioactive GAs, as GA1, and its immediate precursor, GA20. Within the class I clade, HaGA2ox1 formed a sub-clade with LsGA2ox1 and GA 2-oxidase of Chrysanthemum x morifolium (CmGA2ox1). HaGA2ox1 is ubiquitously expressed in sunflower organs but most abundantly in the inflorescence meristem and in the roots. Treatments of sunflower seeds with gibberellic acid showed that HaGA2ox1 was subject to a slight positive feed-forward regulation by an increase of bioactive GAs. The HaGA2ox1 is a candidate gene to manipulate the amount of bioactive GAs in sunflower by a genetic engineering approach.

* Corresponding author: Pugliesi Claudio, Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica,

Università di Pisa, Via Matteotti 1B, I-56124 Pisa, Italy; Tel. +39(0)502216666; Fax. +39(0)502216661; Email address: [email protected]

276 Vania Michelotti, Filippo G. Carzoli, Marco Fambrini et al.

Keywords: Helianthus annuus; Gibberellins biosynthesis; Gibberellin 2-oxidase; Green Revolution; Sunflower

ABBREVIATIONS GA, gibberellin GA2ox, GA 2-oxidase RACE, rapid amplification of cDNA ends RT-PCR, reverse transcriptase polymerase chain reaction

INTRODUCTION The introduction of dwarfing traits into cereal crops (i.e. wheat and rice) was crucial to

the spectacular increase in food production, obtained since the 1960s and known as Green Revolution (Hedden, 2003; Sakamoto and Matsuoka, 2004). Identification of the genes responsible for these traits shows that they control gibberellin (GA) metabolism and/or perception (Hedden, 2003).

GAs form a large family of tetracyclic diterpenoid phytohormones that mediate environmental and developmental signals acting by regulating cellular processes such as cell elongation and division. These processes manifest themselves in many aspects of plant growth and development including, seed germination, stem extension, flower initiation and development, and fruit growth (Hedden, 1999; Olszewski et al., 2002; Fleet and Sun, 2005; Swain and Singh, 2005; Yamaguchi, 2008). The most important bioactive GAs for vegetative growth and development are probably GA1 and GA4, which differ in the presence or absence, respectively, of a hydroxyl group at C-13 (Sakamoto et al., 2004). In most species that have been investigated, including Helianthus annuus (Hutchison et al., 1988; Pearce et al., 1991), GA1 is the predominant bioactive GA, although some species contains more GA4 than GA1 in shoots (Hedden, 1999; Yamaguchi, 2006). GAs derived from the common diterpenoid precursor gernylgeranyl diphosphate via the tetracyclic hydrocarbon, ent-kaurene (Hedden, 1999; Hedden and Phillips, 2000; Yamaguchi, 2008). Formation of the bioactive GAs from ent-kaurene is the result of a series of oxidative reactions catalyzed by membrane-associated mono-oxygenases and soluble dioxygenases (Sakamoto et al., 2004). Microsomal cytochrome P450 mono-oxygenases synthesize GA12 and GA53 in the early reactions. These GAs are converted to physiologically active C19-GAs by GA 20-oxidases (GA20oxs) and GA 3β-hydroxylases (GA3oxs), two principal dioxygenases. The GA20oxs form a C19 skeleton by the sequential oxidation of C-20 of GA12 and GA53. The GA3oxs catalyze 3β-hydroxylation of the C19 skeleton, resulting in the formation of GAs that are biologically active. A major catabolic pathway for the GAs is initiated by 2β-hydroxylation, a reaction catalyzed by GA 2-oxidases (GA2oxs), 2-oxoglutarate-dependent dioxygenases (2ODDs), that introduce a 2β-hydroxyl group, resulting in biologically inactive products that cannot be converted to active forms (Hedden and Phillips, 2000; Olszewski et al., 2002; Yamaguchi, 2008). These enzymes also inactivate immediate precursors of bioactive GAs such as GA20 and GA9 and they belong to class I or II on the basis of the phylogenetic relationships (Lee and Zeevaart, 2005; Serrani et al., 2007; Yamaguchi, 2008). A new type of GA2oxs that accepts only C20-GAs was also

Molecular Analysis of a Candidate Gene to Manipulate Plant Height … 277

reported (Schomburg et al., 2003; Lee and Zeevaart, 2005). These enzymes are categorized into class III, and are likely to play a role in depleting pools of precursor GAs (such as GA12 and GA53) that are otherwise converted to bioactive forms. Thus, GA2oxs have been considered to play an important role in the regulation of plant growth through the reduction of endogenous levels of bioactive GAs (Kloosterman et al., 2007). New GA deactivation enzymes have recently been discovered, including 16α,17-epoxidase (EUI/CYP714D1) that epoxidizes the 16,17-double bond of non13-hydroxylated GAs (Zhu et al., 2006), and GA methyltransferases (GAMTs) that catalyze methylation of the C-6 carboxyl group of GAs using S-adenosine-L-methionine as a methyl donor (Varbanova et al., 2007). GAs can be also inactivated by conjugation (Olszewski et al., 2002; Yamaguchi, 2008), but it remains unclear whether GA conjugations play any regulatory role in the control of bioactive GA levels.

The development of shorter genotypes is of interest in sunflower breeding. One of the major disadvantages of sunflower, particularly of irrigated one, is the increased plant height in relation to a relatively restricted root system (Weiss, 2000). Therefore, when it is grown in areas where strong winds blow during the seed filling period, plants are subjected to lodging or easily uprooted because wet soil gives little support. The reduction in plant height can led to an improved harvest hindex and to enhanced lodging resistance under high nitrogen fertilization. In addition, sunflower varieties would be also used as potted plants for floret markets if height could be effectively controlled. A strategy to control this trait is to modify GA content by genetic engineering (Sakamoto and Matsuoka, 2004). This can be achieved by suppressing expression of biosynthetic genes (e.g. GA20oxs and/or GA3oxs) or by increasing expression of genes that encode GA-deactivation enzymes, such as GA2oxs (Coles et al., 1999; Hedden and Phillips, 2000; Busov et al., 2003; Sakamoto et al., 2003).

Genes encoding most of the GA biosynthetic enzymes have been isolated from numerous plant species (Olszewski et al., 2002; Yamaguchi, 2008). The first GA2ox cDNA was cloned from Phaseolus coccineus by functional screening, and then three GA2ox cDNAs were cloned from Arabidopsis thaliana by database screening (Thomas et al., 1999). Functional analyses have revealed the important role of several GA2ox genes in the control of GA levels (Thomas et al., 1999; Nakaminami et al., 2003; Sakai et al., 2003; Ubeda-Tomás et al., 2006; Kloosterman et al., 2007; Serrani et al., 2007).

In this chapter, we described the molecular analysis of a GA2ox gene from sunflower (HaGA2ox1). Results afford information on the steady state of HaGA2ox1 mRNA during plant development.

MATERIALS AND METHODS

Plant Material and Growth Conditions Sunflower (Helianthus annuus L.) seeds of the inbred line HOR (provided by the Genetic

Section of the Crop Plant Biology Department of the University of Pisa, Italy) were germinated at 23 ± 1°C in Petri dishes in the dark. Germinated seeds were transferred to 20 cm diameter pots containing a mixture of soil and sand. Plants were grown in a growth chamber at 23 ± 1°C under a 16 h photoperiod (100 µmol m-2 s-1). Irradiance was provided by a mercury vapour lamps (Osram HQI-TS 250 W/NDN, Wembley, England). Sunflower seeds


of the inbred line HOR were also imbibed for three days with gibberellic acid (GA3, 10-4 M) or distilled water in the same conditions described above.

RNA Extraction and cDNA Isolation Total RNA was extracted from H. anuuus samples with the TriPure Isolation Reagent,

according to the manufacturer’s instructions (Roche Diagnostics GmbH). The total RNA (4 µg), from vegetative shoots (VS) of three-week-old plants, was used with the Superscript pre-amplification kit (Invitrogen S.R.L., Life Technologies), to produce the first strand cDNA in conditions recommended by manufacturer. A cDNA fragments, named HaGA2ox1 was obtained with the degenerate primers� GAF2 (forward, 5’-TCTTCTT/CCTC/TG/TCG/ CAAA/TCC-3’) and GAR1 (reverse, 5’-TCAGC/GAGGA/GACAGA/GC/GATCCA-3’), placed in conserved regions shared between the GA2ox genes of Lactuca sativa (GenBank accession no. AB031206), Vigna angularis (GenBank accession no. AB181376) and Pisum sativum (GenBank accession no. AF100954) The HaGA2ox1 cDNA fragment sequencing allowed choosing primers to use in the 5’3’RACE (Rapid Amplification of cDNA Ends) approach, according to the manufacturer’s instructions (Invitrogen). 3’RACE for the HaGA2ox1 fragment was performed with the specific primer FIP1 (forward, 5’-CAGATGAATTGAAACTACAACC-3’) with the following PCR conditions: 5 min for 94°C, 35 cycles (30 s at 94°C, 30 s at 58°C, 1 min at 72°C), 72°C for 7 min. In 5’RACE, the HaGA2ox1-specific primers FIP3REV (reverse, 5’-CCAGAAGTGTTATTGGATCGC-3’) and FIP2REV (reverse, 5’-CTCTGTTCATCTTTCAACAGC-3’) were used in the first and second PCR, respectively. The first PCR conditions were: 94°C for 5 min, 35 cycles (30 s at 94°C, 30 s at 57°C, 45 s at 72°C), 72°C for 7 min. The second PCR conditions were: 94°C for 5 min, 35 cycles (30 s at 94°C, 30 s at 54°C, 40 s at 72°C), 72°C for 7 min. The PCR amplifications performed for the HaGA2ox1 fragment yielded product of about 730 bp and 600 bp at 3’ and 5’ end, respectively, which were cloned into the pDrive Cloning Vector (Qiagen) and sequenced.

Full-length HAGA2ox1 cDNA was obtained with the following primers: GA2oxIN (forward, 5’-CGTTTCTCACATTCTTCTTCTAC-3’), and GA2oxFINE (reverse, 5’-GACACAAGTGGAAGTTTACATC-3’). A product of about 1,200 bp was cloned into the pDrive Cloning Vector (Qiagen) and several clones, containing the entire sequence, were sequenced on both strands.

DNA Extraction and Gene Isolation DNA was extracted from leaves of 30-day-old plants of H. annuus (inbred line HOR)

with the Nucleon DNA extraction and purification protocol for plant tissue, according to the manufacturer’s instructions (GE Healthcare, Bio-Sciences).

To isolate the full-length introns/exons region of HaGA2ox1, the gene-specific primer combinations GA2oxIN/FIP4REV (reverse, 5’-CCAGAAGTGTTATTGGATCGC-3’) and FIP2 (forward, 5’-GCTGTTGAAAGATGAACAGAG-3’)/FIN3REV (reverse, 5’-CTACTTCTATACCTATTCCT-3’) were used for PCRs with H. annuus genomic DNA. The


PCR conditions were: 94°C for 5 min, 35 cycles (30 s at 94°C, 30 s at 55°C, 3 min at 72°C), 72°C for 14 min; 1.5 µM each primer, 0.02 U µL-1 Pfu-DNA polymerase (Promega) and 50 ng genomic DNA. Two amplified products of 1,562 bp (GA2oxIN/FIP4REV) and 1,272 bp (FIP2/FIN3REV) were obtained; they were cloned into the pCRII vector (Invitrogen) and sequenced on both strands. The overlapping region between the two clones (162 bp) was confirmed. Sequence data from this article have been deposited in GenBank under the accession numbers FM872397 and FM872398.

Database Searches and Phylogenetic Analysis Database searches were carried out using the Blast program at the National Center for

Biotechnology Information (NCBI) (Altschul et al., 1997). PROSITE and PFAM databases were searched to identify conserved domains (Bateman et al., 2002; Falquet et al., 2002). A data set including the amino acid sequence deduced from the H. annuus GA2ox mRNA (HaGA2ox1) and other 23 amino acid sequences of GA 2-dioxygenase genes (8 GA20oxs, 6 GA3oxs and 9 GA2oxs) was constructed. The amino acid sequence of desacetoxycep-halosporin C synthase (ScDAOCS) from Streptomyces clavuligerus (Kovacevic et al., 1989; Valegård et al., 1998) (GenBank accession no. P18548) was used as outgroup in the phylogenetic analysis. The sequences were aligned by ClustalW version 1.7 (Thompson et al. 1994) and manually adjusted. The HaGA2ox1 amino acid sequence was also compared to representative class I, II and III GA2ox sequences of several dicot and monocot species (Serrani et al., 2007). In this phylogenetic analysis, the A. thaliana Ga20ox1 (GenBank accession no. X83379) was used as outgroup. The sequences were aligned by Muscle (MUltiple Sequence Comparison by Log-Expectation) at the EMBL-EBI site (http://www.ebi.ac.uk/) and manually adjusted (Edgar, 2004). Phylogenetic analyses was performed using programs from the PHYLIP group [PHYLogeny Inference Package, Version 3.67 (Felsenstein, 1985)]. A distance matrix was computed, according to the Jones-Taylor-Thornton model of amino acid replacement by the program Protdist (Jones et al., 1992), and then it was subjected to the program Neighbor, where the UPGMA (Unweighted Pair Group Method with the Arithmetic mean) method of clustering was selected (Nei, 1985). As support for the tree a bootstrap analysis, with 100 replicates, was performed by Seqboot program and strict consensus trees were obtained by Consense program.

Semi-Quantitative RT-PCR Analysis To analyse the HaGA2ox1 transcript levels, total RNA extractions were carried out from

different organs of the inbred line HOR: zygotic embryos (E) collected at 13 days from pollination (DAP); cotyledons (CO) and hypocotyls (H) of 10-day-old plants; roots (R) and VS of three-week-old plants; inflorescence meristems (IM), internodal sections of apical stem (S), young leaves (2-3 cm long, YL) and mature leaves (10 cm long, ML) of 30-day-old plants grown in growth chamber conditions as above. Total RNA extractions were also carried out from three-day-old seedlings treated with GA3 (10-4 M) or distilled water (control, C). First strand cDNAs derived from 1 or 2 µg of total RNA (see RNA isolation) were used in


all PCR amplifications. PCRs were performed using HaGA2ox1-specific primers: GA2oxREL (forward, 5’-ACAGACTTGGCTT GTTCGAA-3’) and GA2oxFINE (reverse). To normalize for equal amounts of total RNA and efficiency of cDNA synthesis from various samples, the different samples were normalized by parallel amplification of the constitutively expressed Haβ-actin transcript. Haβ-actin primers were: ACT5 (forward, 5’-GATTCCGTTGCCCA/TGAGGTC/T-3’) and ACT3, (reverse, 5’-TC/TTCTGGA/TGGA/TGCAACCACC-3’). Primers were designed to yield a 180 bp and 243 bp fragments for HaGA2ox1 and Haβ-actin, respectively. PCRs were carried out with 28 or 30 cycles where the cDNAs were exponentially amplified. PCRs were performed in 20 µL of 1X buffer (Fermentas GMBH) containing 0.2 mM dNTPs, 0.25 or 0.75 µM of each primer, 2 mM MgCl2, 0.4 U Taq DNA polymerase (Fermentas GmbH) and 0.5 µL of single-strand cDNA. Amplification was carried out according to the following temperature profile: 95°C for 4 min for denaturation, then 94°C for 30 s, 58°C for 30 s, 72°C for 20 s and a final extension of 7 min at 72°C. In the experiment of seed imbibition with GA3, the relative amounts of each PCR product were quantified using a Bio-Rad’s Quantity One software (Bio-Rad Laboratories). The relative expression levels of HaGA2ox1 transcripts were expressed as percentage with respect to the Haβ-actin product. Each percentage value is the mean of three independent experiments. The data, after arcsine transformation, were compared by Student’s t-test (p < 0.05).

RESULTS AND DISCUSSION Control of the endogenous level of physiologically active GAs is important for the

regulation of plant growth. This can be achieved by regulating both the biosynthesis and deactivation of active GAs. There are two major pathways for GA deactivation: hydroxylation on C-2β and glycosylation. Hydroxylation on C-2β, catalyzed by GA2oxs (EC 1.14.11.13), results in the formation of inactive products, and this step is thought to be important for controlling the endogenous levels of bioactive GAs (Hedden and Phillips, 2000; Yamaguchi, 2008). On the basis of sequence conservation, a PCR approach has been used to isolate firstly GA2ox cDNAs and then the corrisponding gene(s) in sunflower. The HaGA2ox1 cDNA is 1,257 nucleotides. Sequence analysis showed a 975 bp coding sequence, flanked by a 91 bp 5’-untranslated region (UTR), a 175 bp 3’-UTR and a 16 bp poly(A) tail (Figure 1). The deduced putative peptide is 324 amino acids long (Figure 1), with a calculated molecular mass of about 36,348 Da and a theoretical isoelectric point (pI) of 6.04.

A phylogenetic analysis was carried out with amino acid sequences derived from published GA dioxygenase genes of several plant species, including the HaGA2ox1 gene of sunflower here described and previously published sequences of sunflower GA20ox genes (Carzoli et al., 2009). Three groups of 2ODDs corresponding to GA20oxs, GA3oxs and GA2oxs were found (Figure 2). The presumed amino acid sequence of HaGA2ox1 of sunflower clustered with the GA2ox sequences (Figure 2).

The dioxygenases belong to a large class of Fe-containing enzymes found in plants and fungi that share a common mechanism (Prescott and John, 1996). In HaGA2ox1, the active site core motif of 2ODDs involved in gibberellin metabolism was present, including the His-196, Asp-198, and His-253 (numbers refer to HaGA2ox1 sequence) that bind Fe2+ at the active site and Arg-263 and Ser-


265 that interact with the 5-carboxylate of 2-oxoglutarate (Figure 3). These residues are conserved in all the GA dioxygenases (Hedden and Phillips, 2000), with Ser occasionally replaced by Thr.

Figure 1. The nucleotide sequence and the deduced amino acid sequence of the HaGA2ox1 cDNA of sunflower (GenBank accession numbers FM872397 and FM872398). The amino acid sequence of the putative coding region is shown below the nucleotide sequence. The stop codon in frame with the open reading frame (ORF) is indicated with an asterisk. The arrows indicate the position of introns in the HaGA2ox1 gene.

To better locate the new HaGA2ox1 gene within the large GA2ox family, a phylogenetic analysis was performed with the amino acid sequences predicted from most of the full-length GA2ox genes found in the databases, using the AtGA20ox1 sequence as outgroup to position the root of the tree. This analysis divides the GA 2-oxidase family into three different clades (Figure 4). Members of classes I and II catabolize C19-GAs (Lee and Zeevaart, 2005; Serrani et al., 2007; Yamaguchi, 2008); whereas, members of class III (e.g. SoGA2ox3, AtGA2ox7, and AtGA2ox8) can only 2β-hydroxylate C20-GAs (Schomburg et al., 2003; Lee and Zeevaart, 2005). The phylogenetic analysis indicates that groups I and II are more closely related to each other that the group III (Lee and Zeevaart, 2005; Serrani et al., 2007). The data suggest that group III diverged from all other GA2oxs before the split between groups I and II. Both monocot and dicot genes are present in each of the three groups, indicating that the


gene duplication events that give rise to these three subfamilies occurred before the split between monocots and dicots (Serrani et al., 2007). The amino acid sequence allows to predict that HaGA2ox1, included in the GA2ox class I, would be able to catabolize C19-GAs, including bioactive GAs, as GA1, and its immediate precursor, GA20. Within the class I clade, HaGA2ox1 formed a sub-clade with GA 2-oxidases from L. sativa (LsGA2ox1) (Nakaminami et al., 2003) and Chrysanthemum × morifolium (CmGA2ox1) (Figure 4). Identities of predicted amino acid sequences HaGA2ox1/LsGA2ox1 and HaGA2ox1/CmGA2ox1 are 89%, and 70%, respectively.

Figure 2. Bootstrap consensus UPGMA tree, based on a Jones-Taylor-Thornton distance matrix. Seqboot, Protdist, Neighbor, and Consense programs of the Phylip package (version 3.67) were used (Felsenstein, 1985). Bootstrap replicates were 100 (values are given at the nodes). The deduced amino acid sequence of HaGA2ox1 (GenBank accession number FM872397) was compared to representative GA 2-dioxygenase sequences including GA 2-oxidases (GA2oxs), GA 3β-hydroxylase (GA3oxs) and GA 20-oxidase (GA20oxs). Accession numbers corresponding to the sequences in the tree are the following: BAB12442 for Lactuca sativa LsGA2ox1, BAG48322 for Chrysanthemum × morifolium CmGA2ox1, ABO27636 for Solanum lycopersicum SlGA2ox5, AAT92094 for Nerium oleander NoGA2ox2, NP_177965 for Arabidopsis thaliana AtGA2ox1, NP_174296 for A. thaliana AtGA2ox2, CAB41009 for A. thaliana AtGA2ox3, AAG51528 for A. thaliana AtGA2ox4, AAG00891 for A. thaliana AtGA2ox6, X83379 for A. thaliana AtGA20ox1, X83380 for A. thaliana AtGA20ox2, X83381 for A. thaliana AtGA20ox3, NM_104778 for A. thaliana AtGA20ox4, DQ056484 for A. thaliana AtGA20ox5, BAB12438 for L. sativa LsGA20ox3, CAQ43616 for Helianthus annuus HaGA20ox1, CAQ43617 for H. annuus HAGA20ox2, L37126 for A. thaliana AtGA3ox1, AF070937 for A. thaliana AtGA3ox2, NM_118289 for A. thaliana AtGA3ox3, NM_106682 for A. thaliana AtGA3ox4, BAG48320 for Chrysanthemum × morifolium CmGA3ox1 and BAA37129 for L. sativa LsGA3ox1. The amino acid sequence of deacetoxycephalosporin C synthase (Chain A) (ScDAOCS) of Streptomyces clavuligerus (GenBank accession no. P18548) was used as outgroup.


Figure 3. Amino acid sequence alignment of the active site core of gibberellin (GA) 2-oxidases, 2-oxoglutarate-dependent dioxygenases (2ODDs) involved in GA catabolism. The Helianthus annuus GA 2-oxidase, HaGA2ox1 (GenBank accession number FM872397), was compared to sequences of GA2ox-related peptides of mocot and dicot species. Residues that bind the active-site Fe2+ are marked in red bold and those that interact with the 5-carboxylate of 2-oxoglutarate are marked in blue bold. The GenBank accession numbers are the following: BAB40934 for Oryza sativa OsGA2ox1, BAC16751 for O. sativa OsGA2ox2, BAC16752 for O. sativa OsGA2ox3, BAC10398 for O. sativa OsGA2ox5, CAE03751 for O. sativa OsGA2ox6, EF441351 for Solanum lycopersicum SlGA2ox1, EF441352 for S. lycopersicum SlGA2ox2, EF441353 for S. lycopersicum SlGA2ox3, ABO27635 for S. lycopersicum SlGA2ox4, Q9SQ80 for Pisum sativum PsGA2ox1, AAD45424 for P. sativum PsGA2ox2, CAB41036 for Phaseolus coccineus PcGA2ox1, AAN87571 for Spinacia oleracea SoGA2ox1, AAN87572 for S. oleracea SoGA2ox2, AAX14674 for S. oleracea SoGA2ox3, AAG50945 for Arabidopsis thaliana AtGA2ox7 and CAB79120 for A. thaliana AtGA2ox8. The other accession numbers corresponding to the sequences in the alignment are the same of Figure 2.


We have conduced analyses to evaluate the HaGA2ox1 introns/exons organisation in the H. annuus genome. Based on the cDNA sequence, specific PCR primer pairs were designed to isolate the full-length introns/exons region of HaGA2ox1. The exon/intron positions were deduced from a comparison of HaGA2ox1 nucleotide sequence with that of its relative transcript and other published genomic sequences of class I GA2ox genes (e.g. AtGA2ox1, AtGA2ox3 and OsGA2ox3), according to the GT/AG splicing rule. The reconstructed 2,791 bp full-length exons/introns region of the HaGA2ox1 gene includes three exons and two introns, flanking by a 91 bp 5’-UTR, and a 175 bp 3’-UTR (Figure 5). Class I GA2ox genes from dicot and monocot species show similar structures (Figure 5). Major differences between the genes were attributable to intron lengths.

The GA 2-oxydase enzymes convert biologically active GAs or their precursors to inactive products and therefore play an essential role in determining GA concentration in plant tissues. There is evidence that members of small GA2ox families, within a species, are differently regulated during plant development, suggesting a unique role for each family member (Olszewski et al., 2002; Yamaguchi, 2008). We analyzed the steady state levels of HaGA2ox1 mRNA to obtain clues about its role during development. HaGA2ox1 mRNA is ubiquitously presents in sunflower organs, but most abundantly in the inflorescence meristem and in the root (Figure 6A). It is likely that in sunflower HaGA2ox1 play a key role in the regulation of endogenous levels of bioactive GAs in most of developmental processes.

GA upregulates genes encoding GA2ox by a positive feed-forward loop, whereas GA20x and GA3ox genes are negatively regulated by GA activity through feedback inhibition. These homeostatic mechanisms would maintain the concentration of active GAs within a limited range (Hedden, 1999; Olszewski et al., 2002; Fleet and Sun, 2005). The expression of GA2ox in the shoot apex of a GA-deficient mutant of Arabidopsis is lower than in wild-type apices and it is substantially increased when the mutant is treated with GAs (Thomas et al., 1999). This result indicates that positive feed-forward regulation of GA2ox gene expression contributes to the mechanism for GA homeostasis (Thomas et al., 1999; Sakai et al., 2003; Wang et al., 2004). In sunflower seeds treated with a high concentration (10-4 M) of gibberellic acid, HaGA2ox1 transcript levels was subject to a slight positive feed-forward regulation by the increase of bioactive GAs (Figure 6B-C). Indeed, some GA2ox genes from dicot and monocot species resulted not regulated by changes in endogenous GA level (Sakamoto et al., 2001b; Nakaminami et al., 2003). It has been also shown that in addition to a metabolic regulation by GAs, other factors can impose, in a strict hierarchy, a control on transcription of genes involved GA metabolism (Desgagné-Penix and Sponsel, 2008). Thus, we cannot exclude that spatial (e.g. organ-specific) and developmental regulation of sunflower HaGA2ox1 genes could partially conceal the positive feed-forward regulation by GA activity.

CONCLUSION Considerable progress has been made in recent years in the isolation and characterization

of genomic and cDNA clones encoding enzymes of GA metabolism (Yamaguchi, 2008). These advances are providing essential basic information on the role of GAs in plant development as well the availability of the enzymes for biochemical studies (Fleet and Sun,


2005). These genes is being also investigated as a target for genetic manipulation to control GA metabolism in crops (Hedden, 2003; Sakamato and Matsuoka, 2004).

Figure 4. Bootstrap consensus UPGMA tree, based on a Jones-Taylor-Thornton distance matrix. Seqboot, Protdist, Neighbor, and Consense programs of the Phylip package (version 3.67) were used (Felsenstein, 1985). Bootstrap replicates were 100 (values are given at the nodes). The deduced amino acid sequence of HaGA2ox1 (GenBank accession number FM872397) was compared to representative sequences of class I, II and III of GA 2-oxidases (GA2oxs) of both mono and dycotiledon species. The accession number of the amino acid sequence of OsGA2ox4 of Oryza sativa is AAU03107. The other accession numbers corresponding to the sequences in the tree are the same of Figure 2 and Figure 3. The amino acid sequence of GA 20-oxidase1 of Arabidopsis thaliana (AtGA20ox1) was used as outgroup (GenBank accession no. X83379).

Semi-dwarfism is a desirable trait in agriculture (Sakamoto and Matsuoka, 2004). When suitable dwarf lines are not available, it is a common practice to control stem growth by treating with growth retardants, which inhibit enzymes in the GA-biosynthetic pathway. However, the application of chemicals for such purposes, particularly to food crops, may not be sustainable in the long-term. An alternative strategy is to modify GA content by genetic engineering (Hedden, 2003; Sakamoto and Matsuoka, 2004). Two approaches to reduce endogenous GA content have been described: suppression of GA biosynthesis and enhancement of GA catabolism. The first approach is the antisense expression of GA biosynthetic genes (Coles et al., 1999; Itoh et al., 2002). In sunflower, two genes,


HaGA20ox1 and HaGA20ox2, encoding GA 20-oxidases, have been recently cloned (Carzoli et al., 2009). These GA20ox genes showed to be highly expressed in elongating sunflower stems, and their suppression could be a strategy to reduce the GA content, and thereby induce a dwarf phenotype. The second approach is exemplified by the overproduction or the ectopic expression of GA2ox genes that could provide the means to increase GA catabolism in transgenic plant, thereby reducing hormone content. Because most of the known dwarf genes are recessive, the ectopic expression of genes that encode GA catabolic enzymes is advantageous and, in principle, can be adopted for transformable crop species (Peng et al., 1999; Busov et al., 2003; Sakamoto et al., 2001b; 2003; Sakamoto and Matsuoka, 2004; Appleford et al., 2007; Dijkstra et al., 2008), including sunflower. However, the HaGA2ox1 gene is broadly expressed in all organ tested, thus it is likely that HaGA2ox1 over-expression could also induce pleiotropic and detrimental effects on flower development. Indeed, in other species, some of transformants overproducing GA 2-oxidase showed severe defects in flower and grain development (Sakamoto and Matsuoka, 2004), indicating that GA is essential for the development of reproductive organs (Swain and Singh, 2005). As such defects compromise breeding programs, it is essential to employ organ-specific or tissue-specific promoters when GA 2-oxidase overproduction is used as a tool to produce dwarf phenotypes of agronomical significance. Several dioxygenases genes are expressed in stem tissues but not in flower organs (Yamaguchi et al., 2006). Thus, the promoters of these genes have been efficiently used to specifically over-express GA2ox genes in the stem. For example, when OsGA2ox1 is ectopically expressed in transgenic rice under the control of the D18 promoter of the GA 3β-oxidase (OsGA3ox2) gene, inhibition of GA biosynthesis was restricted to the leaves and stems (Sakamoto et al., 2003). Although the complexity of GA biosynthesis and signalling provides us with several targets for manipulation, the strategy outlined above enables the introduction of a dominant dwarfing gene to modify plant stature without the need for a conventional, long-term breeding program.

Figure 5. GA 2-oxidase (GA2ox) genes from dicot and monocot species show similar structures. Schematic representation of Class I GA2ox genes of Helianthus annuus (HaGA2ox1; GenBank accession number FM872398), Arabidopsis thaliana (AtGA2ox1 and AtGA2ox3; GenBank accession numbers NC_003070.6 and NC_003071.4, respectively) and Oryza sativa (OsGA2ox3; GenBank accession number NC_008394.1). The exons, numbered sequentially from left to right, are boxed. The 5’ and 3’ untranslated region are boxed in blue. The introns, numbered with italic character, are shown as lines. The base pairs are indicated under the depicted exons and introns.


Figure 6. Steady state level of HaGA2ox1 mRNA in sunflower. Transcript of the Haβ-actin gene was used as an internal amplification control. The RT-PCR products were resolved on a TAE 2.0% agarose gel. (A) Semi-quantitative RT-PCR analysis for HaGA2ox1 transcript accumulation in various sunflower organs. Total RNA was extracted from vegetative shoots (VS), inflorescence meristems (IM), apical internodal stems (S), roots (R), young leaves (YL), expanded mature leaves (ML), cotyledons (C), hypocotyls (H), and immature embryos (E) at 13 days from pollination (DAP). (B) Semi-quantitative RT-PCR analysis for HaGA2ox1 transcript accumulation in three-day-old seedlings treated with 10-4 M gibberellic acid (GA3). Seeds treated for three-days with distilled water were used as control (C). (C) The amount of HaGA2ox1 transcripts was estimated by measuring the ethidium bromide staining of the PCR bands. Values are mean ± SD (n = 3). Values with different letters are significantly different based on Student’s t-test (p < 0.05).

The characterization of gene involved in hormonal metabolism would be also useful to define the complex pathways controlling the meristem activity. GAs are implicated in meristem function throughout interactions with KNOTTED1-like homeobox (KNOX) genes (Hake et al., 2004; Swain and Singh, 2005). KNOX proteins directly repress expression of GA20ox genes (Sakamoto et al., 2001a; Hay et al., 2002), that encode the enzymes of the penultimate step in GA biosynthesis. In addition, SHOOTMERISTEMLESS (STM), a KNOX protein of Arabidopsis, upregulates expression of the GA deactivation genes, AtGA2ox4 and AtGA2ox6 at the base of the meristem, similar to the ring-shaped localization pattern of the OsGA2ox1 transcript that accumulates around the vegetative shoot apex (Sakamoto et al., 2001b). This expression would also contribute to the establishment of a low-GA regime by KNOX proteins (Jasinski et al., 2005). Because KNOX proteins also activate citokinin (CK) biosynthesis (Sakamoto et al., 2006), has been suggested that KNOX activity promotes SAM function by orchestrating a gene-expression regime that results in elevated CK and low GA activity (Jasinski et al., 2005). We have recently demonstrated the involvement of KNOX overexpression in the initiation of ectopic meristems from the abaxial leaf surface of the EMB-2 clone of the interspecific hybrid Helianthus annuus × H. tuberosus


(Chiappetta et al., 2006). The cloning of both GA20ox (Carzoli et al., 2009) and GA2ox genes of H. annuus would be also useful to study the interaction between GA activity and KNOX gene function in the epiphylly of Helianthus.

ACKNOWLEDGMENTS This work was supported by a grant from Ministero Italiano dell’Università e Ricerca

(PRIN 2006-2008).

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