Breadfruit (Artocarpus altilis) gibberellin 2-oxidase genes in stem elongation and abiotic stress response

Plant Physiology and Biochemistry 98 (2016) 81e88

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Research article

Breadfruit (Artocarpus altilis) gibberellin 2-oxidase genes in stem elongation and abiotic stress response Yuchan Zhou a, b, *, Steven J.R. Underhill a, b a b

Queensland Alliance for Agriculture and Food Innovation, University of Queensland, St Lucia, QLD 4072, Australia Faculty of Science, Education and Engineering, University of the Sunshine Coast, Sippy Downs, QLD 4556, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 April 2015 Received in revised form 30 October 2015 Accepted 16 November 2015 Available online 23 November 2015

Breadfruit (Artocarpus altilis) is a traditional staple tree crop in the Oceania. Susceptibility to windstorm damage is a primary constraint on breadfruit cultivation. Significant tree loss due to intense tropical windstorm in the past decades has driven a widespread interest in developing breadfruit with dwarf stature. Gibberellin (GA) is one of the most important determinants of plant height. GA 2-oxidase is a key enzyme regulating the flux of GA through deactivating biologically active GAs in plants. As a first step toward understanding the molecular mechanism of growth regulation in the species, we isolated a cohort of four full-length GA2-oxidase cDNAs, AaGA2ox1- AaGA2ox4 from breadfruit. Sequence analysis indicated the deduced proteins encoded by these AaGA2oxs clustered together under the C19 GA2ox group. Transcripts of AaGA2ox1, AaGA2ox2 and AaGA2ox3 were detected in all plant organs, but exhibited highest level in source leaves and stems. In contrast, transcript of AaGA2ox4 was predominantly expressed in roots and flowers, and displayed very low expression in leaves and stems. AaGA2ox1, AaGA2ox2 and AaGA2ox3, but not AaGA2ox4 were subjected to GA feedback regulation where application of exogenous GA3 or gibberellin biosynthesis inhibitor, paclobutrazol was shown to manipulate the first internode elongation of breadfruit. Treatments of drought or high salinity increased the expression of AaGA2ox1, AaGA2ox2 and AaGA2ox4. But AaGA2ox3 was down-regulated under salt stress. The function of AaGA2oxs is discussed with particular reference to their role in stem elongation and involvement in abiotic stress response in breadfruit. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Breadfruit (Artocarpus altilis) Gibberellin 2-oxidase Stem elongation Dwarfing Drought Salinity

1. Introduction Breadfruit [Artocarpus altilis (Parkinson) Fosberg)] is a traditional staple tree crop in the Oceania. Millennia of domestication has resulted in hundreds of cultivars, some of which are fertile or sterile diploids (2n ¼ 2x ¼ 56) and others sterile triploids (2n ¼ 3x ¼ 84) (Ragone, 2001). The species is primarily grown as an energy food, a source of complex carbohydrates, vitamins and minerals, and has long been recognized as a food security crop in the tropics (Maxwell et al., 2013). However, being an evergreen tree from 15 to 30 m, breadfruit is prone to wind damage. Significant tree loss due to intense tropical windstorm in the past decades has driven an increasing interest in developing dwarf varieties of breadfruit (Zhou et al., 2014). In many temperate and tropical fruit

* Corresponding author. Queensland Alliance for Agriculture and Food Innovation, University of Queensland, St Lucia, QLD 4072, Australia. E-mail address: [email protected] (Y. Zhou). http://dx.doi.org/10.1016/j.plaphy.2015.11.012 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.

tree species, dwarfism has been achieved by the widespread use of dwarfing rootstocks, which has revolutionised fruit production by allowing dense cultivation, increasing harvest index and substantially decreasing production costs (Costes and Garci'a-Villanueva, 2007; Foster et al., 2014). Although great diversity among breadfruit cultivars exists, genetic resource showing dwarf traits is still not known (Zhou et al., 2014). Owing to the fact that dominant alleles resulting in dwarf stature are expected to be rare in some tree species (Busov et al., 2003), and breadfruit species is characterised by long juvenile phase, heterogeneous genetic background and predominantly vegetative propagation (Ragone, 1997; Zerega et al., 2004), breeding dwarfing rootstocks through conventional breeding in breadfruit is therefore very slow and difficult. In this context, insertion of dominant transgenes may be an alternative for imparting dwarf phenotype to such tree species (Busov et al., 2008). Various factors cause dwarfism in plants, of which gibberellin (GA) is one of the most important determinants of plant height (Peng et al., 1999; Chandler et al., 2002; Wang and Li, 2008). GAs are a group of more than 100 tetracyclic diterpenes, some of which

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regulate a wide range of growth and development processes in plants, including stem elongation, flowering and fruit development (Hedden and Kamiya, 1997). Naturally dwarf phenotype is often associated with mutation in genes controlling GA concentration or GA signalling (Peng et al., 1999; Chandler et al., 2002). GA metabolic and response genes have been a logical focus for improving crop performance via both conventional breeding and genetic engineering (Sakamoto et al., 2004). In higher plants, the flux of active GAs is regulated by the balance between their rates of biosynthesis and deactivation (Hedden and Kamiya, 1997). Biosynthesis of GAs in plants starts from geranylgeranyl diphosphate (GGDP), a C20 precursor (Hedden and Kamiya, 1997). The pathway includes consecutive steps that occur in the plastid, endoplasmic reticulum (ER) and cytoplasm, with the final stage of the pathway being carried out by 2-oxoglutaratedependent dioxygenases, GA 20-oxidase and GA 3-oxidase (Hedden and Phillips, 2000; Yamaguchi, 2008). On the other hand, the main route for GA deactivation is through 2b-hydroxylation to produce biologically inactive GAs (Hedden and Phillips, 2000; Yamaguchi, 2008). This step is catalysed by 2-oxyglutaratedependent GA2-oxidases (Thomas et al., 1999). GA 2-oxidases (GA2ox) have been characterized in many plant species including Phaseolus coccineus (Thomas et al., 1999), Arabidopsis thaliana (Schomburg et al., 2003), rice (Sakamoto et al., 2004), spinach (Lee and Zeevaart, 2005), pea (Martin et al., 1999) and poplar (Busov et al., 2003). Overexpression of GA2ox enhanced GA inactivation and induced dwarfism in Solanum species (Dijkstra et al., 2008), Nicotiana tabacum (Ubeda-Tomas et al., 2006), Nicotiana sylvestris (Lee and Zeevaart, 2005), Oryza sativa (Sakamoto et al., 2004) Arabidopsis thaliana (Hedden and Phillips, 2000) and Populus (Busov et al., 2003). However, loss-of-function Arabidopsis mutants revealed gibberellin 2-oxidases had diverse functions in plant growth and development, including negative effect on seed germination, vegetative to floral transition and flower development (Rieu et al., 2008a,b). Breadfruit gibberellin 2-oxidases genes have not yet been cloned, their regulation and developmental function in breadfruit is not known. Although GA biosynthesis inhibitors are widely used as an alternative to mechanical pruning in many fruit trees, the effect of GA or GA biosynthesis inhibitors on breadfruit is rarely examined (Zhou and Underhill, 2015). In this context, investigating the role of GA and the regulation of GA2-oxidase genes is an important first step toward understanding the mechanism of growth regulation in breadfruit. The knowledge may provide opportunity to identify potential dwarfing candidate genes, and design strategies for research and genetic engineering of tree stature in breadfruit. Here we reported the isolation of four breadfruit GA2-oxidase genes, AaGA2ox1 e AaGA2ox4 as well as their expression patters under various growth condition, including exogenous application of GA3 or GA biosynthesis inhibitors, drought and high salinity stress. The physiological function of the AaGA2ox paralogs was discussed with particular reference to their role in stem elongation and, possible involvement of abiotic stress response in breadfruit. 2. Materials and methods 2.1. Plant materials and treatments Breadfruit (Artocarpus altilis) cultivar Cannonball was used in this study. The cultivar is descried as a seedless tree up to 20 m (Goebel, 2004; Zhou and Underhill, 2015). Plants, as rooted cuttings obtained from a nursery at Cap Tribulation, Northern Queensland, were grown under glasshouse condition at 25e28  C with natural daylight and daily water supply. Plants were grown in pots containing vermiculite and soil mixture (1:3), with one application of

10 g of 60 d release fertilizer pellets (Osmocote; Scotts Australia Ltd) added to each pot every month. Plants of 9-month-old (postcutting) were used for the experiment. For GA3 or paclobutrazol treatment, both foliage and soil surface were sprayed with 500 mg L1 GA3 or Paclobutrazol (both from Sigma, St. Lois, USA, dissolved in 0.1% ethanol þ0.1% Triton X-100). Plants sprayed with same concentration of ethanol and Triton X-100 were used for comparison. The treatment was applied twice a week for 4 weeks. Plants were monitored for stem elongation by measuring the length of the first internode from the top. The selected internodes were labelled and re-measured at successive time for a period of 9 weeks. For the drought treatment, water was withheld for 2 weeks after normal watering at Day 0. For salinity treatment, both foliage and soil surface were sprayed with 200 mM NaCl every second days for 2 weeks. All plants were able to recover after removal of the stressor. The apical young leaves at specific time points of the above treatments were collected for RNA extraction. 2.2. Cloning of gibberellin 2-oxidase cDNAs Total RNA was extracted from various organs of breadfruit using RNeasy kit (Qiagen, Australia). Extracted RNA was reverse transcribed with SuperScript reverse transcriptase and oligo(dT) (Life Technologies, Australia). The resulting cDNA was subjected to degenerate PCR using primers 50 - GGI TTY GGI GAR CAY ACI GAY CCI C-30 and 50 -GGI GGI CCI YCR AAR SAI ATC AT-30 . The PCR reactions were performed at 35 cycles with annealing temperature at 53  C. The PCR products were cloned into pGEMT vector (Promega, Australia) and sequenced. To isolate full-length GA2-oxidase genes, total RNA was subjected to 50 and 30 SMART RACE RTePCR followed by full-length amplification (Takara Clontech, USA). For each 50 or 30 RACE PCR product, about 10 clones were sequenced, and their sequence information was used to design primers for full-length amplification. For each gene, the absence of PCR introduced mutation was verified by obtaining identical sequences from five independent clones. Five full-length clones of each gene were sequenced in double strands. The resulting sequences were analysed by Sequencher (version 4.1, Gene Codes Corporation, USA). The sequences of the four breadfruit GA2ox genes, AaGA2ox1AaGA2ox4 reported here are available in GenBank (http://www. ncbi.nlm.nih.gov/) under accession numbers KR150259 e KR150262. 2.3. Quantitative real-time PCR Upon removal from plants, organs (leaves, stems and roots) were immediately snap-frozen in liquid N2. Flowers and immature fruits were harvested from about 15-year-old mature breadfruit trees (altilis cv. Cannonball) at the nursery as described above. At harvest, these organs were immediately immersed in RNAlater (Life Technologies, Australia), before stored at 80  C. Total RNA, extracted from various plant organs, was reverse transcribed as described above. Real-time PCR was performed on a Corbett Research Rotor-Gene 6000 cycler with the QuantiFast SYBR Green PCR Kit (Qiagen, Australia) as previously described (Zhou et al., 2010). Thermocycling was initiated with a 5-min incubation at 95  C, followed by 40 cycles (95  C for 10 s; 60  C for 30 s). The specificity of amplification was confirmed by high-resolution melt curve analysis at the end of each run. The efficiency of each primer set was evaluated by standard curves using serial dilutions of plasmid DNA containing its amplified regions. Each reaction was carried out in duplicate (technical repeat) with non-template control. Gene-specific primers for AaGA2ox1 were 50 -CTG TCG AAC CCG GAT GCC G-30 and 50 - CTG GTC ATG AAC TCC ATT GGG A30 ; for AaGA2ox2 were 50 -CTG TCG AAC CCG GAT GCC A-30 and 50 -

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CTG GTC ATG AAC TCC ATT GGG A-30 ; for AaGA2ox3 were 50 - GAA TCC CGG TAG TGG ACC C-30 and 50 - TGG TCA TGA ACT CCA TTG GGG30 , and for AaGA2ox4 were 50 -CTT AAA ACC CAA CAA ACA ATT TAC AAA C-30 and 50 -CTT CAC AGG CTC TCA CTA TCA AC-30 . The breadfruit actin gene was amplified using primers 50 -AATGGAACTGGAATGGTGAAGGC-30 and 50 TGCCAGATCTTCTCCATGTCATCCT-30 . The expression of each gene was an average of five biological replicates. The transcript abundance was normalized to the expression of actin gene. In specified cases, relative gene expression units were presented as a fold change by setting the expression of one control replicate to a value of 1.

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2.4. Statistical analyses

family members in regulation of many aspects of growth and developments. Of the four breadfruit GA2ox genes, transcripts of AaGA2ox1, AaGA2ox2 and AaGA2ox3 were detected in all plant organs including apical sink leaves, source leaves, stems, roots, flowers and immature fruits, but with predominantly higher level of expression in source leaves and stems (Fig. 3). Compared to AaGA2ox1 and AaGA2ox2, AaGA2ox3 was expressed at much lower level in all organs tested (Fig. 3). In contrast to the three AaGA2oxs above, transcript of AaGA2ox4 exhibited highest expression in roots, but low expression in sources leaves, stems and fruit, and barely detestable in apical sink leaves (Fig. 3). AaGA2ox4 also displayed stronger expression in flowers compared with those of AaGA2ox1 e AaGA2ox3 in the same organ (Fig. 3).

Significant differences were tested using analysis of variance (ANOVA) followed by Tukey's multiple comparison test at P < 0.05.

3.3. Response of AaGA2oxs to exogenous treatment of GA3 and paclobutrazol

3. Results

Although GA biosynthesis inhibitors are widely used as an alternative to the high labour cost of mechanical pruning in many fruit trees, the effect of plant growth regulators on breadfruit species is less known. Here, we examined the elongation of the first internode in response to exogenous application of GA3 and a GA inhibitor, paclobutrazol on breadfruit plants (Fig. 4). These plants were 9-month old (post-cutting) with single-stem, displaying the most active stem-growth localized at the upper end of the stems. In the current study, the length of the first internodes was measured every 2 weeks after time 0 (Fig. 4). The growth pattern in breadfruit is characterised by the progressive development of new internodes from the terminal buds (Ragone, 1997), these recently formed internodes, with uppermost leaves immediately above, were considered as the first internodes until new internodes emerged from their apex, and replaced the first internode position. As a result, while measurement for new internodes was initiated every 2 weeks (week 0, 3, 5 & 7), these selected internodes were also remeasured at successive time in order to maintain continuous measurement of the same segments (Fig. 4). When breadfruit plants were treated with GA3 twice a week for 4 weeks, significant increase in the first internode length was shown from the 5th week, with 3.6- fold increase comparing to the control at the same time (Fig 4). As expected, application of paclobutrazol was able to reduce the internode elongation, with a reduction of 29.9% and 27.0% after seven and nine weeks respectively, compared with the controls at the same time (Fig. 4). Although treatment of GA3 or paclobutrazol significantly affected the length of the internodes, no significant difference in the number of new nodes was observed across different treatment, with new nodes emerging on an average of every two weeks for all plants tested. Feedback regulation of GA2oxs has been observed in many organs that actively synthesize GA especially young leaves, a major site of GA biosynthesis (Lee and Zeevaart, 2002; Lo et al., 2008). In breadfruit, apical sink leaves form part of the most active growth units in stem elongation (Ragone, 1997), with the same preferably expressed GA2ox genes as those in stems (Fig. 3). In addition, the organs expressed four of the six isoforms of GA biosynthesis genes, GA20oxidase recently isolated from breadfruit (Zhou and Underhill, 2015). Here, apical sink leaves were examined for the expression of AaGA2oxs in response to GA3 or Paclobutrazol treatment. At the end of the 4-week treatments, transcripts of AaGA2ox1, AaGA2ox2 and AaGA2ox3 were significantly up-regulated by exogenous GA3 with an increase of 4.1-fold, 8.8-fold and 1.0-fold respectively, compared to those of their controls at the same time (Fig. 5). By contrast, expression of these three transcripts were down-regulated by paclobutrazol treatment, with a reduction of 97.8%, 99.1% and 99.4% respectively compared to those of their controls at the same time (Fig. 5). The expression of AaGA2ox4 remained low and was not

3.1. Isolation of breadfruit gibberellin 2-oxidase cDNAs GA2-oxidase genes were cloned from combined organs (leaves, stem and root) of breadfruit by degenerate PCR using primers corresponding to two conserved regions of all known GA2-oxidase, GFGEH(T/S)DPQ and M(I/V)YF(G/A)GPP (Lee and Zeevaart, 2005; Giacomelli et al., 2013). An expected fragment of 230 bp was amplified and 40 degenerate PCR clones were sequenced. Four distinct groups of GA2-oxidase genes were identified by basic local alignment search tool (BLAST) searches. After 50 and 30 RACE PCR, four genes, AaGA2ox1- AaGA2ox4 were isolated in full-length. Sequence analysis showed that the predicted proteins of the AaGA2ox1-AaGA2ox4 genes bear all the hallmarks of functional 2oxoglutarate-dependent dioxygenase family (Fig. 1). These included the presence of putative 2-oxoglutarate binding sites (Arg-273 and Ser-275), and amino acid residues (His-206, Asp-208 and His-263) presumed to bind Fe at the active site (Giacomelli et al., 2013). Phylogenetic analysis (Fig. 2) defined that GA2ox members could be divided into two distinct classes, the C19 GA2ox and C20 GA2ox ((Lee and Zeevaart, 2007; Lo et al., 2008; Rieu et al., 2008a,b). The predicted proteins encoded by AaGA2ox1-AaGA2ox4 were members of the C19 GA2oxs, which included majority of the GA2oxs including Pisum sativum PsGA2ox, Prunus salicina PslGA2ox and Phaseolus coccineus PcGA2ox1, and were clearly separated from the C20 GA2ox which comprised Arabidopsis thaliana AtGA2ox7, Oryza sativa OsGA2ox6 and Spinacia oleracea SoGA2ox3 (Fig. 2). The predicted proteins of AaGA2ox1-AaGA2ox4 clustered together, with AaGA2ox1 and AaGA2ox2 sharing 97% identity, AaGAox2 and AaGA2ox3 sharing 98% identity, and AaGA2ox1 and AaGA2ox4 sharing 91% identity at the amino acid level (Fig. 2). The AaGA2oxs were closely related to the Morus notabilis homolog MnGA2ox4 (Genbank acc. XP_010110967) with 86 ~ 88% identity and the Cucumis sativus homolog CsGA2ox3 (HE582631) with 70 ~ 71% identity (Fig. 2). 3.2. Expression analysis of GA2-oxidase genes in breadfruit In fruit tree crops, dwarf phenotype induced by GA deficiency is an advantage; however, the availability of sufficient GA in appropriate developmental stages is still very important to ensure proper fruit development and production (Hedden and Kamiya, 1997). This is evidenced by findings in other species that down-regulation of GA2ox genes show defects in floral induction, flower structure and fruit development (El-Sharkawy et al., 2012). Examining expression profile of GA2ox genes in various plant organs therefore provides insight into both the specialized and divergent roles of the gene

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Fig. 1. Alignment of AtGA2ox1 (Arabidopsis thaliana), PcGA2ox1 (Phaseolus coccineus), SoGA2ox1 (Spinacia oleracea) and the deduced amino acid sequences of AaGA2ox1, AaGA2ox2, AaGA2ox3 and AaGA2ox4 from breadfruit. Sequence alignments were performed by ClustalW algorithm. Black shading indicates identical residues, dark-grey shading indicates similar residues in six out of seven of the sequences and clear grey shading indicates similar residues in four out of seven of the sequences. Annotated are: putative 2-oxoglutarate binding sites (^), amino acid residues presumed to bind Fe at the active site (*).

changed following either treatment (Fig 5). 3.4. Expression of AaGA2oxs under drought and salt stresses High salinity and drought conditions contribute to the dwarf phenotype of many species (Knight and Knight, 2001; Naidoo, 2006). To better understand this adaptive trait, the response of GA metabolic genes to abiotic stress has been investigated in organs that actively synthesize GA, including proliferating leaves (Niu et al., 2014; Zawaski and Busov, 2014). Here, the young apical leaves of breadfruit were examined for the response of AaGA2oxs to 2-week treatments of drought or high salinity (200 mM NaCl).

While neither drought nor high salinity led to significant phenotypic symptoms in breadfruit plants under current experiment condition, different response of AaGA2oxs to the stress signals was observed. Treatments of both drought and high salinity were found to significantly increase the expression levels of AaGA2ox1, AaGA2ox2 and AaGA2ox4, with 187.9% increase in AaGA2ox1, 116.8% increase in AaGA2ox2 and 214.2% increase in AaGA2ox4 after drought stress and 146.1% increase in AaGA2ox1, 343.1% increase in AaGA2ox2 and 65.8% increase in AaGA2ox4 after salt stress, compared to those of their controls (Fig. 6). By contrast, the expression of AaGA2ox3 was not changed under drought stress, but down-regulated under salinity stress, with 30% reduction after salt

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Fig. 2. Phylogenetic analysis of AaGA2ox1, AaGA2ox2, AaGA2ox3 and AaGA2ox4 in relation to other plant GA 2-oxidases. The tree was constructed based on maximum likelihood method in the PHYLIP package and unrooted by its Drawgram program. All sequences were obtained from NCBI (http://www.ncbi.nlm.nih.gov). Arabidopsis thaliana: AtGA2ox3 (AJ132437); AtGA2ox1 (AJ132435); AtGA2ox7 (NM_103976); AtGA2ox6 (NM_100121); Cucurbita maxima: CmGA2ox (AJ302041); Cucumis sativus: CsGA2ox1 (HE582629); CsGA2ox2 (HE582630); CsGA2ox3 (HE582631); CsGA2ox4 (HE680067); Gossypium hirsutum: GhGA2ox1 (HQ891930); GhGA2ox2 (HQ891931); Hordeum vulgare: HvGA2ox4 (AAT49062); Lactuca sativa: LsGA2ox1 (AB031206); Lolium perenne: LpGA2ox5 (ABV48913); Morus notabilis: MnGA2ox (XP_010110967); Nerium oleander: NoGA2ox1 (AY594291); NoGA2ox2 (AY594292); NoGA2ox3 (AY588978); Nicotiana tabacum: NtGA2ox1 (AB125232); NtGA2ox2 (AB125233); Nicotiana sylvestris: NsGA2ox1 (AY242858); Oryza sativa: OsGA2ox3 (AB092485); OsGA2ox2 (AB092484); OsGA2ox1 (AB059416); OsGA2ox6 (BAF15255); Phaseolus coccineus: PcGA2ox1 (AJ132438); Pisum sativum: PsGA2ox (Q9SQ80); Prunus salicina: PslGA2ox (AEA51242); Populus tomentose: PtGA2ox (AFP58845); Raphanus sativus: RsGA2ox1(BAM73281); Solanum tuberosum: StGA2ox (NM_001288082); Solanum lycopersicum: SlGA2ox (NM_001247409); Spinacia oleracea: SoGA2ox1 (AF506281); SoGAox3 (AY935713); Torenia fournieri: TfGA2ox1 (BAJ65444); Triticum aestivum: TaGA2ox (AEA30111); Vitis Vinifera: VvGA2ox4 (KC898182); VvGA2ox6 (KC898185); Zea mays: ZmGA2ox (NM_001158585).

stress compared to the control (Fig. 6). 4. Discussion In this paper, we report the isolation of four GA2-oxidase genes, AaGA2ox1 e AaGA2ox4 from breadfruit. Phylogenetic analysis suggested that the four AaGA2oxs were members of C19 GA2oxs (Fig. 2). This group of GA2oxs from many plant species can hydroxylate the C-2 of active C19 GAs (GA1 and GA4) or C19 GA precursors (GA20 and GA9) to produce biologically inactive GAs (GA8, GA34, GA29, and GA51, respectively) (Sakamoto et al., 2004). Plant GA2ox genes are encoded by small gene families of various sizes (Hedden and Phillips, 2000). The four AaGA2oxs in breadfruit, compared with the large size of GA2oxs families found in other species with both C19 GA2oxs and C20 GA2oxs, such as A. thaliana with 7 AtGA2oxs (5 C19 GA2oxs and 2 C20 GA2oxs) (Rieu et al., 2008a,b), Rice (Oryza sativa) with 10 OsGA2oxs (7 C19 GA2oxs and 3 C20 GA2oxs) (Lo et al., 2008) and grape (Vitis Vinifera) with 8 VvGA2oxs (5 C19 GA2oxs and 3 C20 GA2oxs) (Giacomelli et al., 2013), suggest that other breadfruit GA 2-oxidase genes remain to be

cloned. However, the expression profile of genes AaGA2ox1 e AaGA2ox3 found to be predominant in sources leaves and stems in the current study corresponds with numbers of GA2-oxidase isoforms expressed in these organs of other species. For example, populus expressed two C19 GA2oxs (Gou et al., 2011), spinach expressed two C19 GA2ox2 (Lee and Zeevaart, 2005), pea expressed 2 C19 GA2oxs (Lester et al., 1999). The overlap expression pattern of the three AaGA2oxs suggests functional redundancy between these AaGA2ox gene family members may be expected in the aerial vegetative organs. Distinct expression patterns were shown between group of AaGA2ox1 e AaGA2ox3 and the AaGA2ox4, with the former preferentially being expressed in source leaves and stems, and the latter in roots and flowers (Fig. 3). The results are different from those in Arabidopsis where five C19 GA2ox genes are nearly ubiquitously expressed in different organs and developmental stages (Rieu et al., 2008a,b), but are in agreement with reports from some other species where different GA2ox members are predominantly expressed only in a subset of organs. For examples, the populus GA2oxs have two PtGA2oxs primarily expressed in aerial organs, and

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Fig. 3. Expression pattern of the breadfruit GA2-oxidase genes, AaGA2ox1, AaGA2ox2, AaGA2ox3 and AaGA2ox4 in plant organs. Relative expression level of AaGA2ox1 e AaGA2ox4 was determined using real-time quantitative RT-PCR normalized to expression of the actin gene. Results are plotted as the ratio to the lowest detected level (i.e. one replicate of AaGA2ox1 in fruit). All values represent mean ± SE from five separate RNA extractions (n ¼ 5). SkL, sink leaves in shoot apex; SoL, source leaves; St, stems; Rt, roots; Fl, flowers, Fr, fruits.

other two PtGA2oxs predominantly expressed in roots (Gou et al., 2011). Diversified expression patterns are also found in rice with one larger group expressed in vegetative tissues and another smaller group expressed in reproductive stage (Lo et al., 2008). 12

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Weeks Fig. 4. Stem elongation in the first internode of the breadfruit plant following exogenous application of GA3 or paclobutrazol. Plants of 9-month-old (post-cutting) were sprayed with 500 mg L1 GA3 or paclobutrazol twice a week for 4 weeks (Time ¼ 0 ~ 4 weeks in the Figure). Measurement of the first internode length was initiated at 0, 3, 5 or 7 weeks after treatment, and continued at successive time. All values represent mean ± SE from five biological replicates (*P < 0.05).

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AaGA2ox4

Fig. 5. Effect of exogenous GA3 or paclobutrazol treatment on the expression of AaGA2ox1, AaGA2ox2, AaGA2ox3 and AaGA2ox4 in the apical leaves of breadfruit plants. Plants of 9-month-old (post-cutting) were sprayed with 500 mg L1 GA3 or paclobutrazol twice a week for 4 weeks. The expression of AaGA2ox1 e AaGA2ox4 was analysed at the end of the 4-week treatment. Expression level of each transcript was normalized to the expression level of actin gene. The relative gene expression units were presented as the fold change by setting the expression of one control replicate before treatment to a value of 1. All values represent mean ± SE from five separate RNA extractions (*P < 0.05).

Furthermore, transgenic analysis of loss-of function mutation confirmed that the different organisation of GA2ox expression pattern was linked to the coordination of functional redundancy and specialization between GA2ox family members (Lo et al., 2008; Gou et al., 2011). In Arabidopsis, knockout-related phenotypes could only be observed in quintuple mutations, with loss-of-function of all five C19 GA2oxs displaying negative effects on seed germination, delayed vegetative to floral transition, and defects in flower development (Rieu et al., 2008a,b). However, in populus the shootand leaf-expressed GA2oxs were found to specifically restrain aerial shoot growth, with their over-expression leading to strong dwarfing phenotype, whereas the root-expressed GA2oxs functioned to promote root development, with their suppression decreasing root biomass (Gou et al., 2011). In this context, the distinct expression patterns between group AaGA2ox1 e AaGA2ox3 and AaGA2ox4 may reflect functional divergence between the two groups of AaGA2ox family members in their relative contribution to GA deactivation in various organs, with the three AaGA2oxs, AaGA2ox1 e AaGA2ox3 being more specialized in the growth of leaves and stems, and AaGA2ox4 in the growth of roots. Feedback regulation of GA metabolic genes has been described in many species (Hedden and Phillips, 2000). The response of AaGA2ox1, AaGA2ox2 and AaGA2ox3, but not AaGA2ox4 to exogenous application of GA3 or paclobutrazol supported the homeostatic model where treatment of exogenous GA3 promoted internode elongation of breadfruit and also imparted a feedback up-regulation of GA catabolism genes, GA2oxs, and treatment of paclobutrazol, a GA biosynthesis inhibitor, suppressed internode elongation, and also exerted feedback inhibition in these genes (Figs. 4 and 5). These results are also consistent with previous findings in other species where only a subset of GA2ox genes are subjected to GA-regulated feedback (Gallego-Giraldo et al., 2008), and reflect different sensitivity of GA metabolic genes to changes of GA concentration and difference in their spatial expression patterns (Gallego-Giraldo et al., 2008; Rieu et al., 2008a,b). Our results that AaGA2ox1 e AaGA2ox3 were subjected to GA feedback regulation suggests that these genes play active role in GA homeostatic regulation, concurrent with their high expression level in stems and leaves (Fig. 3). Taken together, the gene expression profiles of

Y. Zhou, S.J.R. Underhill / Plant Physiology and Biochemistry 98 (2016) 81e88

9

Relative expression

control 8

drought

7

NaCl

*

6 5 4

* *

* *

3 2

*

1

*

0 AaGA2ox1

AaGA2ox2

AaGA2ox3

AaGA2ox4

Fig. 6. Effect of drought and high salinity treatment on the expression of AaGA2ox1, AaGA2ox2, AaGA2ox3 and AaGA2ox4 in the apical leaves of breadfruit plants. Plants of 9-month-old (post-cutting) were used for the drought and high salinity treatments. For the drought treatment, water was withheld for 2 weeks after normal watering at Day 0. For salinity treatment, both foliage and soil surface were sprayed with 200 mM NaCl every second days for 2 weeks. The expression of AaGA2ox1 e AaGA2ox4 was examined at the end of the 2-week treatment. Expression level of each transcript was normalized to the expression level of actin gene. The relative gene expression units were presented as the fold change by setting the expression of one control replicate before treatment to a value of 1. All values represent mean ± SE from five separate RNA extractions (*P < 0.05).

AaGA2oxs, together with the results of GA feedback regulation, suggest that the three AaGA2oxs, AaGA2ox1 e AaGA2ox3 might have a specialised role in the control of the stem elongation by reducing active GAs in above ground vegetative organs, therefore, may be used as potential targets for genetic engineering of tree stature in breadfruit. The evidence that stem elongation of breadfruit can be manipulated by exogenous gibberellin-related growth regulators provides an opportunity for reducing tree stature through application of GA biosynthesis inhibitors in breadfruit cultivation. However, further investigation on the response and sensitivity of these chemicals in breadfruit species is required. In current study, although the shortterm, repeated treatment of paclobutrazol eventually led to significant inhibition on the stem elongation, the slow growth remained non-significant for 7 weeks after the first application (Fig. 4). This lag-period of response to paclobutrazol has also been reported on many woody species, such as Queen palm (Syagras romanzoffiana) and Manila palm (Veitchia memllii) (Hensley and Yogi., 1996), Royal palms (Ali and Bernick, 2010), sweet cherry trees (Jacyna et al., 1989), sweetgum, pine and oak trees (Bai et al., 2004). More experiments need to be conducted to compare the short-term vs. continuous application of growth regulators, such as paclobutrazol, and the different stages at which the chemicals are applied to provide better control over tree size in breadfruit. A better understanding of the nature of the lag response to paclobutrazol may also help improve its efficacy in horticulture practices, given that long-term and repeated application of such chemicals in fruit trees can be costly both commercially and environmentally. The association of dwarf phenotype with abiotic stress has long been recognized in natural environments (Munns, 2002; Achard et al., 2008). Dwarfism is an adaptive trait that benefits plants through avoidance of high energy costs under unfavourable conditions (Knight and Knight, 2001; Achard et al., 2008; Magome et al., 2008). GA is well known for its role in regulation of stem elongation (Hedden and Kamiya, 1997), its involvement in stress response and adaptation has recently attracted attention (Achard

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et al., 2008; Magome et al., 2008). The results that AaGA2ox1, AaGA2ox2 and AaGA2ox4 were up-regulated by drought and salt stresses (Fig. 6) is consistent with previous findings in Arabidopsis where at least six GA2ox genes were elevated under high-salinity stress (Magome et al., 2008). The expression of GA2ox in tobacco was also increased under salt stress (Cong et al., 2008). Other stress signals, such as cold (Achard et al., 2008; Siddiqua and Nassuth, 2011), drought (Dubois et al., 2013) and cadmium stress (Liu et al., 2015) were also shown to induce the expression of GA2ox genes. The mechanism of stress-related increase in GA2ox expression has been investigated in Arabidopsis. It has been demonstrated that the stimulation of GA2ox genes under cold stress involves a transcriptional activator, the C-repeat/drought-responsive element binding factor CBF1 (Achard et al., 2008). An AP2 transcription factor, DDF1 was shown to play a similar role under salt stress in elevating GA2ox expression in Arabidopsis (Magome et al., 2008), whereas under drought stress, the up-regulation of an Arabidopsis GA2ox gene involved a group of transcription factors, ETHYLENE RESPONSE FACTOR (Dubois et al., 2013). On the other hand, it is interesting that not all GA2ox genes are up-regulated under stress condition; the results that the AaGA2ox3 was down-regulated by salt stress but not changed under drought condition suggested that AaGA2ox3 may differ from the rest of AaGA2oxs in its role in drought and salt stress response. As AaGA2ox1 and AaGA2ox2 are expected to play a role in GA deactivation in leaves and stems (Fig. 3), longterm down-regulation of these AaGA2oxs under prolong drought or salt stress would therefore predict growth inhibition in breadfruit. In horticultural practice, controlled water deficit for regulation of stem elongation is used as an alternative to plant growth retardants in poinsettia plants (Alem et al., 2015). Constant exposure to high salinity is also used to produce shorter stem in rose (Wahome et al., 2000; Oki and Lieth, 2004) and tomatoes plants (McCall and Atherton, 1995). In this context, managing abiotic stress condition for control of tree height in breadfruit deserves further investigation. Breadfruit is well adapted to local climates and soils with varying degree of salt and drought tolerance (Ragone, 1997). While some cultivars have adapted to the wet tropics, showing tolerance to salt spray for brief periods; some are often found on saline soils of coral atolls, displaying tolerance to high salinity (Ragone, 1997). Therefore, selection of varieties tolerant to high salinity or draught stress as rootstocks to induce dwarf phenotype may be a feasible approach for breadfruit dwarfism. The information of GA2ox genes and expression profiles may provide opportunity to develop screening strategies to assist in phenotype characterization of adaptive dwarf traits in natural population. Taken together, we isolated a cohort of four breadfruit GA2oxidase genes, AaGA2ox1- AaGA2ox4 in full-length. The different expression patterns between group AaGA2ox1 e AaGA2ox3 and AaGA2ox4, together their GA feedback regulation suggests there may be functional divergence between the two groups, with probably functional redundancy between AaGA2ox1, AaGA2ox2 and AaGA2ox3 for the control of the stem elongation, and specialization of AaGA2ox4 in roots and flowers of breadfruit. The differential regulation of AaGA2oxs by drought and salt stress signals suggests AaGA2ox3 may differ from AaGA2ox1, AaGA2ox2 and AaGA2ox4 in its role in stress response. The results may reflect different regulatory mechanism and roles of AaGA2ox genes in their contribution to the balance of growth and stress response under drought and high salinity condition. Contributions This work was carried out in collaboration between authors. Authors YZ and SJRU conceived the idea and collected plant materials. YZ conducted molecular analysis, drafted the original

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