Environmental control of branching in petunia.

Plant Physiology Preview. Published on April 24, 2015, as DOI:10.1104/pp.15.00486

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Running head: Environmental control of branching in petunia

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Corresponding author:

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Kimberley C Snowden

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Postal Address: Plant & Food Research Private Bag 92169, Auckland Mail Center, Auckland, 1142, New Zealand Physical Address: Plant & Food Research 120 Mt Albert Road, Sandringham, Auckland, 1025, New Zealand

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Telephone: +64 9 925 7180

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E-mail: [email protected]

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Research areas:

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Genes, Development and Evolution

Signaling and Response

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Downloaded from www.plantphysiol.org on1May 24, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Copyright 2015 by the American Society of Plant Biologists

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Title:

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Authors:

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The New Zealand Institute for Plant & Food Research Limited, 120 Mt Albert Road, Sandringham, Auckland, 1025, New Zealand.

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Summary

Environmental control of branching in petunia

Revel SM Drummond, Bart J Janssen, Zhiwei Luo, Carla Oplaat2, Susan E Ledger3, Mark W Wohlers, Kimberley C Snowden*.

Nutrient availability and light quality regulate branching in plants. Both environmental signals modulate the strigolactone signalling pathway affecting production and perception of this hormone.

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Footnotes

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1 This work was supported by funding from the MeriNet programme, New Zealand Foundation for Research, Science and Technology and AgResearch (grant number C10X0816).

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2 Current address: ClearDetections, Nieuwe Kanaal 7H, 6709 PA Wageningen, The Netherlands.

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3 Current address: Charles Perkins Centre, The University of Sydney Camperdown NSW 2006, Australia

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Address correspondence to [email protected]

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Abstract Plants alter their development in response to changes in their environment. This responsiveness has proven to be a successful evolutionary trait. Here we have tested the hypothesis that two key environmental factors, light and nutrition, are integrated within the axillary bud to promote or suppress the growth of the bud into a branch. Using Petunia hybrida as a model for vegetative branching we manipulated both light quality (as crowding and the red: far red ratio) and phosphate availability, such that the axillary bud at node seven varied from deeply dormant to rapidly growing. In conjunction with the phenotypic characterisation we have also monitored the state of the strigolactone (SL) pathway by quantifying SL-related gene transcripts. Mutants in the SL pathway inhibit but do not abolish the branching response to these environmental signals and neither signal is dominant over the other, suggesting the regulation of branching in response to the environment is complex. We have isolated three new putatively SL-related TCP genes from petunia and have identified that these TCP type transcription factors may have roles in the SL signalling pathway both before and after the reception of the SL signal at the bud. We show that the abundance of the receptor transcript is regulated by the light quality such that axillary buds growing in added far red light have greatly increased receptor transcript abundance. This suggests a mechanism whereby the impact of any SL signal reaching an axillary bud is modulated by the responsiveness of these cells to the signal.

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Introduction

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To survive and reproduce, plants must respond to continual changes in their local environment, often having to integrate multiple simultaneous changes. Each individual plant integrates inputs from the environment with endogenous developmental programs to achieve optimal growth, survival and reproduction. To achieve this, plants have evolved phenotypic plasticity (Nicotra et al., 2010), the ability to modify growth and development over their entire life span, in some cases many decades or hundreds of years. The control of branch outgrowth is one of the phenotypes modulated to optimise plant growth. Although the growth of a new branch leads to greater persistence, increased light and carbon capture, and may allow greater production of flowers, it is energetically costly. The decision to produce a new branch therefore likely involves signalling from multiple pathways.

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Branch formation begins with a genetic program that leads to the growth of axillary meristems from the axils of developing leaves (Steeves and Sussex, 1989; Janssen et al., 2014). In many species these meristems are produced in every leaf axil soon after the boundary layer forms between the developing leaf primordia and the apical meristem (Bell et al., 2012; Gendron et al., 2012; Yang et al., 2012; Naz et al., 2013; Janssen et al., 2014). In a number of species the growth of these meristems is suppressed based on their position on the plant. In petunia (Petunia hybrida), for example, the axillary meristems in the nodes of the cotyledons and those of the first two leaves usually do not grow into branches, even in environmental conditions greatly favouring vegetative plant growth (Snowden and Napoli, 2003; Snowden et al., 2005). Stopping these four lowest axillary meristems from growing requires production of the hormone strigolactone (SL) and its reception; in both SL production and signalling mutants these normally suppressed nodes produce fully elaborated branches (Napoli and Ruehle, 1996; Snowden and Napoli, 2003).

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One of the costs of growing a branch is the allocation of mineral nutrients. Two of the major macronutrients that limit growth are nitrogen and phosphorus (Marschner, 1995) and the relationship between N, P and SL is well established (Yoneyama et al., 2007; Yoneyama et al., 2007; Lopez-Raez et al., 2008; Umehara et al., 2010). Even before their identification as SLs, it was shown that a factor involved in Orobanche seed germination was produced in response to low P and N (Nagahashi and Douds, 2000; Yoneyama et al., 2001). Once identified, it was definitively shown that SLs (their production and exudation) were responsive to low N and P (Yoneyama et al., 2007; Yoneyama et al., 2007). SLs affect multiple aspects of root development in response to P and sometimes N (Mayzlish-Gati et al., 2010; Kapulnik et al., 2011; Kapulnik et al., 2011; Ruyter-Spira et al., 2011; Arite et al., 2012; Rasmussen et al., 2012; Sun et al., 2014). In rice, SL may be the primary regulator of root architecture responses to both P and N (Sun et al., 2014). Linking branching to SLs came when it was demonstrated that the d mutants of rice, the rms mutants of pea and the max mutants of Arabidopsis were defective in SL biosynthesis or reception (Gomez-Roldan et al., 2008; Umehara et al., 2008). Additional results from Arabidopsis confirmed that, in response to low P, SL increased in root exudates and in xylem sap and branch outgrowth was reduced (Kohlen et al., 2011).

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The light required for carbon fixation is also a major limiting factor in the production of new branches. The shade avoidance syndrome (SAS) in plants is a collection of responses evolved to


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maximise the chance of outcompeting other plants in the collection of light: plants typically grow rapidly taller at the same time as limiting secondary growth in the stem, they also suppress branching and decrease leaf angle relative to the stem, all responses evolved to lift leaves above those of neighbouring plants (Franklin, 2008; Casal, 2012). The SAS is triggered in plants when the red to far red ratio (R:FR) decreases, for example when plants are grown in crowded conditions. Plants have a number of red light sensors in the form of the phytochrome proteins, but with respect to biotic competition for light in natural environments, phyB is the dominant player in the signalling pathway (Franklin, 2008; Casal, 2012). Branching is suppressed in low R:FR light conditions in a number of species and in the species studied thus far a major component of the far red induced suppression of branching are the Type II TCP (named for Teosinte branched 1, Cycloidia and Proliferating cell factor) transcription factors including TB1 of maize; FC1/OsTB1 of rice; SbTB1 of Sorghum; PsBRC1 from pea; and the BRC1 and BRC2 genes of Arabidopsis (Doebley et al., 1995; Takeda et al., 2003; Aguilar-Martínez et al., 2007; Finlayson, 2007; Kebrom et al., 2010; Minakuchi et al., 2010; Braun et al., 2012). The connection between these genes and phyB signalling is well established, however, whether their mode of action involves SL signalling remains unresolved (Kebrom et al., 2006; Finlayson, 2007; Finlayson et al., 2010; Whipple et al., 2011; Dun et al., 2012; Guan et al., 2012; Gonzalez-Grandio et al., 2013; Guo et al., 2013) reviewed in (Janssen et al., 2014).

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Recently we proposed a model bringing together all of the genes that had been suggested to play a role in the SL pathway to show the interactions that had been described (Janssen et al., 2014). Due to the nature of the data the model was developed incorporating experimental evidence spanning petunia, Arabidopsis, pea, rice, sorghum and maize. SL is derived from carotenoids via the sequential actions of a carotenoid isomerase, two carotenoid cleavage dioxygenases (CCD7 and CCD8) and a cytochrome P450 (Abe et al., 2014; Janssen et al., 2014; Zhang et al., 2014). Following production, SLs are exported by an ABC-type transporter, PDR1 (Kretzschmar et al., 2012). The increase in SL content and exudation by plants in low phosphate environments has been linked to increased expression of these biosynthetic genes (Umehara et al., 2010).

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Recent work has shown that the SL reception system uses a receptor protein (DAD2/D14) that hydrolyses the SL molecule to inactive compounds (Boyer et al., 2012; Hamiaux et al., 2012; Zhao et al., 2013). The SL receptor mutants (dad2, d14) and the mutants in an F-box protein (max2, d3) are completely insensitive to SL (Gomez-Roldan et al., 2008; Umehara et al., 2008; Arite et al., 2009; Hamiaux et al., 2012). The F-Box LRR protein MAX2 is involved in the signalling pathway by direct interaction with DAD2/D14 at the protein level in response to SL, interestingly this F-box protein is also involved in the response to another signal (Woo et al., 2001; Stirnberg et al., 2007; Nelson et al., 2011; Hamiaux et al., 2012; Waters et al., 2012). SL-induced interaction between DAD2/D14 and MAX2 results in ubiquitination and proteosome-dependent degradation of D53, an EAR motif containing protein which in turn can interact with the transcriptional repressor TOPLESS (Jiang et al., 2013; Zhou et al., 2013).The receptor protein is also degraded in response to SL in Arabidopsis in a max2 and proteosome dependant manner (Chevalier et al., 2014). This suggests that axillary meristems will only be restricted in their growth under conditions where both SL and receptor protein are constantly generated. This should allow a rapid return to growth upon exposure to a favourable environment.

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The connection between light, the TCP genes and the SL pathway appears to vary between species. In rice, it has been shown that phytochrome signalling affects the SL receptor’s transcript levels by


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altering the interaction of OsMADS57 and the rice TCP OsTB1 (Guo et al., 2013). This suggests that light quality and TCP proteins are upstream of the SL receptor and that they may control the sensitivity of plants to branch inhibition by SL by altering the availability of the receptor protein. Other reports have suggested a complete disconnect between TCP and SL in maize (Whipple et al., 2011; Guan et al., 2012). By contrast, in Arabidopsis the BRC1 and BRC2 genes seem to have diversified in their function (Aguilar-Martínez et al., 2007; Finlayson, 2007; Gonzalez-Grandio et al., 2013), BRC1 appears to act downstream of the SL signal with transcript levels of BRC1 altered in SL mutants, whilst BRC2 expression is unaffected in SL production or signalling mutants (AguilarMartínez et al., 2007). Both the brc1 and brc2 mutants themselves have an increased branching phenotype, although this is quite mild for brc2. A TCP orthologue from pea, PsBRC1, is responsive to SL (Braun et al., 2012; Dun et al., 2012) with PsBRC1 mRNA levels regulated in a translation independent manner, suggesting SL-induced degradation of a transcriptional repressor of PsBRC1.

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Here we report experiments that alter phosphate and nitrogen availability and quantify the effect on the growth of petunia plants, in both wild type and SL signalling mutants. In parallel, we altered the light quality experienced by vegetative phase petunia plants and measured the affect of this environmental variable. Phenotypic measurements in a two-factor array of treatments confirmed that petunia’s response to the environment integrates the combination of inputs to produce a, presumably, optimised architecture. Against the background of these phenotypic changes, we have measured expression of genes known to be in the SL pathway and tested the hypothesis that light quality, perceived at the axillary bud, alters the availability of the receptor complex thus moderating the effect of nutrient-regulated SL signalling from the roots.

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Results

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Light quality and nutrient availability act in concert to regulate axillary bud outgrowth.

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A series of experiments was carried out to determine the effects of altering phosphate, nitrogen, crowding and light quality (as the red: far red ratio) on the growth of petunia. In these experiments we included a Petunia hybrida SL biosynthetic mutant (ccd8, also known as dad1-1), the SL receptor mutant (dad2) and the isogenic wild-type plants.

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To understand the impacts of nutrients we examined the effects of nutrient status (altering either phosphate or nitrogen availability) and the SL pathway genes on growth responses in hydroponically grown petunia. The three genotypes grew as expected in the control conditions, with the mutant lines being shorter, producing more branches and having delayed leaf senescence (Fig. 1) as shown previously (Napoli and Ruehle, 1996; Snowden and Napoli, 2003; Snowden et al., 2005). In low phosphate (Fig. 1A – C) and low nitrogen (Fig. 1D – F) conditions similar trends were observed. We also quantified shoot and root mass, and provide more detail of branching into primary and secondary types, and in the nitrogen experiment measured the affect on branching angle which is altered in dad mutants (Supplemental Fig. S1 and S2). The wild-type petunia responded to both low phosphate and low nitrogen with a reduction in branching and increased leaf senescence, but no significant change to plant height (Fig. 1A – F). The ccd8 and dad2 mutants both showed an increased number of branches under low nitrogen or low phosphate conditions compared to wildtype; however, both mutant lines were capable of responding to a reduction in nutrients with reduced branching compared with the mutants grown in nutrient sufficient conditions. Hence, SL signalling is required for normal growth in normal and low phosphate and nitrogen conditions but SL pathway mutants in both biosynthesis and perception are still able to respond to low nutrient availability by suppressing branching (Fig. 1B and 1E and Supplemental Fig. 1 and 2).

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To examine the effect of light quality and the SL pathway’s involvement in growth responses we grew petunia in crowded conditions without nutrient limitation. Any changes observed are the combined action of the thigmotactic, light quality and light intensity inputs. The experiment created three classes of crowding, considering the middle, edge and corner plants of a densely planted stand as separate classes (Supplemental Fig. 3). As the number of neighbouring plants increased, all three genotypes had increased plant height and decreased branching (Fig. 1G – H). The impact on primary versus secondary branching is shown in Supplemental Figure 3. Although the ccd8 and dad2 mutants could still respond to crowding conditions, both the branching and height traits were still significantly different from the wild-type phenotype. Hence, whilst the SL pathway contributes to the control of height and branching at the different planting densities tested, both biosynthesis and perception mutants remain competent to respond to the proximity of neighbouring plants. Interestingly, while the corner and edge (with three or five neighbours respectively) treatments showed the previously reported (Snowden et al., 2005) delayed leaf senescence in the ccd8 and dad2 mutants (Fig. 1I), when entirely surrounded by competing plants (middle treatment) both of these mutants had a rate of leaf senescence indistinguishable from wild type. This suggests that some signal in this crowding experiment is able to bypass the mutation in the SL signalling system to induce leaf senescence.

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To isolate light quality (as the red:far red ratio) from other signals in our crowding experiment we altered light quality by specific wavelength addition. Red (660 nm) and far red (735 nm) LED light


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sources were used to supplement daylight in a greenhouse altering the red:far red ratio. The ccd8 mutant displayed the expected decreased height, increased branching and delayed leaf senescence compared with the wild type, and these differences were apparent in both light conditions (Fig. 1J – L). As part of the process of developing a more rapid (early detection) phenotyping technique for branching in petunia we investigated three alternate measures of branching: branch count data (as previously used in the experiments shown in Figure 1B, E and H), summed branch length (in mm) or ‘leaves on branches’ count data. All three methods were able to distinguish wild type from ccd8 plants and detect alterations in branching due to light quality (Supplemental Fig. 4). The ‘leaves on branches’ count data gave a robust branching measure and was easily measurable early in development and in plants with limited branch growth and hence was used for subsequent experiments. Both wild type and ccd8 mutant petunia were taller and had fewer branches in the low R:FR treatment (i.e. far red added treatment, Fig. 1J – K). However, the magnitude of the branching response in the ccd8 plants was suppressed (4.4 fold in wt versus 1.3 fold in ccd8).We measured the diameter of the hypocotyls and stems of the plants in this experiment. In both cases the ccd8 mutant was insensitive to light quality whereas the wild type stem growth decreased with the addition of far red light (Supplemental Fig. 4). Hence, SL signalling is required for normal growth in a range of light quality environments, but some SL independent systems exist that are able to adjust a range of plant growth characteristics in response to altered R:FR ratios.

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Simultaneous changes in nutrient and light quality interact to alter branching

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To determine whether one environmental condition might take priority over another we designed an experiment to alter both nutrient availability and light quality. We regulated both phosphate and the R:FR ratio simultaneously in a 3 by 3 array of treatments. Both normal amounts of phosphate and increased red light led to higher branch numbers (Fig. 2). These affects are additive such that the plants grown in red light with sufficient phosphate are the most branched. By contrast, sufficient phosphate and increased far red light produced the tallest plants (Fig. 2B). Again the impact of the two environment factors was additive. At 250 µM P and 5 µM P far red light increases the height of the plants above that of white light. However, when phosphate is decreased to zero in the media this effect is lost, suggesting the P has become limiting. Only very minor changes were observed in root length across the treatments (Fig. 2C). However, a visual inspection of the plants at harvest suggests that root development may have been altered in this experiment in a manner not revealed by a simple measurement of root length (Supplemental Fig. 5).

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A detailed characterisation of the growth of the axillary buds at each node of the plants was performed (Fig. 2D). The data supported earlier observations that petunia branch in a zone starting from node 3 in growth promoting conditions (Snowden and Napoli, 2003). Nodes 1 and 2 are kept in a dormant state by developmental or positional cues that are largely insensitive to environmental conditions. Nodes above this are increasingly able to grow out to produce branches with the most growth observed at nodes 6 and 7 in our experiment, and less branch growth is observed at nodes closer to the apex. Axillary buds at node 7 show a wide range of growth, from none detected in the 0 µM P/far-red light treatment to maximal in the 250 µM P/red light treatment. Within each light treatment the three phosphate treatments alter the growth of the plants such that three distinct patterns of axillary bud growth are seen. Within the phosphate treatments there is significant overlap in the growth patterns observed in the far red and white treatments.


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CCD7 promoter expression indicates biosynthetic gene expression is predominantly in roots and stems and is responsive to low PO4 in fine roots

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Earlier experiments have shown that the biosynthetic pathway leading to the production of SL is largely localised in the roots and stems of plants (with weak expression in leaves) and that production of SL is responsive to a number of developmental and environmental factors (Lopez-Raez et al., 2008; Umehara et al., 2010; Kohlen et al., 2011; Yoneyama et al., 2012; Foo et al., 2013) and the biosynthetic genes respond to nutrient stress (Umehara et al., 2010). These observations indicated which tissues would be most informative for examination of the responses to environmental signals. To define target tissues further, we examined the CCD7 expression pattern in petunia plants carrying a CCD7 promoter-GUS fusion showing that this promoter is most strongly active in low stem tissue and in proximal tissues of the main root (Fig. 3). Cross sections of the stem suggest that the expression is wide spread near the base but becomes restricted to narrow vascular traces in more acropetal regions of the stem (Fig.3A, D – H). Interestingly there is a tightly localised zone of expression in the vasculature at the junction between the leaf petioles and the stem, immediately adjacent to the axillary bud (Fig. 3B). In conditions of plentiful phosphate very little GUS staining was observed in fine roots, however in low phosphate conditions staining was more readily apparent (Fig. 3C).

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Three TB1-like TCP transcription factors were isolated from Petunia hybrida cDNA

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To investigate whether petunia TCP gene family members could be involved in controlling branch growth, particularly under different light conditions we isolated petunia TCP genes using nested RACE with degenerate primers designed to the conserved TCP domain. cDNA from a mixture of above ground tissues was used as template and three petunia TCP genes (PhTCP1, PhTCP2 and PhTCP3) with sequence similarity to the TB1-like TCP genes were isolated. The TCP gene family is divided into two distinct clades (class I & II) with a shared highly conserved TCP domain. A phlyogenetic analysis of the TB1 sub-clade of class II TCP proteins from rice, Brachypodium, Arabidopsis, tomato, potato, Medicago, date palm, lotus, Physchomitrella and poplar is shown in Figure 4, aligned with the maize TB1, pea BRC1 and the three petunia TCP genes (phylogenetic analysis of the entire TCP class II clade is shown in Supplemental Figure 6, see also Supplemental Data 1). All of the genes known to be involved in branching fall into a single TB1 sub-clade within the class II genes (Fig. 4B). There is little homology within this sub-clade between species except for within the three Solanaceae species where potato and tomato form 6 pairs of similar genes. For three of the tomato and potato pairs there is a corresponding petunia TCP, suggesting that there may be further petunia TCP genes to be identified. All the members of the TB1 sub-clade share a conserved SRXKAR(E/A)RARERT motif in the C-terminal region except for SlTCP25, StTCPo and PhTCP2; however, the overall feature of this class of genes is the lack of sequence conservation between species apart from the conserved TCP domain.

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To examine the involvement of these PhTCP genes in the SL signal transduction pathway qPCR was used to examine the expression of these genes in wild type and dad2 mutant petunia. The expression of the PhTCP1 and PhTCP3 genes is decreased in the dad2 mutant indicating that these genes are likely to be involved in SL signalling (Fig. 4A). The expression of the cell cycle gene CDKB is included as a marker for cell division (growth). In the case of PhTCP2 no changes in expression were observed between wild type and the dad2 mutant. This result limits any function of PhTCP2 in the SL

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pathway to upstream of DAD2, and further suggests that there is no feedback regulation from the SL pathway regulating this gene. Given the nearly 100-fold change in the expression of PhTCP3 in axillary buds between wild type and the dad2 mutant we suggest that PhTCP3 expression is dependent on a functioning SL perception system and our observations place PhTCP3 downstream of DAD2. These results are consistent with PhTCP3 (and to a lesser extent PhTCP1) being similar in function to AtBRC1 and PhTCP2 being similar to AtBRC2 (Aguilar-Martínez et al., 2007; Finlayson, 2007).

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Molecular responses to phosphate availability are predominately in fine roots

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To determine the status of SL signal production and signalling in the plants from the multienvironmental factor experiment we investigated the expression of the petunia TCP genes as well as other known SL pathway genes. We quantified the relative transcript abundance of these genes in a range of organs including those shown to express SL biosynthetic (Fig. 3) and perception genes (Supplemental Data 2). In Figure 5 we present the average relative transcript abundances (normalised per gene) grouped by organ (Fig. 5A), nutrient (Fig. 5B) and light (Fig. 5C) treatment with evidence of any changes determined by ANOVA with significance indicated by a P value of less than 0.05.

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Previous results suggested that fine roots, the main root and the low stem would be the major sites of SL synthesis gene expression (Snowden et al., 2005; Drummond et al., 2009). We detected the greatest abundance of CCD7 and CCD8 and also PDR1 transcripts in these organs (Fig. 5A). The transcript of CCD7 was not detectable in the axillary bud or leaf samples. Both CCD8 and PDR1 were detected in these tissues, although at relatively low levels. Prior results also suggested that the genes for the receptor complex (DAD2 and PhMAX2A) would be most abundant in axillary buds and leaves (Drummond et al., 2012; Hamiaux et al., 2012). This can also be observed in the experiment presented here, with the transcripts for these genes also detectable in the remaining organs (at approximately 10% of their maximum). All three of the newly isolated TCP transcription factors’ transcripts are most abundant in the two axillary bud samples with either low or non-detectable abundance in the remaining organs. The transcript abundance of CDKB, a marker for cell division, was high in axillary bud 7, low stem, main root and fine roots. CDKB transcript levels were also moderate in axillary bud 2 and low in leaf 7. Our results suggest that the blade of leaf 7 has largely finished cell division and that axillary bud 2 (usually dormant) may still contain a pool of dividing cells.

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We detected significant increases in the transcript abundance of the SL synthesis and transport genes in fine roots in response to decreased phosphate (Fig. 5B). Conversely, the DAD2 receptor transcript decreased in fine roots, main roots and axillary bud 2 in response to decreased phosphate. In this analysis the PhMAX2A transcript abundance was not statistically significantly regulated by phosphate availability or light quality. However, a more detailed analysis of the raw transcript abundance data identified positive correlations (Spearman’s rank correlation coefficients (rs) for different organs of 0.60–0.91; P ≤ 0.001) between these two genes in all the organs tested. The fine root samples showed the most significant changes in gene expression with respect to phosphate, these data are shown in more detail in Fig. 6. The CCD7, CCD8 and PDR1 transcript abundances in the low and minus phosphate treatments are all greater than those in the plus phosphate treatment


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(see also Fig. 5). With respect to DAD2 the decrease is only significant in the red light treatments (Fig. 5C).

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Molecular responses to light quality are widespread, but largely absent from axillary bud 2

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In Figure 5C we also present our qPCR data grouped by light treatment. By contrast to the limited number of changes seen in response to phosphate, the affect of light was seen in a broad set of tissues, particularly axillary bud 7 and the main root. In these two organs, as well as leaf 7 and low stem, the transcript abundance of CDKB decreases with decreasing R:FR ratio. This observation indicates that the light treatments have had the expected major impact on the cell cycle in these plants and this is reflected in the growth phenotypes observed (Fig. 2).

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In both the low stem and main root, where overall relative expression of CCD7 and CCD8 are high the transcript abundance of these genes decreases as the R:FR ratio decreases. This suggests that the most branched plants might be producing the most SL (assuming that increased expression of CCD7 and CCD8 leads to increased SL production). This positive correlation (rs = 0.45–0.61; P ≤ 0.007) of CCD7 and CCD8 expression with branching is consistent with earlier reports (Foo et al., 2005; Snowden et al., 2005; Johnson et al., 2006; Arite et al., 2007; Simons et al., 2007), where it has been suggested that the relationship was explained by negative feedback from growing branches.

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The transcripts for the SL receptor complex and the TCP transcription factors also show significantly altered patterns of abundance in response to the light treatments (Fig. 5C and Fig. 7) with increased R:FR ratios correlated with decreased receptor complex gene expression and hence decreased sensitivity to SL. By contrast, in axillary bud 2, whilst the abundances are similar, there is little or no significant regulation by light (Fig. 5C and Supplemental Fig. 7). The relationships between the DAD2 SL receptor transcript and the three TCP transcription factors showed different patterns with PhTCP3 showing a good correlation with DAD2 in axillary bud 7, PhTCP2 better correlated in axillary bud 2 and PhTCP1 poorly correlated in either sample (Table I).

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We examined the inter-relationships of the molecular data and the phenotypic data using principle component analysis (PCA) to analyse and visualise the data (Fig. 8). By graphing dimensions 1 and 4 of the PCA we were able to separate red light (square symbols) from white and far red light and plus phosphate (filled symbols) from low and minus phosphate (Fig. 8A). This suggests that dimension 1, which explains 27.06% of the data can be attributed to the effect of light quality and dimension 4, which explains 8.13% of the variation, can be attributed to phosphate availability. Graphing dimensions 2 (14.44%) and 3 (9.33%) allowed some separation of the three replicates (Fig. 8B). Samples were taken over an 8-hour period over the middle of a ~14 hour daylight period with each replicate completed before the next was started. This PCA analysis suggests that some other minor changes in gene expression in the samples may be occurring that were not directly tested here (e.g. circadian rhythms, temperature, humidity, other light effects).

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Using the same PCA axes we plotted the latent vectors (as terminal points) for the individual factors in the experiment, both molecular and phenotypic (Fig. 8C and D). This graphing showed clustering between growth of branches and expression of CDKB in three of the four dimensions presented as expected for a gene linked to cell cycle (see also Table II). DAD2, PhMAX2A, PhTCP2 and PhTCP3 expression loosely cluster together for dimensions 1 and 4, consistent with the positive correlation results for these genes mentioned above (Fig. 8C, Table I). This cluster of gene expression is


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separated from the branch growth phenotypic data, which can also be seen with negative correlations, indicating the expected negative control of branching by the SL pathway (Table II). PhTCP3 shows the strongest negative correlation to the branch growth data, indicating this gene likely has more effect on branch growth than PhTCP1 or PhTCP2. Additionally, clustering of CCD7 with CCD8 indicates that these two genes are likely co-ordinately expressed in at least root and low stem tissues (as mentioned previously we did not detect CCD7 expression in axillary bud or leaf samples). Correlations between CCD7 and CCD8 were 0.58 for low stem (P = 0.001), 0.87 for main root (P < 0.001) and 0.88 for fine roots (P < 0.001).

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Discussion

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The observation that both light and nutrients affect branch formation is well established. In these experiments we sought to examine the interplay between environmental cues and the SL pathway in modulating growth of plants. We found that alterations in nutrient availability and light quality can additively effect the growth of branches, and that this control is overlaid on the developmental control of axillary meristem responsiveness to give a range of potential phenotypes for a plant. SL mutants showed increased branching under various non-optimal growth conditions, indicating a role for the SL hormone in regulating this process.

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However, both SL production mutants and reception mutants were still able to modify their phenotype in response to the environmental stimuli suggesting (unsurprisingly) that the SL pathway is unlikely to be the only regulatory pathway involved in the response to these environmental signals. For example, the delayed leaf senescence phenotype observed in both SL receptor and production mutants is completely absent in the crowded plants indicating that a leaf senescence signal induced by crowding is independent of the SL pathway. With respect to branching, other signals, including but probably not limited to auxin, also play a role in the plant’s response to light quality and nutrient availability (Casal, 2012; de Jong et al., 2014; Reddy and Finlayson, 2014; Zhang et al., 2014). A complex interplay between different plant hormone signals is to be expected for a process as energetically costly and developmentally significant as branch growth.

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Using conditions identified from preliminary experiments we designed an experiment that modulated both phosphate levels and R:FR ratios together to produce a range of developmental outcomes from extreme inhibition of branching to very active outgrowth of axillary meristems to form branches. We were able to establish conditions where the phenotype was significantly altered and determine which nodes were most consistent in their branching response. The length of treatment for the light x phosphate experiment was 7 days. This treatment time is relatively long for a light treatment as some light responses are known to be fast and transient (Franklin, 2008; Casal, 2012). However, growth of a branch is more likely to be affected by persistent changes in light conditions (such as shading by neighbouring plants) over longer periods of time than by transient changes. Additionally, we aimed to find a time where phenotypic changes to branching were measurable. This also meant that leaf senescence had not occurred within the time frame of this experiment. It should be kept in mind that some additional gene transcription changes could occur in different time frames to those described here. Additional experiments are needed to examine changes over time for the SL pathway genes. An important future goal is to gain an understanding of the dynamics of strigolactone signalling, given that the process of branching at early stages is able to be switched from dormant to actively growing and back (Napoli et al., 1999).

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Based on current models of SL signalling we had some initial predictions, in particular that increased red light might result in a reduction in receptor levels such that the SL pathway was impaired in its response to SL. Our results indicate that light quality does alter receptor complex expression in a manner consistent with the observed phenotype (increasing receptor expression in far red light and decreasing expression in red light), suggesting that in petunia, modulation of receptor complex expression is a significant mechanism for branch regulation by light quality, supporting observations reported in other species (Kebrom et al., 2006; Finlayson, 2007; Finlayson et al., 2010; Whipple et al., 2011; Dun et al., 2012; Guan et al., 2012; Gonzalez-Grandio et al., 2013; Guo et al., 2013).


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Neither light quality nor phosphate limitation were entirely dominant over the other signal for branching. This suggests either that both P and R:FR conditions can regulate branching through an SL independent pathway or that neither environmental condition is able to alter the SL signalling pathway to the point of a completely on or a completely off state. This result suggests that it may be more useful to consider the branching pathway to be regulated by a balance of signals rather than as a simple on/off switch.

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One of the aims of these experiments was to see if an analysis of expression could clarify which, if any, of the petunia TCP genes was involved in SL signaling and perhaps place them in the pathway. Results from rice show that the rice orthologue of TB1 (FC1) can bind to and inhibit a transcriptional repressor of rice D14 transcription (Guo et al., 2013). This observation places the rice TCP protein FC1 upstream of the SL receptor in the response to R:FR. It has previously been shown that the steady state mRNA levels of the Sorghum TB1 homologue (SbTb1) are regulated by R:FR ratios in a manner also shown for the Arabidopsis TB1 homologues BRC1 and BRC2 (Kebrom et al., 2006; Aguilar-Martínez et al., 2007). This regulation of BRC1 and BRC2 by R:FR ratios has been shown in Arabidopsis to be dependent on the PhyB gene (Aguilar-Martínez et al., 2007). The above observations strongly suggest that a TCP gene homologous to TB1 functions to connect R:FR light signalling from the phytochrome system to the regulation of branching by the SL receptor. However, whereas in monocots there are only one or possibly two TB1 orthologues, in eudicots there are several TCP genes with similarity to TB1. Phylogenetic analysis suggests three TCP genes in Arabidopsis are closely related to TB1 and in the Solanaceae (e.g. tomato, potato and petunia) potentially 6 genes homologous to TB1. The presence of multiple TB1-like TCP genes, presumably resulting from gene duplication events, allows for the possibility of diversification in function. In our experiments one TCP gene (PhTCP2) is regulated by R:FR and unaffected by mutations in the SL receptor gene. These observations suggest that PhTCP2 could act to regulate DAD2 levels in response to R:FR light ratios in a manner similar to FC1/OsTB1. It is interesting to note that PhTCP2, as well as homologous tomato and potato proteins, lack the SRXKAR(E/A)RARERT motif in the Cterminal region that is conserved in the rest of the TB1 subclade in both monocots and dicots. Whether this difference plays any role in the function of PhTCP2 in SL signalling is currently unknown.

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Results from pea have shown that expression of a TCP (PsBRC1) in pea is regulated by SL (Braun et al., 2012; Dun et al., 2012). This observation has been interpreted as placing PsBRC1 downstream of the SL receptor in the signal transduction pathway. Our observations show two of the TCPs in petunia have significantly altered expression in dad2 mutants and both respond to P and R:FR treatment in a manner consistent with a role in signal transduction downstream of DAD2. While both PhTCP1 and PhTCP3 respond in a similar manner, the changes in PhTCP3 expression have a stronger correlation to branching in our experiments and PhTCP3 expression is affected more in dad2 mutants suggesting PhTCP3 may be the primary gene involved in downstream responses to SL. However, PhTCP1 may be sufficiently redundant in function to also fill the role. The difference between monocot and dicot TB1-like TCPs suggest that either one or more duplication events occurred in dicots with subsequent diversification of TCP function or that TCP genes were lost from the monocot lineage with the role(s) of the lost genes taken up by unrelated genes, e.g. GT1 (Whipple et al., 2011).


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The observation of multiple TCPs functioning at different points in the SL pathway resolves the apparent paradox in the existing data that suggested that a TCP transcription factor was both upstream and downstream of the DAD2/MAX2 receptor complex. In petunia PhTCP2 appears to act upstream and PhTCP3 downstream of the DAD2/MAX2 receptor complex and perhaps this is the case in other species. However, it may be that any differences in TCP gene function play a role in the different architectures between species.

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In our experiments PhMAX2A expression was present in most tissues and while it did not show statistically significant changes in expression in response to P or R:FR PhMAX2A expression was highly correlated with DAD2 expression. This suggests that while PhMAX2A expression was variable it is still responding to environmental signals co-ordinately with DAD2. Transcriptional responses for DAD2 and MAX2 appear to vary somewhat between species, in petunia PhMAX2A seems somewhat variable but does appear to respond to environmental and developmental signals (this work and (Drummond et al., 2012)) whereas DAD2 expression responds significantly (this work and (Hamiaux et al., 2012)). By contrast, in Arabidopsis and sorghum MAX2 appears to respond significantly to light signals (Kebrom et al., 2010; Chevalier et al., 2014) and in Arabidopsis Chevalier et al. (2014) show that D14 mRNA levels do not respond to developmental or light quality signals.

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In the roots and low stems the gene expression responses were largely as expected from previous reports and models of SL synthesis (Umehara et al., 2010). Interestingly, the increase in expression of SL synthesis and transport genes in response to phosphate starvation was significant only in the fine roots. We also observed a reduction in expression of DAD2 and a correlated change in PhMAX2A in response to phosphate starvation in fine roots, a similar reduction in receptor expression has been reported in rice under phosphate and nitrogen limitation (Sun et al., 2014), suggesting an as yet unidentified negative feedback loop regulates the receptor complex. Significant changes in root gene expression were also observed in response to different light conditions, with synthetic genes downregulated in far-red light where branching is decreased, possibly as a result of some feedback mechanism. Decreased SL biosynthetic gene expression should result in a decrease in SL in far-red light conditions that is presumably balanced by an increase in receptor expression in axillary buds or perhaps by a change in transport of SL. It remains unclear how SL synthetic genes are regulated, however the NSP1 and NSP2 transcription factors are likely to be involved (Liu et al., 2011).

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In these and other experiments (Snowden and Napoli, 2003; Drummond et al., 2012) we observe differential growth of branches within a petunia plant. In this work, the axillary bud at node 2 remains dormant under all conditions, whereas the axillary bud at node 7 is responsive to environmental signals. The basis for this developmental difference is currently unknown. However, the expression of PDR1 and PhTCP3 suggest they play a role in this difference. PDR1 expression does not change in axillary bud 2 and expression overall is higher in this sample than in axillary bud 7. Within axillary bud 7 we do observe significant decreases in PDR1 expression and hence transport of SL under growth permitting conditions. Another interesting observation is the high and unchanging level of expression of PhTCP3 at node 2, which is the only tissue where its expression does not correlate with DAD2. Our results suggest PhTCP3 (like PsBRC1) acts downstream of DAD2/PhMAX2A and the normal response to SL is to increase expression of this TCP (Dun et al., 2012) by proteosomedependent degradation of a repressor of this TCP. With that hypothesis in mind, it is possible that the developmental difference between axillary bud 2 and axillary bud 7 that prevents growth under any environmental condition is that the putative repressor of PhTCP3 is itself absent in this tissue.


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The commitment to the growth of an axillary meristem into a branch is a complex and important point in the development of a plant, especially when environmental conditions are not optimal. The shade avoidance response results in suppression of branch growth in petunia as does the reduction in availability of the key nutrient phosphate. Our understanding of the strigolactone signal production and reception pathway suggested that both nutrient and light quality environmental inputs may be integrated at the point of SL reception, with SL production regulated in response to nutrient availability and the shade avoidance response regulating the availability of the receptor for SL. By modulating both of these environmental inputs together we were able to observe a range of developmental outcomes from extreme inhibition of branching to very active outgrowth of axillary meristems to form branches. Our results show that the SL receptor does act to integrate environmental signals; however, we were not able to completely inhibit the response to either signal, suggesting it is not an all or nothing system where the receptor is completely prevented from responding to input signals.

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These results indicate that the genes in the strigolactone pathway are likely to be good targets for modification of branching in field crops, as the phenotype is robust under diverse conditions, while still allowing plants to respond to non-ideal growth conditions. The retention of the ability to respond to environmental changes is particularly important for plants that grow for many seasons where the regulation of branching must be flexible enough to change growth as seasonal and other environmental inputs change.

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Methods Genetic stocks and plant growth conditions

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The wild type petunia used in these experiments is Petunia hybrida Vilm inbred line V26. The dad mutants, derived from this line, were isolated by Napoli and Ruehle (Napoli and Ruehle, 1996). All experiments were conducted in greenhouse conditions under natural light (for some experiments this was supplemented as described below) and with temperature set at 25±5 °C. All soil-grown plants were grown in commercial potting medium (Yates seed raising mix; Matamata, New Zealand) in six-packs (100-mL size) watered with fertiliser 80 mg L-1 nitrogen, 80 mg L-1 phosphorus, and 60 mg L-1 potassium from Wuxal Super 8-8-6 plus micro liquid fertilizer (Aglukon, Dusseldorf, Germany). For crowding experiments, petunia plants were grown in six-packs placed next to each other to create a 6 by 8 grid with two additional 2 by 3 grids and then grown in the centre of a greenhouse unit to avoid any shading from the walls (Supplemental Fig. 3). For hydroponic growth, petunia seeds were germinated on soil or vermiculite and after 2 weeks seedlings were transferred to 250 mL of 4-8 mm HydrotonTM clay pebbles that were suspended in nutrient media. At most 21 plants were grown in 20 L of constantly aerated (Two AquaOne® 1×1×10 cm airstones) ½ strength HHS media (Hamiaux et al., 2012). The phosphate concentration was altered by the variable addition of phosphoric acid. The Nitrogen concentration was altered by the variable addition of calcium nitrate. The solution volume was maintained at 20 L using media, and replaced only at the time of treatment. The pH of the media was maintained at 5.7 with the daily addition of hydrochloric acid.

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The red: far red (R:FR) ratio was altered by the addition of monochromatic light supplied from LED light sources. All LEDs were purchased from Roithner Lasertechnik GmbH, Austria. To add red light two LED660N-66-60 LEDs were used and to add far red light four LED735-66-60 were used. To achieve appropriate R:FR ratios, for the W (white), R and FR treatments, the irradiance of the


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baseline W light was restricted such that PAR (photosynthetically active radiation) was not greater than 200 µmol photons m-2s-1. During this experiment the day-length was approximately 14 hours (Auckland, NZ; summer). The spectra for the light treatments are shown in Supplemental Figure 8; R:FR ratios were 4–10 for the R treatment, 1 for the W treatment and 0.2–0.3 for the FR treatment. The spectra were measured using an Ocean Optics USB4000 spectroradiometer and data captured using the Spectrasuite software following the manufacturer’s instructions (www.oceanoptics.com).

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Analysis of the CCD7 promoter action in petunia

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Inverse PCR was used to isolate the PhCCD7 promoter using previously described methods (Snowden and Napoli, 1998). A total of 5 kb of sequence upstream of the PhCCD7 ATG was isolated (KR002102), and the entire 5 kb promoter fragment was amplified with PCR using Expand High Fidelity enzyme according to the manufacturer’s instructions and verified by sequencing. The PhCCD7 promoter fragment was cloned into pHEX14, a binary vector containing a promoter-less GUS gene, (Rae et al., 2014) resulting in a PCCD7-GUS construct. Six transgenic lines were generated with this construct, expression patterns for one line is shown (five out of the six lines showed identical GUS expression patterns).

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Cloning of the TCP genes

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To identify TCP genes from petunia, nested RACE was performed using degenerate primers designed to the conserved TCP domain. RNA was isolated from 4-week-old wild-type petunia plants (most vegetative organs, though mature leaves were removed so that the material was enriched for stem, shoot apex and axillary buds). cDNA was generated using a GeneRacer kit from Invitrogen, according to the manufacturer’s recommended protocols. The degenerate primer oTB2 (5’GAYMGICAYWSIAARATITGYACNGC-3’) was first used in combination with the GeneRacer 3’ primer (5’-GCTGTCAACGATACGCTACGTAACG-3’), then a nested PCR used the degenerate primer oTB7 (5’CARGAYATGYTIGGNTTYGAYAARGC-3’) in combination with the GeneRacer 3’ nested primer (5’CGCTACGTAACGGCATGACAGTG-3’). Additional sequence for the three TCP genes was isolated through a combination of RACE from a cDNA template and inverse PCR using a genomic DNA template to obtain the full coding sequence of PhTCP1 (KR002103), PhTCP2 (KR002104) and PhTCP3 (KR002105).

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Quantitative RT-PCR

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The samples from which the data in Figure 4 were produced are: axillary buds (from the four nodes above the highest branch), leaf (exposed small leaf near apex), low stem (the 2 cm of stem above the cotyledons, axillary buds and leaves removed), root (fine roots including tips).

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The samples from which the data in Figures 5–7 and Supplemental Figure 7 and Supplemental Data 2 were produced are: fine roots (including tips), main root (the 2 cm below root hypocotyl boundary), low stem (the 2 cm above cotyledons, axillary buds and leaves removed), the axillary buds from nodes two and seven, and leaf blade from leaf seven. Each sample type is represented by three pools of seven plants (except the minus phosphate samples from axillary buds at node seven where there are two pools of seven). Each biological replicate for all nine treatment conditions was collected over a two hour time period before the next biological replicate was started (replicate 1 was collected from 10 am – 12 pm, replicate 2 from 1:15 pm – 3:10 pm, and replicate 3 from 4:20


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pm – 6:00 pm). On the day of sample collection, sunrise was at 6:01 am and sunset was at 8:42 pm. The timing of the sample collection matches that of the phenotypic characterisation shown in Figure 2: 7 days after the onset of treatments.

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Quantitative RT-PCR was carried out largely as described previously (Drummond et al., 2012). Four reference genes (Actin, Histone, Ef1a and GAPDH) were tested and the two most stable genes as determined by GeNorm were kept for the remaining analysis. The GAPDH primers are described in Kretzschmar et al. (2012). We designed and tested primers to detect the transcripts of PhTCP1 (5’TGGTTCAAATTCCATATGCAGGT-3’, 5’-TCCAGTCTATGATGTCTGAAGGA-3’) PhTCP2 (5’AGCAGCAGTTATTGTAGTACCACT-3’, 5’-TGCTGGAACTGAAACCTCGAA-3’) and PhTCP3 (5’TGCAGTCAAGGAGCTGGAAG-3’, 5’-TATCATTTGTGGCAGATTCGTC-3’). These primers gave sensitive detection of the target transcripts without amplification of interfering primer dimer. For the experiments shown in Figures 4 to 7 the data comes from RNA extracted from three biological replicates of seven pooled individuals, excepting Figure 4 where four individuals were pooled.

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Phylogenetics, alignments and trees

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Sequence data were retrieved from public databases (similarity to existing sequence as determined by BLAST) or generated in this laboratory. All alignments were created using the Geneious Align tool and edited manually. Trees were calculated based on protein sequence alignments using the PhyML plugin for Geneious (Guindon and Gascuel, 2003). Support for the topology was determined by bootstrap with 100 replicates, percent support is indicated at each node. All bioinformatic analysis was carried out in Geneious (Biomatters, Auckland, NZ).

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Statistical analysis

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Most statistical analyses were performed using the GenStat statistical software package (14th Edn). ANOVAs were performed for statistical analyses of phenotypic and expression data. Appropriate transformations were used where necessary to ensure that model assumptions were met. Mean separation tests were performed using Tukey’s least significant difference (LSD) test at the 5% level of significance. For any given character in an experiment, values with no common lowercase identifiers are significantly different from each other. For correlations between data Spearman’s rank correlation test was used. Principle components analysis used a correlation matrix with 4 dimensions. Imputed values for missing data points were calculated in R 3.1.1 using the regularized imputePCA function in the missMDA library (Josse and Husson, 2012).


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Acknowledgements We would like to thank Kevin Reade, Rebecca Turner, Derek White, Sakuntala Karunairetnam, Andrew Gleave and Jo Putterill for materials, technical assistance and helpful discussions.

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Figure 1

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Petunia growth responses to individual environmental signals: light and nutrients.

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All graphs show phenotypic data as indicated. Values are means ±SEM. A–C Petunia (wild type (V26), ccd8 and dad2 mutants) were grown in nutrient controlled hydroponics. After germination the plants were established in standard nutrient media for 4 weeks. The media was replaced with either standard (250 µM P; dark green) or low (5 µM P; light green) nutrient media and growth continued for 4 weeks (n = 9 – 10). D–F Petunia (wild type, ccd8 and dad2 mutants) were grown in nutrient controlled hydroponics. After germination the plants were established in standard nutrient media for 3 weeks. The media was replaced with either standard (750 µM N; dark blue) or low (250 µM N; light blue) nutrient media and growth continued for four weeks (n = 6). G–I Petunia plants (wild type, ccd8 and dad2 mutants) were grown on soil supplemented with fertiliser for 7 weeks at a peak density of 1 plant/ 25 cm2. The plants were subjected to three levels of crowded conditions for 7 weeks: corner (three neighbours; blue), edge (five neighbours; green) or middle (eight neighbours; mustard; n = 11 – 20). J–L Petunia plants (wild type and ccd8 mutants) were germinated then established in hydroponics in white light for 5 weeks. The lighting was then supplemented with red (R:FR = 2.3) or far red light (R:FR = 0.4) for 3 weeks. Bright red bars (red light treatment), dark red bars (far red light treatment; n = 8). Statistical significance of differences was determined by ANOVA, values with no common lowercase identifiers are significantly different from each other (P = 0.05).

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Figure 2 Light quality and nutrient status (phosphate) work in concert to control branching in petunia

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Petunia plants were germinated and grown on soil in a glasshouse for 20 days then transferred to hydroponics for 17 days (R:FR = 1, 250 µM P) before being transferred to nine treatment environments for seven days. The treatments were made up of a 3x3 array of P availability (250, 5, 0 µM P) by light quality (R:FR for R = 4–10, W = 1, FR = 0.2–0.3). A–C The total number of leaves on all branches were counted (as a measure of branching), plant height and root length were measured. Statistical significance of differences was determined by ANOVA, values with no common lowercase identifiers are significantly different from each other (P = 0.05). D The numbers of leaves on branches at each node are plotted individually for the nine treatments. Values are means ±SEM (n = 21).

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Figure 3 Expression of the PhCCD7 gene as visualised from a PCCD7-GUS construct in transgenic petunia plants.

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All plants shown are 6–7 weeks old, plants in A and C were grown in hydroponics, plants shown in B and D–H were soil grown. A, longitudinal section of the bottom half of the stem of a plant and root system of a plant, most leaves, branches, and the top half of the main shoot have been removed. Red star indicates position where the stem was cut. B, longitudinal section through a section of stem showing an axillary bud and subtending leaf. C, fine roots from two plants that were grown in normal (+P, left) or in low (-P, right) phosphate media. D–H, cross sections from a single plant at the upper stem (D), mid-stem (E, approximately node 12), low stem (F), hypocotyl-low stem junction (G), main root (H). Scale bars represent 1 cm (A) or 2 mm (B–H).


Figure 4

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Petunia TCP genes involved in the SL signalling pathway

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The relative transcript abundance in 8-week-old wild-type and dad2 mutant petunia organs (A). Values are means ±SEM (n = 3). Samples were axbud, axillary bud samples from the four nodes above the highest branch; lowstem, 2 cm of stem above the cotyledons (nodes and internodes); leaf, expanded leaf blade; root, fine roots. Relative transcript abundance as rescaled against the sample with the greatest expression for each gene as indicated on the graphs.

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Phylogenetic analysis of the TB1 sub-clade of the classII TCP proteins (B). Sequences with similarity to known TCP proteins were identified in public databases by iterative BLAST search and an initial alignment by using Geneious was manually edited. Phylogenetic relationships were determined using the PhyML plugin to create a maximum likelyhood tree. Numbers are percent bootstrap values for 100 replicates. Names are given as two letters for the species (At, Arabidopsis thaliana; Sl, Solanum lycopersicum; St, Solanum tuberosum; Ph, Petunia hybrida; Bd, Brachypodium distachyon; Os, Oryza sativum; Mt, Medicago truncatulum; Ps, Pisum sativum; Pt, Populus tremula; Pd, Phoenix dactylifera; Pp, Physcomitrella patens; Ap, Anemone pulsatilla; Nn, Nelumbo nucifera; Zm, Zea mays) followed by TCP and a number where the gene has been assigned a TCP number in the literature and a letter where such an assignment has not yet been made. AtTCP18 and AtTCP12 are commonly known as BRC1 and BRC2 respectively. SlTCP18 and AtTCP20 are class I TCP proteins and were used as the outgroup.

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Figure 5 The transcription of the SL synthesis and reception pathway genes is altered in response to environmental cues

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The relative transcript abundance of SL related genes was quantified in six organs from plants treated in an array of light quality by P availability using qRT-PCR. The data have been grouped by organ A, P treatment B and light treatment C, averaged and within each grouping normalised to 1. Each bar represents average abundance on a linear scale from 0 to 1. The transcript abundance of a given gene (organ average) is directly comparable across all organs within the organ grouping (A), but only within an organ when grouped by P or light (B and C). Transcript abundance is not directly comparable between different genes because of normalisation. The statistical significance of any changes seen within the P or light treatments has been tested by ANOVA and the level of significance indicated by P; P is less than 0.05 (*), less than 0.01 (**), or less than 0.001 (***). Where P was greater than 0.05 the bars have been faded to 20% of the original intensity. nd transcript not detected. plus 250 µM P, low 5 µM P, minus 0 µM P, R R:FR >4, W R:FR = 1, FR R:FR < 0.3. Organ average n = 27, P or Light n = 9.

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Details of transcript abundance changes in response to phosphate availability in fine root samples

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Relative transcript abundance of selected genes in fine root samples from wild-type petunia treated with varied P availability and light quality. For each gene the samples are grouped by phosphate treatment, the coloured bars representing light treatment R:FR > 4 (red), R:FR = 1 (grey), R:FR < 0.3 (dark red). Values are means (n = 3), with SEM. The values of abundance for each gene are

Figure 6


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normalised to the greatest abundance of that transcript in any sample. The * next to the gene name indicates the P value in Figure 5.

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Figure 7 Details of transcript abundance changes in response to light quality (R:FR) in axillary bud 7 samples

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Relative transcript abundance of the genes tested in the axillary bud 7 samples from wild-type petunia treated with varied P availability and light quality. The CCD7 transcript was not detected in these samples. For each gene the samples are grouped by light treatment, the coloured bars representing P availability 250 µM P (green), 5 µM P (blue), 0 µM (dark blue). Values are means (n = 3), with SEM. The values of abundance for each gene are normalised to the greatest abundance of that transcript in any sample. The * next to the gene name indicates the P value in Figure 5.

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Figure 8 PCA analysis of growth and molecular data

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Principle components scores (A, B) and latent vectors (C, D) are shown for the first four dimensions from a principle components analysis of growth characteristics (branch growth, root length and plant height) and gene expression. Gene expression for gene/organ combinations where no expression was detected were excluded from this analysis (i.e. CCD7 expression was not detected in axillary bud or leaf samples, PhTCP1 expression was not detected in root samples, and PhTCP3 expression was not detected in fine roots). Dimensions 1 and 4 (A, C) explain 27.06% and 8.13% of the variance in the data respectively and show separation of the environmental treatments, whereas dimensions 2 and 3 (B, D) explain 14.44% and 9.33% of the variance and show some separation of the biological replicates.

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Table I Spearman’s rank correlation coefficients (rs) and corresponding P values for expression of DAD2 and the three PhTCP genes in buds that are responsive (bud 7) and non-responsive (bud 2). PhTCP1

Table I

DAD2

PhTCP2

PhTCP3

rs

P

rs

P

rs

P

Axillary bud 2

0.54

0.002

0.74

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Genetic and Environmental Control of Neurodevelopmental Robustness in Drosophila.

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The characterization of modified starch branching enzymes: toward the control of starch chain-length distributions.