Journal of Plant Physiology 171 (2014) 7–13
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Physiology
Light and nitrogen nutrition regulate apical control in Rosa hybrida L. Pierre-Maxime Furet a , Jérémy Lothier a , Sabine Demotes-Mainard b , Sandrine Travier a , Clémence Henry a , Vincent Guérin b , Alain Vian a,∗ a Université d‘Angers, UMR 1345 Institut de Recherche en Horticulture et Semences (Université d‘Angers, Agrocampus-Ouest, INRA), SFR 4207 QUASAV, PRES L‘UNAM, 2 Bd Lavoisier, F-49045 Angers, France b INRA, UMR 1345 Institut de Recherche en Horticulture et Semences (Université d‘Angers, Agrocampus-Ouest, INRA), SFR 4207 QUASAV, BP 60057, F-49071 Beaucouzé, France
a r t i c l e
i n f o
Article history: Received 11 July 2013 Received in revised form 27 September 2013 Accepted 21 October 2013 Available online 11 December 2013 Keywords: Apical control Bud outgrowth Light intensity Nitrate nutrition Rosa hybrida
a b s t r a c t Apical control is defined as the inhibition of basal axillary bud outgrowth by an upper actively growing axillary axis, whose regulation is poorly understood yet differs markedly from the better-known apical dominance. We studied the regulation of apical control by environmental factors in decapitated Rosa hybrida in order to remove the apical hormonal influence and nutrient sink. In this plant model, all the buds along the main axis have a similar morphology and are able to burst in vitro. We concentrated on the involvement of light intensity and nitrate nutrition on bud break and axillary bud elongation in the primary axis pruned above the fifth leaf of each rose bush. We observed that apical control took place in low light (92 mol m−2 s−1 ), where only the 2-apical buds grew out, both in low (0.25 mM) and high (12.25 mM) nitrate. In contrast, in high light (453 mol m−2 s−1 ), the apical control only operates in low nitrate while all the buds along the stem grew out when the plant was supplied with a high level of nitrate. We found a decreasing photosynthetic activity from the top to the base of the plant concomitant with a light gradient along the stem. The quantity of sucrose, fructose, glucose and starch are higher in high light conditions in leaves and stem. The expression of the sucrose transporter RhSUC2 was higher in internodes and buds in this lighting condition, suggesting an increased capacity for sucrose transport. We propose that light intensity and nitrogen availability both contribute to the establishment of apical control. © 2013 Elsevier GmbH. All rights reserved.
Introduction It is well known that the apical part of the stem inhibits axillary bud outgrowth during the vegetative growth period. This phenomenon is mostly interpreted in terms of apical dominance (Dun et al., 2006). After apical dominance release, apical axillary buds generally break (Ferguson and Beveridge, 2009). It is well established that three classes of phytohormones (auxins, cytokinins and strigolactones) are implicated in the activity of axillary buds (see Domagalska and Leyser, 2011 for a recent review). Even if the mechanism is still a matter of debate, it is understood that auxin synthesized in the growing apex moves basipetally in the polar auxin transport stream and inhibits axillary bud outgrowth.
Abbreviations: 6-BAP, 6-benzylaminopurine; Hi, high-light condition; Hi–Lo, switch from Hi to Lo light condition; Lo, low-light condition; Lo–Hi, switch from Lo to Hi light condition; PAR, photosynthetically active radiation; RhSUC-2, Rosa hybrida sucrose transporter-2. ∗ Corresponding author at: Université d‘Angers, IRHS UMR 1345, Equipe ARCH-E, 2 Bd Lavoisier 49045, Angers Cedex, France. Tel.: +33 241 735 355; fax: +33 241 735 352. E-mail address: [email protected] (A. Vian). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.10.008
Since auxin is unable to enter in the bud, some authors suggest that the basipetal flux of auxin inhibits auxin export from the axillary buds and consequently their outgrowth (Bennett et al., 2006; Prusinkiewicz et al., 2009). An alternative hypothesis suggests that auxin inhibits bud outgrowth through the action of a second messenger, which is able to enter the bud, although the antagonistic effects of cytokinins and strigolactones that respectively promote or inhibit bud outgrowth (Tanaka et al., 2006; Gomez-Roldan et al., 2008; Brewer et al., 2009). Auxin regulates the synthesis of cytokinins at the node by down-regulating the gene coding for adenylate isopentenyl transferase (IPT), which is, in turn, implicated in cytokinin biosynthesis (Tanaka et al., 2006). Auxin also regulates the synthesis of strigolactone by up-regulating transcription of genes encoding proteins implicated in strigolactone biosynthesis (Johnson et al., 2006). However, some observations, namely an apical gradation of bud break along a beheaded axis (Cutter and Chiu, 1975; Chern et al., 1993; Zalewska et al., 2010), suggest that a more complex regulation of bud outgrowth is actually taking place after apical dominance release. This mechanism, designated as the “apical control”, is described as “the growth suppression of an existing sub-dominant branch by a higher dominating shoot” (Cline and
8
P.-M. Furet et al. / Journal of Plant Physiology 171 (2014) 7–13
Harrington, 2007). The regulation of this mechanism is still in debate, but environmental factors, mainly temperature, light and nutrient are likely to be implicated in the establishment and functionality of apical differential growth of axillary buds (De Vries et al., 1986; Kebrom et al., 2006). Temperature was among the first parameter shown to influence bud burst (Partanen et al., 1998). Light could also exerts an action on plant growth both as an energy supply and as a signal (Girault et al., 2010; Smeekens et al., 2010). Light regulates diverse developmental processes such as vegetative bud outgrowth (Anderson et al., 2005; Chao et al., 2007; Rabot et al., 2012), in parallel to hormone action. Modulation of bud outgrowth by different wavelengths is one of the most addressed processes (Ballaré, 1999). These aspects could be under the influence of both light quality and/or low intensity signaling mechanisms (Suzuki, 2003; Girault et al., 2008). As an example, plants grown in low R:FR ratio under high density or in deep canopy, develop fewer lateral branches (Tucic´ et al., 2006; Casal et al., 1986; Ballaré et al., 1987). This ratio is mainly perceived by phytochrome B (phyB) that is known to regulate the auxin and strigolactone signaling pathways, or induce BRC1 a transcription factor involved in bud growth arrest in response to a low R:FR ratio (Finlayson et al., 2010; Gonzalez-Grandio et al., 2013). A recent study by Su et al. (2011) suggests that light intensity could promote bud outgrowth through a partly phyB-independent pathway, which seems to be also independent from the leaf photosynthetic rate. In contrast to the effect of spectral composition, the effect of light intensity on shoot branching remains poorly understood. Axillary buds from most species (tomato, Arabidopsis, poplar) growth out in darkness while in rose bushes, light is required to promote bud burst even at very low intensity (Girault et al., 2008). Plants cultivated under high light intensity show a higher level of branching compared to those under low light (Bartlett and Remphrey, 1998; Demotes-Mainard et al., 2013). Some studies report that nutrient availability interacts with the hormonal network. High N fertilizing induces the up-regulation of IPT genes in the roots, and leads to a high level of endogenous cytokinins, which are likely to result in an increase in bud outgrowth (Takei et al., 2001, 2004; Miyawaki et al., 2004). Nutrient deficiency, especially nitrogen (N) and inorganic phosphorus (P) in soil, is known to reduce shoot branching (Lortie and Aarssen, 1997; Cline et al., 2006; Umehara et al., 2010). Furthermore, phosphorus deficiency increases strigolactone production in roots and its transport to the shoot in the xylem sap, and this represses bud outgrowth (Umehara et al., 2010; López-Ráez et al., 2008). Thus, beside hormonal control, the overall mechanism that involves environmental factors in regulating bud outgrowth through apical control is still poorly understood. Under normal growth conditions, plants must integrate many environmental factors to adjust shoot branching to their light environment and nutritional status. Our aims are to decipher how environmental factors, i.e. light intensity and nitrogen nutrition, could alone or in combination affect the outgrowth of axillary buds along an axis. Indeed, to our knowledge, the effects of more than one environmental factor on the regulation of branching in plants have received very little attention.
(N15-P10-K30, Plant Products, Brampton, Canada). Plants were transferred to one of the experimental designs (growth chamber, Froids et Mesures, Beaucouzé, France, L:D 16 h:8 h, 22 ◦ C:22 ◦ C, RH 65%, Fig. 1) when the third leaf developed (stage 2). Plants were subjected to 92 ± 2 (Lo) or 453 ± 9 mol m−2 s−1 illumination (Hi) photosynthetically active radiation (PAR). These treatments never varied in light intensity or in photoperiod. In the Lo–Hi treatment (Fig. 1A), plants were first grown in Lo light (92 ± 2 mol m−2 s−1 ) until the shoot apex was removed (stage 3) and then subjected to Hi light (453 ± 9 mol m−2 s−1 ) for the rest of the experiment (duration of 35 days). In the Hi–Lo treatment, the plants were treated similarly to the Lo–Hi treatment, except that the lighting conditions were reversed (Fig. 1A). Light intensity was measured at different levels along the stem close to the buds using a Licor quantum sensor (Li 190, LI-COR Instruments, Lincoln, Nebraska, USA). Leaf net CO2 assimilation rate was assayed on the different leaves along the stem using a gas analyzer (Li 6400, LI-COR Instruments, Lincoln, Nebraska, USA), and refereed to the assimilation rate of the third leaf (Supplemental data). Plants cultivated on low nitrate were initially deprived of nitrate by washing the substrate 5 times (5 min) under running tap water (Charpentier et al., 2001) and then watered with low nitrate (0.25 mM) nutrient solution. Plants cultivated on high nitrate were kept watered with the regular, high nitrate (12.25 mM) nutritive solution both under Hi and Lo light conditions. Bud outgrowth was measured daily with a digital caliper. At the end of the experiments (Fig. 1, 35 days), the axes were collected and the dry weight was determined after five days of desiccation in an oven at 60 ◦ C. Growth of excised axillary buds The five axillary buds present at the fully-formed leaves with their underlying internodes were collected at the fifth-developed leaf stage of plants grown in Lo or Hi light condition (stage 3, Fig. 1B) and cultivated in vitro (Henry et al., 2011). The excised stem fragments bearing individual bud and the neighboring region (1 cm both side) were placed vertically in a Petri dish, on Murashige and Skoog (1962) liquid medium supplemented with 50 mM of sucrose. The percentage of bud-burst was determined after 10 days, based upon the bud swelling and new leaf protruding from the scales. Exogenous cytokinin application The plants grown in Lo conditions were beheaded (Fig. 1C, stage 3) and the different buds along the stem were daily soaked with a droplet (15 L) containing 1 mM of the synthetic cytokinin 6benzylaminopurine (6-BAP, Sigma–Aldrich) for 5 days, as described by Cline and Dong-Il (2002). Applications of the buffer minus 6-BAP or only water were performed in parallel as control treatments. The lengths of the outgrowing axes were measured after 11 days. Morphology of axillary buds
Materials and methods
We collected the five axillary buds present along the primary axis of plants grown in Lo treatment (Fig. 1D, stage 3) and counted the number of preformed leaves under a dissecting binocular microscope.
Plant material and growing conditions
RNA isolation and qRT-PCR analysis
Rosa hybrida ‘Radrazz’ rose bushes were obtained from single-node cuttings and placed in 500 mL pots containing a 50/40/10 mixture (v/v/v) of neutral peat, coconut fibers and perlite in a greenhouse (average values: L:D 16 h:8 h, 20 ◦ C:16 ◦ C, 120 mol m−2 s−1 ). They were soaked with Plant Prod® fertilizer
RNA was isolated from the third internode using the RNeasy Qiagen Plant RNA isolation kit (Qiagen, Courtaboeuf, France). The RNA was quantified and verified for integrity on agarose gels. Reverse transcription was performed with oligo(dT)20 primer using SuperScript III Reverse transcriptase kit (Invitrogen, Paris,
P.-M. Furet et al. / Journal of Plant Physiology 171 (2014) 7–13
9
5
4
3 2
Low light intensity (Lo)
1
High light intensity (Hi) Cytokinin application
Stage 1
Stage 2
Rooted cutting
Third leaf appearance
End of experiment
Stage 3 Fifth leaf appearance
A B C D Greenhouse
Growth chamber
Days -30
-7
5
10
16
35
Shoot apex removal Fig. 1. Schematic representation of light treatments for the four experiments. Periods of high light treatment (Hi, white bars, 453 mol m−2 s−1 ) or low light (Lo, black bars, 92 mol m−2 s−1 ) are represented as a function of developmental stage of the primary axis (stages 1–3) and during shoot branching (stage 3 to the end of each experiment). Letters A to D refer to the different experiments (A: effect of light intensity on the length of growing axis; B: excised bud outgrowth; C: effect of 6-BAP application on the length of axes; D: axillary buds morphology). Lo and Hi light treatments (experimental plan A) were either supplied with 12.25 mM or 0.25 mM nitrate. In experimental plan B, C and D nitrogen was supplied as 12.25 mM nitrate.
Table 1 Light intensity measurements along the stem. Photosynthetically active radiation (mol m−2 s−1 ) was measured with a LI-COR quantum sensor 190 at each leaf position along the primary axis, from the basal (#1) to the apical (#5) one, under Hi and Lo light conditions. Each value is the mean of at least 3 independent measurements ± SE. Leaf rank
Light intensity (mol m−2 s−1 ) Hi
5 4 3 2 1
453.2 345.8 270.0 181.4 154.1
± ± ± ± ±
8.6 24.4 25.3 6.7 11.9
%
Lo
100 76.3 59.6 40.0 34.0
91.5 80.4 66.7 58.5 49.5
% ± ± ± ± ±
2.1 3.8 4.8 5.2 3.5
100 87.9 72.9 64.0 54.1
France). Quantitative RT-PCR was conducted using the R. hybrida sucrose transporter-2 (RhSUC2) primers, as described by Henry et al. (2011).
less strongly reduced, ranging from 91.5 mol m−2 s−1 at the plant apex to 49.5 mol m−2 s−1 (about 54% of the apical incident light intensity) at the base of the stem. We measured net CO2 assimilation rate in Lo and Hi conditions under 2 different nitrate supplies (0.25 and 12.25 mM). In low light and low nitrogen supply, only the 2-apical leaves readily incorporated CO2 , while the basal leaves had very low fixation (Table 2). Supplying high level of nitrate (12.25 mM) in low light only slightly but significantly changed CO2 fixation all along the axis. In high light and low nitrogen supply, the CO2 fixation was much higher with a gradation from the apical bud (8.3 mol m−2 s−1 ) to the base (0.1 mol m−2 s−1 ) where it was very low, similar to that observed in Lo condition. This gradient was also observed in Hi condition in the presence of high nitrogen, while the basal buds (#1 and #2) showed significantly greater CO2 fixation (1.9 and 2.2 mol m−2 s−1 , respectively).
Statistical analysis
Carbohydrate content
All statistical analyses were carried out using R statistical package software version 2.12.2 (R Core Team, 2013).
We analyzed the amounts of fructose, glucose, sucrose and starch in the leaves and internodes of the primary axis in Lo and Hi light conditions (Fig. 2). While the amounts of fructose and glucose were low and nearly identical in Lo and Hi light conditions in leaves (about 5–8 mg g−1 of dry matter, Fig. 2A), they were significantly higher in Hi light compared to Lo light in the internodes (Fig. 2B, about 10–12 and 20 mg g−1 dry matter, respectively). Sucrose was much more abundant in leaves and stem (90 and 50 mg g−1 of dry matter, respectively) in Lo and Hi light. In contrast, starch was much higher in Hi light in leaves (about 2.5 fold) while the difference stayed statistically significant, but markedly reduced in the stem (Fig. 2B). The capacity of sucrose transport was evaluated by the measurement of the sucrose transporter RhSUC2 expression in the 3rd internode and in adjacent bud. Transcript accumulation was
Results PAR and net CO2 assimilation rates We measured the light intensities (PAR) in the region neighboring the buds along the primary axis in Hi and Lo light conditions (Table 1). In Hi light, the intensity displayed a continuous gradient with the maximum value measured at the plant apex (453 mol m−2 s−1 ). The light intensity was attenuated about 3fold at the base (154 mol m−2 s−1 , about 34% of the apical incident light intensity). In Lo light, the light gradient along the stem was
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P.-M. Furet et al. / Journal of Plant Physiology 171 (2014) 7–13
Table 2 Net CO2 assimilation rate of the different leaves along the primary axis under different light and nitrogen supply conditions. CO2 fixation (mol m−2 s−1 ) was measured using a LI-COR 6400 instrument on each leaf along the primary axis, from the basal (#1) to the apical (#5) one in Lo (92 mol m−2 s−1 ) and Hi (453 mol m−2 s−1 ) light condition, under low (−N, 0.25 mM) and high (+N, 12.25 mM) nitrate supply. Each value is the mean of at least 3 independent measurements ± SE. Different letters indicate significant differences between the different culture conditions for the same leaf rank (p < 0.05), according to HSD Tukey test. CO2 assimilation rate (mol m−2 s−1 )
Leaf rank
Lo −N 5 4 3 2 1
3.56 1.39 0.67 0.31 0.02
± ± ± ± ±
100
mg g -1 of dry matter
Hi −N
Lo +N 0.09 (a) 0.07 (a) 0.05 (a) 0.03 (a) 0.00 (a)
3.92 1.49 0.67 0.53 0.41
A
± ± ± ± ±
0.09 (b) 0.07 (a) 0.05 (a) 0.05 (b) 0.03 (b)
*
8.28 3.55 2.22 1.05 0.07
*
± ± ± ± ±
Hi +N 0.16 (c) 0.25 (b) 0.21 (b) 0.04 (c) 0.01 (c)
9.70 4.09 2.75 2.17 1.94
± ± ± ± ±
0.19 (d) 0.19 (b) 0.26 (b) 0.08 (d) 0.15 (d)
B
80
*
60 40
*
20 0
*
*
* Fructose Glucose Sucrose Starch
Fructose Glucose Sucrose Starch
Fig. 2. Sugars and starch content in leaves (A) and internodes (B) of primary axes. Fructose, glucose, sucrose and starch were quantified as mg g−1 dry matter in leaves (A) and stem (B) of plants cultivated under low (92 mol m−2 s−1 , black bars) and high (453 mol m−2 s−1 , white bars) light. Each value is the mean of at least 3 independent experiments ± SE. Stars indicate significant differences between light treatments (p < 0.05), according to HSD Tukey test.
promoted in Hi light treatment, and was higher in the internode (Fig. 3A, 5-fold) compared to the bud (Fig. 3B, 2.5-fold). Bud development and length of new branches
RhSUC2 mRNA relative quantity
Table 3 gives the number of preformed leaves in all the buds along the stem at stage 3 (Fig. 1) in Lo light. In these conditions, all five buds had a similar structure with statistically comparable number of preformed leaves (6.8–7.6). We analyzed the length of secondary axes produced from bud outgrowth along the stem under the 4 light treatments, and under
7
A
6
*
B
5 4
*
3 2 1 0
Lo
Hi
Lo
Hi
Fig. 3. RhSUC2 mRNA accumulation in the third internode (A) and adjacent bud (B) under low and high light intensities. RhSUC2 mRNA was quantified using qRTPCR in the third internode (A) and adjacent bud (B) of plants grown under low (92 mol m−2 s−1 , black bars) and high (453 mol m−2 s−1 , white bars) light treatments. Each point is the average of at least three independent experiments (±SE), normalized to SAND1 (Henry et al., 2011) and expressed relative to low light condition. Stars indicate significant differences between light treatments (p < 0.05), according to HSD Tukey test.
2 different nitrogen supplies. In our conditions, bud swelling that stayed below 3 mm never gave rise to a developing axis and was therefore not considered an outgrowth. In Lo condition (and low or high nitrate supply), only the apical buds (#4 and #5) grew out and produced actively growing axes that reached respectively about 15–20 cm (bud #4) and 29–32 cm (bud #5) at the end of the experiment (Fig. 4A). In marked contrast, virtually no outgrowth of basal buds occurred (about 2 mm at the end of the experiment) and was very similar in low and high nitrogen supplies. In the Hi–Lo condition, the apical buds (#4 and #5) broke and had strong axes elongation (23.3 and 27.3 cm, Fig. 4D) while the other buds (#1 to #3) did not grow out and behaved very similarly to those observed in the Lo condition. In the Hi condition and high nitrate supply, all 5 buds along the stem grew out (Fig. 4B, +N). The 4 apical ones (#2 to #5) formed, at the end of the experiment, axes whose lengths were comparable to those observed for the 2 apical buds in Lo treatment (25.6–30.7 cm). However, the very basal #1 bud showed significantly reduced growth (14.5 cm) that was, however, considerably higher than that observed for the same bud in the Lo condition (
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Physiology
Light and nitrogen nutrition regulate apical control in Rosa hybrida L. Pierre-Maxime Furet a , Jérémy Lothier a , Sabine Demotes-Mainard b , Sandrine Travier a , Clémence Henry a , Vincent Guérin b , Alain Vian a,∗ a Université d‘Angers, UMR 1345 Institut de Recherche en Horticulture et Semences (Université d‘Angers, Agrocampus-Ouest, INRA), SFR 4207 QUASAV, PRES L‘UNAM, 2 Bd Lavoisier, F-49045 Angers, France b INRA, UMR 1345 Institut de Recherche en Horticulture et Semences (Université d‘Angers, Agrocampus-Ouest, INRA), SFR 4207 QUASAV, BP 60057, F-49071 Beaucouzé, France
a r t i c l e
i n f o
Article history: Received 11 July 2013 Received in revised form 27 September 2013 Accepted 21 October 2013 Available online 11 December 2013 Keywords: Apical control Bud outgrowth Light intensity Nitrate nutrition Rosa hybrida
a b s t r a c t Apical control is defined as the inhibition of basal axillary bud outgrowth by an upper actively growing axillary axis, whose regulation is poorly understood yet differs markedly from the better-known apical dominance. We studied the regulation of apical control by environmental factors in decapitated Rosa hybrida in order to remove the apical hormonal influence and nutrient sink. In this plant model, all the buds along the main axis have a similar morphology and are able to burst in vitro. We concentrated on the involvement of light intensity and nitrate nutrition on bud break and axillary bud elongation in the primary axis pruned above the fifth leaf of each rose bush. We observed that apical control took place in low light (92 mol m−2 s−1 ), where only the 2-apical buds grew out, both in low (0.25 mM) and high (12.25 mM) nitrate. In contrast, in high light (453 mol m−2 s−1 ), the apical control only operates in low nitrate while all the buds along the stem grew out when the plant was supplied with a high level of nitrate. We found a decreasing photosynthetic activity from the top to the base of the plant concomitant with a light gradient along the stem. The quantity of sucrose, fructose, glucose and starch are higher in high light conditions in leaves and stem. The expression of the sucrose transporter RhSUC2 was higher in internodes and buds in this lighting condition, suggesting an increased capacity for sucrose transport. We propose that light intensity and nitrogen availability both contribute to the establishment of apical control. © 2013 Elsevier GmbH. All rights reserved.
Introduction It is well known that the apical part of the stem inhibits axillary bud outgrowth during the vegetative growth period. This phenomenon is mostly interpreted in terms of apical dominance (Dun et al., 2006). After apical dominance release, apical axillary buds generally break (Ferguson and Beveridge, 2009). It is well established that three classes of phytohormones (auxins, cytokinins and strigolactones) are implicated in the activity of axillary buds (see Domagalska and Leyser, 2011 for a recent review). Even if the mechanism is still a matter of debate, it is understood that auxin synthesized in the growing apex moves basipetally in the polar auxin transport stream and inhibits axillary bud outgrowth.
Abbreviations: 6-BAP, 6-benzylaminopurine; Hi, high-light condition; Hi–Lo, switch from Hi to Lo light condition; Lo, low-light condition; Lo–Hi, switch from Lo to Hi light condition; PAR, photosynthetically active radiation; RhSUC-2, Rosa hybrida sucrose transporter-2. ∗ Corresponding author at: Université d‘Angers, IRHS UMR 1345, Equipe ARCH-E, 2 Bd Lavoisier 49045, Angers Cedex, France. Tel.: +33 241 735 355; fax: +33 241 735 352. E-mail address: [email protected] (A. Vian). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.10.008
Since auxin is unable to enter in the bud, some authors suggest that the basipetal flux of auxin inhibits auxin export from the axillary buds and consequently their outgrowth (Bennett et al., 2006; Prusinkiewicz et al., 2009). An alternative hypothesis suggests that auxin inhibits bud outgrowth through the action of a second messenger, which is able to enter the bud, although the antagonistic effects of cytokinins and strigolactones that respectively promote or inhibit bud outgrowth (Tanaka et al., 2006; Gomez-Roldan et al., 2008; Brewer et al., 2009). Auxin regulates the synthesis of cytokinins at the node by down-regulating the gene coding for adenylate isopentenyl transferase (IPT), which is, in turn, implicated in cytokinin biosynthesis (Tanaka et al., 2006). Auxin also regulates the synthesis of strigolactone by up-regulating transcription of genes encoding proteins implicated in strigolactone biosynthesis (Johnson et al., 2006). However, some observations, namely an apical gradation of bud break along a beheaded axis (Cutter and Chiu, 1975; Chern et al., 1993; Zalewska et al., 2010), suggest that a more complex regulation of bud outgrowth is actually taking place after apical dominance release. This mechanism, designated as the “apical control”, is described as “the growth suppression of an existing sub-dominant branch by a higher dominating shoot” (Cline and
8
P.-M. Furet et al. / Journal of Plant Physiology 171 (2014) 7–13
Harrington, 2007). The regulation of this mechanism is still in debate, but environmental factors, mainly temperature, light and nutrient are likely to be implicated in the establishment and functionality of apical differential growth of axillary buds (De Vries et al., 1986; Kebrom et al., 2006). Temperature was among the first parameter shown to influence bud burst (Partanen et al., 1998). Light could also exerts an action on plant growth both as an energy supply and as a signal (Girault et al., 2010; Smeekens et al., 2010). Light regulates diverse developmental processes such as vegetative bud outgrowth (Anderson et al., 2005; Chao et al., 2007; Rabot et al., 2012), in parallel to hormone action. Modulation of bud outgrowth by different wavelengths is one of the most addressed processes (Ballaré, 1999). These aspects could be under the influence of both light quality and/or low intensity signaling mechanisms (Suzuki, 2003; Girault et al., 2008). As an example, plants grown in low R:FR ratio under high density or in deep canopy, develop fewer lateral branches (Tucic´ et al., 2006; Casal et al., 1986; Ballaré et al., 1987). This ratio is mainly perceived by phytochrome B (phyB) that is known to regulate the auxin and strigolactone signaling pathways, or induce BRC1 a transcription factor involved in bud growth arrest in response to a low R:FR ratio (Finlayson et al., 2010; Gonzalez-Grandio et al., 2013). A recent study by Su et al. (2011) suggests that light intensity could promote bud outgrowth through a partly phyB-independent pathway, which seems to be also independent from the leaf photosynthetic rate. In contrast to the effect of spectral composition, the effect of light intensity on shoot branching remains poorly understood. Axillary buds from most species (tomato, Arabidopsis, poplar) growth out in darkness while in rose bushes, light is required to promote bud burst even at very low intensity (Girault et al., 2008). Plants cultivated under high light intensity show a higher level of branching compared to those under low light (Bartlett and Remphrey, 1998; Demotes-Mainard et al., 2013). Some studies report that nutrient availability interacts with the hormonal network. High N fertilizing induces the up-regulation of IPT genes in the roots, and leads to a high level of endogenous cytokinins, which are likely to result in an increase in bud outgrowth (Takei et al., 2001, 2004; Miyawaki et al., 2004). Nutrient deficiency, especially nitrogen (N) and inorganic phosphorus (P) in soil, is known to reduce shoot branching (Lortie and Aarssen, 1997; Cline et al., 2006; Umehara et al., 2010). Furthermore, phosphorus deficiency increases strigolactone production in roots and its transport to the shoot in the xylem sap, and this represses bud outgrowth (Umehara et al., 2010; López-Ráez et al., 2008). Thus, beside hormonal control, the overall mechanism that involves environmental factors in regulating bud outgrowth through apical control is still poorly understood. Under normal growth conditions, plants must integrate many environmental factors to adjust shoot branching to their light environment and nutritional status. Our aims are to decipher how environmental factors, i.e. light intensity and nitrogen nutrition, could alone or in combination affect the outgrowth of axillary buds along an axis. Indeed, to our knowledge, the effects of more than one environmental factor on the regulation of branching in plants have received very little attention.
(N15-P10-K30, Plant Products, Brampton, Canada). Plants were transferred to one of the experimental designs (growth chamber, Froids et Mesures, Beaucouzé, France, L:D 16 h:8 h, 22 ◦ C:22 ◦ C, RH 65%, Fig. 1) when the third leaf developed (stage 2). Plants were subjected to 92 ± 2 (Lo) or 453 ± 9 mol m−2 s−1 illumination (Hi) photosynthetically active radiation (PAR). These treatments never varied in light intensity or in photoperiod. In the Lo–Hi treatment (Fig. 1A), plants were first grown in Lo light (92 ± 2 mol m−2 s−1 ) until the shoot apex was removed (stage 3) and then subjected to Hi light (453 ± 9 mol m−2 s−1 ) for the rest of the experiment (duration of 35 days). In the Hi–Lo treatment, the plants were treated similarly to the Lo–Hi treatment, except that the lighting conditions were reversed (Fig. 1A). Light intensity was measured at different levels along the stem close to the buds using a Licor quantum sensor (Li 190, LI-COR Instruments, Lincoln, Nebraska, USA). Leaf net CO2 assimilation rate was assayed on the different leaves along the stem using a gas analyzer (Li 6400, LI-COR Instruments, Lincoln, Nebraska, USA), and refereed to the assimilation rate of the third leaf (Supplemental data). Plants cultivated on low nitrate were initially deprived of nitrate by washing the substrate 5 times (5 min) under running tap water (Charpentier et al., 2001) and then watered with low nitrate (0.25 mM) nutrient solution. Plants cultivated on high nitrate were kept watered with the regular, high nitrate (12.25 mM) nutritive solution both under Hi and Lo light conditions. Bud outgrowth was measured daily with a digital caliper. At the end of the experiments (Fig. 1, 35 days), the axes were collected and the dry weight was determined after five days of desiccation in an oven at 60 ◦ C. Growth of excised axillary buds The five axillary buds present at the fully-formed leaves with their underlying internodes were collected at the fifth-developed leaf stage of plants grown in Lo or Hi light condition (stage 3, Fig. 1B) and cultivated in vitro (Henry et al., 2011). The excised stem fragments bearing individual bud and the neighboring region (1 cm both side) were placed vertically in a Petri dish, on Murashige and Skoog (1962) liquid medium supplemented with 50 mM of sucrose. The percentage of bud-burst was determined after 10 days, based upon the bud swelling and new leaf protruding from the scales. Exogenous cytokinin application The plants grown in Lo conditions were beheaded (Fig. 1C, stage 3) and the different buds along the stem were daily soaked with a droplet (15 L) containing 1 mM of the synthetic cytokinin 6benzylaminopurine (6-BAP, Sigma–Aldrich) for 5 days, as described by Cline and Dong-Il (2002). Applications of the buffer minus 6-BAP or only water were performed in parallel as control treatments. The lengths of the outgrowing axes were measured after 11 days. Morphology of axillary buds
Materials and methods
We collected the five axillary buds present along the primary axis of plants grown in Lo treatment (Fig. 1D, stage 3) and counted the number of preformed leaves under a dissecting binocular microscope.
Plant material and growing conditions
RNA isolation and qRT-PCR analysis
Rosa hybrida ‘Radrazz’ rose bushes were obtained from single-node cuttings and placed in 500 mL pots containing a 50/40/10 mixture (v/v/v) of neutral peat, coconut fibers and perlite in a greenhouse (average values: L:D 16 h:8 h, 20 ◦ C:16 ◦ C, 120 mol m−2 s−1 ). They were soaked with Plant Prod® fertilizer
RNA was isolated from the third internode using the RNeasy Qiagen Plant RNA isolation kit (Qiagen, Courtaboeuf, France). The RNA was quantified and verified for integrity on agarose gels. Reverse transcription was performed with oligo(dT)20 primer using SuperScript III Reverse transcriptase kit (Invitrogen, Paris,
P.-M. Furet et al. / Journal of Plant Physiology 171 (2014) 7–13
9
5
4
3 2
Low light intensity (Lo)
1
High light intensity (Hi) Cytokinin application
Stage 1
Stage 2
Rooted cutting
Third leaf appearance
End of experiment
Stage 3 Fifth leaf appearance
A B C D Greenhouse
Growth chamber
Days -30
-7
5
10
16
35
Shoot apex removal Fig. 1. Schematic representation of light treatments for the four experiments. Periods of high light treatment (Hi, white bars, 453 mol m−2 s−1 ) or low light (Lo, black bars, 92 mol m−2 s−1 ) are represented as a function of developmental stage of the primary axis (stages 1–3) and during shoot branching (stage 3 to the end of each experiment). Letters A to D refer to the different experiments (A: effect of light intensity on the length of growing axis; B: excised bud outgrowth; C: effect of 6-BAP application on the length of axes; D: axillary buds morphology). Lo and Hi light treatments (experimental plan A) were either supplied with 12.25 mM or 0.25 mM nitrate. In experimental plan B, C and D nitrogen was supplied as 12.25 mM nitrate.
Table 1 Light intensity measurements along the stem. Photosynthetically active radiation (mol m−2 s−1 ) was measured with a LI-COR quantum sensor 190 at each leaf position along the primary axis, from the basal (#1) to the apical (#5) one, under Hi and Lo light conditions. Each value is the mean of at least 3 independent measurements ± SE. Leaf rank
Light intensity (mol m−2 s−1 ) Hi
5 4 3 2 1
453.2 345.8 270.0 181.4 154.1
± ± ± ± ±
8.6 24.4 25.3 6.7 11.9
%
Lo
100 76.3 59.6 40.0 34.0
91.5 80.4 66.7 58.5 49.5
% ± ± ± ± ±
2.1 3.8 4.8 5.2 3.5
100 87.9 72.9 64.0 54.1
France). Quantitative RT-PCR was conducted using the R. hybrida sucrose transporter-2 (RhSUC2) primers, as described by Henry et al. (2011).
less strongly reduced, ranging from 91.5 mol m−2 s−1 at the plant apex to 49.5 mol m−2 s−1 (about 54% of the apical incident light intensity) at the base of the stem. We measured net CO2 assimilation rate in Lo and Hi conditions under 2 different nitrate supplies (0.25 and 12.25 mM). In low light and low nitrogen supply, only the 2-apical leaves readily incorporated CO2 , while the basal leaves had very low fixation (Table 2). Supplying high level of nitrate (12.25 mM) in low light only slightly but significantly changed CO2 fixation all along the axis. In high light and low nitrogen supply, the CO2 fixation was much higher with a gradation from the apical bud (8.3 mol m−2 s−1 ) to the base (0.1 mol m−2 s−1 ) where it was very low, similar to that observed in Lo condition. This gradient was also observed in Hi condition in the presence of high nitrogen, while the basal buds (#1 and #2) showed significantly greater CO2 fixation (1.9 and 2.2 mol m−2 s−1 , respectively).
Statistical analysis
Carbohydrate content
All statistical analyses were carried out using R statistical package software version 2.12.2 (R Core Team, 2013).
We analyzed the amounts of fructose, glucose, sucrose and starch in the leaves and internodes of the primary axis in Lo and Hi light conditions (Fig. 2). While the amounts of fructose and glucose were low and nearly identical in Lo and Hi light conditions in leaves (about 5–8 mg g−1 of dry matter, Fig. 2A), they were significantly higher in Hi light compared to Lo light in the internodes (Fig. 2B, about 10–12 and 20 mg g−1 dry matter, respectively). Sucrose was much more abundant in leaves and stem (90 and 50 mg g−1 of dry matter, respectively) in Lo and Hi light. In contrast, starch was much higher in Hi light in leaves (about 2.5 fold) while the difference stayed statistically significant, but markedly reduced in the stem (Fig. 2B). The capacity of sucrose transport was evaluated by the measurement of the sucrose transporter RhSUC2 expression in the 3rd internode and in adjacent bud. Transcript accumulation was
Results PAR and net CO2 assimilation rates We measured the light intensities (PAR) in the region neighboring the buds along the primary axis in Hi and Lo light conditions (Table 1). In Hi light, the intensity displayed a continuous gradient with the maximum value measured at the plant apex (453 mol m−2 s−1 ). The light intensity was attenuated about 3fold at the base (154 mol m−2 s−1 , about 34% of the apical incident light intensity). In Lo light, the light gradient along the stem was
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P.-M. Furet et al. / Journal of Plant Physiology 171 (2014) 7–13
Table 2 Net CO2 assimilation rate of the different leaves along the primary axis under different light and nitrogen supply conditions. CO2 fixation (mol m−2 s−1 ) was measured using a LI-COR 6400 instrument on each leaf along the primary axis, from the basal (#1) to the apical (#5) one in Lo (92 mol m−2 s−1 ) and Hi (453 mol m−2 s−1 ) light condition, under low (−N, 0.25 mM) and high (+N, 12.25 mM) nitrate supply. Each value is the mean of at least 3 independent measurements ± SE. Different letters indicate significant differences between the different culture conditions for the same leaf rank (p < 0.05), according to HSD Tukey test. CO2 assimilation rate (mol m−2 s−1 )
Leaf rank
Lo −N 5 4 3 2 1
3.56 1.39 0.67 0.31 0.02
± ± ± ± ±
100
mg g -1 of dry matter
Hi −N
Lo +N 0.09 (a) 0.07 (a) 0.05 (a) 0.03 (a) 0.00 (a)
3.92 1.49 0.67 0.53 0.41
A
± ± ± ± ±
0.09 (b) 0.07 (a) 0.05 (a) 0.05 (b) 0.03 (b)
*
8.28 3.55 2.22 1.05 0.07
*
± ± ± ± ±
Hi +N 0.16 (c) 0.25 (b) 0.21 (b) 0.04 (c) 0.01 (c)
9.70 4.09 2.75 2.17 1.94
± ± ± ± ±
0.19 (d) 0.19 (b) 0.26 (b) 0.08 (d) 0.15 (d)
B
80
*
60 40
*
20 0
*
*
* Fructose Glucose Sucrose Starch
Fructose Glucose Sucrose Starch
Fig. 2. Sugars and starch content in leaves (A) and internodes (B) of primary axes. Fructose, glucose, sucrose and starch were quantified as mg g−1 dry matter in leaves (A) and stem (B) of plants cultivated under low (92 mol m−2 s−1 , black bars) and high (453 mol m−2 s−1 , white bars) light. Each value is the mean of at least 3 independent experiments ± SE. Stars indicate significant differences between light treatments (p < 0.05), according to HSD Tukey test.
promoted in Hi light treatment, and was higher in the internode (Fig. 3A, 5-fold) compared to the bud (Fig. 3B, 2.5-fold). Bud development and length of new branches
RhSUC2 mRNA relative quantity
Table 3 gives the number of preformed leaves in all the buds along the stem at stage 3 (Fig. 1) in Lo light. In these conditions, all five buds had a similar structure with statistically comparable number of preformed leaves (6.8–7.6). We analyzed the length of secondary axes produced from bud outgrowth along the stem under the 4 light treatments, and under
7
A
6
*
B
5 4
*
3 2 1 0
Lo
Hi
Lo
Hi
Fig. 3. RhSUC2 mRNA accumulation in the third internode (A) and adjacent bud (B) under low and high light intensities. RhSUC2 mRNA was quantified using qRTPCR in the third internode (A) and adjacent bud (B) of plants grown under low (92 mol m−2 s−1 , black bars) and high (453 mol m−2 s−1 , white bars) light treatments. Each point is the average of at least three independent experiments (±SE), normalized to SAND1 (Henry et al., 2011) and expressed relative to low light condition. Stars indicate significant differences between light treatments (p < 0.05), according to HSD Tukey test.
2 different nitrogen supplies. In our conditions, bud swelling that stayed below 3 mm never gave rise to a developing axis and was therefore not considered an outgrowth. In Lo condition (and low or high nitrate supply), only the apical buds (#4 and #5) grew out and produced actively growing axes that reached respectively about 15–20 cm (bud #4) and 29–32 cm (bud #5) at the end of the experiment (Fig. 4A). In marked contrast, virtually no outgrowth of basal buds occurred (about 2 mm at the end of the experiment) and was very similar in low and high nitrogen supplies. In the Hi–Lo condition, the apical buds (#4 and #5) broke and had strong axes elongation (23.3 and 27.3 cm, Fig. 4D) while the other buds (#1 to #3) did not grow out and behaved very similarly to those observed in the Lo condition. In the Hi condition and high nitrate supply, all 5 buds along the stem grew out (Fig. 4B, +N). The 4 apical ones (#2 to #5) formed, at the end of the experiment, axes whose lengths were comparable to those observed for the 2 apical buds in Lo treatment (25.6–30.7 cm). However, the very basal #1 bud showed significantly reduced growth (14.5 cm) that was, however, considerably higher than that observed for the same bud in the Lo condition (
