Photosynthetic sucrose acts as cotyledon-derived long-distance signal to control root growth during early seedling development in Arabidopsis

Stefan Kircher1 and Peter Schopfer1

Department of Plant Physiology, Faculty of Biology, Albert-Ludwigs-University, D-79104 Freiburg, Germany

Edited by Philip N. Benfey, Duke University, Durham, NC, and approved May 29, 2012 (received for review March 2, 2012) The most hazardous span in the life of green plants is the period This conclusion was further supported by amputation experiments after germination when the developing seedling must reach the in which root growth during global irradiation was followed over state of autotrophy before the nutrients stored in the seed are a period of 3 d after dissecting either the visible leaf primordia exhausted. The need for an economically optimized utilization of (“apex”), one cotyledon, the entire apex plus one cotyledon, both limited resources in this critical period is particularly obvious in cotyledons, or the entire apex plus both cotyledons (Fig. 3). species adopting the dispersal strategy of producing a large amount The results shown in Figs. 1 and 2 demonstrate that light pro- of tiny seeds. The model plant Arabidopsis thaliana belongs to this duces a reversible growth-promoting signal in the cotyledons, category. Arabidopsis seedlings promote root development only raising questions about the nature of this signal and its transport in the light. This response to light has long been recognized and from the cotyledons to the root tip, where growth occurs. Using recently discussed in terms of an organ-autonomous feature of photomorphogenic mutants, we first tested the previously sug- photomorphogenesis directed by the red/blue light absorbing gested possibility (3, 5–7) that the established photomorphogenic photoreceptors phytochrome and cryptochrome and mediated by pigments phytochrome (Phy) and cryptochrome (Cry) serve as hormones such as auxin and/or gibberellin. Here we show that the photoreceptors for root growth responses. Compared with the primary root of young Arabidopsis seedlings responds to an inter- wild type (WT, Columbia-0), the quadruple mutant phyA,B/cry1,2 organ signal from the cotyledons and that phloem transport of lacks the most prominently acting photomorphogenic photo- photosynthesis-derived sugar into the root tip is necessary and suf- receptors and thus shows skotomorphogenesis of the shoot also in ficient for the regulation of root elongation growth by light. the light (10). As illustrated in Fig. 4A, phyA,B/cry1,2 mutant seedlings produce short roots both in light and darkness, arguing he storage materials of the Arabidopsis seed (∼30 μg) support against the possible involvement of photoreceptors besides PhyA, Tseedling development for not more than 4–5 d (25 °C). After PhyB, Cry1, and Cry2 in the root growth response to light. To germination in the soil (i.e., in darkness), stored nutrients are further investigate whether well established light-signaling cas- primarily invested in the elongation of the shoot axis (hypocotyl) cades of these sensors are responsible for root elongation in the while root growth remains repressed (skotomorphogenesis). light (11, 12), we used the mutant constitutive photomorphogenic1 When the hypocotyl reaches the light, its growth stops and the (cop1-4). COP1 represses the default program of photomorpho- cotyledons are converted into photosynthetically active leaves genesis in darkness resulting in skotomorphogenesis. Accordingly, (photomorphogenesis). Concomitantly, elongation of the root is cop1 mutants show photomorphogenesis of the shoot also in induced, allowing the exploration of the soil for water and min- darkness (13), and its genome-expression profile in darkness is erals. In contrast to photomorphogenesis of shoot organs, the similar to that of light-grown WT seedlings (14). Thus, if root impact of light on the development of the root has so far not been growth is part of this developmental program, cop1-4 seedlings extensively investigated (1, 2). Recent investigators have con- should produce long roots also in darkness. Instead, Fig. 4A shows BIOLOGY

cluded that the effect of light on root development in seedlings is that the roots of cop1-4 seedlings are short, indicating that primary DEVELOPMENTAL an organ-autonomous feature of photomorphogenesis controlled root growth does not follow the photomorphogenic program ex- by the red/blue light absorbing photoreceptors phytochrome and ecuted in the shoot via the light-mediated elimination of COP1 cryptochrome (3–7) and mediated by hormones such as auxin and/ function, in line with observations of Miséra et al. (15). or gibberellin (7–9). Our conclusion that the conventional photoreceptors Phy and Cry may not be directly involved in the light-mediated root growth Results response directed our attention to another plant light-sensitive Fig. 1 shows the time course of primary root elongation of seedlings system that is often disregarded in photomorphogenesis research: grown on vertical agar plates transferred from the light chamber to photosynthesis. Sugars synthesized in the green parts of plants are darkness or vice versa. For investigating the reversible changes in known to have important roles as signaling molecules in addition growth rate in more detail, we determined root elongation with an to their metabolic functions (16–18), for example, acting as a sys- infra-red video camera system allowing monitoring growth kinetics temic signal for promoting root development in response to in light and darkness with a high temporal and spatial resolution. phosphorus starvation (19). We tested the hypothesis that the light Fig. 2A shows that growth attenuation induced by the transition stimulus emerging in the cotyledons and transmitted to the root is from light to dark and growth enhancement by dark to light photosynthetically produced sugar. In a first experiment, we grew transfer can be detected with this technique after a lag of 60–90 min. As shown in Fig. 2B, a light spot of 5 mm diameter of similar fl uence rate directed to the whole shoot (trace 1), a single cotyle- Author contributions: S.K. and P.S. designed research, performed research, analyzed data, don (trace 2), or the cotyledon of a seedling from which the other and wrote the paper. cotyledon including the entire shoot apex was removed (trace 3), The authors declare no conflict of interest. was similarly effective in inducing root elongation after dark ad- This article is a PNAS Direct Submission. aptation. When the light spot was directed to various regions of the 1To whom correspondence may be addressed. E-mail: [email protected]. seedling, the root started to grow only if the cotyledons received de or [email protected]. the light (Fig. 2C). These results indicate that the light stimulus This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. inducing root growth is perceived specifically by the cotyledons. 1073/pnas.1203746109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1203746109 PNAS | July 10, 2012 | vol. 109 | no. 28 | 11217–11221 Downloaded by guest on September 25, 2021 Fig. 1. Root growth in light and darkness 3–8 d after initiation of germi- nation (daig). After 3 d in the light, seedlings were kept in continuous light (cL) or darkness (cD) and transferred from light to darkness (D) or darkness to light (L) after 4 and 5 daig as indicated.

WT, phyA,B/cry1,2, and cop1-4 seedlings on agar plates containing sucrose. Fig. 4B shows that, in all cases, the short-root morpho- types demonstrated in Fig. 4A could be converted to long-root morphotypes by supplementary sucrose. To further test the hy- pothesis, we used three physiological treatments for inhibiting various sections of the photosynthetic apparatus in WT seedlings: (i) removal of CO2 from the atmosphere in the light, (ii) pre- venting greening with the carotenoid-biosynthesis inhibitor nor- flurazone in the light (20), and (iii) preventing greening, but not PhyA-mediated photomorphogenesis, by growing the seedlings in far-red light (Fig. 5). In all three treatments, the inhibition of photosynthetic assimilation produced short roots that could be converted to long roots by adding sucrose to the medium. Root growth in the light and hypocotyl growth in light and darkness were not affected by the sucrose treatment (except a slight pro- motion in far-red light). Thus, sucrose promotes growth specifi- cally in the root where it interacts synergistically with light. The concentration-effect curve for sucrose (Fig. 6) indicates that the sucrose effect on root growth in darkness can be detected already at 1 mM and may not yet be saturated at 100 mM, a sucrose concentration that produced about 90% of the root length in the Fig. 2. Short-term kinetics of root growth (representative single measure- light without sucrose. However, because the hypocotyl growth was ments). (A) Intact light-grown seedlings were transferred to darkness 3 daig inhibited at ≥100 mM, we routinely used 30 mM as a standard (D) and back to the light (broad field) 4 daig (L). (B) Perception of the light concentration in this type of experiment. We also tested the pos- stimulus by the cotyledons. Light-grown seedlings were transferred to sibility that the potentially growth-promoting plant hormones darkness 5 daig. At day 6 (L), a spotlight beam was directed to the whole auxin and gibberellin contribute to root growth (8) in addition to shoot (1), a single cotyledon of an intact seedling (2), or the cotyledon of sucrose. We found that auxin (IAA) inhibits root elongation in a seedling from which the other cotyledon + the shoot apex were dissected 5 ≥ μ daig (3). The dark control seedling (4) was growing next to the seedling of darkness at concentrations 0.01 M (as known from light-grown trace 1. (C) Selective irradiation of seedling parts. Intact seedlings grown as fi seedlings), whereas gibberellin (GA3) had no signi cant effect up in B were partially irradiated with a spot light beam as outlined. In seedling to 10 μM(Table S1). 1, the visible leaf primordia at the shoot apex were dissected 5 daig. The The data presented so far suggest that root growth depends on distance between measuring points was 32 min. photosynthetic sugar, presumably sucrose, which is delivered from the cotyledons to the root. Alternatively, it could be hypothesized that sugar gives rise to a growth factor X in the cotyledons, which from the cotyledons to the root. In agreement with this conclusion, can be transported to the root tip. To discriminate between these we found that the roots respond to high sucrose concentrations in possibilities, we fed sucrose specifically either to the cotyledons or the range normally present in the sieve tube sap (up to 1 M). Note the root (close to the growth zone) of light-grown, dark-adapted that, in these experiments, the growth zone of the root was not in seedlings and determined root elongation in darkness. Fig. 7 touch with the applied sucrose, avoiding osmotic side effects. shows that root growth can be induced by feeding sucrose to the A critical step in sugar transport to the root is phloem loading cotyledons and that sugar acts as a direct growth stimulus in the in the cotyledons. Arabidopsis is known as an “apoplastic loader” root. Thus, conventional source-to-sink transport of sucrose in the in which sucrose secreted by the mesophyll cells can be pumped phloem seems to be sufficient to explain the signal transmission into the sieve tubes by the H+-sucrose transporter SUC2 (21).

11218 | www.pnas.org/cgi/doi/10.1073/pnas.1203746109 Kircher and Schopfer Downloaded by guest on September 25, 2021 Fig. 3. Effect of cotyledon and apex amputation on root growth. After 5 daig in the light, the following seedling parts were dissected: visible leaf primordia (−apex), one cotyledon (−1 cot), entire apex and one cotyledon (−apex, 1 cot), two cotyledons (−2 cots), entire apex and two cotyledons (−apex, 2 cots). Root elongation was followed for further 3 d in the light. If present, the leaf pri- mordia started to expand between day 1 and day 2 after amputation.

We used a SUC2-deficient mutant for testing the involvement of this transporter in the growth-signal transfer to the root. As expected (22, 23), a fraction of the segregating progeny of het- erozygous (SUC2/suc2) plants grown on sucrose-free medium in the light showed a strong retardation of root growth (Fig. 8 A Fig. 5. Effect of photosynthesis inhibition on root and hypocotyl growth and B). This mutant phenotype could be rescued by growing the and its reversal by sucrose. Seedlings were grown for 4 daig in light (L) or seedlings on sucrose medium (Fig. 8C). darkness (D) with (gray bars) or without (white bars) 30 mM sucrose. (A) Without further treatment (control). (B)InaCO-depleted atmosphere Discussion 2 (−CO2). (C) With 0.1 μM norflurazone (San 9789) in the medium (20) (+NF). Our results suggest a hierarchical order of steps in the execution of (D) Under far-red light (FR) with or without CO2. the light-dependent developmental program during early seedling development (Fig. 9). In darkness, the limited resources are mainly allocated to hypocotyl growth for pushing the cotyledons toward the from direct photoreceptor signaling and put under the command of light, whereas growth of the nearly functionless root is stalled. After photosynthesis. Moreover, the emerging role of Phy/Cry-mediated reaching the light, photoreceptor signaling effects photomorpho- photomorphogenesis as a prerequisite for photosynthetic activity genesis in the shoot, including the establishment of photosynthesis may explain why the involvement of these photoreceptors shows up in the cotyledons. Subsequently, photosynthetically generated su- under particular experimental conditions (3–7). crose acts as an interorgan signal as well as fuel to initiate growth of In contrast to general belief and in disagreement with published BIOLOGY

the root that is now needed for capturing nutrients from the soil. results (8, 24), our amputation experiments (Figs. 2 and 3) provide DEVELOPMENTAL This interpretation explains why root growth control is uncoupled

Fig. 4. Effect of light on root elongation of mutants impaired either in the photomorphogenesis program (phyA,B/cry1,2) or the skotomorphogenesis program (cop1-4) grown in the absence (a) or presence (b)of30mMsucrose Fig. 6. Root and hypocotyl growth in darkness as a function of sucrose (suc). WT and mutant seedlings were kept either in darkness (D) or light (L) concentration in the medium. Seedlings were grown for 4 daig on media for 4 daig. Arrowheads indicate root tip. containing 0–100 mM sucrose.

Kircher and Schopfer PNAS | July 10, 2012 | vol. 109 | no. 28 | 11219 Downloaded by guest on September 25, 2021 Fig. 9. Summarizing scheme to illustrate the dual role of light in early seedling development. After seed germination, seedlings follow the skoto- morphogenic program of growth in darkness (1). Near the soil surface, in- cident light is perceived by phytochrome (Phy) and cryptochrome (Cry) Fig. 7. Effect of sucrose application to the root (A) or the cotyledons (B). photoreceptor systems (2) leading to onset of photomorphogenic de- Sucrose (0–1 M) or mannitol (0.1–1 M, osmotic control) were applied to the velopment including establishment of the photosynthetic apparatus. In cotyledons or the root (above the growth zone) of seedlings grown for 5 consequence, photosynthesis generates sugars (S) acting as interorgan signal daig in the light plus 1 d in darkness and subsequently kept in darkness for and as fuel to drive root growth (3). further 4 d.

The dominant role of sugar in the control of root growth may be no evidence that the elongation growth of the root of light-grown restricted to the cotyledon stage of seedling development. With Arabidopsis seedlings is controlled by auxin derived from the shoot the onset of leaf development additional factors may come into fi apex. However, this nding does not preclude a general require- play, indicated by the observation that external sucrose supply ment of auxin (or cytokinin and gibberellin) as permissive, rather cannot replace light with respect to side root formation that than regulating, factors with essential functions in meristem ac- commences 5–6 d after initiation of germination (daig) (Fig. S1). tivity or cell elongation (24). A permissive role of gibberellin is Light required for promoting chlorophyll synthesis and other light- evident from the finding that root growth in the light is impaired in dependent processes in addition to photosynthesis may then be- the gibberellin-deficient ga1-3 mutant (8). come increasingly important also for root development (5, 6). There is scattered evidence in the literature that sucrose feeding can have beneficial effects on root development, for instance by dampening the decrease in growth rate during the daily night in rhythmically entrained plants (25) or promoting the emergence of lateral root primordia (26). Sugar application has been shown to increase root growth in the dark, reaching about 30% of the effect of light (27). However, as in many other studies with Arabidopsis seedlings, the routine of including sucrose into the culture medium may have hindered the full recognition of its specific morphogenic role in root growth. In this paper, we show that the allocation of photosynthesis-derived sugar limits root growth already during the first few days after germination and serves directly as a long-dis- tance signal for coupling root growth to shoot growth in the light. Materials and Methods Materials. Wild-type Arabidopsis thaliana (Columbia-0), cop1-4, and phyA,B/ cry1,2 mutants were as described (10, 13, 28). Mutant Suc2 seeds (22) were a gift from Brian Ayre (University of North Texas, Denton, TX). Fig. 8. Phenotypes of SUC2-deficient mutant seedlings grown in the ab- sence (A and B) or presence (C) of sucrose. Seeds from the progeny of het- Growth Conditions. Seeds were sterilized with 70% ethanol and sown on half- erozygous SUC2/suc2 plants were germinated on medium without sucrose. strength MS medium + vitamins (Duchefa) with 1% agar and 10 mM Mes After 4 daig in the light, seedlings differing from WT (10–12 mm root length) buffer (pH 6.1) and additions as indicated. After 2 d storage at 5 °C, the plates by having 1- to 2-mm-long roots (apparent homozygous individuals (A), were incubated in vertical orientation in continuous white light (fluorescent −2 −1 −2 −1 were selected and kept on medium without (B) or with (C)30mMsucrose tubes, 100 μmol m s ), far-red light (λmax = 730 nm; 25 μmol m s ;ref. for further 4 d in the light. 29), or darkness at 25 °C (after 5 h white light). CO2 depletion was achieved

11220 | www.pnas.org/cgi/doi/10.1073/pnas.1203746109 Kircher and Schopfer Downloaded by guest on September 25, 2021 by enclosing the plates in hermetically closed, transparent plastic bags con- with a focusing lens. Picture sequences were captured with AVT software taining 10 g of NaOH/CaO pellets. For local sucrose application, 50-μldropsof (AVT Universal Package 2.0) and analyzed using ImageJ software (NIH). sterilized solutions fortified with boiled starch slurry (60 mg ml−1)wereplaced on cotyledons or a 10-mm region of the root 5 mm above the tip. Amputation Experiments. Cotyledons were excised under a stereomicroscope using fine scissors or scalpels. Microknives prepared by flattening 0.3-mm Growth Measurements. Macroscopic root elongation was measured by injection needles were used for excising the visible leaf primordia at the marking root tips daily at the backside of the plates. For kinematic time-lapse shoot apex. measurements, the plates were exposed from behind to weak IR radiation −2 −1 (880-nm LED array, Advanced Illumination; 0.5 μmol m s ). Pictures were Statistics. Data points are means of 20 seedlings from four to six independent taken automatically with an IR-sensitive CCD camera (Marlin F146B, AVT) experiments. SEs were between 3% and 10%. equipped with a 720-nm cutoff filter (Hoya R-72, Tokina) following the set- up proposed by Edgar Spalding (Phytomorph; http://phytomorph.wisc.edu/ ACKNOWLEDGMENTS. We thank B. Ayre for providing mutant suc2 seeds hardware/fixed-cameras.php). The seedlings were irradiated with white LED − − and E. Schäfer and T. Kretsch for critical discussions and reading of the light (100 μmol m 2 s 1) either globally or partially with a spot beam of 5- manuscript. S.K. is funded by SFB 592 of the Deutsche Forschungsgemein- mm diameter emitted from a LED source attached to fiber optics equipped schaft (DFG) and the Wissenschaftliche Gesellschaft, Freiburg.

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