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

Abstract

The most hazardous span in the life of green plants is the period after germination when the developing seedling must reach the state of autotrophy before the nutrients stored in the seed are exhausted. The need for an economically optimized utilization of limited resources in this critical period is particularly obvious in species adopting the dispersal strategy of producing a large amount of tiny seeds. The model plant Arabidopsis thaliana belongs to this category. Arabidopsis seedlings promote root development only in the light. This response to light has long been recognized and recently discussed in terms of an organ-autonomous feature of photomorphogenesis directed by the red/blue light absorbing photoreceptors phytochrome and cryptochrome and mediated by hormones such as auxin and/or gibberellin. Here we show that the primary root of young Arabidopsis seedlings responds to an interorgan signal from the cotyledons and that phloem transport of photosynthesis-derived sugar into the root tip is necessary and sufficient for the regulation of root elongation growth by light.

The storage materials of the Arabidopsis seed (∼30 μg) support seedling development for not more than 4–5 d (25 °C). After germination in the soil (i.e., in darkness), stored nutrients are primarily invested in the elongation of the shoot axis (hypocotyl) while root growth remains repressed (skotomorphogenesis). When the hypocotyl reaches the light, its growth stops and the cotyledons are converted into photosynthetically active leaves (photomorphogenesis). Concomitantly, elongation of the root is induced, allowing the exploration of the soil for water and minerals. In contrast to photomorphogenesis of shoot organs, the impact of light on the development of the root has so far not been extensively investigated (1, 2). Recent investigators have concluded that the effect of light on root development in seedlings is an organ-autonomous feature of photomorphogenesis controlled by the red/blue light absorbing photoreceptors phytochrome and cryptochrome (3–7) and mediated by hormones such as auxin and/or gibberellin (7–9).

Results

Fig. 1 shows the time course of primary root elongation of seedlings grown on vertical agar plates transferred from the light chamber to darkness or vice versa. For investigating the reversible changes in growth rate in more detail, we determined root elongation with an infra-red video camera system allowing monitoring growth kinetics in light and darkness with a high temporal and spatial resolution. Fig. 2A shows that growth attenuation induced by the transition from light to dark and growth enhancement by dark to light 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 fluence rate directed to the whole shoot (trace 1), a single cotyledon (trace 2), or the cotyledon of a seedling from which the other cotyledon including the entire shoot apex was removed (trace 3), was similarly effective in inducing root elongation after dark adaptation. When the light spot was directed to various regions of the seedling, the root started to grow only if the cotyledons received the light (Fig. 2C). These results indicate that the light stimulus inducing root growth is perceived specifically by the cotyledons. This conclusion was further supported by amputation experiments in which root growth during global irradiation was followed over a period of 3 d after dissecting either the visible leaf primordia (“apex”), one cotyledon, the entire apex plus one cotyledon, both cotyledons, or the entire apex plus both cotyledons (Fig. 3).

Fig. 1.

Root growth in light and darkness 3–8 d after initiation of germination (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.

Fig. 2.

Short-term kinetics of root growth (representative single measurements). (A) Intact light-grown seedlings were transferred to darkness 3 daig (D) and back to the light (broad field) 4 daig (L). (B) Perception of the light stimulus by the cotyledons. Light-grown seedlings were transferred to darkness 5 daig. At day 6 (L), a spotlight beam was directed to the whole shoot (1), a single cotyledon of an intact seedling (2), or the cotyledon of a seedling from which the other cotyledon + the shoot apex were dissected 5 daig (3). The dark control seedling (4) was growing next to the seedling of trace 1. (C) Selective irradiation of seedling parts. Intact seedlings grown as in B were partially irradiated with a spot light beam as outlined. In seedling 1, the visible leaf primordia at the shoot apex were dissected 5 daig. The distance between measuring points was 32 min.

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 primordia started to expand between day 1 and day 2 after amputation.

The results shown in Figs. 1 and 2 demonstrate that light produces a reversible growth-promoting signal in the cotyledons, raising questions about the nature of this signal and its transport from the cotyledons to the root tip, where growth occurs. Using photomorphogenic mutants, we first tested the previously suggested possibility (3, 5–7) that the established photomorphogenic pigments phytochrome (Phy) and cryptochrome (Cry) serve as photoreceptors for root growth responses. Compared with the wild type (WT, Columbia-0), the quadruple mutant phyA,B/cry1,2 lacks the most prominently acting photomorphogenic photoreceptors and thus shows skotomorphogenesis of the shoot also in the light (10). As illustrated in Fig. 4A, phyA,B/cry1,2 mutant seedlings produce short roots both in light and darkness, arguing against the possible involvement of photoreceptors besides PhyA, PhyB, Cry1, and Cry2 in the root growth response to light. To further investigate whether well established light-signaling cascades of these sensors are responsible for root elongation in the light (11, 12), we used the mutant constitutive photomorphogenic1 (cop1-4). COP1 represses the default program of photomorphogenesis in darkness resulting in skotomorphogenesis. Accordingly, cop1 mutants show photomorphogenesis of the shoot also in darkness (13), and its genome-expression profile in darkness is similar to that of light-grown WT seedlings (14). Thus, if root growth is part of this developmental program, cop1-4 seedlings should produce long roots also in darkness. Instead, Fig. 4A shows that the roots of cop1-4 seedlings are short, indicating that primary root growth does not follow the photomorphogenic program executed in the shoot via the light-mediated elimination of COP1 function, in line with observations of Miséra et al. (15).

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) of 30 mM sucrose (suc). WT and mutant seedlings were kept either in darkness (D) or light (L) for 4 daig. Arrowheads indicate root tip.

Our conclusion that the conventional photoreceptors Phy and Cry may not be directly involved in the light-mediated root growth response directed our attention to another plant light-sensitive system that is often disregarded in photomorphogenesis research: photosynthesis. Sugars synthesized in the green parts of plants are known to have important roles as signaling molecules in addition to their metabolic functions (16–18), for example, acting as a systemic signal for promoting root development in response to phosphorus starvation (19). We tested the hypothesis that the light stimulus emerging in the cotyledons and transmitted to the root is photosynthetically produced sugar. In a first experiment, we grew WT, phyA,B/cry1,2, and cop1-4 seedlings on agar plates containing sucrose. Fig. 4B shows that, in all cases, the short-root morphotypes demonstrated in Fig. 4A could be converted to long-root morphotypes by supplementary sucrose. To further test the hypothesis, 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) preventing greening with the carotenoid-biosynthesis inhibitor norflurazone 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 promotion in far-red light). Thus, sucrose promotes growth specifically 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 light without sucrose. However, because the hypocotyl growth was inhibited at ≥100 mM, we routinely used 30 mM as a standard concentration in this type of experiment. We also tested the possibility that the potentially growth-promoting plant hormones auxin and gibberellin contribute to root growth (8) in addition to sucrose. We found that auxin (IAA) inhibits root elongation in darkness at concentrations ≥0.01 μM (as known from light-grown seedlings), whereas gibberellin (GA₃) had no significant effect up to 10 μM (Table S1).

Fig. 5.

Effect of photosynthesis inhibition on root and hypocotyl growth and its reversal by sucrose. Seedlings were grown for 4 daig in light (L) or darkness (D) with (gray bars) or without (white bars) 30 mM sucrose. (A) Without further treatment (control). (B) In a CO₂-depleted atmosphere (−CO₂). (C) With 0.1 μM norflurazone (San 9789) in the medium (20) (+NF). (D) Under far-red light (FR) with or without CO₂.

Fig. 6.

Root and hypocotyl growth in darkness as a function of sucrose concentration in the medium. Seedlings were grown for 4 daig on media containing 0–100 mM sucrose.

The data presented so far suggest that root growth depends on 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 can be transported to the root tip. To discriminate between these possibilities, we fed sucrose specifically either to the cotyledons or the root (close to the growth zone) of light-grown, dark-adapted seedlings and determined root elongation in darkness. Fig. 7 shows that root growth can be induced by feeding sucrose to the cotyledons and that sugar acts as a direct growth stimulus in the root. Thus, conventional source-to-sink transport of sucrose in the phloem seems to be sufficient to explain the signal transmission from the cotyledons to the root. In agreement with this conclusion, we found that the roots respond to high sucrose concentrations in the range normally present in the sieve tube sap (up to 1 M). Note that, in these experiments, the growth zone of the root was not in touch with the applied sucrose, avoiding osmotic side effects.

Fig. 7.

Effect of sucrose application to the root (A) or the cotyledons (B). Sucrose (0–1 M) or mannitol (0.1–1 M, osmotic control) were applied to the cotyledons or the root (above the growth zone) of seedlings grown for 5 daig in the light plus 1 d in darkness and subsequently kept in darkness for further 4 d.

A critical step in sugar transport to the root is phloem loading in the cotyledons. Arabidopsis is known as an “apoplastic loader” in which sucrose secreted by the mesophyll cells can be pumped into the sieve tubes by the H⁺-sucrose transporter SUC2 (21). 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 heterozygous (SUC2/suc2) plants grown on sucrose-free medium in the light showed a strong retardation of root growth (Fig. 8 A and B). This mutant phenotype could be rescued by growing the seedlings on sucrose medium (Fig. 8C).

Fig. 8.

Phenotypes of SUC2-deficient mutant seedlings grown in the absence (A and B) or presence (C) of sucrose. Seeds from the progeny of heterozygous SUC2/suc2 plants were germinated on medium without sucrose. After 4 daig in the light, seedlings differing from WT (10–12 mm root length) by having 1- to 2-mm-long roots (apparent homozygous individuals (A), were selected and kept on medium without (B) or with (C) 30 mM sucrose for further 4 d in the light.

Discussion

Our results suggest a hierarchical order of steps in the execution of 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 light, whereas growth of the nearly functionless root is stalled. After reaching the light, photoreceptor signaling effects photomorphogenesis in the shoot, including the establishment of photosynthesis in the cotyledons. Subsequently, photosynthetically generated sucrose acts as an interorgan signal as well as fuel to initiate growth of the root that is now needed for capturing nutrients from the soil. This interpretation explains why root growth control is uncoupled from direct photoreceptor signaling and put under the command of photosynthesis. Moreover, the emerging role of Phy/Cry-mediated photomorphogenesis as a prerequisite for photosynthetic activity may explain why the involvement of these photoreceptors shows up under particular experimental conditions (3–7).

Fig. 9.

Summarizing scheme to illustrate the dual role of light in early seedling development. After seed germination, seedlings follow the skotomorphogenic program of growth in darkness (1). Near the soil surface, incident light is perceived by phytochrome (Phy) and cryptochrome (Cry) photoreceptor systems (2) leading to onset of photomorphogenic development including establishment of the photosynthetic apparatus. In consequence, photosynthesis generates sugars (S) acting as interorgan signal and as fuel to drive root growth (3).

In contrast to general belief and in disagreement with published results (8, 24), our amputation experiments (Figs. 2 and 3) provide no evidence that the elongation growth of the root of light-grown Arabidopsis seedlings is controlled by auxin derived from the shoot apex. However, this finding does not preclude a general requirement of auxin (or cytokinin and gibberellin) as permissive, rather than regulating, factors with essential functions in meristem activity or cell elongation (24). A permissive role of gibberellin is evident from the finding that root growth in the light is impaired in the gibberellin-deficient ga1-3 mutant (8).

The dominant role of sugar in the control of root growth may be restricted to the cotyledon stage of seedling development. With the onset of leaf development additional factors may come into play, indicated by the observation that external sucrose supply cannot replace light with respect to side root formation that commences 5–6 d after initiation of germination (daig) (Fig. S1). Light required for promoting chlorophyll synthesis and other light-dependent processes in addition to photosynthesis may then become 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-distance 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).

Growth Conditions.

Seeds were sterilized with 70% ethanol and sown on half-strength MS medium + vitamins (Duchefa) with 1% agar and 10 mM Mes buffer (pH 6.1) and additions as indicated. After 2 d storage at 5 °C, the plates were incubated in vertical orientation in continuous white light (fluorescent tubes, 100 μmol m⁻² s⁻¹), far-red light (λ_max = 730 nm; 25 μmol m⁻² s⁻¹; ref. 29), or darkness at 25 °C (after 5 h white light). CO₂ depletion was achieved by enclosing the plates in hermetically closed, transparent plastic bags containing 10 g of NaOH/CaO pellets. For local sucrose application, 50-μl drops of sterilized solutions fortified with boiled starch slurry (60 mg ml⁻¹) were placed on cotyledons or a 10-mm region of the root 5 mm above the tip.

Growth Measurements.

Macroscopic root elongation was measured by marking root tips daily at the backside of the plates. For kinematic time-lapse measurements, the plates were exposed from behind to weak IR radiation (880-nm LED array, Advanced Illumination; 0.5 μmol m⁻² s⁻¹). Pictures were taken automatically with an IR-sensitive CCD camera (Marlin F146B, AVT) equipped with a 720-nm cutoff filter (Hoya R-72, Tokina) following the set-up proposed by Edgar Spalding (Phytomorph; http://phytomorph.wisc.edu/hardware/fixed-cameras.php). The seedlings were irradiated with white LED light (100 μmol m⁻² s⁻¹) either globally or partially with a spot beam of 5-mm diameter emitted from a LED source attached to fiber optics equipped with a focusing lens. Picture sequences were captured with AVT software (AVT Universal Package 2.0) and analyzed using ImageJ software (NIH).

Amputation Experiments.

Cotyledons were excised under a stereomicroscope using fine scissors or scalpels. Microknives prepared by flattening 0.3-mm injection needles were used for excising the visible leaf primordia at the shoot apex.

Statistics.

Data points are means of 20 seedlings from four to six independent experiments. SEs were between 3% and 10%.