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.