Photophobic behavior of maize roots

Abstract

Primary roots of young maize seedlings showed peculiar growth behavior when challenged by placing them on a slope, or if whole seedlings were turned upside down. Importantly, this behavior was dependent on the light conditions. If roots were placed on slopes in the dark, they performed “crawling” behavior and advanced rapidly up the slope. However, as soon as these roots were illuminated, their crawling movements along their horizontal paths slowed down, and instead tried to grow downwards along the gravity vector. A similar light-induced switch in the root behavior was observed when roots were inverted, by placing them in thin glass capillaries. As long as they were kept in the darkness, they showed rapid growth against the gravity vector. If illuminated, these inverted roots rapidly accomplished U-turns and grew down along the gravity vector, eventually escaping from the capillaries upon reaching their open ends. De-capped roots, although growing vigorously, did not display these light-induced photophobic growth responses. We can conclude that intact root cap is essential for the photophobic root behavior in maize.

Introduction

Francis Darwin discovered negative root phototropism in 1879 and he, together with his famous father Charles, published numerous experiments in their well-known book “Power of Movements in Plants” one year later.^1,2 Although this book is cited mainly in respect of experiments performed with coleoptiles³—which subsequently led to the discovery of their mobile chemical signal known today as the plant hormone auxin⁴—the major part of their book is devoted to roots and their tropisms.² The Darwins used roots of several species, including roots of maize which can be easily de-capped² but which retain their vigorous growth rates intact. They were among the first authors characterizing also thigmotropism and nutations of plant organs including roots.²

Light affects all aspects of plant biology. Photomorphogenesis, together with phototropism and nutations, belongs presently to one of ther best characterized branches of plant physiology, as well as plant sensory and developmental biology.^3-10

In nature, roots evolved and live in soil, in more-or-less complete darkness. But roots continue their growth even when illuminated and this feature has resulted in the common practice of growing Arabidopsis seedlings in transparent Petri dishes.¹¹ However, our previous results¹² and those reported here strongly argue that this long-established procedure—which has been common practice for > 30 y¹¹—should be stopped. After only a few seconds of illumination, root apices of Arabidopsis generate massive amounts of reactive oxygen species (ROS)¹² and their growth rate increases.^12,13 In fact, ROS species act as stress-induced signals, regulating—among other processes—root development and growth.^14-17 Importantly, stress-induced ROS signals spread rapidly throughout plant bodies, integrating stress-induced adaptation across the whole plant.¹⁷ Arabidopsis roots are too sensitive for experiments into their growth behavior, especially as related to the root cap, as they are not able to fully regenerate removed root caps. Therefore, we have switched to roots of maize as our experimental system of choice. Maize roots served Charles and Francis Darwin well in their seminal works on root tropisms when removal of root caps was used as important novel aspect of their approach.^{1,2,5,6,18-20} Darwins showed that the intact root apex act as a brain-like command center controlling behavior of the whole root.^2,5,6,18-23 This conclusion was causing controversy at that time,^24,25 and remains controversial also presently.^3,4,20,23 Crawling behavior of maize roots was discovered only few years ago.²⁶ This unique root behavior supports the Darwinian view of roots,^20,21 documenting that plant roots display rich repertoire of behavior. In fact, we predict that this complexity is just the ‘tip of iceberg’^27,28 as far as root behavior is concerned. Roots are only exceptionally studied in situ, within the soil, in the natural context of the underground and dark environment.

Results

Crawling behavior of horizontal maize roots and its sensitivity to light

Maize roots placed horizontally, or on a slope, performed so-called crawling-like behavior (Fig. 1A–D and Vid. S1, see also refs. 21 and 26). As shown in our study, this crawling behavior of maize roots is root cap-dependent (Vid. S2) and sensitive to light. Roots accomplish forward crawling in darkness, or if exposed to dim light (Fig. 1A–D and Vid. S1). But when maize roots are eposed to white light, the “touch-down” phase becomes immediately prolonged (Fig. 1F and Vid. S2) and is associated with the exertion of pressure by the root apex on the substrate (Fig. 1F). If several roots are crawling in darkness side-by-side, brief (20 min) illumination with white light elicits an immediate and coordinated change in crawling behavior (Fig. 1D; Vid. S2) with growing root apices performing persistent downward movement (Fig. 1F). As the substrate is too tough to be penetrated, the roots and attached seedlings are raised up via the force exerted by the root apices on the substrate (Fig. 2).

Figure 1. Still images taken from movies of crawling maize roots. (A) Intact root crawling up the slope. (B) De-capped roots growing, but without showing crawling behavior. (C) Intact roots crawling up the slope under dim white light. (D) 20 min illumination with white light results in rapid bending of roots due to stronger gravitropism. (E) Intact roots starting crawling up the slope under white light. (F) Intact roots illuminated for 6 h with white light are not crawling effectively as they try to grow downwards along the gravity vector. Images are taken from movies shown in the Supplemental data.

Figure 2. Inverted maize roots crawl against the gravity in darkness but turn down in light. (A) Intact inverted maize roots crawling against the gravity vector within glass capillaries if kept in darkness. (B) The same situation under white light—all roots performed U-turns within capillaries and grew down along the gravity vector. (C) After 12 h, the roots grew out of the capillaries if kept continually in white light (C1 shows whole seedlings, C2 shows escaped root apices in higher magnification). (D) Summary of all situations. (1 and 2) Roots inverted in darkness; (3 and 4) roots inverted in white light; (5) white light illuminated roots performing U-turns; (6) De-capped roots grow upwards in white light. Even after 2 d within capillaries, roots are still able to accomplish U-turns if suddenly illuminated (data not shown).

Inverted maize roots perform crawling behavior in darkness, but accomplish light-induced U-turn bending-morphogenesis upon illumination

Roots of maize inserted into thin glass capillaries in the inverted position continue in crawling behavior if kept in darkness, growing against the gravity vector (Fig. 2 and Vid. S3). Similarly, as in the case of horizontal root crawling, the de-capped inverted roots do not display crawling behavior but continue in their upward growth (Fig. 2D). In order to test if illumination would change the root crawling behavior also in the inverted roots, we kept roots in white light after their inversion. Surprisingly, such roots stopped their upward movement and initiated U-turn behavior (Figs. 2B and D, 3 and Vid. S4). Clearly, light exposure changed the root behavior not only where roots were placed horizontally but also if they were inverted with respect to the gravity vector. In order to accomplish this complicated turning (Fig. 1D) within the narrow glass capillaries (the diameter of roots is around 1 mm, whereas the inner diameter of capillaries is around 2 mm), the diameter of the root apices was rapidly reduced from about 1 to 0.5 mm. This thinning of root apices allowed them to accomplish U-turn behavior. These thinned roots then increased their rate of downward growth and rapidly emerged from the narrow glass capillaries (Fig. 2C).

Figure 3. Maize roots inverted and inserted in glass capillaries. Percentage of roots which performed U-turns in darkness and in white light. Three independent experiments were performed for both light and dark situation. From 103 roots, only one performed U-turn in the darkness (SD = 1,65) and from 89 roots, 68 performed U-turn in the white light (SD = 17,38).

Discussion

We have demonstrated that roots have an unexpectedly sophisticated strategy to escape from light, not just negative phototropism. Arabidopsis roots speed-up their growth rate if illuminated and this so-called escape-tropism is based on actin polymerization²⁹ and auxin transport.^13,30,31 Maize roots inserted into thin glass capillaries in inverted position continue in their crawling against the gravity vector as long as they are kept in darkness. After illumination, such roots have immediately and effectively performed U-turns, requiring also significant thinning of their apices. This U-turn represents new, until now not reported type of root behavior, which we characterize as the root photophobism.

An intact apex with root cap is essential for root crawling behavior. This implies that the root cap is essential for sensory detection process and signal transduction processes?not only of gravity forces but also of light stimulation. Importantly, the root cap is not essential for vigorous maize root growth. A similar result was published for maize roots recently when de-capped roots were shown to be unresponsive to ethylene.³² Besides a direct role in the perception, as is implicated in gravisensing, the intact root cap is known to be essential also for the basipetal (shootward) auxin transport which is based on PIN2 auxin efflux carriers.^30-33 Importantly, Arabidopsis roots of the pin2 mutant resemble maize roots devoid of their root caps as they are not able to perform either gravi- or phototropism.^29,31,33

In combination with the negative phototropism, the ROS-mediated speeding-up of root growth after illumination of roots of Arabidopsis is interpreted as an additional strategy of stressed roots to relieve the stress by escaping from the light.¹² In maize, illuminated roots increase their positive gravitropism. We propose a new term for this escape behavior of illuminated roots—root photophobism. This Darwinian view of plant roots, with the root apex acting as brain-like “command center”,^2,6,20-23 will allow a better understanding of the true communicative and behavioral nature of plants and their roots,^{5,27,28,34-36} which is essential for the sustainable agriculture of the future^5,36

Materials and Methods

After 2 h of imbibition in distilled water, grains of maize (Zea mays L., cv Tasty Sweet; provided by Hild samen GmbH, Marbach, Germany) were allowed to germinate in moistened filter paper rolls for 3–4 d at 20°C in darkness. Seedlings with straight primary roots 2 cm long were selected for further study. For the root inversion experiments, the seedlings were placed up-side down for 24 h, the root tip facing upwards, into transparent glass capillaries made of chemically inert glass with an external diameter of about 5 mm and an internal diameter of about 2 mm. For the root crawling experiments, the germinating grains were put on a slope (about 30 degrees) made of wet block of Styrofoam covered by wet filter paper for 24 h. The whole experimental set-up was placed in a glass container filled with water to keep the grains and young seedlings in optimal humid conditions. In order to prevent strong interactions between gravitropism and phototropism, the light source for root illumination was placed above the glass container (illumination from the top). As a source of the light served Osram T8 L36W/954 LUMILUX DE LUXE Daylight G13, color temperature: 5,400 k. Roots were decapped by carefully removing their caps by using a sharp razor blade to remove surgically the root cap withour damaging the apical meristem. Such decapped roots regenerated new root capse within 30–36 h completely. Decapped and regenerating roots accomplished vigorous growth (data now shown). Images were taken using a CANON EOS 1000D SLR digital camera or with a standard PC scanner. Image processing was done using Adobe Photoshop CS3 or Image-J (http://rsb.info.nih.gov/ij/).