Root cap-mediated evaluation of soil resistance towards graviresponding roots of maize (Zea mays L.) and the relevance of ethylene

Root cap-mediated evaluation of soil resistance towards graviresponding roots of maize (Zea mays... Abstract Background and Aims Besides biological and chemical impacts, mechanical resistance represents an important obstacle that growing roots face. Graviresponding roots must assess the mechanical resistance of the substrate and take decisions on whether they change growth direction and grow around obstacles or tolerate growth conditions impaired to varying degrees. To test the significance of the root cap, we measured pressure and growth behaviour of single intact, as well as decapped, roots encountering diverse mechanical obstacles. We examined ethylene emission in intact roots as well as roots without a root cap, thereby lacking the capacity to deviate. Methods Roots of fixed seedlings were grown vertically onto diverse mechanical obstacles. Developing pressure profiles of vertically growing roots encountering horizontal mechanical obstacles were measured employing electronic milligram scales, with and without root caps in given local environmental conditions. The evolution of root-borne ethylene was measured in intact roots and roots without the root cap. Key Results In contrast to decapped roots, intact roots develop a tentative, short-lasting pressure profile, the resolution of which is characterized by a definite change of growth direction. Similarly, pressure profiles and strengths of roots facing gradually differing surface resistances differ significantly between the two. This correlates in the short term with root cap-dependent ethylene emission which is lacking in roots without caps. Conclusions The way gravistimulated and graviresponding roots cope with exogenous stimuli depends on whether and how they adapt to these impacts. With respect to mechanical hindrances, roots without caps do not seem to be able to evaluate soil strengths in order to respond adequately. On encountering resistance, roots with intact caps emit ethylene, which is not observed in decapped roots. It therefore appears that it is the root cap which specifically orchestrates the resistance needed to overcome mechanical resistance by specifically inducing ethylene. Mechanical impedance, root growth, root cap, soil resistance, root ethylene emission, root pressure, Zea mays, mechanical resistance, ethylene, graviresponding, evaluation of soil INTRODUCTION In order to ensure a successful development, plants have to accomplish a variety of basic tasks. One such task is to perceive the gravity vector and direct root growth relative to it. Equally important is either to overcome soil-borne mechanical impediments, or to avoid the obstacles eventually by deviating root growth in an apparently intelligent way (Trewavas, 2014), i.e. temporarily disengaging the gravity-dependent mode of growth. In fact, the capacity of plants to perceive and respond adequately towards gravity represents one of the earliest and longest standing problems of plant physiology. Dating back to the early 19th century, studies dealing with graviregulated plant growth (Knight, 1806) were generally carried out with shoots or roots exposed to humid air, in order to be able to document and analyse the gravitropic growth behaviour (Rawitscher, 1932; Blancaflor and Masson, 2003). In this way, over many decades, a range of parameters were determined for different species, employing operationally defined experimental conditions. These studies included growth behaviours depending on differing angles relative to the vertical under 1 g conditions, using fast- and slow-rotating, 2-D and 3-D clinostats (Barjaktarovic et al., 2007), but also, in more recent studies, under conditions of weightlessness, i.e. microgravity conditions in orbit (e.g. Volkmann, 1992; Volkmann and Tewinkel, 1996). In most of these studies, the main focus was on one of the three formal processes of a hypothetical causal sequence, namely graviperception, gravitransduction and graviresponse. Based on the chosen experimental conditions, these studies delivered a substantial range of sound physiological data, in some way relevant to the mechanism of gravitropic growth regulation. Parameters such as stimulus latency, stimulus intensity and stimulus summation (Tewinkel and Volkmann, 1994; Mullen et al., 2000) were determined. These studies allow a precise analysis and characterization of root behaviour. Yet, these operationally defined, experimental ‘free space’ circumstances, i.e. mechanically unhindered, do not apply to ‘natural’ conditions. In contrast, roots are typically growing in conditions entailing mechanical impediments of various densities. Therefore, the eco-physiological significance of most studies, i.e. their relevance for in situ conditions, may be rather restricted, since plant roots are constantly exposed to soil-typical profiles of constricting conditions. Growth and the underlying directing processes are then mainly dictated by the mechanically restricting conditions imposed by soil. They will either occur on top of ongoing gravi-induced processes, or in alternation with – temporarily disengaged – graviresponses, giving way to appropriate growth behaviour, as dictated by the mechanical environment of the roots. In addition to and apart from the mechanical hindrance (Bengough and Mullins, 1990), conditions in soil, especially with respect to root surfaces, may be very different compared with roots freely exposed to the humid gas phase. Apart from different light conditions, which have been demonstrated to affect ethylene metabolism (Edelmann, 1996; Edelmann et al., 2002), both the emission and the uptake of gases is fundamentally affected. As discussed in a review (Wenke et al., 2010), roots emit a broad range of volatiles. Depending on soil densities, they will accumulate in a higher range of concentrations as compared with roots growing in free space. This difference between free space and typical natural conditions also applies to the intake of gases, the uptake of liquids and temperature exchange, which can be strongly affected by convection. Apart from plagiotropically growing roots, roots generally grow vertically downwards. According to the classical models, this behaviour represents the physiological consequence of adequately positioned statoliths within the statocytes in root caps and thus correlated radial symmetric indole acetic acid (IAA) redistribution (also known as the ‘inverted fountain model’). In principle, therefore, a vertically downward growing root hindered in its elongation growth from the plumb-line by a horizontal mechanical obstacle does not perceive signals induced by the repositioning of the statocytes on deviating from verticality. It would be expected to keep its vertical orientation, since neither a change in gravistimulus nor a redistribution of statoliths occurs, and therefore no gravidependent change in IAA redistribution. However, it is obvious that roots generally overcome such barriers by either keeping growing in the original direction at a more or less unchanged growth rate or by changing into a growth mode characterized and regulated via ethylene – also known as the ‘triple-response’ of the roots (Bleecker et al., 1988; Abeles et al., 1992). This is characterized by three typical, ethylene-dependent root growth responses, namely inhibition of elongation growth, increase in (root) diameter and diagravitropic root elongation. By these means, the root eventually overcomes the hindrances typical of different soil qualities. It is to be expected, therefore, that roots have to possess a means of evaluating the resistance strength of the soil they are faced with, in order to trigger changes in growth rate as well as growth direction in an adequate, i.e. intelligent, way in order to survive. By this, growth inhibition can be avoided in substrates which can be mechanically overcome – yet it will be inhibited and redirected on surfaces which are impenetrable. Hence, roots must take decisions and measures based on and originating from tip-borne physiological scenarios induced by their mechanical environment. The eminent role of the tip-borne tissues in such conditions has already been attributed to root tips by Darwin (1881). He described the root cap ‘as comparable to the brain of the lower animals’ (Barlow, 2003; Baluska et al., 2009; Kutschera and Niklas, 2009). In the present study, we fixed dark-grown, 2-day-old maize seedlings and analysed the behaviours of the roots exposed to various conditions with respect to the mechanical impediments they faced. In addition, we examined the effect of mechanical impediments on ethylene emission. MATERIALS AND METHODS Maize kernels (Hybridmais, Ronaldinio, KWS) were germinated in darkness at room temperature (approx. 21–24 °C) by rolling them in moistened sheets of filter paper (MN 710; 580 × 580 mm). For this, 20 kernels were placed in rows at interval distances of 1–1.5 cm on chromatography paper sheets (40 × 10 cm). The rolled sheets were placed vertically in 200 mL glass beakers, and filled with distilled water to a depth of 1 cm. The beakers were then covered with aluminium foil. After 2–3 d, the germinated seedlings, exhibiting roots with lengths in the range of 2–3 cm, were selected for the experiments. Pressure/mass profiles of the extending roots of fixed maize seedlings In order to position roots in a standardized way, they were fixed in a device, as shown in Fig. 1. Kernels with on average 2 cm long roots were placed in a chamber with downwardly oriented roots and fixed into a hollow column filled with water-soaked rolled filter paper, the vertical position of which was adjustable (Fig. 1). Fig. 1. View largeDownload slide Sketch of the gadget for measuring mass (in grams) of roots of 3-day-old maize seedlings towards horizontal obstacles. For sufficient water supply, the middle column was filled with water-imbibed filter paper, delivering sufficient liquid to the fixed seedling (not shown); the measured values were continuously electronically recorded. Fig. 1. View largeDownload slide Sketch of the gadget for measuring mass (in grams) of roots of 3-day-old maize seedlings towards horizontal obstacles. For sufficient water supply, the middle column was filled with water-imbibed filter paper, delivering sufficient liquid to the fixed seedling (not shown); the measured values were continuously electronically recorded. This cylindrical device was mounted on electronic balances (Kern KB 120-3N) with sensitivities of 1 mg. Petri dishes containing either moistened (with a defined volume of water) filter paper or layers of agar gels with defined concentrations were placed on the surfaces of balance pans. Based on the fact that the measured values of the balance over time represent weight/mass and not pressure, we nevertheless omitted a general conversion of these values into pressure units. Our argument for this is that the area of contact of the root with the hindrance is changing as growth occurs. Taking as a first approximation 1 mm2 as the average area of the root tips, the weights as measured in milligrams are equivalent to a range between 0 and 200 kPa. Measurement of ethylene emission For the determination of ethylene emission, seedlings were placed in glass beakers, which allowed differentiation between root- and shoot-borne ethylene emission (Fig. 2). Ethylene was analysed with an ‘ETD 300’ (‘Sensor Sense’). Depending on head space and seedling numbers, the flow rates (stop and flow operation mode) were adjusted to an average of 1 L h–1. Fig. 2. View largeDownload slide Glass gadget for measuring ethylene emission of single maize seedlings, separated by a small ‘bottle neck’ between the shoot and root part. Tightly insulated by application of ‘Fitnis SH medium’ (Kaniedenta), shoot- and root-emitted ethylene emissions were measured separately, employing the real-time sub-ppb analyser ETD 300 (Sensor Sense); the scale bar represents 1 cm in length. Fig. 2. View largeDownload slide Glass gadget for measuring ethylene emission of single maize seedlings, separated by a small ‘bottle neck’ between the shoot and root part. Tightly insulated by application of ‘Fitnis SH medium’ (Kaniedenta), shoot- and root-emitted ethylene emissions were measured separately, employing the real-time sub-ppb analyser ETD 300 (Sensor Sense); the scale bar represents 1 cm in length. RESULTS In order to ensure vertical downward growth of the seedling roots, the kernels were fixed in such a way that the root tips ended approx. 5 mm above the horizontal hindrance of the weighing surface of the electronic balance (Fig. 1). Roots on average grew vertically downwards for 3–4 h, before impinging on the horizontal surface. Despite the outlined precautions and standardized conditions, the maximum values of the two observed peaks were fairly heterogeneous, due to a range of potential issues discussed below. On average, intact roots were characterized by an initial prominent peak value, lasting in some cases for just a few minutes. Thereafter, this peak was followed by a pronounced steep drop in pressure, after which strongly decreased and less pronounced, smaller peaks were observed in sequence (Fig. 3A). On average, the extent of the first peak for intact and decapped roots did not differ greatly. Thereafter growth behaviour clearly differed, being characterized by 6–8 h long oscillating, zig–zag-like pressure profiles for decapped roots (Fig. 3B). Their pressure profiles also differed in that they generally developed a second, higher or similar peak compared with the initial one. The decapped roots, over time, exhibited a bow-like distortion, which was responsible for the zig–zag-shaped trace, caused by intermittent sideways slipping of the root tips on the mechanical surface. In contrast, intact roots bowed and then actively changed growth direction, eventually resulting in a horizontal growth along the moist surface, similar to the ‘crawling behaviour’ described previously (Hahn et al., 2006). This crawling behaviour was not observed in decapped roots, as the zig–zag oscillations imply. Therefore, the oscillations originated from a jerky sideways slippage of the roots, building a kind of tensed bow in the root rather than an adapted change of the growth direction as observed in intact roots Similar profiles were observed in roots faced with NaCl solution-soaked filter paper on the balance weighing surface (Fig. 3C). However, as compared with water-supplied roots (Fig. 3A, B), the first peak values were strongly enhanced, on average by 2- to 3-fold, in both intact and decapped roots. In addition to this, on average decapped roots faced with NaCl solution exhibited a second peak larger than the first peak (Fig. 3D). Similar to decapped, water-faced roots, they did not change growth direction, but slid the bow-shaped roots over the filter paper surface. Under natural conditions, roots are not necessarily faced with large flat smooth obstacles, but rather unfavourable spatially complicated structures. We therefore let – as a first approximation to these conditions – roots grow into small conically shaped ‘bowls’, obtained by cutting off the bottoms of Eppendorf tubes, filled with 50 µL liquid and fixed vertically on the balance weighing surface. Under these conditions (Fig. 4), mass/pressure profiles of intact, water-faced roots were generally higher and did not exhibit the steep drop seen in the vertical plane conditions shown in Fig. 3A, but maintained pressure for on average 6 h or more. Intact roots actively changed growth direction, eventually reaching the edge of the Eppendorf tube then redirecting growth direction once again vertically downwards, i.e. they escaped the encased conditions they were faced with. In contrast to this, decapped roots developed and maintained an increased, in trend ascending, mass/pressure profile. Eventually the roots adopted a corkscrew-like appearance (Fig. 5). After ≥3 d, in a few cases, decapped roots behaved similarly to intact roots, once the root cap had regenerated, by then despiralizing the corkscrew architecture (data not shown). In NaCl solution, pressure increases were generally less steep, but kept gradually increasing or were maintained at the pressures they had attained (Fig. 4C, D). Fig. 3. View largeDownload slide Representative mass/(pressure) profiles as measured with 5–7 independent maize seedling roots during the first 12–15 h after impinging of the roots on the horizontal hindrance; various grey scales are adequate to differentiate seedlings. Profiles of (A) intact roots impinging on water-imbibed filter paper; (B) decapped roots/water; (C) intact roots impinging on NaCl solution-imbibed filter paper; (D) decapped roots/NaCl solution, dissolved in filter paper. Please note the differing vertical scales. Fig. 3. View largeDownload slide Representative mass/(pressure) profiles as measured with 5–7 independent maize seedling roots during the first 12–15 h after impinging of the roots on the horizontal hindrance; various grey scales are adequate to differentiate seedlings. Profiles of (A) intact roots impinging on water-imbibed filter paper; (B) decapped roots/water; (C) intact roots impinging on NaCl solution-imbibed filter paper; (D) decapped roots/NaCl solution, dissolved in filter paper. Please note the differing vertical scales. Fig. 4. View largeDownload slide Representative mass/(pressure) profiles of maize seedling roots (6–15 samples) exposed to bowl-shaped hindrances. Profiles of (A) intact roots growing into 100 µL of water; (B) decapped roots growing into 50 µL of water; (C) intact roots growing into 100 µL of 17 mm NaCl solution; (D) decapped roots growing into 100 µL of 17 mm NaCl solution. Fig. 4. View largeDownload slide Representative mass/(pressure) profiles of maize seedling roots (6–15 samples) exposed to bowl-shaped hindrances. Profiles of (A) intact roots growing into 100 µL of water; (B) decapped roots growing into 50 µL of water; (C) intact roots growing into 100 µL of 17 mm NaCl solution; (D) decapped roots growing into 100 µL of 17 mm NaCl solution. Fig. 5. View largeDownload slide Images of dark-grown, 3-day-old maize seedlings exposed to bowl-shaped hindrances with either intact roots (four seedlings on the left, A) or decapped roots (four seedlings on the right, B) after 48 h of exposure to vertical, narrowing tubes. In some cases, decapped roots regenerated the root cap and then actively despiralized the root architecture (data not shown). The arrow indicates the gravivector; the horizontal bar applies to a length of 1 cm. Fig. 5. View largeDownload slide Images of dark-grown, 3-day-old maize seedlings exposed to bowl-shaped hindrances with either intact roots (four seedlings on the left, A) or decapped roots (four seedlings on the right, B) after 48 h of exposure to vertical, narrowing tubes. In some cases, decapped roots regenerated the root cap and then actively despiralized the root architecture (data not shown). The arrow indicates the gravivector; the horizontal bar applies to a length of 1 cm. In order to test how roots behave towards different surface strengths in the presence or absence of the root cap, we let intact and decapped roots grow onto plates of differing agar concentrations placed on the weighing surfaces of the balances. Under such conditions, downward growing intact roots exposed above agar plates of concentrations of 0.5 %, i.e. with small mechanical resistance, were characterized by very faint profiles. Thereafter, they exhibited short-lasting pressure increases with steep drops, due to penetration of the root tip into the substrate (Fig. 6A). Subsequently, pressure again quickly increased, eventually leading in most cases to pronounced oscillations due to the crawling behaviour of the roots over the bottoms of the Petri dishes, and submerged in less dense agar. As a rule, such behaviour was not observed in decapped roots. Once they reached the surface of the agar, they exhibited rapid increases in pressure, leading in a short time to sideways slippages, i.e. they built up a pressure increase and then either slipped aside or grew in some cases into the substrate. In the case of 1 % agar plates, again intact roots developed a steep, yet small initial pressure rise, which abruptly decayed and was followed by a similar second increase once the roots reached the bottom of the Petri dish. Decapped roots developed relatively higher mass/pressure values before they slipped aside or, again, in some cases, penetrated the agar. Generally, intact roots penetrated agar substrates more frequently than decapped roots at agar concentrations of ≤2 %. At 4 % agar, the vast majority of roots, irrespective of the presence of the root cap, did not penetrate the substrate, but either slipped aside (in the case of decapped roots, Fig. 6) or actively changed growth direction by a crawling behaviour (in the case of intact roots). Fig. 6. View largeDownload slide Typical mass/pressure profiles of roots of 3-day-old maize seedlings vertically growing onto horizontal layers of different nutrient agar concentrations dependent on the presence of the root cap. Left column, intact roots; right column, decapped roots. Percentage values refer to nutrient agar concentrations. Fig. 6. View largeDownload slide Typical mass/pressure profiles of roots of 3-day-old maize seedlings vertically growing onto horizontal layers of different nutrient agar concentrations dependent on the presence of the root cap. Left column, intact roots; right column, decapped roots. Percentage values refer to nutrient agar concentrations. In general, variability of root mass/pressure behaviour was pronounced during these experiments, due to the fact that not all roots eventually came into contact with the hydrated surface in an exactly vertical orientation, i.e. the slick moist root surface also contributed to the pronounced heterogeneity of the measured profiles. Nevertheless, the root pressure behaviour in the chosen conditions was generally similar in the different treatments as represented by the single cases shown in Fig. 6, i.e. the majority penetrated the substrate at a concentration of ≤1 %. A summary of the dependence on nutrient agar concentration of the mean mass/pressure values of the root caps is given in Fig. 7. As shown, in all cases decapped roots developed initially higher mass/pressure values than intact roots. The difference increased according to the concentration of the agar (i.e. the lower the concentration, the lower the resistance of the substrate). Most obviously this difference is seen in roots impinging 1 % agar plates, in which the ratio was in the range of 1:4. Fig. 7. View largeDownload slide Mean mass/pressure values of roots of 3-day-old maize seedlings with and without root caps on nutrient agar plates with different concentrations, ± s.e. (n = 7). Fig. 7. View largeDownload slide Mean mass/pressure values of roots of 3-day-old maize seedlings with and without root caps on nutrient agar plates with different concentrations, ± s.e. (n = 7). Since it is, on the one hand, known that roots exposed to mechanical hindrances emit ethylene – generally known as an inhibitor of elongation growth (Moss et al., 1988) – and on the other, that the diffusion coefficient of ethylene is strongly decreased in liquids, we were interested in how pressure profiles looked during the two differing conditions of root environment. We therefore repeated the above-mentioned experiments, letting the root tips either grow into Eppendorf tubes filled with small volumes of water to a depth of 1 cm or grow in water-saturated air before impinging on the bottoms of the tubes. It turned out that intact roots impinging mechanical hindrances while their tips were submerged developed much less pronounced pressures compared with roots impinging the hindrance in moist air (Fig. 8). On average these developed 2- to 3-fold higher pressure (Fig. 8, bottom image), i.e. the ‘clash’ of intact root tips was much more strongly dampened in water than in air. In addition, they were generally characterized by a more pronounced oscillating behaviour. Fig. 8. View largeDownload slide Comparison of typical mass/pressure profiles of roots of 3-day-old maize seedlings the tips of which were either incubated in a total volume of 100 µL of water (upper figure) or kept in humid air (lower figure).The bottom diagram shows mean average mass/pressure values ± s.e. Fig. 8. View largeDownload slide Comparison of typical mass/pressure profiles of roots of 3-day-old maize seedlings the tips of which were either incubated in a total volume of 100 µL of water (upper figure) or kept in humid air (lower figure).The bottom diagram shows mean average mass/pressure values ± s.e. Employing the ETD, we analysed the effect of roots impinging on the horizontal surface as compared with controls, i.e. roots growing unhindered vertically downwards (Fig. 9). For this, ethylene emission of single seedlings was measured at intervals within the shoot part and the root part of the seedling separately. Ethylene content was measured in flow-through volumes received during sequential 10 min measurement time periods, regularly alternated with control/reference measurements, i.e. measurement of gases without seedlings in the chambers. From a typical result for intact roots, the impingement of the root tip resulted in a strong increase of ethylene emission as compared with unimpaired root tips (Fig. 9, left panel). In contrast, this effect was not observed in decapped roots in which ethylene emission is not affected by root tip impingement during a time period of 8–12 h on average. Similar to previously reported results (Alarcon et al., 2009), the individual seedlings were characterized by pronounced fluctuating absolute values of ethylene emission. Removal of the root cap resulted in increased emission of ethylene from the shoot (Fig. 9, right panel). Fig. 9. View largeDownload slide Representative ethylene emission profiles of intact (left) and decapped (right) roots of 3-day-old maize seedlings mechanically hindered in their vertical growth of the root tip by glass plateaus, as compared with controls within the gadget shown in Fig. 2. Vertically growing roots reached the hindrance during the initial zone, i.e. the beginning of the abscissa. Measurements were carried out in flow-through intervals. Fig. 9. View largeDownload slide Representative ethylene emission profiles of intact (left) and decapped (right) roots of 3-day-old maize seedlings mechanically hindered in their vertical growth of the root tip by glass plateaus, as compared with controls within the gadget shown in Fig. 2. Vertically growing roots reached the hindrance during the initial zone, i.e. the beginning of the abscissa. Measurements were carried out in flow-through intervals. DISCUSSION Under natural growing conditions, roots are constantly exposed to exogenous physical as well as chemical impacts which exert direct effects on root growth, which in turn affect per se root-borne processes (Moss et al., 1988; Barlow and Baluska, 2000). Based on the conditions encountered, they have to take decisions which will eventually dictate their fate – and their proliferation and productivity. As a consequence, the way roots manage to cope with given circumstances determines their developmental success (Trewavas, 2014). Since the root tip, with its functionally versatile root cap (Baluska et al., 2009; Kutschera and Niklas, 2009), represents the dominant unit of a growing root, we were interested in what impact its removal has on the pressure profile of roots faced with mechanical obstructions. As shown in Fig. 3, intact roots develop a steep brief pressure increase followed by a decrease, after which they actively change growth direction characterized by a crawling behaviour demonstrated earlier (Hahn et al., 2006). In decapped roots, this behaviour is not observed; such roots slip aside, leading to a slow and gradual oscillatory-like decrease in pressure. They apparently lack a mechanism to enable the root to adapt to the local conditions. Such an active response to mechanical obstruction observed in intact roots must inevitably be characterized by pressure-dependent signal transduction chains, which are, in principle, not affected by the impact of either Na or Cl ions. The presence of NaCl on the roots at the point of contact resulted in a strong general pressure increase, yet did not change the principal mass/pressure profiles observed in controls with only water. This might be explained by an enhanced osmotic strength of the root cells after ion uptake. However, we do not yet know the reason why decapped NaCl-exposed roots generally develop a significant second pronounced pressure increase (Fig. 3D), not observed in water-exposed decapped roots. It would therefore be worthwhile to measure turgor pressure within the root tip cells. In contrast to other reports (Neumann, 1995), the enhanced pressure profiles in fact indicate an apparent enhanced vitality and not an impairment of the root growth capacity – at least not within the measured time and at the given concentrations. More complex surfaces, as mimicked by bowl-shaped Eppendorf tubes, are again characterized by slightly less steep yet eventually higher pressure increases, in both intact and decapped roots (Fig. 4A, B). In these measurements, heterogeneity was most pronounced. This may be mainly interpreted as the complex reversion growth behaviour of the roots. Again, intact roots apparently possess the sophisticated capacity not only to divert growth direction, but also to reverse growth direction repeatedly despite unchanged gravistimulation (Fig. 5). This response is not observed in decapped specimens, which generally drilled their roots onto the bottom of the Eppendorf tube, in the absence of a rebuilt root cap. The root cap must, therefore, possess a means of directing growth, in both a positive and negative gravitropic mode, favouring the positive mode when it is possible, constantly adjusting this behaviour in real time as a function of the close mechanical environment. Such a situation-sensitive versatile mechanism is supported by the results presented in Figs 6 and 8. Below substrate strengths of 4 % agar, intact roots penetrate agar layers at much lower mass/pressure values and do not slip aside; this is surprising, since intact root tips are characterized by high surface moisture due to the hygroscopic quality of the root cap. However, at concentrations of 4 % agar, intact roots change direction of growth, whereas decapped roots again slip aside across the surface. In a way, therefore, the cap takes the decision depending on the strength of the hindrance. These mechanical impacts per se may be mediated via cytoskeletal rearrangements, which were demonstrated to depend on ethylene as one effect of its mediated triple-response (Roberts et al., 1985; Baluska et al., 1992; Cyr, 1994) as well as wall peripheral distortions (Barlow and Baluska, 2000). A crucial difference from the free air root experiments as compared with natural conditions consists of markedly different diffusion conditions above the root surfaces. It is to be expected that when surrounded by soil-emitted gases, such as ethylene or other compounds (Wenke et al., 2010), the roots reach a several times higher concentration compared with roots exposed to the free space/gas phase. In the case of ethylene, as a hormone very relevant to development (Abeles et al., 1992; Davies, 1995), it is to be expected that in roots embedded within the substrate, i.e. surrounded by mechanically dense soil particles, or with the root surface in contact with liquids, its concentration will increase to values far beyond those of roots exposed to the free space. During such non-restraining conditions, gases will diffuse away with little or no effect on the root cells. Being aware that various other processes may be affected, it would be expected that root tips impinging on mechanical surfaces during conditions of immersion in water would be more strongly inhibited than roots faced with moist air. A strong indication of such scenarios is given in results typical for the two different treatments (Fig. 8). The conditions underlying the two representative curves differ only by the phase (liquid/air) surrounding the root tips. Immersed roots develop slight pressure profiles as compared with roots pressing on the mechanical surface exposed to moist air. Additional support arises from the results shown in Fig. 9 based on single root ethylene emission measurements. Only root tips with intact caps display ethylene emission from roots impinging on mechanical surfaces. It is therefore not only compatible, but also very likely, that root growth orchestration and management of growth response dependent on mechanical impacts is via root cap-mediated ethylene emission. This phenomenon appears as ‘automatically’ modulated and independent of the root surroundings. In fact, one could, in principle, argue that the mechanical and chemical surroundings determine graviresponsivity. It appears plausible that a means for this gravimodulating environment is endogenous ethylene, the impact of which on the root itself represents a (biochemically printed) ‘picture’ of the mechanical surroundings – on top of other parameters integrated into the growth behaviour, as suggested earlier (Edelmann and Roth, 2006). On the other hand, it has been shown that inhibition of ethylene synthesis inhibits graviresponsive growth (Edelmann and Roth, 2006) and mechanical impacts induce ethylene synthesis (Fig. 9). For those reasons, it appears that it is enhanced ethylene which, during these substrate-penetrating conditions, may be responsible for enhanced gravitropic growth (Edelmann, 2002; Hahn et al., 2008). The way in which the demonstrated root cap-dependent ethylene acts may be through modification of the cytoskeleton within the elongation zone in such a way that associated cell extension processes are inhibited (Cyr, 1994). It may also interfere in such a way that growth-directing processes within the elongation zone of the roots are practically disengaged from gravity-dependent growth. However, the phenomenon of root cap-dependent ethylene release observed during mechanical hindrance may reflect correlation rather than causation. For future studies, the use of efficient and reliable inhibitors of ethylene synthesis applied to intact and decapped roots may deliver more unequivocal evidence in support of the suggested potential causalities. In strong support of a causal role for ethylene in changes in growth direction is the earlier reported finding where it was shown that in intact roots inhibition of ethylene synthesis inhibits gravidependent changes in root growth direction of maize seedlings, which was not observed in decapped roots (Hahn et al., 2008). Based on the scenario outlined, dense soils and soil water will ‘automatically’ restrain root expansion and temporarily shift the cap-based ‘root management’ to conditions controlled by exogenous parameters – in addition to or independent of gravity. As well as the mechanical root environments, local water conditions may also induce adequate root behaviour by enhanced ethylene accumulation, which in air (i.e. during mechanically and chemically unimpaired conditions) volatilizes into the gas phase. Although our ethylene measurements differentiated between root- and shoot-borne emissions, we are aware that this organ-specific localization does not allow us to decide whether or not ethylene originated from more mature and therefore possibly lignifying or otherwise affected parts of the roots (Ingemarsson et al., 1991; Huang et al., 2013). In ecophysiological terms, a hypothesized ‘stand-by’ mode of root ethylene emission may allow the plant to sense parameters in its vicinity in addition to those other(wise) perceived and other growth-relevant parameters. ACKNOWLEDGEMENTS The authors would like to thank Editor in Chief Professor Pat Heslop-Harrison as well as the peer reviewers for their suggestions and critical, constructive comments, which are highly appreciated. Thanks also to Dr Keith Baverstock, who has carefully proofread and corrected the manuscript for errors. LITERATURE CITED Abeles FB, Morgan PW, Saltveit ME. 1992. Ethylene in plant biology . San Diego: Academic Press. Alarcón MV, Lloret-Salamanca A, Lloret PG, Iglesias DJ, Talon M, Salguero J. 2009. Effects of antagonists and inhibitors of ethylene biosynthesis on maize root elongation. Plant Signaling and Behavior  4: 1154– 1156. Baluska F, Parker JS, Barlow PW. 1992. 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In: Baluska F, Ciamporova M, Gasparíková O, Barlow PW, eds. Structure and function of roots. Proceedings of the Fourth International Symposium on Structure and Function of Roots , June 20–26, 1993, Stará Lesná, Slovakia. Dordrecht: Kluwer Academic Publishers, 299– 304. Rawitscher F. 1932. Der Geotropismus der Pflanzen . Jena: Fischer Verlag. Roberts IN, Lloyd CW, Roberts K. 1985. Ethylene-induced microtubule reorientations: mediation by helical arrays. Planta  164: 439– 447. Tewinkel M, Volkmann D. 1994. Gravisensitivity of cress roots: determination of threshold values under reduced gravity during the Spacelab Mission D-2. In: Oser H, Guyenne TD, eds. Proceedings of the Fifth European Symposium on Life Science Research in Space , Arcachon, France. Noordwijk: ESA SP-366, ESA Publications Division, ESTEC, 139– 144. Trewavas A. 2014. Plant behaviour and intelligence . Oxford: Oxford University Press. Volkmann D. 1992. Gravitationsbiologie. Biologie in unserer Zeit  22: 323– 329. Volkmann D, Tewinkel M. 1996. Gravisensitivity of cress roots: investigations of threshold values under specific conditions of sensor physiology in microgravity. Plant, Cell and Environment  19: 1195– 1202. Wenke K, Kai M, Piechulla B. 2010. Belowground volatiles facilitate interactions between plant roots and soil organisms. Planta  231: 499– 506. © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Botany Oxford University Press

Root cap-mediated evaluation of soil resistance towards graviresponding roots of maize (Zea mays L.) and the relevance of ethylene

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Abstract Background and Aims Besides biological and chemical impacts, mechanical resistance represents an important obstacle that growing roots face. Graviresponding roots must assess the mechanical resistance of the substrate and take decisions on whether they change growth direction and grow around obstacles or tolerate growth conditions impaired to varying degrees. To test the significance of the root cap, we measured pressure and growth behaviour of single intact, as well as decapped, roots encountering diverse mechanical obstacles. We examined ethylene emission in intact roots as well as roots without a root cap, thereby lacking the capacity to deviate. Methods Roots of fixed seedlings were grown vertically onto diverse mechanical obstacles. Developing pressure profiles of vertically growing roots encountering horizontal mechanical obstacles were measured employing electronic milligram scales, with and without root caps in given local environmental conditions. The evolution of root-borne ethylene was measured in intact roots and roots without the root cap. Key Results In contrast to decapped roots, intact roots develop a tentative, short-lasting pressure profile, the resolution of which is characterized by a definite change of growth direction. Similarly, pressure profiles and strengths of roots facing gradually differing surface resistances differ significantly between the two. This correlates in the short term with root cap-dependent ethylene emission which is lacking in roots without caps. Conclusions The way gravistimulated and graviresponding roots cope with exogenous stimuli depends on whether and how they adapt to these impacts. With respect to mechanical hindrances, roots without caps do not seem to be able to evaluate soil strengths in order to respond adequately. On encountering resistance, roots with intact caps emit ethylene, which is not observed in decapped roots. It therefore appears that it is the root cap which specifically orchestrates the resistance needed to overcome mechanical resistance by specifically inducing ethylene. Mechanical impedance, root growth, root cap, soil resistance, root ethylene emission, root pressure, Zea mays, mechanical resistance, ethylene, graviresponding, evaluation of soil INTRODUCTION In order to ensure a successful development, plants have to accomplish a variety of basic tasks. One such task is to perceive the gravity vector and direct root growth relative to it. Equally important is either to overcome soil-borne mechanical impediments, or to avoid the obstacles eventually by deviating root growth in an apparently intelligent way (Trewavas, 2014), i.e. temporarily disengaging the gravity-dependent mode of growth. In fact, the capacity of plants to perceive and respond adequately towards gravity represents one of the earliest and longest standing problems of plant physiology. Dating back to the early 19th century, studies dealing with graviregulated plant growth (Knight, 1806) were generally carried out with shoots or roots exposed to humid air, in order to be able to document and analyse the gravitropic growth behaviour (Rawitscher, 1932; Blancaflor and Masson, 2003). In this way, over many decades, a range of parameters were determined for different species, employing operationally defined experimental conditions. These studies included growth behaviours depending on differing angles relative to the vertical under 1 g conditions, using fast- and slow-rotating, 2-D and 3-D clinostats (Barjaktarovic et al., 2007), but also, in more recent studies, under conditions of weightlessness, i.e. microgravity conditions in orbit (e.g. Volkmann, 1992; Volkmann and Tewinkel, 1996). In most of these studies, the main focus was on one of the three formal processes of a hypothetical causal sequence, namely graviperception, gravitransduction and graviresponse. Based on the chosen experimental conditions, these studies delivered a substantial range of sound physiological data, in some way relevant to the mechanism of gravitropic growth regulation. Parameters such as stimulus latency, stimulus intensity and stimulus summation (Tewinkel and Volkmann, 1994; Mullen et al., 2000) were determined. These studies allow a precise analysis and characterization of root behaviour. Yet, these operationally defined, experimental ‘free space’ circumstances, i.e. mechanically unhindered, do not apply to ‘natural’ conditions. In contrast, roots are typically growing in conditions entailing mechanical impediments of various densities. Therefore, the eco-physiological significance of most studies, i.e. their relevance for in situ conditions, may be rather restricted, since plant roots are constantly exposed to soil-typical profiles of constricting conditions. Growth and the underlying directing processes are then mainly dictated by the mechanically restricting conditions imposed by soil. They will either occur on top of ongoing gravi-induced processes, or in alternation with – temporarily disengaged – graviresponses, giving way to appropriate growth behaviour, as dictated by the mechanical environment of the roots. In addition to and apart from the mechanical hindrance (Bengough and Mullins, 1990), conditions in soil, especially with respect to root surfaces, may be very different compared with roots freely exposed to the humid gas phase. Apart from different light conditions, which have been demonstrated to affect ethylene metabolism (Edelmann, 1996; Edelmann et al., 2002), both the emission and the uptake of gases is fundamentally affected. As discussed in a review (Wenke et al., 2010), roots emit a broad range of volatiles. Depending on soil densities, they will accumulate in a higher range of concentrations as compared with roots growing in free space. This difference between free space and typical natural conditions also applies to the intake of gases, the uptake of liquids and temperature exchange, which can be strongly affected by convection. Apart from plagiotropically growing roots, roots generally grow vertically downwards. According to the classical models, this behaviour represents the physiological consequence of adequately positioned statoliths within the statocytes in root caps and thus correlated radial symmetric indole acetic acid (IAA) redistribution (also known as the ‘inverted fountain model’). In principle, therefore, a vertically downward growing root hindered in its elongation growth from the plumb-line by a horizontal mechanical obstacle does not perceive signals induced by the repositioning of the statocytes on deviating from verticality. It would be expected to keep its vertical orientation, since neither a change in gravistimulus nor a redistribution of statoliths occurs, and therefore no gravidependent change in IAA redistribution. However, it is obvious that roots generally overcome such barriers by either keeping growing in the original direction at a more or less unchanged growth rate or by changing into a growth mode characterized and regulated via ethylene – also known as the ‘triple-response’ of the roots (Bleecker et al., 1988; Abeles et al., 1992). This is characterized by three typical, ethylene-dependent root growth responses, namely inhibition of elongation growth, increase in (root) diameter and diagravitropic root elongation. By these means, the root eventually overcomes the hindrances typical of different soil qualities. It is to be expected, therefore, that roots have to possess a means of evaluating the resistance strength of the soil they are faced with, in order to trigger changes in growth rate as well as growth direction in an adequate, i.e. intelligent, way in order to survive. By this, growth inhibition can be avoided in substrates which can be mechanically overcome – yet it will be inhibited and redirected on surfaces which are impenetrable. Hence, roots must take decisions and measures based on and originating from tip-borne physiological scenarios induced by their mechanical environment. The eminent role of the tip-borne tissues in such conditions has already been attributed to root tips by Darwin (1881). He described the root cap ‘as comparable to the brain of the lower animals’ (Barlow, 2003; Baluska et al., 2009; Kutschera and Niklas, 2009). In the present study, we fixed dark-grown, 2-day-old maize seedlings and analysed the behaviours of the roots exposed to various conditions with respect to the mechanical impediments they faced. In addition, we examined the effect of mechanical impediments on ethylene emission. MATERIALS AND METHODS Maize kernels (Hybridmais, Ronaldinio, KWS) were germinated in darkness at room temperature (approx. 21–24 °C) by rolling them in moistened sheets of filter paper (MN 710; 580 × 580 mm). For this, 20 kernels were placed in rows at interval distances of 1–1.5 cm on chromatography paper sheets (40 × 10 cm). The rolled sheets were placed vertically in 200 mL glass beakers, and filled with distilled water to a depth of 1 cm. The beakers were then covered with aluminium foil. After 2–3 d, the germinated seedlings, exhibiting roots with lengths in the range of 2–3 cm, were selected for the experiments. Pressure/mass profiles of the extending roots of fixed maize seedlings In order to position roots in a standardized way, they were fixed in a device, as shown in Fig. 1. Kernels with on average 2 cm long roots were placed in a chamber with downwardly oriented roots and fixed into a hollow column filled with water-soaked rolled filter paper, the vertical position of which was adjustable (Fig. 1). Fig. 1. View largeDownload slide Sketch of the gadget for measuring mass (in grams) of roots of 3-day-old maize seedlings towards horizontal obstacles. For sufficient water supply, the middle column was filled with water-imbibed filter paper, delivering sufficient liquid to the fixed seedling (not shown); the measured values were continuously electronically recorded. Fig. 1. View largeDownload slide Sketch of the gadget for measuring mass (in grams) of roots of 3-day-old maize seedlings towards horizontal obstacles. For sufficient water supply, the middle column was filled with water-imbibed filter paper, delivering sufficient liquid to the fixed seedling (not shown); the measured values were continuously electronically recorded. This cylindrical device was mounted on electronic balances (Kern KB 120-3N) with sensitivities of 1 mg. Petri dishes containing either moistened (with a defined volume of water) filter paper or layers of agar gels with defined concentrations were placed on the surfaces of balance pans. Based on the fact that the measured values of the balance over time represent weight/mass and not pressure, we nevertheless omitted a general conversion of these values into pressure units. Our argument for this is that the area of contact of the root with the hindrance is changing as growth occurs. Taking as a first approximation 1 mm2 as the average area of the root tips, the weights as measured in milligrams are equivalent to a range between 0 and 200 kPa. Measurement of ethylene emission For the determination of ethylene emission, seedlings were placed in glass beakers, which allowed differentiation between root- and shoot-borne ethylene emission (Fig. 2). Ethylene was analysed with an ‘ETD 300’ (‘Sensor Sense’). Depending on head space and seedling numbers, the flow rates (stop and flow operation mode) were adjusted to an average of 1 L h–1. Fig. 2. View largeDownload slide Glass gadget for measuring ethylene emission of single maize seedlings, separated by a small ‘bottle neck’ between the shoot and root part. Tightly insulated by application of ‘Fitnis SH medium’ (Kaniedenta), shoot- and root-emitted ethylene emissions were measured separately, employing the real-time sub-ppb analyser ETD 300 (Sensor Sense); the scale bar represents 1 cm in length. Fig. 2. View largeDownload slide Glass gadget for measuring ethylene emission of single maize seedlings, separated by a small ‘bottle neck’ between the shoot and root part. Tightly insulated by application of ‘Fitnis SH medium’ (Kaniedenta), shoot- and root-emitted ethylene emissions were measured separately, employing the real-time sub-ppb analyser ETD 300 (Sensor Sense); the scale bar represents 1 cm in length. RESULTS In order to ensure vertical downward growth of the seedling roots, the kernels were fixed in such a way that the root tips ended approx. 5 mm above the horizontal hindrance of the weighing surface of the electronic balance (Fig. 1). Roots on average grew vertically downwards for 3–4 h, before impinging on the horizontal surface. Despite the outlined precautions and standardized conditions, the maximum values of the two observed peaks were fairly heterogeneous, due to a range of potential issues discussed below. On average, intact roots were characterized by an initial prominent peak value, lasting in some cases for just a few minutes. Thereafter, this peak was followed by a pronounced steep drop in pressure, after which strongly decreased and less pronounced, smaller peaks were observed in sequence (Fig. 3A). On average, the extent of the first peak for intact and decapped roots did not differ greatly. Thereafter growth behaviour clearly differed, being characterized by 6–8 h long oscillating, zig–zag-like pressure profiles for decapped roots (Fig. 3B). Their pressure profiles also differed in that they generally developed a second, higher or similar peak compared with the initial one. The decapped roots, over time, exhibited a bow-like distortion, which was responsible for the zig–zag-shaped trace, caused by intermittent sideways slipping of the root tips on the mechanical surface. In contrast, intact roots bowed and then actively changed growth direction, eventually resulting in a horizontal growth along the moist surface, similar to the ‘crawling behaviour’ described previously (Hahn et al., 2006). This crawling behaviour was not observed in decapped roots, as the zig–zag oscillations imply. Therefore, the oscillations originated from a jerky sideways slippage of the roots, building a kind of tensed bow in the root rather than an adapted change of the growth direction as observed in intact roots Similar profiles were observed in roots faced with NaCl solution-soaked filter paper on the balance weighing surface (Fig. 3C). However, as compared with water-supplied roots (Fig. 3A, B), the first peak values were strongly enhanced, on average by 2- to 3-fold, in both intact and decapped roots. In addition to this, on average decapped roots faced with NaCl solution exhibited a second peak larger than the first peak (Fig. 3D). Similar to decapped, water-faced roots, they did not change growth direction, but slid the bow-shaped roots over the filter paper surface. Under natural conditions, roots are not necessarily faced with large flat smooth obstacles, but rather unfavourable spatially complicated structures. We therefore let – as a first approximation to these conditions – roots grow into small conically shaped ‘bowls’, obtained by cutting off the bottoms of Eppendorf tubes, filled with 50 µL liquid and fixed vertically on the balance weighing surface. Under these conditions (Fig. 4), mass/pressure profiles of intact, water-faced roots were generally higher and did not exhibit the steep drop seen in the vertical plane conditions shown in Fig. 3A, but maintained pressure for on average 6 h or more. Intact roots actively changed growth direction, eventually reaching the edge of the Eppendorf tube then redirecting growth direction once again vertically downwards, i.e. they escaped the encased conditions they were faced with. In contrast to this, decapped roots developed and maintained an increased, in trend ascending, mass/pressure profile. Eventually the roots adopted a corkscrew-like appearance (Fig. 5). After ≥3 d, in a few cases, decapped roots behaved similarly to intact roots, once the root cap had regenerated, by then despiralizing the corkscrew architecture (data not shown). In NaCl solution, pressure increases were generally less steep, but kept gradually increasing or were maintained at the pressures they had attained (Fig. 4C, D). Fig. 3. View largeDownload slide Representative mass/(pressure) profiles as measured with 5–7 independent maize seedling roots during the first 12–15 h after impinging of the roots on the horizontal hindrance; various grey scales are adequate to differentiate seedlings. Profiles of (A) intact roots impinging on water-imbibed filter paper; (B) decapped roots/water; (C) intact roots impinging on NaCl solution-imbibed filter paper; (D) decapped roots/NaCl solution, dissolved in filter paper. Please note the differing vertical scales. Fig. 3. View largeDownload slide Representative mass/(pressure) profiles as measured with 5–7 independent maize seedling roots during the first 12–15 h after impinging of the roots on the horizontal hindrance; various grey scales are adequate to differentiate seedlings. Profiles of (A) intact roots impinging on water-imbibed filter paper; (B) decapped roots/water; (C) intact roots impinging on NaCl solution-imbibed filter paper; (D) decapped roots/NaCl solution, dissolved in filter paper. Please note the differing vertical scales. Fig. 4. View largeDownload slide Representative mass/(pressure) profiles of maize seedling roots (6–15 samples) exposed to bowl-shaped hindrances. Profiles of (A) intact roots growing into 100 µL of water; (B) decapped roots growing into 50 µL of water; (C) intact roots growing into 100 µL of 17 mm NaCl solution; (D) decapped roots growing into 100 µL of 17 mm NaCl solution. Fig. 4. View largeDownload slide Representative mass/(pressure) profiles of maize seedling roots (6–15 samples) exposed to bowl-shaped hindrances. Profiles of (A) intact roots growing into 100 µL of water; (B) decapped roots growing into 50 µL of water; (C) intact roots growing into 100 µL of 17 mm NaCl solution; (D) decapped roots growing into 100 µL of 17 mm NaCl solution. Fig. 5. View largeDownload slide Images of dark-grown, 3-day-old maize seedlings exposed to bowl-shaped hindrances with either intact roots (four seedlings on the left, A) or decapped roots (four seedlings on the right, B) after 48 h of exposure to vertical, narrowing tubes. In some cases, decapped roots regenerated the root cap and then actively despiralized the root architecture (data not shown). The arrow indicates the gravivector; the horizontal bar applies to a length of 1 cm. Fig. 5. View largeDownload slide Images of dark-grown, 3-day-old maize seedlings exposed to bowl-shaped hindrances with either intact roots (four seedlings on the left, A) or decapped roots (four seedlings on the right, B) after 48 h of exposure to vertical, narrowing tubes. In some cases, decapped roots regenerated the root cap and then actively despiralized the root architecture (data not shown). The arrow indicates the gravivector; the horizontal bar applies to a length of 1 cm. In order to test how roots behave towards different surface strengths in the presence or absence of the root cap, we let intact and decapped roots grow onto plates of differing agar concentrations placed on the weighing surfaces of the balances. Under such conditions, downward growing intact roots exposed above agar plates of concentrations of 0.5 %, i.e. with small mechanical resistance, were characterized by very faint profiles. Thereafter, they exhibited short-lasting pressure increases with steep drops, due to penetration of the root tip into the substrate (Fig. 6A). Subsequently, pressure again quickly increased, eventually leading in most cases to pronounced oscillations due to the crawling behaviour of the roots over the bottoms of the Petri dishes, and submerged in less dense agar. As a rule, such behaviour was not observed in decapped roots. Once they reached the surface of the agar, they exhibited rapid increases in pressure, leading in a short time to sideways slippages, i.e. they built up a pressure increase and then either slipped aside or grew in some cases into the substrate. In the case of 1 % agar plates, again intact roots developed a steep, yet small initial pressure rise, which abruptly decayed and was followed by a similar second increase once the roots reached the bottom of the Petri dish. Decapped roots developed relatively higher mass/pressure values before they slipped aside or, again, in some cases, penetrated the agar. Generally, intact roots penetrated agar substrates more frequently than decapped roots at agar concentrations of ≤2 %. At 4 % agar, the vast majority of roots, irrespective of the presence of the root cap, did not penetrate the substrate, but either slipped aside (in the case of decapped roots, Fig. 6) or actively changed growth direction by a crawling behaviour (in the case of intact roots). Fig. 6. View largeDownload slide Typical mass/pressure profiles of roots of 3-day-old maize seedlings vertically growing onto horizontal layers of different nutrient agar concentrations dependent on the presence of the root cap. Left column, intact roots; right column, decapped roots. Percentage values refer to nutrient agar concentrations. Fig. 6. View largeDownload slide Typical mass/pressure profiles of roots of 3-day-old maize seedlings vertically growing onto horizontal layers of different nutrient agar concentrations dependent on the presence of the root cap. Left column, intact roots; right column, decapped roots. Percentage values refer to nutrient agar concentrations. In general, variability of root mass/pressure behaviour was pronounced during these experiments, due to the fact that not all roots eventually came into contact with the hydrated surface in an exactly vertical orientation, i.e. the slick moist root surface also contributed to the pronounced heterogeneity of the measured profiles. Nevertheless, the root pressure behaviour in the chosen conditions was generally similar in the different treatments as represented by the single cases shown in Fig. 6, i.e. the majority penetrated the substrate at a concentration of ≤1 %. A summary of the dependence on nutrient agar concentration of the mean mass/pressure values of the root caps is given in Fig. 7. As shown, in all cases decapped roots developed initially higher mass/pressure values than intact roots. The difference increased according to the concentration of the agar (i.e. the lower the concentration, the lower the resistance of the substrate). Most obviously this difference is seen in roots impinging 1 % agar plates, in which the ratio was in the range of 1:4. Fig. 7. View largeDownload slide Mean mass/pressure values of roots of 3-day-old maize seedlings with and without root caps on nutrient agar plates with different concentrations, ± s.e. (n = 7). Fig. 7. View largeDownload slide Mean mass/pressure values of roots of 3-day-old maize seedlings with and without root caps on nutrient agar plates with different concentrations, ± s.e. (n = 7). Since it is, on the one hand, known that roots exposed to mechanical hindrances emit ethylene – generally known as an inhibitor of elongation growth (Moss et al., 1988) – and on the other, that the diffusion coefficient of ethylene is strongly decreased in liquids, we were interested in how pressure profiles looked during the two differing conditions of root environment. We therefore repeated the above-mentioned experiments, letting the root tips either grow into Eppendorf tubes filled with small volumes of water to a depth of 1 cm or grow in water-saturated air before impinging on the bottoms of the tubes. It turned out that intact roots impinging mechanical hindrances while their tips were submerged developed much less pronounced pressures compared with roots impinging the hindrance in moist air (Fig. 8). On average these developed 2- to 3-fold higher pressure (Fig. 8, bottom image), i.e. the ‘clash’ of intact root tips was much more strongly dampened in water than in air. In addition, they were generally characterized by a more pronounced oscillating behaviour. Fig. 8. View largeDownload slide Comparison of typical mass/pressure profiles of roots of 3-day-old maize seedlings the tips of which were either incubated in a total volume of 100 µL of water (upper figure) or kept in humid air (lower figure).The bottom diagram shows mean average mass/pressure values ± s.e. Fig. 8. View largeDownload slide Comparison of typical mass/pressure profiles of roots of 3-day-old maize seedlings the tips of which were either incubated in a total volume of 100 µL of water (upper figure) or kept in humid air (lower figure).The bottom diagram shows mean average mass/pressure values ± s.e. Employing the ETD, we analysed the effect of roots impinging on the horizontal surface as compared with controls, i.e. roots growing unhindered vertically downwards (Fig. 9). For this, ethylene emission of single seedlings was measured at intervals within the shoot part and the root part of the seedling separately. Ethylene content was measured in flow-through volumes received during sequential 10 min measurement time periods, regularly alternated with control/reference measurements, i.e. measurement of gases without seedlings in the chambers. From a typical result for intact roots, the impingement of the root tip resulted in a strong increase of ethylene emission as compared with unimpaired root tips (Fig. 9, left panel). In contrast, this effect was not observed in decapped roots in which ethylene emission is not affected by root tip impingement during a time period of 8–12 h on average. Similar to previously reported results (Alarcon et al., 2009), the individual seedlings were characterized by pronounced fluctuating absolute values of ethylene emission. Removal of the root cap resulted in increased emission of ethylene from the shoot (Fig. 9, right panel). Fig. 9. View largeDownload slide Representative ethylene emission profiles of intact (left) and decapped (right) roots of 3-day-old maize seedlings mechanically hindered in their vertical growth of the root tip by glass plateaus, as compared with controls within the gadget shown in Fig. 2. Vertically growing roots reached the hindrance during the initial zone, i.e. the beginning of the abscissa. Measurements were carried out in flow-through intervals. Fig. 9. View largeDownload slide Representative ethylene emission profiles of intact (left) and decapped (right) roots of 3-day-old maize seedlings mechanically hindered in their vertical growth of the root tip by glass plateaus, as compared with controls within the gadget shown in Fig. 2. Vertically growing roots reached the hindrance during the initial zone, i.e. the beginning of the abscissa. Measurements were carried out in flow-through intervals. DISCUSSION Under natural growing conditions, roots are constantly exposed to exogenous physical as well as chemical impacts which exert direct effects on root growth, which in turn affect per se root-borne processes (Moss et al., 1988; Barlow and Baluska, 2000). Based on the conditions encountered, they have to take decisions which will eventually dictate their fate – and their proliferation and productivity. As a consequence, the way roots manage to cope with given circumstances determines their developmental success (Trewavas, 2014). Since the root tip, with its functionally versatile root cap (Baluska et al., 2009; Kutschera and Niklas, 2009), represents the dominant unit of a growing root, we were interested in what impact its removal has on the pressure profile of roots faced with mechanical obstructions. As shown in Fig. 3, intact roots develop a steep brief pressure increase followed by a decrease, after which they actively change growth direction characterized by a crawling behaviour demonstrated earlier (Hahn et al., 2006). In decapped roots, this behaviour is not observed; such roots slip aside, leading to a slow and gradual oscillatory-like decrease in pressure. They apparently lack a mechanism to enable the root to adapt to the local conditions. Such an active response to mechanical obstruction observed in intact roots must inevitably be characterized by pressure-dependent signal transduction chains, which are, in principle, not affected by the impact of either Na or Cl ions. The presence of NaCl on the roots at the point of contact resulted in a strong general pressure increase, yet did not change the principal mass/pressure profiles observed in controls with only water. This might be explained by an enhanced osmotic strength of the root cells after ion uptake. However, we do not yet know the reason why decapped NaCl-exposed roots generally develop a significant second pronounced pressure increase (Fig. 3D), not observed in water-exposed decapped roots. It would therefore be worthwhile to measure turgor pressure within the root tip cells. In contrast to other reports (Neumann, 1995), the enhanced pressure profiles in fact indicate an apparent enhanced vitality and not an impairment of the root growth capacity – at least not within the measured time and at the given concentrations. More complex surfaces, as mimicked by bowl-shaped Eppendorf tubes, are again characterized by slightly less steep yet eventually higher pressure increases, in both intact and decapped roots (Fig. 4A, B). In these measurements, heterogeneity was most pronounced. This may be mainly interpreted as the complex reversion growth behaviour of the roots. Again, intact roots apparently possess the sophisticated capacity not only to divert growth direction, but also to reverse growth direction repeatedly despite unchanged gravistimulation (Fig. 5). This response is not observed in decapped specimens, which generally drilled their roots onto the bottom of the Eppendorf tube, in the absence of a rebuilt root cap. The root cap must, therefore, possess a means of directing growth, in both a positive and negative gravitropic mode, favouring the positive mode when it is possible, constantly adjusting this behaviour in real time as a function of the close mechanical environment. Such a situation-sensitive versatile mechanism is supported by the results presented in Figs 6 and 8. Below substrate strengths of 4 % agar, intact roots penetrate agar layers at much lower mass/pressure values and do not slip aside; this is surprising, since intact root tips are characterized by high surface moisture due to the hygroscopic quality of the root cap. However, at concentrations of 4 % agar, intact roots change direction of growth, whereas decapped roots again slip aside across the surface. In a way, therefore, the cap takes the decision depending on the strength of the hindrance. These mechanical impacts per se may be mediated via cytoskeletal rearrangements, which were demonstrated to depend on ethylene as one effect of its mediated triple-response (Roberts et al., 1985; Baluska et al., 1992; Cyr, 1994) as well as wall peripheral distortions (Barlow and Baluska, 2000). A crucial difference from the free air root experiments as compared with natural conditions consists of markedly different diffusion conditions above the root surfaces. It is to be expected that when surrounded by soil-emitted gases, such as ethylene or other compounds (Wenke et al., 2010), the roots reach a several times higher concentration compared with roots exposed to the free space/gas phase. In the case of ethylene, as a hormone very relevant to development (Abeles et al., 1992; Davies, 1995), it is to be expected that in roots embedded within the substrate, i.e. surrounded by mechanically dense soil particles, or with the root surface in contact with liquids, its concentration will increase to values far beyond those of roots exposed to the free space. During such non-restraining conditions, gases will diffuse away with little or no effect on the root cells. Being aware that various other processes may be affected, it would be expected that root tips impinging on mechanical surfaces during conditions of immersion in water would be more strongly inhibited than roots faced with moist air. A strong indication of such scenarios is given in results typical for the two different treatments (Fig. 8). The conditions underlying the two representative curves differ only by the phase (liquid/air) surrounding the root tips. Immersed roots develop slight pressure profiles as compared with roots pressing on the mechanical surface exposed to moist air. Additional support arises from the results shown in Fig. 9 based on single root ethylene emission measurements. Only root tips with intact caps display ethylene emission from roots impinging on mechanical surfaces. It is therefore not only compatible, but also very likely, that root growth orchestration and management of growth response dependent on mechanical impacts is via root cap-mediated ethylene emission. This phenomenon appears as ‘automatically’ modulated and independent of the root surroundings. In fact, one could, in principle, argue that the mechanical and chemical surroundings determine graviresponsivity. It appears plausible that a means for this gravimodulating environment is endogenous ethylene, the impact of which on the root itself represents a (biochemically printed) ‘picture’ of the mechanical surroundings – on top of other parameters integrated into the growth behaviour, as suggested earlier (Edelmann and Roth, 2006). On the other hand, it has been shown that inhibition of ethylene synthesis inhibits graviresponsive growth (Edelmann and Roth, 2006) and mechanical impacts induce ethylene synthesis (Fig. 9). For those reasons, it appears that it is enhanced ethylene which, during these substrate-penetrating conditions, may be responsible for enhanced gravitropic growth (Edelmann, 2002; Hahn et al., 2008). The way in which the demonstrated root cap-dependent ethylene acts may be through modification of the cytoskeleton within the elongation zone in such a way that associated cell extension processes are inhibited (Cyr, 1994). It may also interfere in such a way that growth-directing processes within the elongation zone of the roots are practically disengaged from gravity-dependent growth. However, the phenomenon of root cap-dependent ethylene release observed during mechanical hindrance may reflect correlation rather than causation. For future studies, the use of efficient and reliable inhibitors of ethylene synthesis applied to intact and decapped roots may deliver more unequivocal evidence in support of the suggested potential causalities. In strong support of a causal role for ethylene in changes in growth direction is the earlier reported finding where it was shown that in intact roots inhibition of ethylene synthesis inhibits gravidependent changes in root growth direction of maize seedlings, which was not observed in decapped roots (Hahn et al., 2008). Based on the scenario outlined, dense soils and soil water will ‘automatically’ restrain root expansion and temporarily shift the cap-based ‘root management’ to conditions controlled by exogenous parameters – in addition to or independent of gravity. As well as the mechanical root environments, local water conditions may also induce adequate root behaviour by enhanced ethylene accumulation, which in air (i.e. during mechanically and chemically unimpaired conditions) volatilizes into the gas phase. Although our ethylene measurements differentiated between root- and shoot-borne emissions, we are aware that this organ-specific localization does not allow us to decide whether or not ethylene originated from more mature and therefore possibly lignifying or otherwise affected parts of the roots (Ingemarsson et al., 1991; Huang et al., 2013). In ecophysiological terms, a hypothesized ‘stand-by’ mode of root ethylene emission may allow the plant to sense parameters in its vicinity in addition to those other(wise) perceived and other growth-relevant parameters. ACKNOWLEDGEMENTS The authors would like to thank Editor in Chief Professor Pat Heslop-Harrison as well as the peer reviewers for their suggestions and critical, constructive comments, which are highly appreciated. Thanks also to Dr Keith Baverstock, who has carefully proofread and corrected the manuscript for errors. LITERATURE CITED Abeles FB, Morgan PW, Saltveit ME. 1992. Ethylene in plant biology . San Diego: Academic Press. Alarcón MV, Lloret-Salamanca A, Lloret PG, Iglesias DJ, Talon M, Salguero J. 2009. Effects of antagonists and inhibitors of ethylene biosynthesis on maize root elongation. Plant Signaling and Behavior  4: 1154– 1156. Baluska F, Parker JS, Barlow PW. 1992. 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Mechanical impedance to root growth: a review of experimental techniques and root growth responses. Journal of Soil Science  41: 341– 358. Blancaflor EB, Masson PH. 2003. Plant gravitropism. Unraveling the ups and downs of a complex process. Plant Physiology  133: 1677– 1690. Bleecker AB, Estelle MA, Sommerville C, Kende H. 1988. Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science  241: 1086– 1089. Cyr RJ. 1994. Microtubules in plant morphogenesis: role of the cortical array. Annual Review of Cell Biology  10: 153– 180. Darwin C. 1881. The power of movements in plants . New York, D. Appleton and Company. Davies PD. 1995. Plant hormones: physiology, biochemistry and molecular biology . Dordrecht: Springer Netherlands. Edelmann HG. 1996. Coleoptiles are gravi-guiding systems vital for gravi-insensitive shoots of germinating grass seedlings. Planta  200: 281– 282. Edelmann HG. 2002. Ethylene perception generates gravicompetence in gravi-incompetent leaves of rye seedlings. Journal of Experimental Botany  53: 1825– 1828. Edelmann HG, Roth U. 2006. Gravitropic plant growth regulation and ethylene: an unsought cardinal coordinate for a disused model. Protoplasma  229: 183– 191. Edelmann HG, Gudi G, Kühnemann F. 2002. The gravitropic setpoint angle of dark-grown rye seedlings and the role of ethylene. Journal of Experimental Botany  53: 1627– 1634. Hahn A, Firn R, Edelmann HG. 2006. Interacting signal transduction chains in gravity-stimulated maize roots. Signal Transduction  6: 449– 455. Hahn A, Zimmermann R, Wanke D, Harter K, Edelmann HG. 2008. The root cap determines ethylene-dependent growth and development in maize roots. Molecular Plant  1: 359– 367. Huang WN, Liu HK, Zhang HH, Chen Z, Guo YD, Kang YF. 2013. Ethylene-induced changes in lignification and cell wall-degrading enzymes in the roots of mungbean (Vigna radiata) sprouts. 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Volkmann D, Tewinkel M. 1996. Gravisensitivity of cress roots: investigations of threshold values under specific conditions of sensor physiology in microgravity. Plant, Cell and Environment  19: 1195– 1202. Wenke K, Kai M, Piechulla B. 2010. Belowground volatiles facilitate interactions between plant roots and soil organisms. Planta  231: 499– 506. © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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Published: Jan 23, 2018

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