Differential growth and hormone redistribution in gravireacting maize roots

Environmental and Experimental Botany, Vol. 29, No. 1, pp. 37-45, 1989.

0098 8472/89 $3.00 + O.00 ~) 1989. Pergamon Press plc

Printed in Great Britain.

D I F F E R E N T I A L G R O W T H A N D H O R M O N E R E D I S T R I B U T I O N IN GRAVIREACTING MAIZE ROOTS PAUL-EMILE PILET Institute of Plant Biology and Physiology, University of Lausanne, 1015 Lausanne, Switzerland

(Received 18 January 1988; accepted in revisedform 17 February 1988) PILET P.-E. Differential growth and hormone redistribution in gravireacting maize roots. ENVIRONMENTAL AND EXPERIMENTALBOTANY 29, 37--45, 1989. When growing roots are placed in a horizontal position gravity induces a positive curvature. It is classically considered to be the consequence of a faster elongation rate by the upper side compared to the lower side. A critical examination indicates that the gravireaction is caused by differential cell extension depending on several processes. Some of the endogenous regulators which may control the growth and gravitropism of elongating roots are briefly presented. The growth inhibitors produced or released from the root cap move preferentially in a basipetal direction and accumulate in the lower side of the elongation zone of horizontally maintained roots. The identity of these compounds is far from clear, but one of these inhibitors could be abscisic acid (ABA). However, indol-3yl acetic acid (IAA) is also important for root growth and gravitropism. ABA may interact with IAA. Two other aspects of root cell extension have also to be carefully considered. An elongation gradient measured from the tip to the base of the root was found to be important for the growth of both vertical and horizontal gravireactive roots. It was changed significantly during the gravipresentation and can be considered as the origin of the differential elongation. Sephadex beads have been used as both growth markers and as monitors of surface pH changes when they contain some pH indicator. This technique has shown that the distribution of cell extension along the main root axis is related to a pH gradient, the proton efflux being larger for faster growing parts of roots. A lateral movement of calcium is obtained when Ca ~+ is applied across the tips of horizontally placed roots with a preferential transport towards the lower side. Endogenous calcium, which may accumulate inside the endoplasmic reticulum of some cap ceils, may also act in the gravireception. These observations and several others strongly suggest that calcium may play an essential r61e in controlling root growth and several steps of the root gravireaction. INTRODUCTION

inhibitors formed or released in the c a p cells, their content of endogenous indol-3yl-acetic acid (IAA) a n d abscisic acid (ABA), a n d their surface p H which is d e p e n d e n t on an H + p u m p a n d C a 2+ transport.

ELONGATIONa n d g r a v i r e a c t i o n of roots are consequences of the irreversible increase in the volume of their cells. This is due to the changes in the extensibility of their walls, their p e r m e a b i l i t y to water, a n d their osmotic potential./8'41/ P l a n t hormones m a y control root growth by means of several processes/11'42/which include proton extrusion a n d calcium a v a i l a b i l i t y (see Fig. 1). T h e aim of the present r e p o r t is to discuss briefly some results concerning the g r o w t h a n d gravireaction of maize roots in relation to growth

GROWTH

T h e elongation of roots m a y be first analysed by length m e a s u r e m e n t s each h o u r (kinetic analyses). T h e d a t a , r e p o r t e d in Fig. 2, indicate that the g r o w t h rate of maize roots is higher for seed37

38

P.-E. PILET ROOT V

1.0 GROWTH

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0.7 WALL EXTENSIBILITY

DIFFERENCE IN OSMOTIC POTENTIAL ( INSIDE - - OUTSIDE)

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FIG. 1. Scheme showing the several levels of regulation (cellular, subcellular, molecular, etc.) implicated in the control of the root growth. Adapted from PILET and BARLOW. (27)

Q: 0.7 a. 0.6 E E 0.5

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lings kept in the d a r k than in the light, a n d in a vertical r a t h e r than a horizontal position. G r o w t h m a y also be studied in a large p o p u lation of roots selected at zero time for uniform length. I n d i v i d u a l elongation rates were measured over an 8 hr period in the vertical position (in h u m i d air and in darkness). As can be seen in Fig. 3, the v a r i a t i o n in the growth rate is expressed as the n u m b e r of i n d i v i d u a l roots in each of 17 classes (each class representing an a d d i t i o n a l 0.09 m m / h r ) . T h e m e d i a n growth rate can be found, as well as the percentage of roots exhibiting a given elongation rate. GRAVIREACTION

AND

DIFFERENTIAL

GROWTH Positive c u r v a t u r e o f horizontally placed prim a r y roots is the consequence of a faster rate of elongation by the u p p e r side c o m p a r e d to the lower side (Fig. 4). Several possibilities have to be considered as to how these differences in growth could be obtained. F o r example, if elongation is stimulated in the u p p e r part, then it could be arrested, slowed or u n c h a n g e d in the lower p a r t

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Fro. 2. Rates of elongation (mm/hr) of intact primary roots of maize (cv. LG 11) vertically maintained in darkness (A) and in light (B), in vertical (V) and horizontal (H) positions, over a 10 hr period. Vertical bars represent S.E. x 2. Adapted from BEFFA and PILET.(5)

(see T a b l e 1). T h e p r o m o t i o n of cell extension within the u p p e r h a l f o f g r a v i r e a c t i v e roots could be the m a i n cause of bending, (13'14'3°'31~ b u t the reduction of extension for cells in the lower h a l f has also to be considered. I n this connection the redistribution of growth inhibitors a n d hormones (l) will be discussed later on. It is certain that the rate at which a root bends has to be related to the m a g n i t u d e of the differential elongation. Using p r i m a r y roots of maize (cv L G 11), a difference in extension rate of 0.0127 m m / m i n between u p p e r and lower sides resulted in a 1°/min rate of bending.(4) G R O W T H INHIBITORS It has been shown m a n y times that r e m o v a l of root cap largely eliminates gravicurvature.

H O R M O N E R E D I S T R I B U T I O N AND R O O T G R A V I R E A C T I O N 45O

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Fzo. 3. Rates of elongation (mm/hr measured over an 8 hr period) of intact primary roots of maize (cv. LG 11). A total of 2508 roots were tested. Values for individual roots are reported for 17 growth-rate classes (each having a range of 0.09 mm/hr). Roots were maintained vertically, after being selected, for 8-F 0.25 hr in darkness. The mean total root lengths at zero time, and after 8 hr were 15.3___0.9 and 21.2___1.4 mm, respectively. Adapted from PILET. (26) Its excision m a y cause some injury effect/l°J4) due p a r t l y to ethylene g e n e r a t e d in response to wounding./48) O n e of the reasons w h y d e c a p i t a t i n g or d e c a p p i n g delays or prevents gravireaction is because it removes the site of gravity perception. (2/ M a n y microsurgical experiments (25'45) readily lead to the conclusion that some growth inhibiting substances m a y pass from the cap cells to the elongation zone of the horizontal root a n d a c c u m u l a t e in its lower part. Results of several experiments are s u m m a r i z e d in Fig. 5, showing some of the physiological properties of these growth inhibitors. These d a t a a n d some others (2°'38~provide strong evidence that gravity m a y induce a redistribution of these inhibiting factors which are formed or released in the c a p and move preferentially in the basal direction. T h e lower h a l f o f the c a p produces m o r e inhibitors t h a n the upper, a n d their d o w n w a r d lateral t r a n s p o r t can also be observed in the apex of horizontally m a i n t a i n e d

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FIO. 4. Rates of gravicurvature (A) and of elongation (B) of maize (cv. LG 11) roots (upper and lower sides). Each datum point is the mean + S.E. of, respectively, 180 (A) and 70 (B) roots. Adapted from PILET and NEY.( TM

Table 1. Positive root gravitropism occurs when the initially uppermost side elongates faster than the lowermost side. Six ways in which this could arise are given below

Side

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( + ) Elongation stimulated; ( - ) elongation slowed; ( × ) elongation arrested; (0) no change in elongation rate. Adapted t?om JACKSON and BARLOW.'tt' roots. These growth inhibitors not only act on cell extension a n d hence control root growth and gravireaction, b u t it has also been r e p o r t e d (9) that they inhibit cell differentiation. I n the lower half of the root, m a t u r a t i o n of some organelles a p p e a r e d to be delayed, whereas in the upper hal£ m a t u r a t i o n of other organelles was t~ster than in vertical roots. F u r t h e r analysis of the e x t r a c t a b l e growth

40

P.-E. PILET CZ

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Fio. 5. Curvature (in degrees _ S.E.) after 14 +0.5 hr of half-decapitated primary roots of maize (cv. Kelvedon 33) with (B, C) or without (A) a mica barrier inserted, and after 6 hr of root segments into which mica was inserted vertically (D) or horizontally (E) inside the cap or tip. The broken arrows indicate the direction of transport of the growth inhibitor(s) from the root cap (rc), through the apex (a), to the elongating zone (ez). Adapted from data in PILET. (20'24'25) inhibitors is required to ascertain their chemical structure. E t h e r extracts of maize caps contain several different c o m p o u n d s separable by TLC./46) O n e of the first candidates proposed was ABA. (21) It is present in the cap (36"47) a n d m a y induce an inhibition of root growth./2~) O t h e r inhibiting substances found in the root tip m a y also regulate the differential growth of gravireacting roots. J u d g i n g by their physiological properties, as revealed by microsurgical experiments, they could also include ABA or its derivatives as well as other compounds. (4°/ H O R M O N A L R E G U L A T I O N OF G R O W T H

It is now certain that all the known p l a n t hormones are potentially c a p a b l e of influencing root growth u n d e r some circumstance or other. A l t h o u g h they m a y never act separately in vivo, it is p o s s i b l e ~ a t least t h e o r e t i c a l l y - - t o characterize

Ct Ct

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FIG. 6. Theoretical scheme showing the several ways in which a hormone might be implicated in growth regulation. Lines A and B indicate that the hormone acts in the control of root elongation while this is not tile case ibr lines C and D. When the effect is obtained, it can be positively (1, 2) or negatively (3, 4) related to the increasing hormone concentration. When changes in the hormone level (5, 6) caused no variation in the growth rate which stays constant (Ct), or when the content of the hormone (7, 8) stays constant (C t) but the growth rate was changing, this indicates that the hormone has no effect in the growth regulation. some possible types of relations between changes in the level o f one p a r t i c u l a r h o r m o n e and changes in the elongation rate of roots containing this h o r m o n e (Fig. 6). W e can c o m p a r e the effect o f I A A and A B A when applied to growing roots. Some results given in Fig. 7 clearly indicate that I A A (10 -8 to 10 -4 M) caused in 6 hr the i n c r e m e n t of root elongation to decrease from a b o u t 7 to 2.5 mm, while for the same concentration range o f ABA, the decrease was only from 6 to 4 mm. T h e level of I A A and ABA has been analysed by G C - M S in maize roots from several of the growth classes depicted in Fig. 3. A strong relationship between the content of these hormones a n d the elongation rate can be observed (Fig. 8): the higher the level of hormone, the lower the g r o w t h rate. I n the elongation zone of the fast growing roots, the level of the two hormones was almost similar. A small difference in the growth of the slowest growing roots corresponds to a large change in A B A level. This attests to the v a r i a b i l i t y o f the A B A content in maize roots grown u n d e r similar conditions, and to the difference between I A A and A B A in their relation with growth. Results on w h e t h e r an a s y m m e t r i c a l redistribution of I A A occurs d u r i n g root gravireaction

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IAA ng/g fresh weight

IAA ng/g dry weight

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Fro. 7. Elongation ( m m _ S.E.) of intact primary roots of maize (cv. LG 11) pretreated for 3 0 ± 5 min in buffered solution (3,3-dimethyl glutaric acid at 10 -3 M, pH 6.0±0.1) containing IAA (A) or ABA (B) at different concentrations. Roots of 12_+ 1 mm in length (at zero time) were partly immersed (the distal 10± 1 mm) in a vertical position. Following washing in the buffer, they were kept for 6 hr in the dark (19.0 ± 0.5°C). Data are the mean of five experiments, each with 4 0 ± 5 roots. Adapted from PILET and SAUGY.

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* Cortex only. t Stele and cortex. Adapted from SAUOVand PILET. 137) Table 3. ABA content (in relative values and in ng per 100 segments+_S.E.) in a 4_+0.1 mm segment of rootfrom which the first mm (tip, cap and apex) has been removed. Apical segments of 10 mm in length were initially maintained for 2 hr in a horizontal position (in white light). Data for the three kinds of root gravireaction are shown Gravireaction Positive

(33)

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GROWTH RATE IN ram PER h FIG. 8. Variation in the level of IAA and ABA (ng-+ S.E. per 30 segments) as a function of the elongation rate during an 8 hr period (mm/hr-+ S.E.). The hormone content is given for segments of the elongation zone (2.5-4.0 mm). Adapted from PILET and SAUGV.(3~/

a n d thereby causes the differential growth have now to be considered. Some d a t a are given in T a b l e 2. T h e y indicate that I A A , measured by a G C - M S technique, is p r e d o m i n a n t l y located in the stele. W h e n roots are placed for 2 h r in a horizontal position, the I A A content in the elongation zone was slightly b u t significantly e n h a n c e d in the cortex of the lower p a r t of the root. I A A is not the only growth r e g u l a t o r involved in the gravireaction. Endogenous A B A is also a s y m m e t r i c a l l y redistributed after 2 hr of gravipresentation, as can be seen in T a b l e 3. F r o m these d a t a it is clear that a positive gravireaction is related to a higher A B A level in the lower half o f the roots than in the u p p e r half. W h e t h e r ABA plays any direct r61e as an endogenous r e g u l a t o r in root g r o w t h is still an open question. (3'27~'External factors can change A B A content in roots and

42

P.-E. PILET

this may modulate essential root functions, namely, their water uptake and ion movement. Thus, differential elongation, on which the gravireaction of roots depends, may be regulated indirectly by ABA. For both IAA and ABA the results obtained are all significant, but the actual levels of the two hormones may appear relatively low. Two comments, at least, have to be made. Ill The data relate to large parts of roots, while if they were to be expressed on a per cell basis, the level could well be somewhat higher for the cells of the lower side (which are relevant to the growth differential), and lower for the corresponding cells of the upper side./2/ Only a part of the total IAA or ABA was assayed by G C - M S (i.e. the free hormones). Bound IAA or ABA might also be redistributed and they probably play some r61e in the differential elongation of gravireactive roots through equilibrium with the free fraction. Special attention is given in this present report to IAA and ABA, but it is clear that other endogenous growth regulators may also play an important r61e in regulating differential elongation in gravireacting roots. 02'27) For instance, by using microinjection techniques, it has been observed that gibberellic acid labelled with 14C shows some preferential upward movements in horizontal maize roots. (44) Furthermore under non-stressful conditions the level of ethylene is quite low in maize roots, tT/ but in a stressful environment ethylene content may increase greatly, leading to significant changes in root growth and gravitropism. I'll SURFACE pH AND PROTON

EFFLUX

As shown by qualitative/]91 and quantitative ~34/ measurements, maize roots develop a higher proton efflux in their growing zone. When roots were placed horizontally, the "acidification ability" of their upper side was stronger than that of their lower side. (]9'43) Some data will be given here for maize roots maintained in a vertical or horizontal position. The local growth rate and proton fluxes were measured simultaneously using Sephadex beads containing a pH indicator. The transmittance of each bead was measured using a microdensitometer; after appropriate calibration, pH and H + fluxes can be calculated. As can be seen

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FIG. 9. Mean gradient of the local growth rate (in relative values per hr-t- S.E.) along the lower and upper parts of maize (cv. LG 11) roots horizontally placed (A) and along a similar sample of vertical roots (B). Adapted from VERSELand PILET. (43/ in Fig. 9, when the roots were kept horizontal, the growth of the lower side was strongly stimulated, as compared with vertical roots. Data in Fig. 10 indicate that the proton extrusion, which was greatest in the elongation zone, was strongly reduced on the lower side and slightly increased on the upper side as compared to that of vertical roots. Thus, the differential elongation, of the gravireacting maize roots, has to be related to the asymmetry of their proton fluxes. The observed changes in H + extrusion probably reflect differences in the acidification of the walls of the cells from epidermis and cortex. An increase in the elongation rate of the cells in the upper side could result from a stimulation of proton extrusion across the plasmalemma. (35/ Consequently, a decrease in proton extrusion at the lower side of horizontal roots and an increase at the upper side would induce, at least in part, the root gravireaction. CALCIUM TRANSPORT

During the last few years there has been increasing evidence that calcium may control root

H O R M O N E R E D I S T R I B U T I O N AND R O O T G R A V I R E A C T I O N i

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Table 4. Photodependence of gravity-induced lateral transport

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* Mean + S.E. from five experiments with five roots each. Donor blocks contained 27,300 cpm of 45Ca2+ (as 45CAC12).Cpm shown are for the tissue-halfopposite the donor block• 1"Ratio cpm in tissue-half opposite the donor block when the donor is on top to cpm in the tissue-half opposite where the donor is on bottom. Adapted from LEE and EVANS./15)

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DISTANCE FROM THE TIP IN mm

FIG. 10. Mean local acid flux (in microequivalents ___S.E. of proton concentration given per hr and per bead). Results concern the lower and upper sides of maize (cv. LG 11 ) roots maintained either horizontally (A) or vertically (B). Values are to be read in con.junction with those shown in Fig. 9. Adapted from VERSEL and PILET. (43) growth and gravitropism. Several observations can be briefly s u m m a r i z e d from experiments recently carried out using intact maize roots. A p o l a r t r a n s p o r t of a p p l i e d 45Ca2+ across the tips of horizontal roots is o b t a i n e d with preferential m o v e m e n t t o w a r d the lower side./15) This calcium t r a n s p o r t is significantly r e d u c e d for roots treated with some a u x i n - t r a n s p o r t inhibitors./~8/ W h e n Ca 2+ was a p p l i e d to one side of the c a p of roots kept vertical it caused a c u r v a t u r e t o w a r d the side on which it was given. 1~6)W h e n the caps of intact roots were treated with c a l c i u m - c h e l a t i n g substances, the roots did not respond to gravity. R e p l a c i n g these chelators with calcium restored the gravireaction. !17/ I n a n o t h e r series of experiments, it was shown (see T a b l e 4) that there was little p o l a r m o v e m e n t of 45Ca2+ a c r o s s the elongation zone o f g r a v i s t i m u l a t e d d a r k - g r o w n roots, while there was substantial redistribution of labelled calcium across the growing zone of gravistimulated light-grown roots. Some previous

conclusions have to be mentioned here. I t has been well established that roots of some cultivars of maize when grown in light exhibit normal gravireaction, while in darkness they show little or no bending. 122,23~ C a l c i u m t r a n s p o r t m a y also p l a y an essential r61e in the perception of gravity, i.e. the first step ofgravitropism./39) Some experiments done when using Lepidiurn roots will be briefly discussed. I t is well established (1/ that g r a v i p e r c e p t i o n m a y be due, at least in part, to the action of gravity on the amyloplasts (statoliths) contained in specialized root cap cells (statocytes). T h e perception of the gravistimulus m a y result from the pressure which these statoliths exert on the endoplasmic reticulum of the statocytes. O n the other hand, gravistimulation is tbllowed by alterations in the symmetry of the external current, ca) T h e resting potential of statocytes in vertical roots is a b o u t - 1 18 mV. U p o n gravistimulation the m e m b r a n e potential for the statocytes located in the lower flank of the cap was - 9 3 m V , while that in the u p p e r flank was - 13 mV. These d a t a indicate that ion fluxes across the p l a s m a l e m m a are altered. W h e n the endoplasmic reticulum of these roots was isolated a n d i n c u b a t e d with Ca 2+ (at m i c r o m o l a r concentrations), it was observed that the vesicular m e m b r a n e fraction a c c u m u l a t e d Ca2+./39) Such a c c u m u l a t i o n was found to be A T P - d e p e n d e n t . Thus, in analogy to the sarcoplasmic reticulum of muscle, it has been con-

44

P.-E. PILET

cluded (39/ that regulation of the reticulum-localized Ca 2+ c o m p a r t m e n t might be an essential step in the stimulus-transduction of root gravireaction.

CONCLUSION

G r o w t h and gravitropism can be analysed for a group of selected roots for which the m e a n values are discussed, or for a large p o p u l a t i o n of roots distributed in several classes each characterized by a m e a n elongation rate. In the latter case, it is possible to compare, for instance, the properties of slow and fast growing roots. I t is clear t h a t gravireaction is the consequence of a differential extension between the u p p e r and lower sides of horizontally placed roots. Special attention was given to I A A and A B A although several authors are now claiming that these growth hormones have little or nothing to do with root gravireactivity. It is clear that the d a t a showing the h o r m o n e action are sometimes conflicting and the concepts not completely satisfactory. H o w e v e r a very large n u m b e r of facts can nevertheless be accepted as showing some evident implication of I A A a n d ABA; a n d some recent reports that d e n y a r61e for I A A a n d ABA have to be considered as inconclusive. O t h e r hormones such as gibberellins, cytokinins a n d ethylene m a y interact with I A A a n d ABA a n d m o d ify their action. Several endogenous factors also regulate elongation and gravireaction. T h e proton efflux, larger in the p a r t of roots having a higher rate of growth a n d g r a v i c u r v a t u r e , could p l a y a crucial r61e. Calcium, the lateral transport of which depends on gravity, is surely an essential growth regulator and is now considered a second messenger in plants. Acknowledgmen~I wish to thank Dr P. W. Barlow for his helpful comments.

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1. AuDus L. J. (1975) Geotropism in roots. Pages 327-363 in J. G. TORREY and D. T. CLARKSON, eds The development and function of roots. Academic Press, London.

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