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Journal of Experimental Botany, Vol. 48, No. 311, pp. 1229-1239, June 1997 Journal of Experimental Botany Growth of Zea mays L. plants with their seminal roots only. Effects on plant development, xylem transport, mineral nutrition and the flow and distribution of abscisic acid (ABA) as a possible shoot to root signal W. Dieter Jeschke1'3, Margita Holobrada2 and Wolfram Hartung1 1 Julius-von-Sachs-lnstitut fOr Biowissenschaften, Lehrstuhl fiir Botanik I, Universita't Wurzburg, Mittlerer Dallenbergweg 64, D-97082 WQrzburg, Germany 2 Institute of Botany, Slovak Academy of Sciences, Dubravska' cesta 14, SK-84223 Bratislava, Slovak Republic Received 1 November 1996; Accepted 20 February 1997 Abstract Maize l£ea mays L.) was grown in quartz sand culture either with a normal root system (controls) or with seminal roots only ('single-rooted'). Development of adventitious roots was prevented by using plants with an etiolated mesocotyl and the stem base was positioned 5 - 8 cm above the sand. Even though the roots of the single-rooted plants were sufficiently supplied with water and nutrients, the leaves experienced water deficits and showed decreased transpiration as transpirational water flow was restricted by the constant number of xylem vessels present in the mesocotyl. As a consequence of this restriction, transpirational water flow velocities in the metaxylem vessels reached mean values of 270 m h " 1 and phloem transport velocities of 5.2 m h " ' . Despite limited xylem transport mineral nutrient concentrations in leaf tissues were not decreased in single-rooted plants, but shoot and particularly stem development was somewhat inhibited. Due to the lack of adventitious roots the shoot:root ratio was strongly increased in the single-rooted plants, but the seminal roots showed compensatory growth compared to those in control plants. Consistent with decreased leaf conductance, ABA concentrations in leaves of single-rooted plants were elevated up to 10-fold, but xylem sap ABA concentrations in these plants were lower than in controls, in good agreement with the well-watered conditions experienced by the seminal roots. Surprisingly, however, ABA concentrations in tissues of the seminal roots of the singleJ To whom correspondence should be addressed: Fax: +49 931 8886156. Oxford University Press 1997 rooted plants were clearly increased compared to the controls, presumably due to increased ABA import via phloem from the water-stressed leaves. The results are discussed in relation to the role of ABA as a shoot to root signal. Key words: Zea mays, seminal roots, plant development, xylem transport, mineral nutrition, ABA, shoot-to-root signal. Introduction Growth and development of the shoot of a whole plant is strongly dependent on concomitant and unrestricted development of the root system, as evident from the effects of root pruning (Jesko, 1972a, b; Carmi and Koller, 1978; Milligan and Dale, 1988) and the restriction of the rooting volume Carmi et ah, 1983; Robbins and Pharr, 1988; Creswell and Causton, 1988; Peterson et al, 1991a). In both of these cases the capacity of a major assimilate sink in the plant was decreased by restricting the size of the root and this led to a commensurate decrease in shoot growth and, usually, in the rate of leaf photosynthesis. In the case of monocotyledons which lack secondary thickening the size of the root system can be severely restricted if adventitious nodal roots are excised (Jesko, 1972a; Jeschke, 1984) or their development is prevented (Passioura, 1972; Passioura and Ashford, 1974) and the root system is then confined to the primary root. Passioura used single-rooted wheat plants, in which the hydraulic 1230 Jeschke et al. resistance to water flow was greatly increased, as a means to decrease water consumption by the whole plant. In this way he was able substantially to increase the grain yield in comparison with plants having the full root system, if both the single-rooted and the control plants were dependent on a limited water resource (Passioura, 1972). While effects of a restricted root size on shoot and leaf development and photosynthesis have been widely studied (Carmi et al., 1983; Robbins and Pharr, 1988; Cresswell and Causton, 1988; Peterson et al., 1991a), any possible water deficit and restriction of the mineral nutrition of the shoot, which may also arise, have attracted somewhat less attention (Richards and Rowe, 1977; Hameed et al., 1987). In preliminary experiments with single-rooted maize {Zea mays L.) plants continually supplied with water and nutrients not only was decreased water uptake (see Passioura, 1972) observed, but also signs of severe water deficits. The aim of the present paper is firstly to study the effects of a severely restricted root system, consisting only of the primary and adventitious seminal roots, on the water relations, the xylem sap composition and the mineral supply of maize plants. Secondly, it is intended to reveal possible root to shoot or shoot to root signals acting in maintaining economical water consumption in the whole plant with an inadequately small root system which is, nevertheless, exposed to adequate soil moisture. In this case, neither biosynthesis of abscisic acid (ABA), which seems to play a well established role as a root-to-shoot stress signal (Davies and Zhang, 1991), should be increased in the root nor its concentration in the xylem sap elevated, because roots are well supplied with water and nutrients and because root volume is not restricted. Restricted root volume was shown by Liu and Latimer (1995) and Ternesi et al. (1994) to increase ABA concentrations in the xylem. One could expect therefore an increased ABA formation in the leaves. Part of this ABA could be translocated in the phloem back to the roots. Materials and methods Plant culture Zea mays L., cv. Garant, FAO 240 hybrid maize seeds were soaked and germinated in 10 1 pots in quartz sand moistened with 1/16 Hoagland nutrient solution at different light conditions. Control plants (control) were allowed to germinate in the greenhouse in the light; nodal roots developed in due course at the base of the stem. In order to produce plants having their seminal roots only, some of the pots were kept in the dark and these seedlings developed a strongly etiolated, elongated mesocotyl. The coleoptile node was about 5-8 cm above-ground. Throughout cultivation the shoot of these plants was supported so that it remained above-ground and though nodal roots were initiated, they were unable to reach the quartz sand substrate (Plate 1). In the text these plants will be termed 'single-rooted plants'. Starting from 6d afteT sowing (DAS) all plants were initially cultivated in the greenhouse with 16 h d " 'supplemental Plate 1. Basal part of the shoot of a single rooted maize plant. The arrow denotes the etiolated mesocotyl; note the undeveloped initials of adventitious roots. Age of the plant: 11 weeks. illumination (Osram HQL 400, 300-500 ^onol photon m 2 s '), 15-25 CC and 45-70% relative humidity and later both variants were grown outside the greenhouse under ambient light, temperature and humidity conditions. Plants were grown in quartz sand culture and watered daily with nutrient solution. The concentration was gradually increased from an initial 1/16 to a final 1/2 strength of Hoagland solution (2.5 mM Ca(NO3)2, 2.5 mM KNO3) 1 mM MgSO4, 1 mM KH2PO4, 0.5 mM H3BO3) with additional micronutrients and Fe-citrate. Measurement of total plant transpiration and leaf conductance Total plant transpiration was measured gravimetrically by determining the daily or sometimes 2 h water loss and correcting for the loss from the substrate by weighing a pot without a plant. Leaf conductance of all leaves of control and singlerooted plants was measured with a LiCor 1600 steady-state porometer under ambient conditions. Measurements were done at the same time of the day (1-3 p.m.), but only under sunny conditions. Harvesting of plants and collection of root pressure xylem exudate Harvests were made in experiment 1 at 77 DAS at early fruiting and in experiment 2 at 68 DAS at the time of tasseUing. Plants were harvested and separated into roots, stem and individual Maize plant growth leaves. Leaf laminae were further subdivided into basal (b) and apical (a) parts. Roots were carefully washed to remove quartz sand particles. Samples of all plant parts were weighed, freezedried and dry weight was determined. After fine grinding, sample aliquots of dry matter were used for chemical analyses. Xylem sap was collected at the day of harvesting. After excision of the shoots silicon rubber tubing was connected to the tops of the mesocotyl of the single-rooted plants or to the stump of the stem base of control plants. The xylem sap collected during the first few min was discarded to avoid contamination resulting from cut tissues (Else et al., 1994). Collected xylem sap volumes were recorded and samples were frozen before use for further analyses. 1231 mesocotyl plus root was obtained from the sum of all individual conductivities and the pressure difference Ap needed for driving the measured volume flow (total water loss from the plant) was obtained from (2). Using (1), the volume flow [cm 3 h"'] in each of the vessels at the prevailing pressure difference Ap was then estimated and from this volume and the cross-sectional vessel area [cm2] theflowvelocity [cm h - 1 ] or [m h" 1 ] occurring in each individual vessel was calculated. Flow velocities in the phloem were estimated on the basis of the measured sieve tube area, the quantities of carbon needed for root growth and respiration and an assumed sucrose concentration of 20% in the phloem sap, for details see below in the Discussion. Chemical analyses Cations in tissues and in the xylem sap were determined using atomic absorption spectrometry (FMD 3, Carl Zeiss, Oberkochen), anions by anion chromatography (Anionlenchromatograph, Biotronik Co., Maintal, Germany). Amino acids were determined using an amino acid analyser (Biotronik Co., Maintal, Germany). Analysis of free and conjugated ABA Freeze-dried tissue samples were homogenized and extracted in 80% methanol. Extracts were passed through a Sep Pak C18cartridge. Methanol was removed under reduced pressure and the aqueous residue partitioned three times against ethyl acetate at pH 3.0. The ethyl acetate of the combined organic fractions was removed under reduced pressure. The residue was taken up in TBS-buffer (TRIS-buffered saline; 150mmol I" 1 NaCl lmmol I" 1 MgCl2> and 50mmol I" 1 TRIS; pH 7.8) and subjected to an immunological ABA assay (ELISA) as described earlier (Peuke et al., 1994; Mertens et al. 1985). For xylem saps the Sep-Pak C18 purification step was omitted. The aqueous phase after partitioning against ethyl acetate was hydrolysed for 1 h at room temperature with 1 M NaOH. This fraction was acidified with concentrated hydrochloric acid to pH 3, and partitioned three times against ethyl acetate. ABA which had been released from conjugates (predominantly ABA-glucose ester) was analysed by ELISA in the organic phase. The accuracy of the ELISA was verified for Zea in earlier investigations (Hartung et al., 1994). Recoveries of ABA during purification procedures were checked routinely using radioactive ABA and found to be more than 95%. The immunochemicals were generously supplied by Professor Weiler, Ruhr Universitfit Bochum (Germany). Evaluation of volume flow conductivities and flow velocities of the xylem vessels Using transverse sections of the mesocotyl (Plate 2) or the basal stem of control plants the radii of xylem vessels r, were measured. According to Hagen-Poiseuilles law (Stuart and Klages, 1988; Nobel, 1991) the volume flow J, [cm3 s"1] through a capillary vessel with the radius r, is Jv = A V/At = - ( (1) and for a series of parallel vessels (2) with A V the volume flowing in the interval A t under the pressure difference Ap, I the length of the vessel and TJ the viscosity of the solution (for water 77=0.01002 dynes cm" 2 ). For each vessel the individual volume flow conductivity Arf/S-q I was estimated using 30 cm for the length / from the soil to the upper end of the mesocotyl. The total conductivity of the Results Plant growth and development The single-rooted plants developed an etiolated 8-15 cm long and 2-3 mm wide mesocotyl. The anatomical structure of the etiolated mesocotyl in its mature state was distinguished from other stem parts by a tertiary and in parts double endodermis with strongly thickened walls (Plate 2). The central cylinder showed the arrangement of xylem and phloem as in the root but contained further mechanical elements in the outer part of the central pith. Interestingly one or two lacunae were also present (Plate 2) which resembled the protoxylem lacunae in the stem bundles. Amongst the vessels (Table 1) there were 7-11 larger late metaxylem vessels on average 59 ^m wide which were responsible for 89% of the water transport, compared to c. 450 vessels 87 ±3 fun wide in the basal stem above the uppermost nodal roots of the control plants (not counting the protoxylem lacunae). Until 45 DAS, plant height was not affected, but later on it was decreased by about 10% in the single-rooted plants. Similarly, leaf area expansion in the uppermost leaves, which completed expansion after 52 DAS, was depressed in response to the reduced root system (Fig. 1A, leaves younger than leaf 8). During the 68 or 77 d of growth in the two experiments the primary seminal root plus the adventitious seminal roots (Jesko, 1991) grew profusely and branched remarkably to reach final dry weights of 3.4 or 4.0 g. Nevertheless, at harvest, root biomass was the most strongly decreased compared to that of the control, followed by that of the stem (Table 2), while leaf dry matter production was least inhibited in single-rooted plants (93% or 78% of the controls in experiments one and two respectively). The severe restriction of root development, caused by the lack of nodal root growth, led to strongly elevated shoot-to-root ratios of 15.7 or 11.9 compared to 3.5 or 6.1 in controls in experiments one and two, respectively. Inflorescence development was delayed and probably decreased, since it occurred during and after the first signs of water deficits were visible in the single-rooted plants (see Fig. IB). In the first experiment the fruiting inflorescences of single- 1232 Jeschke et al. Plate 2. Transverse cross-section of the etiolated mesocotyl of Zea mays L. at the age of 68 days after sowing (DAS). (A) Inner part of the cortex (Co) and stele. (B) Detail of stele and inner part of cortex. En = endodermis, ph = phloem, MX = metaxylem vessel, Sc = sclerenchyma, Pi = pith, Lac=>lacunae. The length of the bar corresponds to 250 ^m in (A) and to 100 (im in (B). Fixing: ethanol-fonnaldehyde-acetic acid, 6-8 fun thick sections (HN34 microtome, Jung, Germany). Staining has been performed by R Wacker, University of WOrzburg, Biozentrum, Physiological Chemistry, D-97218 Gerbrunn. rooted plants weighed only 13% of those of the control, but this was also due to a delay in flowering. Plants were harvested too early to judge effects of single-rootedness on yield (Passioura, 1972). Whilst specific signs of severe mineral deficiency and leaf necroses were not visible, rolling of the leaves of single-rooted plants indicated incipient water deficits and with time slightly chlorotic bands developed along the leaves. Water relations and leaf photosynthesis of control and single-rooted plants As was to be expected, the dependence of maize plants on one single root had the most substantial effects on their water relations (Table 3, items i to vii). As a direct consequence of the much smaller root biomass, the water uptake and hence loss per plant (i) was severely decreased (by 72% or 41% in experiments one and two, respectively). Since leaf area was somewhat smaller, water loss relative to leaf area (ii) was less depressed (by 42% or 27%), but leaf conductance was substantially lower in single-rooted than in control plants (Fig. 2; iii in Table 3). Remarkably, however, these differences between singlerooted and control plants disappeared or were even inverted, if the transpirational water loss was related to the root size and the water uptake per root weight was calculated (neglecting the small amounts of water used for growth). Relative to root fresh weight, water uptake Maize plant growth means stable, but it decreased with time as shoot growth and size progressively exceeded that of the root. CO2 assimilation by leaves of single-rooted plants was decreased (viii in Table 3) as has similarly been found in plants with a restricted rooting volume (Robbins and Pharr, 1988), but not in sorghum after excision of nodal roots (Jesko, 1972a). The difference most probably was due to the much shorter (39 d) duration of the experiments with sorghum. 500 control 400E 300- e 200- u 100- L6 L7 tuOy expanded at: 4 2 47 L8 L9 leaf number 52 57 L10 82 Mineral element concentrations in root and leaf tissues of control and single-rooted plants L11 66 DAS 100 B 75 I o 1 20 r—— watf loss led area 50 15 10 o 25 *>a' 0 50 60 As seen in Fig. 3, for no element, except for Zn in roots, was the concentration in root or leaf tissues decreased significantly in single-rooted compared to control plants. On the contrary, even in leaf tissues, which would have been more likely to become inadequately supplied as a consequence of the restricted root size, the concentrations of all major and minor nutrient elements more or less exceeded those in leaves of the control plants. This was also true for Mg, which might have been deficient, as suggested by slightly chlorotic bands along the leaves. 5 pressure gradient o—""' 40 1233 70 days after sowing (D A S) Fig. 1. (A) Final leaf area of leaves No. 6 to 11 of control (—D—) and single-rooted ( V ) plants of Zea mays L. The numbers below the x-axis show the time in days after sowing when expansion of each leaf was completed and indicate that the x-axis also is a time-axis, which is almost synchronous with that of (B). (B) Time-dependence of the water loss per leaf area of single-rooted maize plants, given as % of the controls (—•—) and time-dependence of the pressure gradient needed to drive xylem sap at the prevailing rate through the mesocotyl of single-rooted plants of Zea mays L. ( — D — ) (right hand scale). Bars indicate SE of the mean. in single-rooted plants even slightly exceeded that in the controls (iv in Table 3). Interestingly, the same result was seen in the volume flows of root pressure exudates (vii) which, though much slower than transpirational water flow, were 14% higher in single-rooted than control plants. As seen in Fig. 3B, in relation to the control plants the water supply available from the single root was by no Concentration of free and conjugated ABA in root, tassel and leaf tissues of control and single-rooted plants In Fig. 4 concentrations of abscisic acid in root, tassel and leaf tissues of the apical half of control and singlerooted plants are shown. Whereas almost constant ABA levels of 0.05 to O.lnmolg" 1 FW were found in all control leaves, ABA concentrations were twice as high in young leaves of single-rooted plants and increased with leafage to reach in older leaves 12-fold higher levels than in the controls. Tassel ABA concentration was increased by 60% and roots contained an 8-fold elevated ABA level (Fig. 4). Root ABA concentrations are in a range similar to that found by Zhang and Davies (1989). Concentrations of conjugated ABA, which consists mainly of ABA-glucose ester (ABA-GE) were also determined and slightly lower concentrations than those of free ABA were found. Similar to free ABA, young leaves of single-rooted plants contained 2-fold higher levels of conjugated ABA than controls and concentrations increased with leaf age to reach 4-fold higher levels compared to the Table 1. Number, diameter and volume flow conductivity of the xylem vessels in the etiolated mesocotyl of single-rooted maize plants Type of vessels Late metaxylem vessels Smaller vessels* Experiment Number of vessels Mean diameter Range (fun) Conductivity* 11 9 ±0.4 112 36 + 3 59 ±4.2 55.8 ±4.2 21.9± 1.7 23.6 ±1.2 41-85 35-93 9-37 10-45 1.45x10 0.2x10 ' Mostly early metaxylem vessels. * Estimated with Hagen-Poiseuille's law as -n^/i^l for all individual vessels. The length / of vessels was assumed to be 30 cm from the top of the mesocotyl down to within the primary root. The choice of / has no effect on the relative contribution of the differently wide vessels to total conductivity. 1234 Jeschke et al. Table 2. Dry matter content of control and single-rooted Zea mays plants recorded at the day of harvesting, Le. 68 d after sowing The results are presented as mean values ±SE of 4 control and 10 single-rooted plants in experiment 2. Organs Experiment 2 Experiment 1* Control plants (gDW plant" 1 ) Leaves Tassel Cob Stem Shoot Root Shoot/root Single-rooted plants % of Control (gDW plant" 1 ) 13.1 ±0.2 3.4 ±0.1 12.1 ±0.4 2.6±0.2 40.3 ±3.9 56.8 ±4.0 9.3±1.1 6.10 25.3 ±0.9 40.1 ±2.3 3.4±0.3 11.90 % of Control 93 78 — 63 71 36 78 — 13 58 56 16 * For comparison, data for the single-rooted plants in experiment 1 are given as % of the respective control. Since the harvest was later (at 77 DAS) in this experiment, dry weights of all organs in the single-rooted plants were more depressed than in experiment 2 and a cob was formed. controls in the oldest leaves. Conjugated ABA was also higher in roots (8-fold) and the tassel (2-fold) of singlerooted plants (Fig. 5). The increased leaf ABA concentrations of single-rooted plants were well related to the observed decreases in leaf conductances (Fig. 2). ing from the fully developed root system was two times faster than from the single primary root (vi in Table 3). If volume flows were related to the root fresh weight, however, single-rooted plants bled 14% more rapidly than the controls, a quite remarkable result (vii in Table 3) which agrees, however, with the high water uptake capacity of maize seminal roots observed by Navara et al. (1994). In full agreement with this somewhat higher volume flow and owing to the much higher hydraulic resistance Xylem sap flow and composition in control and singlerooted plants Both control and single-rooted maize plants produced root pressure exudate after shoot excision, although bleed- Table 3. Water loss and transport parameters and leaf photosynthesis in control and single-rooted plants o/Zea mays L. Item Experiment 1* Control Experiment 2 Single-rooted' %of Control Control Single-rooted' %of Control Water relations Mean water loss per plant [gh" 1 plant" 1 ] Water loss per leaf area Leaf conductance g Water uptake per root fresh weight vi vii Flow velocity in large metaxylem vessels [mh-'Htcms- 1 ]) Xylem sap flow (root pressure) per plant [cm'h" 1 plant" 1 ] Xylem sap flow (root pressure) [cm 3 gFW" 1 h" 1 ] 29.2±6 8.2 ±2.4 28 14.2±3.0 8.4 ±1.0 59 1460±31 840±70 58 1290±27 940± 11 73 n.d.' 1.3±0.2 (0.3) — n.d' 0.21 ±0.02 1.4 ±0.4 107 e 2400 270±35 (7.5) 0.1 ±0.009 50 0.4 ±0.1 0.44 ±0.05 110 3.2±0.2 1.6±0.2 50 0.028 ±0.006 0.032 ±0.003 114 53.2±3.0 45.5 ±2.4 86 Photosynthesis CO2 assimilation [/Imol m- 2 s~ 1 ] n.d.' n.d.' n.d.' 5.2' Phloem transport Flow velocity in the sieve tubes [rnh" 1 ] * Measurements of water loss and transport in experiment 1 are means over the period 40-60 DAS. * Measurements of water loss in experiment 2 were made over the period 36-67 DAS. ' Single-rooted maize plants with etiolated mesocotyl. ' n.d. = Not determined. ' Mean flow velocity estimated for the mean water loss (i) = 8.2, the range was 193-374 m h " ' depending on the size of the vessels. This mean flow velocity increased with the size of the transpiring leaf area and reached, e.g. 510 m h" 1 or 14 cms" 1 at 63 DAS. 1 Mean flow velocity was estimated on the basis of the measured sieve tube area, the carbon use in the root and a sucrose concentration of 20%. Maize plant growth 1235 0.6- O) 0 4 - o < ; 0.2-1 V 0 CD 8 increasing leaf age Leaf 9 10 1 1 5x root number Fig. 2. Leaf conductance g as measured in leaves of control (—•—) and single-rooted ( V ) plants of Zea mays L. as a function of leaf number (and age). 4+5 leaf number 7 8 9 10 11 + 1 2tassel Fig. 4. Concentration of abscisic acid (ABA) in tissues of the apical leaf half, roots and tassel of control (—•—) and single-rooted ( V ) plants of Zea mays L. -0.25- T o E0.15< CD < 0.1- 220.05^ V 3 o D 0—Q5x root 4+5 leal number 7 8 9 10 11 + 12tassel Fig. 5. Concentration of abscisic acid conjugates (ABA-GE) in tissues of the apical leaf half, roots and tassel of control (—•—) and singlerooted ( V ) plants of Zea mays L. Fig. 3. Element concentrations in ^mol g ' FW in root and leaf tissues of control (ED) and single-rooted plants (Q) of Zea mays L. Note that major elements are given at a 20- or 10-fold reduced scale and minor elements at a 5- or 10-fold enlarged scale and that the y-axis scale for the root is 2-fold enlarged. to flow (see below) the concentrations of some solutes, notably K + , NOf, Mg2 + , SO*" and amino acids, in the exudate from single-rooted plants were substantially higher than in that from control plants (Fig. 6). Owing to the slightly increased volume flow (Table 3), solute flows of nutrients were even more increased than were their concentrations. Levels of other ions such as Ca2 + , H2POJ and malate were the same in exudates from the two types of plants (Fig. 6). Consistent with results of Fig. 3, however, none of the nutrient ions showed lower levels in xylem sap of single-rooted than control plants. In addition to an overall higher concentration, the amino acid composition in exudates from single-rooted plants was also altered compared to that of the controls (Table 4). The concentrations of basic amino acids and in particular of amides were increased while those of acidic amino acids were lower. Concentrations of ABA in xylem exudates from the mesocotyl of single-rooted plants were on average 7 times lower than in those from the control stem base. Concentrations of conjugated ABA, however, were 2.5-fold higher in single-rooted than control plants (Table 5). The changes in xylem sap ABA and ABA-GE concentrations clearly were not due to the slightly higher flow rates (see Else et al., 1994) in single-rooted plants, since (a) ABA and ABA-GE levels changed in opposing 1236 Jeschke et al. 40-, control single-rooted 8 n 20 10 O O> <3 CO O Fig. 6. Concentrations of ions and of total amino acids in the root pressure xylem exudate collected from control (ED) and single-rooted plants (Of) of Zea mays L. Solute flow rates may be obtained from concentrations and the volume flow rates given in Table 3. Table 4. N-compounds in xylem sap of control and single-rooted plants of Zea. mays L.; data are means ±SEM Acidic amino acids Basic amino acids Neutral amino acids NH 3 Amides NO3 Control plants (mmol m" 3 ) Single-rooted plants (mmol m~3) 0.18 ±0.02 0.76 ±0.23 3.13±1.45 0.67 ±0.13 4.68± 1.15 8.11 ±3.79 0.15 ±0.02 1.02±0.11 4.10±0.22 1.52±0.13 16.47 ±1.29 34.09 ±2.77 direction and since (b) root pressure exudates and not pressure-induced sap was used here. Similar differences were observed when solute flows of ABA and ABA-GE relative to the root weight were calculated using the volume flows in Table 3. Discussion Roof growth and water transport Restriction of root development to that of the seminal roots eventually led to a reduction in shoot growth but it also favoured growth of these roots. Their dry matter (3.4 or 4 g at 68 or 7 DAS, respectively) was clearly larger than in normally grown maize plants in which the sum of the primary and adventitious seminal roots had a dry weight of 2.3 g (64 DAS) and 2.8 g (79 DAS) (Jesko, personal communication), indicating a compensatory though limited additional growth when nodal roots failed to develop. This corresponds with pronounced compensatory growth particularly in the seminal roots of Zea mays in response to soil water deficits (Jesko et al, 1996). The root treatment used in the present experiments, i.e. limiting the root system to the seminal root(s) in a monocotyledon is principally distinguished (A) from the restriction of the rooting volume (Carmi et al, 1983; Robbins and Pharr, 1988; Cresswell and Causton, 1988; Peterson et al., 1991a) and also (B) from root pruning in a dicotyledon (Carmi and Koller, 1978; Milligan and Dale, 1988). In contrast to (A), the primary and adventitious seminal roots were allowed to grow in a fairly unrestricted volume (the only restriction being rooting depth) and had free access to water, minerals and oxygen (for effects of root constriction see Peterson et al, 19916) while its development was limited by internal factors, such as the lack of secondary thickening and possibly an insufficient assimilate supply. In contrast to (B), the primary root was fully intact. As opposed to both (A) and (B) where vascular bundles between root and shoot can develop freely and do not excessively hinder xylem transport, in the single-rooted maize plants, similar to wheat (Passioura, 1972), all of the water transport is largely confined to the few major vessels in the stele of the mesocotyl (Plate 2), so that the hydraulic resistance is greatly increased in comparison to the controls having numerous and larger vessels in their nodal roots and the adjacent stem interaodes. This results in enormous mean flow velocities of 270 mh" 1 (7.5 cms" 1 ) which by far exceed those in the controls (11 mh" 1 ), those reported for herbaceous plants (10-60mh" 1 ) or even in lianas (150mb" 1 , Strasburger, 1991). The pressure gradients required for these flow rates were estimated on the basis of vessel diameters (Nobel, 1991) and were 5 kPa cm" 1 for the flow through the mesocotyl of single-rooted plants compared to O.HkPacm" 1 for the controls or 0.09 kPa cm" 1 for trees (Nobel, 1991). Assuming a length of 30 cm from the top of the mesocotyl through the primary root, a pressure of about 150 kPa or 1.5 bar is required for water transport in addition to that needed for other hydraulic resistances. Moreover, since the number of vessels in the primary root and the mesocotyl was constant whilst the size of the supplied shoot and its water loss increased, flow rates (Table 3) and the required pressure gradients were substantially elevated with time as seen in Fig. IB. Phloem transport A corresponding constriction as for xylem flow exists for the descending phloem transport with a restricted total phloem area in the mesocotyl of about 4.7 x 10"5cm2. Table 5. Concentration of free and conjugated ABA in the root pressure xylem sap collected from control and single-rooted 7JZ2L mays L. plants Plants ABA [nM] Conjugated ABA [nM] Controls Single-rooted plants 30.9 ±6.1 4.1 ±0.4 0.40 ±0.08 1.0±0.2 Maize plant growth Assuming a relative growth rate of the single root of 0.022 d ~' and using the measured C concentration of 42% in root dry matter, the daily carbon increment of the root was 31.9 mg C. When assuming further that root respiration amounted to 70% of the C increment (Jeschke et ai, 1996), then the root required 54.2 mg C or 128.7 mg sucrose or 0.6 ml of a phloem sap containing 20% sucrose per day. Together with the sieve tube area this required a flow velocity in the sieve tubes of 5.2 mh" 1 . These data correspond to a sieve tube area of 1.35 x 10~5cm2 and transport velocities o f 6 m h " ' reported for single-rooted wheat (Passioura and Ashford, 1974) and are much higher than usual (0.5-1 mh" 1 , Strasburger, 1991). Whereas in wheat the single root reached 60% of the control dry weight and root growth was therefore not likely to be limited by assimilate transport (Passioura and Ashford, 1974), the stronger inhibition down to 16% in the present experiments with maize suggests that resistance to assimilate transport in the mesocotyl restricted root development and led to the high shoot to root ratio of 11.9 or 15.7 as found in the two experiments with maize. Indirect indications for a build-up of assimilates above the mesocotyl as a bottle neck were seen from the appearance of highly thickened but non-growing initials for nodal roots (Fig. 1). A build-up of assimilates in the shoot also occurred in plants with restricted rooting volume Robbins and Pharr, 1988). Mineral nutrition Given the small primary root and the high shoot-to-root ratio, the capacity of the root to take up nutrient ions might have limited shoot growth. In this case, the concentration in shoot tissues of limiting nutrients would be expected to lower than of those in the controls. However, all nutrient concentrations in leaves of the single-rooted maize were equal to or exceeded those in the controls, indicating that ion uptake by the branched seminal root was adequate. Similar conclusions were reached for bean plants grown with a restricted root volume (Carmi and Heuer, 1981) and for similarly treated peach (Richards and Rowe, 1977), although in this latter case root restriction led to somewhat decreased N, P and Ca levels in leaves. Adequate nutrient ion uptake follows also from the composition of the root pressure xylem exudates. The composition of the control xylem sap was well comparable with published data (Engels and Marschner, 1993), but it was altered distinctly in single-rooted plants. In essence, the concentrations of three solutes, K + , NO3~, and amino acids were substantially elevated, while Mg 2+ , Cl" and SO2," were somewhat increased, which altogether led to a doubling of the osmotic concentration of the exudate from 50 (control) to 98 mM (single-rooted). This increase was obviously needed to enable xylem exudation despite 1237 the higher hydraulic resistance due to much lower root surface area and to the restricted number of vessels. On the basis of the flow rates of 1.6 ml h" 1 in single-rooted and 3.2 ml h" 1 in control shoot stumps, an additional pressure of 27 kPa was needed to drive the exudate through the mesocotyl compared to only 1.8 kPa in the controls. The measured water potential differences between external medium and exudate were —190 and — 78 kPa, respectively, for single-rooted and control plants and hence well adequate to overcome the radial and longitudinal resistance within the root up to the base of the shoot. Characteristically the concentrations of NO3~ and K + were elevated (Fig. 6), both of which are the basic ions for xylem transport (Triplett et ai, 1980; Rufty et ai, 1981). When NO3~~ was provided as the major anion, xylem sap volume flow was twice as high as with SO2." (Triplett et ai, 1980) and also under saline conditions K + and NO3" contributed decisively to the generation of a positive root pressure (Jeschke et ai, 1995). Besides these two ions, amino acids showed substantially higher xylem sap concentrations as similarly found for Leptochloa fusca under saline conditions (Jeschke et ai, 1995), indicating that synthesis of amino acids in the root and their release into the xylem vessels contribute to generating root pressure. Based on the ratio of reduced to total N in the xylem sap (Pate, 1973), the single root contributed somewhat less (62%) to nitrate reduction in the plant than the control roots (82%). Although these ratios were somewhat higher than found for maize by Rufty et ai (1981), the apparently depressed nitrate reduction in the single compared to control roots may be attributed to lower assimilate import through the mesocotyl. Whereas single-rooted wheat produced higher grain yield than controls with a fully developed root system (Passioura, 1972) single-rooted maize showed decreased shoot growth, smaller leaves and decreased photosynthetic rate. The decisive difference was that control and single-rooted wheat plants grew on a limited water supply and the single-rooted ones conserved water during vegetative growth and had larger resources during the later stages (Passioura, 1972). In the present case with continuous water supply, however, the control plants had no disadvantage in the later stages of growth, whereas in the single-rooted maize plants the much smaller root led to a reduction in photosynthesis, presumably due to a sinkinduced limitation (Herold, 1980; Plaut et ai, 1987) and in later stages to a progressively severe reduction in the water supply to the shoot (Fig. 1). ABA and a possible shoot-to-root signal Roots that are exposed to drying soil seem to be able to measure the soil water content and as a response to 1238 Jeschke et al. synthesize and release a chemical stress signal to the xylem and hence to the shoot. Here this signal regulates leaf conductance and leaf growth. It has been shown that ABA is that stress signal in maize plants (Davies and Zhang, 1991; Zhang and Davies, 1991; Hartung et al., 1994). In the present experiments, the shoots of the singlerooted maize plants were experiencing water shortage although their roots were exposed to conditions of optimal water and nutrient supply and of sufficient rooting volume, conditions which are likely to result in low ABA biosynthesis rates within the root. Indeed ABA was not increased, but rather significantly reduced in the xylem sap from seminal roots of single-rooted plants. Lebreton et al. (1995) using QTL (quantitative trait loci) analysis have shown that in Zea a significant portion of xylem sap ABA derives from nodal rather than the seminal roots. This agrees with the findings. Under these present conditions the leaves experienced water deficits and were hence the site to trigger and perform synthesis of additional ABA as a regulator of stomatal movement and this led to clearly increased ABA levels in young and particularly in adult leaves and concomitantly reduced leaf conductances. Surprisingly in this context, however, ABA in root tissues of single-rooted plants was substantially increased compared to the controls. Since, as stated above, an increase of ABA biosynthesis in the well-watered seminal roots seems very unlikely, their elevated ABA levels must have resulted from increased ABA phloem import from the shoot. In contrast to other plants where shoot-derived ABA was in part recirculated back to the shoot (Peuke et al. 1994), in the single-rooted maize plants ABA apparently was retained in root tissues and not released to the xylem vessels. Thesefindingsagree with conclusions of Slovik et al. (1995) drawn from computer simulations. They predicted that a large proportion of ABA synthesized in leaves is loaded to the phloem and transported towards the root as a hormonal shoot to root signal. At present it is only possible to speculate about a stress physiological role of this signal. Two options can be envisaged: (1) ABA could be responsible for the observed enhancement of growth and branching of the seminal roots. It has been shown earlier that ABA can enhance the formation of root hairs and lateral roots (Biddington and Dearman, 1982; Hartung and Heilmeier, 1992) and hence increase the uptake area of a root system. (2) ABA might be instrumental in stimulating water and solute flow through the root into the xylem as is the case of the single-rooted plants (Table 3). There are many reports in the literature of ABA-stimulated water and volume flow through root systems of a wide range of species (Karmoker and van Steveninck, 1978; Ludewig et al., 1988; Bassirirad and Radin, 1992; Zhang et al. 1995, and literature cited therein). Conjugated ABA, which consists mainly of ABA glu- cose ester (ABA-GE), was relatively high in leaves of control plants (the ABA/ABA-GE ratio was (2). Its concentration was slightly elevated in single-rooted plants, although a large difference between treatments was observed in the root (approximately 8-fold increase in single-rooted plants). In contrast to free ABA, the concentration of conjugated ABA was doubled in the xylem of single-rooted plants. It seems that a small portion of extra ABA which had been deposited in roots after phloem import was released to the xylem in the form of ABA-GE and transported to the leaves. After hydrolysis this compound to a certain degree may contribute to the elevated ABA levels in the leaves. The physiological significance of this sequence of events is unclear, although a function of ABA-GE as a hormonal stress signal has been postulated recently by Bano et al. (1993). Acknowledgements This work was supported by SFB 251 of the Deutsche Forschungsgemeinschaft. M Holobrada especially would like to thank the Deutsche Forschungsgemeinschaft for a travel grant and the Julius-von-Sachs Institute fur Biowissenschaften der Universitat Wurzburg, Lehrstuhl Botanik I, Germany for enabling the experimental work during the visit. Thanks are extended to Mrs Andrea Hilpert and Barbara Dierich for skilled technical assistance, to Mrs Marion Reinhardt, Elfriede Reisberg and Eva Wirth for analyses and to Mr Robin Wacker, Wurzburg, for help with microtomy and staining of mesoctyl sections. References Bano A, Dorffling K, Bettin D, Hahn H. 1993. 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