Mineral Nutrition and Growth Responses of Pisum Roots

Short Communication Mineral Nutrition and Growth Responses of Pisum Roots H. G.

BURST ROM

Department of Plant Physiology, University of Lund, Box 7007, 5-22007, Lund, Sweden Received December 4, 1980 . Accepted February 14, 1981

Summary The actions of K+ and Ca 2 + in roOt growth have been studied on Pisum seedlings in 24 hours experiments. Growth is specified as cell multiplication longitudinally and cell elongation. Excision of roots from shoots reduces growth to 1/4 owing to a lack of organic nutrients. A full mineral nutrient solution increases growth X 2. Ca is of special importance for cell elongation, it promotes growth under a reduction of cell wall tensility. A nutrient solution without Ca decreased growth below that in distilled water. Splitting of root tips leads to curvatures with epidermis convex under deformation of tissues and reduction of growth to < Ifs. Ca inverses the radial polarity of these reactions. There is little correlation between curvatures and growth.

Key words: Pisum sativum, root growth, cell elongation, tissue curvatures, mineral nutrition.

Introduction The current literature on growth mainly deals with actions of hormones, and it is no exaggeration to say that general and elementary priciples of growth have been somewhat negleoted. The pattern of organic syntheses in shoot growth have been described by Burstrom (1974), and the hormone balance in root growth was extensively investigated by Pilet (1978). Some basic information is still lacking, such as the specific requirements of mineral nutrient elements in relation to different phases of growth and tissue differentiation. It has generally been neglected that growth in length of any plant axes depends upon two histologically and chemically different elements: Mitoses with cytokineses leading to synthesis of cytoplasm, and cell elongation in a restricted sense responsible for the bulk increase in volume, due mainly to the growth of the cell wall. This distinction ought to be made innstudies of the chemical and physical control of growth, and has been made in the present attempt to demonstrate some growth functions of K and Ca.

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Material and Methods The plant material was Pisum sativum cv. Stivo, Svalov, and the standard growth procedure as follows; Day 1: Seeds soaked in quartzdistilled water; day 2: seeds spread on filter paper in Petri dishes; day 4: plants transferred to test solutions with oxygen aeration; day 5: experiment finished. In each test 20 plants were kept in 600 ml solution containing as a standard in mmol . I-I K 0.6, Ca 0.3, Mg 0.15, N0 3 0.9, P0 4 0.5, and 50 4 0.2, and in [tmol' tl Fe 0.3 and Mn 0.15. The pH was about 6.5. Entire plants were used unless otherwise stated. The experiments were carried out in darkness; the experimental time was 24 hours. The determinations of cell lengths and number of cells longitudinally in the epidermis of the roots were made as in Burstrom (1949). It was striking that when the actions of two salts with the same cation were directly compared, the agreement in cell lengths, or cell number, or both were much better than could be expected from the mean errors, as exemplified in Table 1. This means that there is a regular variation in the epidermis structure such as described earlier in other plant material. Not even in a complete nutrient solution do the roots grow at a constant velocity. The Pisum roots started with a velocity of 0.6 mm . h- I , increasing to 1.3 after 16 h, and then stabilized at 1.5 after 30 hours. An attempt to use Zea was less successful; the starting value was 0.9 mm . h- 1 , it passed a maximum of 3.2 and declined to 2.8 at 26 hours in spite of an ample supply of nutrients. An increasing deficiency of the neglected micronutrient elements can hardly have such an effect. I have not found this technical difficulty mentioned in the literature. Growth is always expressed in mm per 24 hours which was the standard experimental tIme. The osmoticum used for changing the water potential was polyethyleneglycol (PEG) 400, MODO PEG Berol Chemie. It is not an ideal osmoticum since it is slowly taken up by the roots (Lawlor, 1970). Terry et al. (1971) found PEG 6000 to cause considerable decreases in cell lengths in Beta, probably more than caused osmotically. It has successfully been used in short-term tests by Erlandsson (1975). Apical, longitudinal incisions were made to a depth of 6 mm from the root apex; this was done under a 15 cm magnifying lens. The angle of the tangent of the roOt half to the root base was taken as a measure of the curvature, called positive (+) with epidermis convex, negative (-) with epidermis concave.

Results and Discussion It is necessary at first to consider a few technical problems. One is whether the nutrient solutions selected are satisfactory for a uniform nutrient supply during the experimental period. They are somewhat less diluted than usually, but the accessible volume per plant is low. A comparison has been made in Table 1 between the growth responses of a full nutrient medium and the same diluted to 1/;;. The growth responses are the same, meaning that the consumption of nutrients is negligible. Addition of the osmoticum (PEG) in the concentration used has some negative effect, but only on cell multiplication. It is often found in the literature that growth experiments have been made with excised roots, for example pieces 10 mm in length, also that growth is studied with roots in distilled water, even when this was not required for the actual problem studied (e.g. Baehler et ai., 1979). Growth responses under such conditions are shown in Table 2, compared with roots attached to the seeds, both in distilled water and in a nutrient solution. For reasons given later a full nutrient solution lacking only Ca was employed as a third medium.

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Table I: Growth of roots during 24 h with two concentrations of nutrient solution, with and without PEG 400 (- 5 bar). Four replicates each with 20 plants of each treatment. Medium

Growth mm (± 1.4)

Epidermis cell lengths !-1m

Numberof cells longitudinally

Full solution Full solution + PEG 'Is solution 'Is solution + PEG

44 41 48 40

248 ± 5.5 248 ± 6.2 251 ±5.8 251 ± 8.1

177± 7.2 165 ± 7.9 191 ± 9.3 159± 7.1

Table 2: Growth of intact and excised Pisum roots in different media. Four replicates, each with 20 plants of each treatment. Growth mm/24 hours. Material

Water

Nutrient solution without Ca

Full nutrient solution

Intact seedlings Excised roots

22.0 ± 1.2 5.8 ± 0.3

16.8 ± 1.0 4.2±0.4

33.6± 1.2 6.1 ± 0.3

The results are quite clear. Excised roots, used in a large number of studies recorded in the literature, permit only a fraction of the growth maJe by attached roots. The explanation is probably simple. Growth is identical with a reproduction of anatomical structures (Burstrom, 1974, 1979), new cells are formed and grow in size, tissues grow by the syntheses of cell constituents: Carbohydrates, proteins, lipids et eet., and excised root tOps do not contain stOred nutrients. This is an elementary anatOmical experience. Excised roots are for these reasons not suitable for growth studies unless they are artificially supplied with organic nutrients, which meets with obvious technical obstacles: The velocity of growth woulJ not approach that common in intact plants or seedlings, which is well known from tissue culture studies, and aseptic conditions would be required.

In an attempt to distinguish the growth effects of some cations and anions, Ca 2 + and K+ were combined with CI- and N0 3 - (Table 3). Ca supplied alone has the same growth effect as a full nutrient solution neither K nor N0 3 had any Table 3: Pisum root growth for 24 hours in different media. Six replicates, each with 22 plants of each treatment.

2 Medium

Growth mm

3 Epidermis cell length !-1m

4 Cell number longitudinally

Water Nutrient solution CaC1 2 1O- 1 mol Ca(NO J )2 IO- J mol KCIIO- 1 mol KNO J 10-.1 mol

17.9 ± 3.1 37.1 ± 5.2 41.0±1.9 39.5 ± 4.3 18.3 ± 4.0 15.2 ± 2.2

172±3.2 301 ± 5.2 281 ± 6.4 270 ± 6.1 148±3.6 126±3.0

104 ± 123 ± 146 ± 146 ± 124 ± 121 ±

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influence on the growth. The response to Ca is surpnsmg, because the roots are indirectly attached to the cotyledons, which obviously provide the seedlings with both organic and inorganic matter, to judge from the rapid root growth. One hypothetical possibility might be that Ca is required in comparatively large amnounts in the external medium for providing the root surface with an appropriate charge or hydration, required in this very unbalanced solution. This is only a suggestion without direct support. Ca mainly increases the cell elongation, which could be used as a starting point for a further study of its action. Cell elongation and cell divisions as two parameters of growth are here as in earlier articles based only on the growth pattern of the epidermis. The motivation is not only that epidermis is easily accessible for a microscopic study, but it is one of the few cell layers with a regular growth pattern without intercellular spaces or cell deformations; perhaps the only other layer fulfilling the same requirements is endodermis, but this is not easily accessible for a an study of the living root. An old method of studying growth hormone responses was to make a longitudinal incision in the top of a stem, and to study the curvatures performed by the two halves (Went and Thimann, 1937). They curved spontaneously with the epidermis concave (-curvature) and added auxin caused the growing tip to curve back in the + direction under growth. Actually, the spontaneous reactions might be of more interest than those induced by hormones, because they could disclose something of the internal tensions during normal apical growth. Such operations were made on Pisum root tips with the results in Table 4. The treatments were the same as in Table 3. The actions of the mineral salts were obtained by subtracting the water value of + 99, from the curvatures in column 2, shown in column 3. In the corresponding way the actions of PEG, column 5, were obtained by subtracting column 2 from column 4, = column 5. Columns 3 and 5 show the essential results. Ca causes a strong negative curvature and alone among the treatments a decrease in the PEG effect, which means a reduced tensility of the tissues. This could be Table 4: Curvatures of Pisum roots with 6 mm apical incisions in different media. Time 24 hours. Six replicates, number of plants of each treatment 125-150. Curvatures in degrees, + with epidermis convex, - with epidermis concave; with and without PEG 400 (- 5 bar). Medium

2 Curvatures degrees

3 Actions of salts degrees

Water Nutrient solution CaC!] 10-) mol Ca(NOJh 10-) mol KCI )0-) mol KNO, )0-) mol

+ 99 ± 5.5 +118±7.3 - 26±6.0 - 60 ± 6.3 + 35 ± 6.0 + 38 ± 6.5

0 + 19 -125 -159 - 64 - 61

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4 5 Curvatures Actions of with PEG degrees PEG -5 bar -

40 ± 6.5

- 51 ± 8.5 - 132 ± 8.7 -J32±7.0 -141±8.8 -146±8.5

- J39± 8.5 -169±10.1 - J06 ± 11. 1 - 72± S.2 - 176 ± 10.7 - 184 ± 10.7

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expected of a Ca-induced reaction. Column 3, however, does not agree with Table 3. Curvatures and growth are far from accordant. The full nutrient solution in Table 3 caused maximal growth but no effect on the curvature. The two K-salts equal distilled water as far as growth is concerned, but they cause significant curvatures in Table 4. There must be some non-identified component of the mineral nutrient solution with a negligible growth effect but causing strong positive curva,tures, which means a unilateral reaction in the tissues. The osmotic actions of PEG are seemingly unusually large for the three underlined top values in Table 5, but practically none in all other cases. Such conditions may be possible if there are strong mechanical resistances to free expansion, and strains caused osmotically or by growth. The most interesting action is, however, that Ca has caused a complete reversal of the radial polarity in the roots, for which no explanations can be offered. Table 5: The nature of convex (+) and concave (-) curvatures of split Pisum roots without and with PEG 400 - 5 bar. Cell lengths in !!m, - Maximal length values without PEG in Italics. Salts in 10- 1 mol. Cell lengths of non-split roOt epidermis average 142 ± 2,9!!m; + epidermis convex, - epidermis concave, Six replicates, each with 20 plants of each treatment. Medium

Water Water + PEG Ca(NO,)l Ca(NO))l + PEG KCl KCl+PEG

Curvature

(+) (-) (-) (-) (+) (-)

Cell lengths in the curvature !!m Epidermis

Cut stele

81± L7 28 ± 3.0 32± L7 33 ± 1.3 86 ± 1.3 28±0]

23±L8 24± L9 60±0,8 49±0] 46 ± 2.5 48 ± 2.9

General conclusions Ca has a unique position among the essential mineral nutrient elements. It has been connected with several obviously little related growth actions. It is usually supplied in large amounts, and it is easy to show that these are required, but it may resemble a micronutrient element with regard to its physiological action (Burstrom, 1968). This is underlined by determinations of the contents of Ca in relation to those of other essential elements (Burstrom, 1973). In rapidly growing materials such as coleoptiles, seedlings, and stem internodes the Ca content could amount to between < 1 to 10 Ofo of the required content of K. Cohen and Nadler (1976) have emphasized the generally unspecific action of divalent cations without known biochemical functions. This does not explain the observed Ca-induced change in radial polarity, but it may indicate that this should be sought rather in physical than in biochemical conditions in the cells. This is a field which generally

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seems to be little explored, despite the fact that radial gradients in cell multiplication and cell elongation are the bases for the tissue differentiation (Burst rom, 1979), most strikingly in stems, but obvious also in roots. That the gradients go in the opposite directions in roots and stems and that these can be changed nutritionally does not detract from the interest these patterns deserve.

References BAEHLER, W. and P. E. PILET: Z. Pflanzenphysiol. 93, 265-271 (1979). BURSTROM, H. G.: Physiol. Plant. 2, 197-209 (1949). - BioI. Rev. 43, 287-316 (1968). - Z. Pflanzenphysiol. 74, 1-13 (1974). - Plant and Cell Physiol. 14,941-951 (1973). - Amer. J. Bot. 66, 98-104 (1979). COHAN, J. D. and K. D. NADER: Plant and Cell Physiol. 57, 347-350 (1976). ERLANDSSON, G.: Physiol. Plant. 35, 256-262 (1975). LAWLOR, D. W.: New Phytol. 69, 501-543 (1970). PILET, P. E.: Z. Pflanzenphysiol. 89, 411-426 (1978). TERRY, N., MALDRON, L. J., and ULRICH, A.: Planta 97, 281-289 (1971). WENT, F. W. and THIMANN, K. V.: Phytohormones, New York, 1973.

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