Protoplasma 86, 135--140 (1975) 9 by Springer-Verlag 1975
Centre in Excised
Roots of Pisum sativum L.
RONALD D. MAcLEoD 1 and DENIS W. F. SCADENG 2 Department of Plant Biology, University of Newcastle upon Tyne, Ridley Building, Newcastle upon Tyne, U.K. Received February 11, 1975
Summary Excised roots of Pisum sativurn were cultured in White's medium supplemented with various concentrations of sucrose or IAA and exposed to aH-TdR for 24 hours, three days after excision. Varying the concentration of sucrose or IAA in the culture medium had no effect on the size of the quiescent centre in the apical meristem of these roots. It is concluded that the existence of a quiescent centre in angiosperm roots is not due to carbohydrate starvation or to the super-abundance, or lack of, IAA.
1. Introduction The mosz prominent feature of the apical meristem of angiosperm roots is the quiescent centre (CLowEs 1956) in which deoxyribonucleic acid (DNA) synthesis seldom occurs (CLowEs 1959). Although a considerable amoun,t is known about the mechanisms involved in the control of quiescence and cell proliferation in whole meristems of cul~tured roots (VAN'T HOF and KovAcs 1972, VAN'T HOF 1973), little in.format'ion is available concerning in.tact ones. It is known, however, that treatments which suppress cell proliferation in meristematic cells somehow induce D N A synthesis and mitosis in the cells of the quiescent centre. Such treatments include exposure to radiation (CLOWES 1970 a, 1972 a), low temperature (CLOwES and STEWART 1967) or colchicine (DAvlDSON 1961), subjecting excised roots to short periods of carbohydrate starvation (WEBSTER and LANGENAUER 1973), or removing the root cap. (CLowEs 1972 b). The virtual absence of D N A synthesis and mitosis in the quiescent centre could be a result .of .one o.r more factors. Quiescence can be readily induced 1 Department of Plant Biology, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, U.K. 2 Department of Science, College of Arts and Technology, Newcastle upon Tyne, NE4 7SE, U.K.
R . D . MAcL~oD and D. W. F. SCADENG
in the apical meristem of excised roots of Pisum sativum, for example, by culturing them in a medium which lacks carbohydrate (VAN'T Hoi~ 1966). Similarly, dividing cells in intact roots of Vicia faba may cease to proliferate following treatment with indol-3yl-acetic acid (IAA) (MacLEoD and DaWDSON 1966). It follows, therefore, that the cells of the quiescent centre may be non-meristematic as a result of carbohydrate starvation, or a deficiency in some other essential metabolite (CLowns 1970 b), or because of a local supraoptimal concentration of one or more hormones (ToRRtY 1963, MAcLeoI) and DaVIDSON 1966, PHILLIVSand TOR~,EY 1971). In the experiments described in the present paper, we have examined the effects of various concentrations of sucrose and IAA on the size of the quiescent centre in excised roots of P. sativum. 2. Materials and M e t h o d s Peas (Pisum sativurn L. cv. Alaska) were surface sterilized in 6~ sodium hypochlorite for five minutes, following which they were germinated and grown on in sterile vermiculite for three days. 1 cm long root apices were then excised under aseptic conditions and transferred into flasks containing 50ml of sterile White's (1943) medium. This medium was supplemented with sucrose to give a final concentration of 0.5, 1, 1.5, 2, 4, 6, 12, 15, 18, 21, 24, and 270/0, or 2~ sucrose and 104 , 10- 5 , 10_8 , 10- s , 10_20 , or 10--~2M IAA. A few flasks contained no sucrose or IAA. Each batch of excised roots was grown for three days at 20 ~ [see VAN'T HoF (1966) for details]. 0.05 ml of tritiated thymidine (aH-TdR) was then injected into each flask with a syringe to give a final concentration of 1 ~tCi/ml (specific activity 5 Ci/m mol). The roots were exposed to '~H-TdR for 24 hours before being fixed in 3 : 1 v/v absolute ethanol: glacial acetic acid. Roots from intact peas, which had also been treated with aH-TdR for 24 hours, were fixed at the same time. [The concentration of aH-TdR used in these experiments has been widely used by VAN'T HoF (e.g., 1966, 1968 a) in his studies of cell proliferation in excised pea roots.] Feulgen stained apices, 5 mm in length, were then dehydrated in ethanol and infiltrated with hard paraffin wax (melting point 56 ~ and 6 gm thick longitudinal sections cut. The wax was removed with xylol, 1 : 1 v/v xylol : absolute ethanol and absolute ethanol, before the slides were dipped in celloidin, dried, and rehydrated by passing them down the alcohols to water. Each slide was then dipped in Ilford K 2 liquid emulsion, dried, exposed in the dark for 14 days at 4 ~ and finally prepared as permanent autoradiographs [see MAcLEoD (1972) for details of the latter procedure]. Measurements were made of the size of the quiescent centre from the most median iongitudinal section of each root apex, either from camera lucida drawings or by using an eyepiece graticule calibrated against a stage micrometer. Statistical tests were carried out using Student's t, the 5o/0 level being regarded as significant.
3. R e s u l t s
The quiescent centre varied in shape, even in the intact roots, but it appeared most often as a crescen:t-shaped object with the horns of the crescent extending up into the root cortex. The distal edge of the quiescent centre, relative to the position of the cap initials, was no,t fixed, even when medi~an longitudinal sections from the same batch of peas were compared. This was a result of the
The Quiescent Centre in Excised Roots of Pisum sativurn L.
presence of labelled nuclei extending into the quiescent centre from the inner cortex. These labelled nuclei sometimes appeared to split the quiescent centre into two distinct parers. Width of the quiescent centre could be determined relatively easily in most of the roots (Table 1). However, in those roo,ts which had been cultured in media supplemented with ~ 1~ sucrose, > 12~ sucrose or 20/0 sucrose and 10-5 M or 10-4 M IAA, too few nuclei incorporated aH-TdR into D N A Table 1. Width of Quiescent Centre (Q.C.)-+ Standard Error of the Mean in Apical
Meristems of Excised Roots of Pisum sativum Cultured in Sucrose, or 2~ Sucrose and IAA, Supplemented Media. The Various Concentrations of Sucrose or IAA Present in Each Medium are Given in the Table. These Roots Were Exposed to aH-TdR for 24 Hours. Each Determination is Based on Six Roots, the Most Median Longitudinal Section of Each Being Used to Obtain the Required Measurement % Sucrose in medium
Width of Q.C. (b~m)
[IAA] in medium
Width of Q.C. (btm)
1.0 1.5 2.0 4.0 6.O 12.0
156 162 160 158 170 166
lO-lgM 10-1OM 10-8M 10-OM
120 155 138 140
• • • • • •
4.3 11.7 6.3 6.3 7.1 7.5
• • ~ •
17.3 6.2 15.3 5.2
for the boundaries of the quiescent centre to be clearly defined. The width of the quiescent centre was not significantly different in excised roots cultured in media containing from 1 to 12~ sucrose (Table 1, P = > 0.10). Similarly, excised roots grown in media containing 2~ sucrose and between 10-0 M and 10-1~ IAA had quiescent centres with statistically similar widths (Table 1, P - > 0.10). However, the width of the quiescent centre in the roots grown in a medium containing 10-1eM IAA was marginally less than that of those cultured in 10-1~ M IAA (Table 1, P = 0.05). Mean width of the quiescent centre in the intact pea roots (157 ,am + 14.9) was no,t significantly differer~t from the mean values obtained for all of the excised roots cultured in the various sucrose (162 um +_ 2.1) or IAA (138 gm _+ 7.2) containing media (P = > 0.10) or from the results obtained separately for each group of excised roots (Table 1, P = > 0.10). Variabil,ity in the height and shape of the quiescent centre made it extremely difficult to calculate volume with any degree of accuracy in all of the roots and, consequently, this was determined in only a few cases. Volume of the quiescent centre did not seem to be affected to any marked extent by the concentration of sucrose or IAA in the culture medium and there was little difference between the values obtained for the intact or excised roots. How-
R . D . MAcL:eoD and D. W. F. SCADENG
ever, this measurement could not be determined with sufficient accuracy in enough roots to carry out a statistical comparison. Mean volume of the quiescent centre was about 7 5< 10"~tm a, although values as low as 3.5 5< 105 gm a and as high as 12.5 5< 105 ~tm~ were occasionally found. 4. D i s c u s s i o n
Quiescence can readily be induced in all of the cells of the apical meristem of excised roots by growing them in the absence of carbohydrate (VAN'T HoF 1966). However, the absence of any effect of sucrose concentration on the size of the quiescent centre in excised pea roots (Table 1) suggests that these cells are not quiescen,t because of the absence of a carbohydrate source of energy. Exogenously supplied sucrose, like thymidine (CLowEs 1970 b), seems to be available to the cells of the quiescent centre, but is not used by them (THO~aAS 1967). IAA is present in root apical meristems, where it may be synthesized (_kt3ERG 1957, DAVIDSON 1960, TORR~Y 1963), and atthough it is necessary for D N A synthesis, mitosis, and cytokinesis (KLEIN and VOGEL 1956, PATAU, DAS, and SKOOG 1957), high concentrations reduce mitotic activity and extend the duration .of the mitotic cycle (DAvlDSON and MAcLEoD 1966, MAcL~oD and DAVIDSON 1966, W~BST~R and DAVlDSON 1969). Moreover, treatment with IAA can result in an increase in the proportion of quiescent cells in root meristems. VAN'T HOF (1968 b), for example, has demonstrated that treatment with 10-v M IAA markedly reduces the number of cells which enter the period of D N A synthes.is (S) from G 1 (we-synthetic interphase), following ,the provisi~on of sucrose to carbohydrate starved ro.ots, even after 48 hours con.tinuous exp.osure to aH-TdR. However, with the possible exception of 10-12 M IAA the quiescent centre did not increase or decrease in size, relative to that of the controls (Table 1), when IAA was present in the cukure medium. Thus, the quiescent centre does not appear to con~tain too much or too little IAA to maintain normal cell proliferation. It is possible that the cells of the quiescent cen'tre are starved of some metabolite other than sucrose (CLowEs 1970 b) or that the level of a growth factor other than IAA is supra-optimal for normal meristematic activity. Cy.tokinins, for example, are present in pea roots, the greatest concentration being found in the apical mm (SHORT and TORR~Y 1972). The results reported in the present paper, however, suggest that the initiation and subsequen,t maintenance ,of the quiescent centre is neither due to starvation or the super-a.bundance, .or la& of, one or more hormones. Moreover, since the level of sucrose or iAA in the external medium is known to affect the proportion of quiescent cells in ,the root apical meristem as a whole (vAN'T Hoi~ 1966, 1968 b), it seems probable that cell quiescence outside the quiescent cen,tre is controlled in a different way from that wi,thin the quiescent centre.
The Quiescent Centre in Excised Roots of Pisurn sativurn L.
Acknowledgement One of us (R.D.M.) is grateful to the Science Research Council (U.K.) for support (S.R.C. B/RG/14316).
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MAcL~oDet al.: The Quiescent Centre in Excised Roots of Pisum sativurn L.
WEBSTER, P. L., and D. DAVIDSON, 1969: Changes in the duration of the mitotic cycle induced by colchicine and indol-3yl-acetic acid in Vicia faba roots. J. expt. Bot. 20, 671--685. and H. D. LANGENAUER, 1973: Experimental control of the activity of the quiescent centre in excised root tips of Zea mays. Planta 112, 91--100. WHITE, P. R., 1943: A Handbook of Plant Tissue Culture. Lancaster, Pa.: Cattell. -
Authors' address: Dr. R. D. MAcLEoD, Department of Plant Biology, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, U.K.