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Role of endogenous cytokinin in the development of hollowing in the root of Japanese radish (Raphanus sativus L.)
SCIENTIA HORTICULTull& ELSEVIER
Scientia Horticulturae 65 (1996) 105-l 15
Role of endogenous cytokinin in the development of hollowing in the root of Japanese radish ( Raphanus sativus L.) Yasutaka Kano
*,
Nobuyuki Fukuoka
Ishikawa Agricultural College, Norwichi. Ishikawa 921, Japan Accepted 9 October
1995
Abstract
The size of the hollow cavity increased with time in plants of the early sown plot (ESP), whereas its size remained almost unchanged throughout the growth period in plants of the late sown plot (LSP). The daily maximum soil temperatures in the ESP were 6- 12°C higher than those in the LSP. A much higher level of cytokinin was detected in the ammonia eluate fraction than in the acidic ethyl acetate fraction and none was detected in the effluent and washings. Cytokinin levels in the ammonia eluate fraction and the acidic ethyl acetate fraction were low in roots from the ESP in comparison with those from the LSP. A much larger size of hollow cavity was observed at any days after sowing (DAS) in control roots, that were exposed to temperatures of 30°C and above for 30 days, than in roots grown at lower soil temperatures between 25°C and 30°C throughout the growth period. Cytokinin levels in control roots grown at high soil temperature were consistently lower than those in the roots grown at low soil temperature. The production of endogenous cytokinin in the roots of cvs. Sobuto and Fukumi, cultivars that were prone to hollowness, was reduced at higher soil temperatures, while in cultivars that were resistant to hollowness cytokinin production was higher. The role of endogenous cytokinin in the development of hollowing is discussed. Keywords: Hollowing;
Abbreviations: * Corresponding
Endogenous
cytokinin
activity; Cell division; Lignin formation
DAS, day(s) after sowing; ESP, early sown plot; LSP, late sown plot. author.
03044238/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0304-4238(95)00853-5
106
Y. Kano, N. Fukuoka / Scientia Horticulturae 65 (1996) 105-I 15
1. Introduction
Hollow root in Japanese radish, a conspicuous lengthwise hollow cavity in the centre of the root, has been frequently associated with summer-sown crops. This physiological disorder originates from the intercellular spaces formed in the central region of the pith (Kano, 1989); spaces that are normally filled with large cellular elements as some of the protruding cells formed within the space continue cell division and enlargement (Fukuoka and Kano, 1992). Recent work (Kano, 1989; Kano and Fukuoka, 1995) involving anatomical observations of the development of hollowing revealed that heat treatment, in which the soil temperature was kept around 35°C during the middle of the growth period caused the intercellular spaces to coalesce into longitudinal cavities as a result of the obstruction of cell formation within the space. Furthermore, the lowering of meristematic activity in xylem parenchymatous cells in the pith region was one of the most important factors causing the retardation of inner cell formation (Kano and Fukuoka, 1995). To promote continuous, active mitosis and cell division, cytokinin was indispensable (Skoog and Miller, 1957; Miller, 1963). The production of this phytohormone significantly varies depending on a number of environmental factors such as soil moisture (Itai and Vaadia, 19651, fertilizer levels (Wagner and Michael, 1971) and aeration (Burrows and Carr, 1969). The temperature of the roots also appears to affect endogenous hormone activity; in Nicotiana rustica and Phaseolus vulgaris, root systems subjected to heat treatment showed lower amounts of cytokinin in their exudates (Itai et al., 1973). These facts strongly suggest that a high temperature lowers cytokinin activity in roots which, in turn, decreases cell division of meristematic tissue inside the intercellular space, resulting in the formation of a large hollow gap. The present study was carried out to clarify the correlation between endogenous cytokinin activity and the development of hollowing in the root, by varying the sowing dates, soil temperatures and cultivars. 2. Materials and methods
2.1. Experiment I
Seeds of cv. Gensuke were sown on July 1 (early sown plot, ESP) and August 30 (late sown plot, LSP), 1989, at the Ishikawa Agricultural College Experimental Farm to determine the effect of sowing date on the development of hollowing in the root. The size of the hollow cavity was determined at 30, 40, 50 and 60 days after sowing (DAS) as described elsewhere (Kano and Fukuoka, 1995). For each sampling date there were 15 replications. Endogenous cytokinin activity in 30- and 40-day-old roots was determined as follows. Each root sample was stored at - 30°C after harvest until extraction. The mid-portion of the roots was excised and the tissue was homogenized with 2 ml 70% methanol g- ’ tissue using a blender. After extraction for 24 h at 4°C the mixture was filtered and the residue was extracted twice with 80% methanol in a similar manner. The three extracts were combined and evaporated in vacua to an aqueous phase, then fractioned according to the procedure described in Fig. 1 (Kano and Asahira, 1979). The
Y. Kano. N. Fukuoku / Scientia Horticulturae 65 (1996) 105-I 15
107
Methanol extracts Concentrated in vucuo at 40 “C t
Aqueous concentrate Adjusted to pH 3.0 with 2N-HCI Extracted with ethyl acetate Ahueous phase (combinedaqueous phase) Treated with Dowex 50W x 8
Washings7
~~~~~~~~~~~ Ethyl acetate phase Evaporated to dryness in vacua Chromatographed on
t
at pH 3.0 - Washed with distilled water to pH 5.5
silicic acid-Celite - Eluted with 3N-WOH
column Eluted with mixture of
NH, OH eluate
benzen, ethyl acetate and methanol Each eluate Solvents were removed in vucuo at 40 “C
I
NH4 OH was removed in vucuo at 40 “C
t
Ammonia eluate fraction Fig.
1.Fractionation
procedure
Effluent and washings for cytokinins
Ethyl acetate fraction
in the extract of the root of Japanese radish.
combined aqueous extract was percolated through a column of Dowex W X 8 cation exchange resin (H+ form, 150 mesh and resin volume: 1 ml for the extract of 1 g of fresh roots) at a flow rate of 20 ml h-l. The column was washed with distilled water until the pH of the washings was neutral and the effluent and washings were combined. The absorbed substances were eluted with 3N-NH,OH of three times the volume of the resin, and the ammonia eluate was evaporated in vacua to remove ammonia. The residual aqueous phase was used for a bioassay. This phase was designated the ammonia eluate fraction. The ethyl acetate phase in Fig. 1 was evaporated in vacua to dryness and the residue was chromatographed on a silicic acid-Celite column (solvent system: benzene-ethyl acetate-methanol as described in Table 1). To each eluate 50 ml of distilled water was added and the mixture was evaporated in vacua to an aqueous phase. The residual aqueous phase was used for a bioassay. This phase was designated the ethyl acetate fraction. Cytokinin activity in the extract of roots was bioassayed using the
Y. Kano, N. Fuktwka / Scientia Horticulturae 65 (1996) 105-l 15
108 Table 1 Percentage
of benzene,
ethyl acetate and methanol
for each eluate chromatography
of ethyl acetate fraction
Solvents
1
2
3
4
5
6
I
8
9
10
11
Benzene Ethyl acetate Methanol
100 0 0
95 5 0
90 10 0
75 25 0
50 50 0
25 75 0
0 100 0
0 95 5
0 90 10
0 50 50
0 0 100
method described by Miller (1963). Thirty three ml of Miller’s medium in a 50-ml flask were used for culturing five pieces of soybean callus, with one test lot consisting of three flasks. The cultures were kept at 28°C under continuous illumination by cool white fluorescent lamps (4.4 W m-‘>. At the end of the culturing period, the fresh weight of callus was recorded. 2.2. Experiment 2 Seeds of cv. Gensuke and Taibyosobutori (designated as ‘Sobuto’) were sown on July 23, 1988, as described in Experiment 1, to determine the effect of lower soil temperature. For the lower soil temperature treatment, the soil temperature was kept between 25°C and 30°C by circulating water in l&mm calibre tubes laid at 7 cm and 14 cm below the surface of radish-planted rows. The control plants were grown without any treatment and the soil temperature was above 30°C. The size of the hollow cavity in the root was measured at 28, 35, 42 and 56 DAS. For each sampling date of each treatment of each cultivar there were 15 replications. Cytokinin activity in the ammonia eluate fraction of the roots sampled 21 and 35 DAS was determined as described above.
.
Fig. 2. Effect of sowing date on the development of hollowing in the root of cv. Gensuke. ‘, Days after sowing; n , early sown plot; 0, late sown plot; *, significant difference at 5% level between ESP and LSP at the same DAS.
Y. Karw, N. Fukuoka / Scientia Horhxlturae
65 (1996) 105-I 15
109
2.3. Experiment 3 Seeds of the cvs. Sobuto and Fukumi, representing cultivars that were liable to develop hollowness, and cvs. Kaishinsobuto (designated as ‘Kaishin’) and Seieiaokubi (designated as ‘Seiei’), representing cultivars that were resistant to hollowness, were sown on July 14, 1989, as described in Experiment 1, to determine cultivar differences in the occurrence of hollowing using replications for each cultivar. The size of the hollow cavity was measured at 35, 45 and 58 DAS. Cytokinin activity in the ammonia eluate fraction was investigated using roots sampled 35 DAS.
t & 8oo .3 a ‘M e 600-
-cl Z ‘G400 w 3
z-i 200 O-
800 & ‘6 600 3 = 400 .i? h z 200 -
3 O-
40 DAS 30 DAS Effluent and washings
Amtsonia eluate fraction
%a oddd Kinetin(mg/l) 323
a
l-l
I
I1
ud!PliLi \fj 11 1
6
1J
Elution number Elution number
1
6
Elution
30 DAS
Ethyl acetate
11
Elution
40 DAS
Is, odd&
Kinetin(*rl
fraction
Fig. 3. Effect of sowing date on cytokinin activity in the root of cv. Gensuke. ‘, Days after sowing. n , early sown plot; 0, late sown plot. ‘The activity of an extract representing 30 g was bioassayed. The callus was cultured for 21 days. ‘, Significant difference at 5% level between ESP and LSP at the same DAS. S, Significant difference at 5% level between corresponding elutions from ESP and LSP at the same DAS.
Y. Kano, N. Fukuoh / Scientia Horticulturae 65 (1996) 105-l 15
110
3. Results 3.1. Experiment
1
The daily maximum soil temperatures in the ESP were above 32°C from 10 to 23 DAS, and were 6-12°C higher than during the same period in the LSP. The size of the hollow cavity increased as the plants grew and reached 1591 mm2 at 50 DAS in the ESP, whereas cavity size in the LSP was zero at any DAS, with exception of 60 DAS, when it was 57 mm2 (Fig. 2). Much greater activity was detected in the ammonia eluate fraction than in the acidic ethyl acetate fraction, but none was detected in the effluent and washings (Fig. 3). Although the activity in the ammonia eluate fraction reduced as roots matured, the activity was always lower in roots from the ESP in comparison with those from the LSP. The activity in the ammonia eluate fraction of roots from the ESP was equivalent to 0.150 mg 1-l kinetin on the 30th DAS and 0.015 mg 1-l kinetin on the 40th DA& whereas that in roots from the LSP was 0.500 mg 1-l and 0.475 mg l-‘, respectively (Fig. 3). The activity in the acidic ethyl acetate fraction on the 30th and 40th DAS was nearly equivalent to or less than 0.050 mg 1-l kinetin regardless of sowing dates. The activity from the ESP on the 30th DAS was much lower than that from LSP (fraction No. 2, 0.006 vs. 0.095; No. 4, 0.006 vs. 0.011; No. 5, 0.003 vs. 0.004) (Fig. 3). The activity in elutions 4, 5 and 6 from the ESP on the 40th DAS was also lower than that in the corresponding elutions from the LSP. 3.2. Experiment 2 The root system of the plants in the control treatment was exposed to temperatures of 30°C and above for 30 days. The temperatures in the lower soil temperature plot ranged
28 DS’ 35 DA!342 DAS56 DAS cv. Censuke
28 DAS 35 DA!342 DAS 56 Dti
cv. Sobuto
Fig. 4. Effect of soil temperature on the development of hollowing in the roots of cvs. Gensuke and Sobuto. ‘, Days after sowing; n , control; 0, lower soil temperature; ‘, significant difference at 5% level between the control and the lower soil temperature plot at the same DAS.
Y. Kano, N. Fukuoka / Scientia Horticulturae 65 (1996) 105-I 15
111
i
i’ I
cv. Censuke
cv. Sobuto
Kinetinbdl)
Fig. 5. Effect of soil temperature on cytokinin activity in the roots of cvs. Gensuke and Sobuto. ‘, Days after sowing; n , control; 0, lower soil temperature. The activity of an extract representing 30 g was bioassayed. The callus was cultured for 21 days. *, Significant difference at 5% level between the control and the lower soil temperature plot at the same DAS.
from 25°C to 30°C throughout the growth period. A much larger size of hollow cavity was observed in the control root than in the root grown at lower soil temperature at any DAS (Fig. 4). Cavity sizes in both cvs. Gensuke and Sobuto reached their maximum at 56 DAS. In cv. Gensuke they were 899 mm* in the control and 532 mm* in the lower temperature plot, and in cv. Sobuto they were 1020 mm* and 405 mm*, respectively.
cv. Fuhwi
I
cv. Seiei
35 MS’
C”. Kaidlin lx. sobut n.Fukui cv.Seiei
45 DAS
cv.Kaishin C”.Sobut cv. Pukwi cv. Seiei
58
DAS
Fig. 6. Cultivar differences in the development of hollowing in roots grown under high soil temperature.. ‘, Days after sowing. Means with the same letter at the top of the column at the same DAS are not significantly different at the 5% level.
Y. Kam, N. Fukuoka/Scientia
112
Horticulturae 65 (1996) 105-115
ab
3 600c
-1
I
cv. Sobuto
cv. Fukumi
I cv. Kaishin cv. Sciei
i Y;
au2 0
c
LT
odc’d iiinetin(mg;IJ
Fig. 7. Cultivar differences in cytokmin activity in roots grown under high soil temperature for 35 days. The activity of an extract representin 30 g was bioassayed. The callus was cultured for 21 days. Means with same letter at the top of the column f, not significantly different at the 5% level.
Cytokinin activity in the control root was consistently lower than that in roots grown at the lower soil temperature (Fig. 5). The activity in the 21-day old root was not significantly affected by soil temperature and cultivar. However, the activity in the 35-day old root of the lower soil temperature plot was equivalent to 0.450 mg I-’ kinetin in cv. Gensuke and 0.048 mg 1-l kinetin in cv. Sobuto, while that in the control was lower, 0.045 mg l- ’ and 0.004 mg l- ‘, respectively. 3.3. Experiment
3
The daily maximum soil temperature reached a maximum of 34.4”C 10 DAS, and the temperature was above 30°C for 50 DAS. The size of the hollow cavities of cvs. Sobuto and Fukumi (cultivars prone to hollowness) 35 DAS, were 135 mm2 and 32 mm2, respectively, while those of cvs. Kaishin and Seiei (cultivars resistant to hollowness) were 50 mm2 and 0 mm*, respectively. Cavity sizes of cvs. Sobuto and Fukumi at 58 DAS were 180 mm* and 343 mm2, respectively, whereas those for cvs. Kaishin and Seiei were only 75 mm* and 58 mm2, respectively (Fig. 6). Cytokinin activities in cvs. Sobuto and Fukumi were equivalent to 0.050 mg 1-l and 0.048 mg I-’ kinetin, respectively, while those in cvs. Kaishin and Seiei were equivalent to 0.370 mg l- ’ and 0.350 mg l- ’ kinetin, respectively (Fig. 7).
4. Discussion Cytokinin activity in radish roots was found to be greatest in the aqueous phase. Direct extraction, purification and identification by paper or thin layer chromatography,
Y. Kane, N. Fukuoka / Scientia Horticulturae 65 (1996) 105-l 15
113
coupled with a bioassay specific cytokinin, has been reported for a number of plants (Letham and Williams, 1969; Short and Torrey, 1972). The ammonia eluate fraction of the extract from young strawberry fruits (Kane and Asahira, 1979) and the xylem exudate of decapitated sunflower plants (Kende, 1965) contain zeatin-based cytokinins. Zeatin and its derivatives were found to be the major cytokinin extractable from radish roots (Radin and Loomis, 1971). Thus, the major cytokinin activities found in the ammonia eluate fraction in the present study may be either zeatin or zeatin with zeatin derivatives. High cytokinin activity was also detected in the acidic ethyl acetate fraction. High cytokinin activity occurred in the ethyl acetate fraction of the extract from young tomato fruits when its acidic aqueous concentrate was partitioned with ethyl acetate (Asahira et al., 1968). Furthermore, the active compound was considered to be quite different from 6-aminopurine derivatives and to be a new type of cell division promoting factor containing an ester, a carboxyl and a glycol group together with another oxygenated group (Koshimizu et al., 1976). Though further purification was not carried out for the active compound in radish roots in the present study, it is possible that the active compound(s) in the acidic ethyl acetate fraction of the extract from radish roots is similar to the active compound(s) in tomato fruits. Experimental results revealed that changes in soil environment modified cytokinin production in roots. The extract of roots grown under high temperature conditions showed less cytokinin activity than in roots grown in a more suitable temperature range. In general, root systems in plants subjected to various climatic stresses such as water (Itai and Vaadia, 1965; Mizrahi and Richimond, 1972) and flooding (Burrows and Carr, 1969) have shown a reduction in the amounts of cytokinin-like substances. High temperature lowers cytokinin activity in leaf cuttings of Begonia (Heide, 1965). Cytokinin levels in the xylem exudate of Nicotiana rustica and Phase&us exposed to high soil temperature decrease to one-sixth of that of untreated plants (Itai et al., 1973). Furthermore, at different root temperatures qualitative differences are confirmed in the cytokinins of xylem sap from Vitis uiniferu L. vines (Skene and Kerridge, 1967). In the present study, high soil temperature promoted extreme hollowness concomitant with a decrease in cytokinin activity in all three experiments. Although the hollow cavity in the root is formed by intercellular spaces that develop in the pith region (Fukuoka and Kano, 19921, these spaces are usually filled with large and globular cellular elements because parenchymatous cells on the surfaces of the central gap preserve their meristematic potencies to a considerable degree (Kane and Fukuoka, 1992). Lowered meristematic activity caused by high soil temperature results in induction of lignin formation around the intercellular spaces, and this prevents the proliferation and intrusion of the cells into the spaces (Kane and Fukuoka, 1995). To promote continuous, active mitosis and cell division in vitro in excised tobacco pith tissues (Skoog and Miller, 1957) and soybean cotyledons (Miller, 19631, cytokinins are indispensable. Furthermore, coconut milk, which contains cell division promoting factors (Shantz and Steward, 19551, reduces the lignin content in excised carrot tissues cultured in vitro (Koblitz, 1962). Consequently, it can be concluded that high soil temperature reduces cytokinin activity in the roots and this reduction in cytokinin activity prevents xylem parenchymatous cells from dividing and/or enlarging. A lowering of meristematic activity causes
114
I’. Kano, N. Fukuoka / Scientia Horticulturae
65 (1996) 105-I I5
lignin to form in the cells abutting on the intercellular space. Eventually, cell proliferation around the intercellular space is suppressed and the spaces coalesce into a large hollow gap. In addition, the production of endogenous cytokinin in the root and the effect of heat treatment may be affected by the cultivar used. In the case of cvs. Sobuto and Fukumi, marked hollowing develops possibly due to a reduction in cytokinin production following exposure to a high soil temperature. However, for cvs. Kaishin and Seiei high soil temperature may decrease cytokinin production only slightly, and thus no hollowing develops.
Acknowledgements The authors are grateful to Dr. Shuichi Iwahori, Professor of University of Tsukuba for his critical reading of the manuscript. This work was partly supported by a Grant-in-Aid for Scientific Research (B) No. 02556005 from Ministry of Education, Science and Culture of Japan.
References Asabira, T., Takagi, H., Takeda, Y. and Tsukamoto, Y., 1968. Studies on fruit development in tomato. 11. Cytokinin activity in extracts from pollinated, auxin and gibberellin-induced parthenocarpic tomato fruits and its effect on histology of the fruit. Mem. Res. Inst Food Sci., Kyoto Univ., 29: 24-54. Burrows, W.J. and Carr, D.J., 1969. Effects of flooding the root system of sunflower plants on the cytokinin content in the xylem sap. Physiol. Plant., 22: 1105- 1112. Fukuoka, N. and Kane, Y., 1992. The difference in the development of hollowness in roots of ‘Gensuke’ radish between the early and late sowing of seeds. J. Jpn. Sot. Hortic. Sci., 60: 881-887. Heide, O.M., 1%5. Interaction of temperature, auxins, and kinins in the regeneration ability of Begonia leaf cuttings. Physiol. Plant., 18: 891-920. Itai, C. and Vaadia, Y., 1965. Kinetin-like activity in root exudate of water-stressed sunflower plants. Physiol. Plant., 18: 941-944. Itai, C., Ben-Zioni, A. and Grdin, L., 1973. Correlative changes in endogenous hormone levels and shoot growth induced by short heat treatments to the root. Physiol. Plant., 29: 355-360. Kane, Y., 1989. Effects of time of high and low soil temperature treatments on the growth of Japanese radish cv. Gensuke and the occurrence of hollow root. J. Jpn. Sot. Hortic. Sci., 57: 626-632. Kane, Y. and Asahira, T., 1979. Effect of endogenous cytokinin in strawberry fruits on their maturing. J. Jpn. Sot. Hortic. Sci., 47: 463-472. Kane, Y. and Fukuoka, N., 1992. Relationship between the occurrence of hollowing and lignification of parenchymatous cells in the root of Japanese radish cv. Gensuke. J. Jpn. Sot. Hortic. Sci., 61: 359-366. Kane, Y. and Fukuoka, N., 1995. Effect of soil temperature on hollowness in Japanese radish (Rap/tams sativus L. cv. Gensuke). Scientia Hortic., 61: 157-166. Kende, H., 1965. Kmetin-like factors in the root exudate of sunflowers. Proc. Nat. Acad. Sci., 53: 1302-1307. Koblitx, H., 1962. Die Chemie der Zellwand in vitro kultivierter pflanzlicher Gewebe III. Die Wirkung von Cocosmilch, Kinetin und Auxinen auf die Zellwand Karottengeweben. Faserforschung turd Textiltechnik, 13: 270-275. Koshimizu, K., Hayashi, H. and Asahira, T., 1976. Substances with cytokinin activity in tomato fruits. In: Collected Abstracts of the 9th International Conference on Plant Growth Substance. Lausanne, SpringerVerlag, Berlin, pp. 200-202. L&am, D.S. and Williams, M.W., 1969. Regulators of cell division in plant tissues. VIII. The cytokinins of the apple fruit. Physiol. Plant., 22: 925-936.
Y. Kane, N. Fukuoka/Scientia
Horticulturae
65 (1996) 105-115
115
Miller, C.O., 1963. Kinetin and kinetin-like compounds. In: H.F. Linskins and H.V. Tracey (Editors), Modem Methods of Plant Analysis 6. Springer-Verlag, Berlin, pp. 194-202. Mizrahi, Y. and Richimond, A.E., 1972. Hormonal modification of plant response to water stress. Aust. J. Biol. Sci., 25: 437-442. Radin, J.W. and Loomis, RX, 1971. Changes in the cytokinins of radish roots during maturation. Physiol. Plant., 25: 220-244. Shantz, E.M. and Steward, F.C., 1955. The identification of compound A from coconut milk as 1, 3-diphenyhtrea. J. Am. Chem. See., 77: 635 l-6353. Short, K.C. and Torrey, J.G., 1972. Cytokinins in seedling roots of pea. Plant Physiol., 49: 155-160. Skene, K.G.M. and Kenidge, G.H., 1967. Effect of root temperature on cytokmin activity in root exudate of Vitis oinifera L. Plant Physiol., 42: 113 1- 1139. Skoog, F. and Miller, C.O., 1957. Chemical regulation of growth and organ formation in plant tissues cultured in uitro. Symp. See. Exp. Biol., 11: 118-131. Wagner, H. and Michael, G., 1971. Dcr EinfluO unterschiedlicher Stickstoffversorgung auf die Cytokininbildung in Wurzeln von Sonnenbhrmenpflanzen. B&hem. Physiol. Pflanzen, 162: 147-158.
Scientia Horticulturae 65 (1996) 105-l 15
Role of endogenous cytokinin in the development of hollowing in the root of Japanese radish ( Raphanus sativus L.) Yasutaka Kano
*,
Nobuyuki Fukuoka
Ishikawa Agricultural College, Norwichi. Ishikawa 921, Japan Accepted 9 October
1995
Abstract
The size of the hollow cavity increased with time in plants of the early sown plot (ESP), whereas its size remained almost unchanged throughout the growth period in plants of the late sown plot (LSP). The daily maximum soil temperatures in the ESP were 6- 12°C higher than those in the LSP. A much higher level of cytokinin was detected in the ammonia eluate fraction than in the acidic ethyl acetate fraction and none was detected in the effluent and washings. Cytokinin levels in the ammonia eluate fraction and the acidic ethyl acetate fraction were low in roots from the ESP in comparison with those from the LSP. A much larger size of hollow cavity was observed at any days after sowing (DAS) in control roots, that were exposed to temperatures of 30°C and above for 30 days, than in roots grown at lower soil temperatures between 25°C and 30°C throughout the growth period. Cytokinin levels in control roots grown at high soil temperature were consistently lower than those in the roots grown at low soil temperature. The production of endogenous cytokinin in the roots of cvs. Sobuto and Fukumi, cultivars that were prone to hollowness, was reduced at higher soil temperatures, while in cultivars that were resistant to hollowness cytokinin production was higher. The role of endogenous cytokinin in the development of hollowing is discussed. Keywords: Hollowing;
Abbreviations: * Corresponding
Endogenous
cytokinin
activity; Cell division; Lignin formation
DAS, day(s) after sowing; ESP, early sown plot; LSP, late sown plot. author.
03044238/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0304-4238(95)00853-5
106
Y. Kano, N. Fukuoka / Scientia Horticulturae 65 (1996) 105-I 15
1. Introduction
Hollow root in Japanese radish, a conspicuous lengthwise hollow cavity in the centre of the root, has been frequently associated with summer-sown crops. This physiological disorder originates from the intercellular spaces formed in the central region of the pith (Kano, 1989); spaces that are normally filled with large cellular elements as some of the protruding cells formed within the space continue cell division and enlargement (Fukuoka and Kano, 1992). Recent work (Kano, 1989; Kano and Fukuoka, 1995) involving anatomical observations of the development of hollowing revealed that heat treatment, in which the soil temperature was kept around 35°C during the middle of the growth period caused the intercellular spaces to coalesce into longitudinal cavities as a result of the obstruction of cell formation within the space. Furthermore, the lowering of meristematic activity in xylem parenchymatous cells in the pith region was one of the most important factors causing the retardation of inner cell formation (Kano and Fukuoka, 1995). To promote continuous, active mitosis and cell division, cytokinin was indispensable (Skoog and Miller, 1957; Miller, 1963). The production of this phytohormone significantly varies depending on a number of environmental factors such as soil moisture (Itai and Vaadia, 19651, fertilizer levels (Wagner and Michael, 1971) and aeration (Burrows and Carr, 1969). The temperature of the roots also appears to affect endogenous hormone activity; in Nicotiana rustica and Phaseolus vulgaris, root systems subjected to heat treatment showed lower amounts of cytokinin in their exudates (Itai et al., 1973). These facts strongly suggest that a high temperature lowers cytokinin activity in roots which, in turn, decreases cell division of meristematic tissue inside the intercellular space, resulting in the formation of a large hollow gap. The present study was carried out to clarify the correlation between endogenous cytokinin activity and the development of hollowing in the root, by varying the sowing dates, soil temperatures and cultivars. 2. Materials and methods
2.1. Experiment I
Seeds of cv. Gensuke were sown on July 1 (early sown plot, ESP) and August 30 (late sown plot, LSP), 1989, at the Ishikawa Agricultural College Experimental Farm to determine the effect of sowing date on the development of hollowing in the root. The size of the hollow cavity was determined at 30, 40, 50 and 60 days after sowing (DAS) as described elsewhere (Kano and Fukuoka, 1995). For each sampling date there were 15 replications. Endogenous cytokinin activity in 30- and 40-day-old roots was determined as follows. Each root sample was stored at - 30°C after harvest until extraction. The mid-portion of the roots was excised and the tissue was homogenized with 2 ml 70% methanol g- ’ tissue using a blender. After extraction for 24 h at 4°C the mixture was filtered and the residue was extracted twice with 80% methanol in a similar manner. The three extracts were combined and evaporated in vacua to an aqueous phase, then fractioned according to the procedure described in Fig. 1 (Kano and Asahira, 1979). The
Y. Kano. N. Fukuoku / Scientia Horticulturae 65 (1996) 105-I 15
107
Methanol extracts Concentrated in vucuo at 40 “C t
Aqueous concentrate Adjusted to pH 3.0 with 2N-HCI Extracted with ethyl acetate Ahueous phase (combinedaqueous phase) Treated with Dowex 50W x 8
Washings7
~~~~~~~~~~~ Ethyl acetate phase Evaporated to dryness in vacua Chromatographed on
t
at pH 3.0 - Washed with distilled water to pH 5.5
silicic acid-Celite - Eluted with 3N-WOH
column Eluted with mixture of
NH, OH eluate
benzen, ethyl acetate and methanol Each eluate Solvents were removed in vucuo at 40 “C
I
NH4 OH was removed in vucuo at 40 “C
t
Ammonia eluate fraction Fig.
1.Fractionation
procedure
Effluent and washings for cytokinins
Ethyl acetate fraction
in the extract of the root of Japanese radish.
combined aqueous extract was percolated through a column of Dowex W X 8 cation exchange resin (H+ form, 150 mesh and resin volume: 1 ml for the extract of 1 g of fresh roots) at a flow rate of 20 ml h-l. The column was washed with distilled water until the pH of the washings was neutral and the effluent and washings were combined. The absorbed substances were eluted with 3N-NH,OH of three times the volume of the resin, and the ammonia eluate was evaporated in vacua to remove ammonia. The residual aqueous phase was used for a bioassay. This phase was designated the ammonia eluate fraction. The ethyl acetate phase in Fig. 1 was evaporated in vacua to dryness and the residue was chromatographed on a silicic acid-Celite column (solvent system: benzene-ethyl acetate-methanol as described in Table 1). To each eluate 50 ml of distilled water was added and the mixture was evaporated in vacua to an aqueous phase. The residual aqueous phase was used for a bioassay. This phase was designated the ethyl acetate fraction. Cytokinin activity in the extract of roots was bioassayed using the
Y. Kano, N. Fuktwka / Scientia Horticulturae 65 (1996) 105-l 15
108 Table 1 Percentage
of benzene,
ethyl acetate and methanol
for each eluate chromatography
of ethyl acetate fraction
Solvents
1
2
3
4
5
6
I
8
9
10
11
Benzene Ethyl acetate Methanol
100 0 0
95 5 0
90 10 0
75 25 0
50 50 0
25 75 0
0 100 0
0 95 5
0 90 10
0 50 50
0 0 100
method described by Miller (1963). Thirty three ml of Miller’s medium in a 50-ml flask were used for culturing five pieces of soybean callus, with one test lot consisting of three flasks. The cultures were kept at 28°C under continuous illumination by cool white fluorescent lamps (4.4 W m-‘>. At the end of the culturing period, the fresh weight of callus was recorded. 2.2. Experiment 2 Seeds of cv. Gensuke and Taibyosobutori (designated as ‘Sobuto’) were sown on July 23, 1988, as described in Experiment 1, to determine the effect of lower soil temperature. For the lower soil temperature treatment, the soil temperature was kept between 25°C and 30°C by circulating water in l&mm calibre tubes laid at 7 cm and 14 cm below the surface of radish-planted rows. The control plants were grown without any treatment and the soil temperature was above 30°C. The size of the hollow cavity in the root was measured at 28, 35, 42 and 56 DAS. For each sampling date of each treatment of each cultivar there were 15 replications. Cytokinin activity in the ammonia eluate fraction of the roots sampled 21 and 35 DAS was determined as described above.
.
Fig. 2. Effect of sowing date on the development of hollowing in the root of cv. Gensuke. ‘, Days after sowing; n , early sown plot; 0, late sown plot; *, significant difference at 5% level between ESP and LSP at the same DAS.
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2.3. Experiment 3 Seeds of the cvs. Sobuto and Fukumi, representing cultivars that were liable to develop hollowness, and cvs. Kaishinsobuto (designated as ‘Kaishin’) and Seieiaokubi (designated as ‘Seiei’), representing cultivars that were resistant to hollowness, were sown on July 14, 1989, as described in Experiment 1, to determine cultivar differences in the occurrence of hollowing using replications for each cultivar. The size of the hollow cavity was measured at 35, 45 and 58 DAS. Cytokinin activity in the ammonia eluate fraction was investigated using roots sampled 35 DAS.
t & 8oo .3 a ‘M e 600-
-cl Z ‘G400 w 3
z-i 200 O-
800 & ‘6 600 3 = 400 .i? h z 200 -
3 O-
40 DAS 30 DAS Effluent and washings
Amtsonia eluate fraction
%a oddd Kinetin(mg/l) 323
a
l-l
I
I1
ud!PliLi \fj 11 1
6
1J
Elution number Elution number
1
6
Elution
30 DAS
Ethyl acetate
11
Elution
40 DAS
Is, odd&
Kinetin(*rl
fraction
Fig. 3. Effect of sowing date on cytokinin activity in the root of cv. Gensuke. ‘, Days after sowing. n , early sown plot; 0, late sown plot. ‘The activity of an extract representing 30 g was bioassayed. The callus was cultured for 21 days. ‘, Significant difference at 5% level between ESP and LSP at the same DAS. S, Significant difference at 5% level between corresponding elutions from ESP and LSP at the same DAS.
Y. Kano, N. Fukuoh / Scientia Horticulturae 65 (1996) 105-l 15
110
3. Results 3.1. Experiment
1
The daily maximum soil temperatures in the ESP were above 32°C from 10 to 23 DAS, and were 6-12°C higher than during the same period in the LSP. The size of the hollow cavity increased as the plants grew and reached 1591 mm2 at 50 DAS in the ESP, whereas cavity size in the LSP was zero at any DAS, with exception of 60 DAS, when it was 57 mm2 (Fig. 2). Much greater activity was detected in the ammonia eluate fraction than in the acidic ethyl acetate fraction, but none was detected in the effluent and washings (Fig. 3). Although the activity in the ammonia eluate fraction reduced as roots matured, the activity was always lower in roots from the ESP in comparison with those from the LSP. The activity in the ammonia eluate fraction of roots from the ESP was equivalent to 0.150 mg 1-l kinetin on the 30th DAS and 0.015 mg 1-l kinetin on the 40th DA& whereas that in roots from the LSP was 0.500 mg 1-l and 0.475 mg l-‘, respectively (Fig. 3). The activity in the acidic ethyl acetate fraction on the 30th and 40th DAS was nearly equivalent to or less than 0.050 mg 1-l kinetin regardless of sowing dates. The activity from the ESP on the 30th DAS was much lower than that from LSP (fraction No. 2, 0.006 vs. 0.095; No. 4, 0.006 vs. 0.011; No. 5, 0.003 vs. 0.004) (Fig. 3). The activity in elutions 4, 5 and 6 from the ESP on the 40th DAS was also lower than that in the corresponding elutions from the LSP. 3.2. Experiment 2 The root system of the plants in the control treatment was exposed to temperatures of 30°C and above for 30 days. The temperatures in the lower soil temperature plot ranged
28 DS’ 35 DA!342 DAS56 DAS cv. Censuke
28 DAS 35 DA!342 DAS 56 Dti
cv. Sobuto
Fig. 4. Effect of soil temperature on the development of hollowing in the roots of cvs. Gensuke and Sobuto. ‘, Days after sowing; n , control; 0, lower soil temperature; ‘, significant difference at 5% level between the control and the lower soil temperature plot at the same DAS.
Y. Kano, N. Fukuoka / Scientia Horticulturae 65 (1996) 105-I 15
111
i
i’ I
cv. Censuke
cv. Sobuto
Kinetinbdl)
Fig. 5. Effect of soil temperature on cytokinin activity in the roots of cvs. Gensuke and Sobuto. ‘, Days after sowing; n , control; 0, lower soil temperature. The activity of an extract representing 30 g was bioassayed. The callus was cultured for 21 days. *, Significant difference at 5% level between the control and the lower soil temperature plot at the same DAS.
from 25°C to 30°C throughout the growth period. A much larger size of hollow cavity was observed in the control root than in the root grown at lower soil temperature at any DAS (Fig. 4). Cavity sizes in both cvs. Gensuke and Sobuto reached their maximum at 56 DAS. In cv. Gensuke they were 899 mm* in the control and 532 mm* in the lower temperature plot, and in cv. Sobuto they were 1020 mm* and 405 mm*, respectively.
cv. Fuhwi
I
cv. Seiei
35 MS’
C”. Kaidlin lx. sobut n.Fukui cv.Seiei
45 DAS
cv.Kaishin C”.Sobut cv. Pukwi cv. Seiei
58
DAS
Fig. 6. Cultivar differences in the development of hollowing in roots grown under high soil temperature.. ‘, Days after sowing. Means with the same letter at the top of the column at the same DAS are not significantly different at the 5% level.
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Horticulturae 65 (1996) 105-115
ab
3 600c
-1
I
cv. Sobuto
cv. Fukumi
I cv. Kaishin cv. Sciei
i Y;
au2 0
c
LT
odc’d iiinetin(mg;IJ
Fig. 7. Cultivar differences in cytokmin activity in roots grown under high soil temperature for 35 days. The activity of an extract representin 30 g was bioassayed. The callus was cultured for 21 days. Means with same letter at the top of the column f, not significantly different at the 5% level.
Cytokinin activity in the control root was consistently lower than that in roots grown at the lower soil temperature (Fig. 5). The activity in the 21-day old root was not significantly affected by soil temperature and cultivar. However, the activity in the 35-day old root of the lower soil temperature plot was equivalent to 0.450 mg I-’ kinetin in cv. Gensuke and 0.048 mg 1-l kinetin in cv. Sobuto, while that in the control was lower, 0.045 mg l- ’ and 0.004 mg l- ‘, respectively. 3.3. Experiment
3
The daily maximum soil temperature reached a maximum of 34.4”C 10 DAS, and the temperature was above 30°C for 50 DAS. The size of the hollow cavities of cvs. Sobuto and Fukumi (cultivars prone to hollowness) 35 DAS, were 135 mm2 and 32 mm2, respectively, while those of cvs. Kaishin and Seiei (cultivars resistant to hollowness) were 50 mm2 and 0 mm*, respectively. Cavity sizes of cvs. Sobuto and Fukumi at 58 DAS were 180 mm* and 343 mm2, respectively, whereas those for cvs. Kaishin and Seiei were only 75 mm* and 58 mm2, respectively (Fig. 6). Cytokinin activities in cvs. Sobuto and Fukumi were equivalent to 0.050 mg 1-l and 0.048 mg I-’ kinetin, respectively, while those in cvs. Kaishin and Seiei were equivalent to 0.370 mg l- ’ and 0.350 mg l- ’ kinetin, respectively (Fig. 7).
4. Discussion Cytokinin activity in radish roots was found to be greatest in the aqueous phase. Direct extraction, purification and identification by paper or thin layer chromatography,
Y. Kane, N. Fukuoka / Scientia Horticulturae 65 (1996) 105-l 15
113
coupled with a bioassay specific cytokinin, has been reported for a number of plants (Letham and Williams, 1969; Short and Torrey, 1972). The ammonia eluate fraction of the extract from young strawberry fruits (Kane and Asahira, 1979) and the xylem exudate of decapitated sunflower plants (Kende, 1965) contain zeatin-based cytokinins. Zeatin and its derivatives were found to be the major cytokinin extractable from radish roots (Radin and Loomis, 1971). Thus, the major cytokinin activities found in the ammonia eluate fraction in the present study may be either zeatin or zeatin with zeatin derivatives. High cytokinin activity was also detected in the acidic ethyl acetate fraction. High cytokinin activity occurred in the ethyl acetate fraction of the extract from young tomato fruits when its acidic aqueous concentrate was partitioned with ethyl acetate (Asahira et al., 1968). Furthermore, the active compound was considered to be quite different from 6-aminopurine derivatives and to be a new type of cell division promoting factor containing an ester, a carboxyl and a glycol group together with another oxygenated group (Koshimizu et al., 1976). Though further purification was not carried out for the active compound in radish roots in the present study, it is possible that the active compound(s) in the acidic ethyl acetate fraction of the extract from radish roots is similar to the active compound(s) in tomato fruits. Experimental results revealed that changes in soil environment modified cytokinin production in roots. The extract of roots grown under high temperature conditions showed less cytokinin activity than in roots grown in a more suitable temperature range. In general, root systems in plants subjected to various climatic stresses such as water (Itai and Vaadia, 1965; Mizrahi and Richimond, 1972) and flooding (Burrows and Carr, 1969) have shown a reduction in the amounts of cytokinin-like substances. High temperature lowers cytokinin activity in leaf cuttings of Begonia (Heide, 1965). Cytokinin levels in the xylem exudate of Nicotiana rustica and Phase&us exposed to high soil temperature decrease to one-sixth of that of untreated plants (Itai et al., 1973). Furthermore, at different root temperatures qualitative differences are confirmed in the cytokinins of xylem sap from Vitis uiniferu L. vines (Skene and Kerridge, 1967). In the present study, high soil temperature promoted extreme hollowness concomitant with a decrease in cytokinin activity in all three experiments. Although the hollow cavity in the root is formed by intercellular spaces that develop in the pith region (Fukuoka and Kano, 19921, these spaces are usually filled with large and globular cellular elements because parenchymatous cells on the surfaces of the central gap preserve their meristematic potencies to a considerable degree (Kane and Fukuoka, 1992). Lowered meristematic activity caused by high soil temperature results in induction of lignin formation around the intercellular spaces, and this prevents the proliferation and intrusion of the cells into the spaces (Kane and Fukuoka, 1995). To promote continuous, active mitosis and cell division in vitro in excised tobacco pith tissues (Skoog and Miller, 1957) and soybean cotyledons (Miller, 19631, cytokinins are indispensable. Furthermore, coconut milk, which contains cell division promoting factors (Shantz and Steward, 19551, reduces the lignin content in excised carrot tissues cultured in vitro (Koblitz, 1962). Consequently, it can be concluded that high soil temperature reduces cytokinin activity in the roots and this reduction in cytokinin activity prevents xylem parenchymatous cells from dividing and/or enlarging. A lowering of meristematic activity causes
114
I’. Kano, N. Fukuoka / Scientia Horticulturae
65 (1996) 105-I I5
lignin to form in the cells abutting on the intercellular space. Eventually, cell proliferation around the intercellular space is suppressed and the spaces coalesce into a large hollow gap. In addition, the production of endogenous cytokinin in the root and the effect of heat treatment may be affected by the cultivar used. In the case of cvs. Sobuto and Fukumi, marked hollowing develops possibly due to a reduction in cytokinin production following exposure to a high soil temperature. However, for cvs. Kaishin and Seiei high soil temperature may decrease cytokinin production only slightly, and thus no hollowing develops.
Acknowledgements The authors are grateful to Dr. Shuichi Iwahori, Professor of University of Tsukuba for his critical reading of the manuscript. This work was partly supported by a Grant-in-Aid for Scientific Research (B) No. 02556005 from Ministry of Education, Science and Culture of Japan.
References Asabira, T., Takagi, H., Takeda, Y. and Tsukamoto, Y., 1968. Studies on fruit development in tomato. 11. Cytokinin activity in extracts from pollinated, auxin and gibberellin-induced parthenocarpic tomato fruits and its effect on histology of the fruit. Mem. Res. Inst Food Sci., Kyoto Univ., 29: 24-54. Burrows, W.J. and Carr, D.J., 1969. Effects of flooding the root system of sunflower plants on the cytokinin content in the xylem sap. Physiol. Plant., 22: 1105- 1112. Fukuoka, N. and Kane, Y., 1992. The difference in the development of hollowness in roots of ‘Gensuke’ radish between the early and late sowing of seeds. J. Jpn. Sot. Hortic. Sci., 60: 881-887. Heide, O.M., 1%5. Interaction of temperature, auxins, and kinins in the regeneration ability of Begonia leaf cuttings. Physiol. Plant., 18: 891-920. Itai, C. and Vaadia, Y., 1965. Kinetin-like activity in root exudate of water-stressed sunflower plants. Physiol. Plant., 18: 941-944. Itai, C., Ben-Zioni, A. and Grdin, L., 1973. Correlative changes in endogenous hormone levels and shoot growth induced by short heat treatments to the root. Physiol. Plant., 29: 355-360. Kane, Y., 1989. Effects of time of high and low soil temperature treatments on the growth of Japanese radish cv. Gensuke and the occurrence of hollow root. J. Jpn. Sot. Hortic. Sci., 57: 626-632. Kane, Y. and Asahira, T., 1979. Effect of endogenous cytokinin in strawberry fruits on their maturing. J. Jpn. Sot. Hortic. Sci., 47: 463-472. Kane, Y. and Fukuoka, N., 1992. Relationship between the occurrence of hollowing and lignification of parenchymatous cells in the root of Japanese radish cv. Gensuke. J. Jpn. Sot. Hortic. Sci., 61: 359-366. Kane, Y. and Fukuoka, N., 1995. Effect of soil temperature on hollowness in Japanese radish (Rap/tams sativus L. cv. Gensuke). Scientia Hortic., 61: 157-166. Kende, H., 1965. Kmetin-like factors in the root exudate of sunflowers. Proc. Nat. Acad. Sci., 53: 1302-1307. Koblitx, H., 1962. Die Chemie der Zellwand in vitro kultivierter pflanzlicher Gewebe III. Die Wirkung von Cocosmilch, Kinetin und Auxinen auf die Zellwand Karottengeweben. Faserforschung turd Textiltechnik, 13: 270-275. Koshimizu, K., Hayashi, H. and Asahira, T., 1976. Substances with cytokinin activity in tomato fruits. In: Collected Abstracts of the 9th International Conference on Plant Growth Substance. Lausanne, SpringerVerlag, Berlin, pp. 200-202. L&am, D.S. and Williams, M.W., 1969. Regulators of cell division in plant tissues. VIII. The cytokinins of the apple fruit. Physiol. Plant., 22: 925-936.
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Miller, C.O., 1963. Kinetin and kinetin-like compounds. In: H.F. Linskins and H.V. Tracey (Editors), Modem Methods of Plant Analysis 6. Springer-Verlag, Berlin, pp. 194-202. Mizrahi, Y. and Richimond, A.E., 1972. Hormonal modification of plant response to water stress. Aust. J. Biol. Sci., 25: 437-442. Radin, J.W. and Loomis, RX, 1971. Changes in the cytokinins of radish roots during maturation. Physiol. Plant., 25: 220-244. Shantz, E.M. and Steward, F.C., 1955. The identification of compound A from coconut milk as 1, 3-diphenyhtrea. J. Am. Chem. See., 77: 635 l-6353. Short, K.C. and Torrey, J.G., 1972. Cytokinins in seedling roots of pea. Plant Physiol., 49: 155-160. Skene, K.G.M. and Kenidge, G.H., 1967. Effect of root temperature on cytokmin activity in root exudate of Vitis oinifera L. Plant Physiol., 42: 113 1- 1139. Skoog, F. and Miller, C.O., 1957. Chemical regulation of growth and organ formation in plant tissues cultured in uitro. Symp. See. Exp. Biol., 11: 118-131. Wagner, H. and Michael, G., 1971. Dcr EinfluO unterschiedlicher Stickstoffversorgung auf die Cytokininbildung in Wurzeln von Sonnenbhrmenpflanzen. B&hem. Physiol. Pflanzen, 162: 147-158.