Relationship between transpiration and amino acid accumulation in Brassica leaf discs treated with cytokinins and fusicoccin

Transcription

1 Plant & Cell Physiol. 18: (1977) Relationship between transpiration and amino acid accumulation in Brassica leaf discs treated with cytokinins and fusicoccin Susumu Kuraishi 1 and Fumio Ishikawa 2 Department of Biology, College of General Education, University of Tokyo, Komaba, Meguro, Tokyo 153, Japan (Received May 26, 1977) Both cytokinins and fusicoccin (FC) stimulated the transpiration and the amino acid accumulation in leaf discs of Brassica catnptstris var. komatsuna. Enhancement effects were of the same magnitude. Both the accumulation and the transpiration were similarly inhibited when vaseline was smeared on the leaf surface. Abscisic acid (ABA) also inhibited those cytokinin-induced effects. The accumulation of amino acid-j^c was at the cytokinin- or FC-trcated site unless the leaf surface was smeared with vaseline. These facts suggest that cytokinin- or FC-induced amino acid accumulation in leaf is caused by the stimulation of transpiration. Cytokinin-induced chlorophyll retention was only seen when the stomatal surface of the floated leaf was exposed to air (5). Moreover, the cytokinin-induced chlorophyll retention was parallel to the cytokinin-stimulated transpiration (5). These facts indicated the importance of the stomatal opening in the chlorophyll retention of leaf discs. Cytokinins accumulated amino acid at the cytokinin-treated site on the detached leaf (3, 12, 13). If the cytokinin-induced amino acid accumulation is caused by the increased rate of transpiration, any chemical which regulates the stomatal opening should also modify the rate of amino acid accumulation at the treated site. The present experiments were performed to see whether the growth regulatorinduced amino acid accumulation has any relation to the regulator-induced stimulation of transpiration. Materials and methods Leaves of Brassica campestris var. komatsuna purchased locally were used in the study. Ten leaf discs, each with a diameter of 1.0 cm, were cut with a cork borer, and floated on 20 ml of solution in a Petri dish (8.5 cm in diameter). The leaf discs were floated in such a way that the lower surface was in contact with the Abbreviations: BAP, 6-benzylaminopurine; FC, fusicoccin; ABA, abscisic acid. Present addresses: * Department of Environmental Studies, College of Integrated Arts & Sciences, Hiroshima University, Higashisenda-machi, Hiroshima 730, Japan. Mitsui Memorial Hospital, 1-Kanda-Iiumicho, Chiyoda-ku, Tokyo 101, Japan. 1273

2 1274 S. Kuraiihi and F. Iihikawa solution to expose the upper surface to the air. Special care was taken to see that the upper surface of the leaf discs was not covered with water. After 18-hr incubation at 27 C in the dark, the leaf dbcs were rapidly blotted dry on filter paper and the rate of transpiration was measured. The rate of transpiration was measured by determining the decrease in fresh weight of ten leaf discs every lomin, at 25 C in the dark and at 60% relative humidity, with a semi-micro analytical balance. The uptake 1 of a-aminoisobutyric acid-l-^c (specific activity 5.6 mci/mmole, New England Nuclear) was studied by floating ten leaf discs on 10 ml of solution containing 0.1 /*Ci a-aminoisobutyric acid- 14 C for 2 hr at 27 C in the dark. The discs were then washed thoroughly with running water and rapidly dried at 60 C. The dried discs were oxidised with a Packard Tri-carb Sample Oxidiser, and the CO 2-14 C evoked was measured with a Packard Tri-carb Scintillation Spectrometer. For the study of amino acid accumulation at the site of application of growth regulators, leaves of ca. 100 cm 2 were used. Growth regulator at 10~ 4 M with 0.1 % Tween 20 as a wetting reagent was applied to the right side of both upper and lower surfaces of the leaves with a fine paint brush. After 24 hr at 27 C in the dark, the leaves were again similarly treated with growth regulators. Non-treated leaves were painted with 0.1% Tween 20 solution. After the final treatment with regulator, 8 ml of a-aminoisobutyric acid-^c solution (1 pci/ml) was applied by keeping excised leaves with petioles dipped in the radioactive solution at 27 C in the dark for another 24 hr. The petioles were then cut off and dried quickly with a heating iron and autoradiographed for one month using Fuji X-ray film. FC used in the experiment was kind gift from Prof. E. Marre of the University of Milan, Italy. Results Transpiration of ten leaf discs floated on either BAP or FC solution ia shown Fig. 1. Time course study of the transpiration of leaf discs treated with BAP and FC. Standard deviations are indicated. 1 The uptake of amino acid from die medium is described aj the amino add accumulation in the discs, since studies on the uptake were performed as a preliminary experiment on amino acid accumulation in die regulator-treated locus. Thus, here, "uptake" and "accumulation" are used as synonyms.

3 Transpiration and amino acid accumulation 1275 in Fig. 1. Discs floated on water for 1-3 hr had less transpiration than freshly cut discs. After 3 hr of incubation 2X10-«M BAP and 2X10-?M FC stimulated transpiration of discs by 42% and 87%, respectively. The stimulation was clearly seen even 1 hr after treatment with both growth regulators. BAP stimulated both the accumulation (uptake) of amino acid in leaf discs (Fig. 2 bottom) and the transpiration of the discs (Fig. 2 upper) at higher concentrations (IO^M and more). On the other hand, FC, at far lower concentration than BAP, stimulated both the amino acid accumulation and the transpiration of the discs. At the maximum response concentration of both chemicals, the effect of FC was far greater than that of BAP. The dose-response curves for transpiration and accumulation were similar when the same growth regulator was used. This suggests a close relation between the transpiration and the amino acid accumulation in the leaf discs. 10' 10"' 10" CONCENTRATION(M) Fig. 2. P 3h -k WTTH WITH BAP WITHOUT BAP BAP WITHOUT BAP 10' ICT 6 ABA CONCENTRATION(M) Fig ,-4 Fig. 2. Effect of BAP and FC on the transpiration of leaf discs (upper) and the accumulation of radioactivity due to a-aminoisobufyric acid- 14 C in leaf discs (lower). Leaf discs were treated for 2 hr in the dark. Standard deviations are indicated. Fig. 3. Effect of ABA on the cytokinin-induced transpiration of leaf discs (upper) and the cytokinin-induud acatmulaiion of radioactivity due to a-aminoisobutyric acid- u C (lower). Leaf discs were treated for 2 hr in the dark. Standard deviations are indicated.

4 1276 S. Kuraishi and F. Ishikawa ABA is a well-known growth regulator which induces stomatal closure (2, 11, 15, 16). ABA alone, at concentrations from 10~ 8 M to 10~ 4 M, did not severely inhibit the transpiration of leaf discs (Fig. 3), although very weak inhibition by 1_ Fig. 4. Auioradiogram of the accumulation of radioactive a-aininoisobuiyric acid at the growth regidator-trcated site. Only the right half of the leaf was treated with growth regulator. Upper two leaves: water treatment. Bottom left: BAP treatment. Bottom right: FC treatment. Part of the regulatortreated side of the leaf was smeared with vaseline. The decrease in water in vials of water-, water- FC-, and BAP-treated leaves was 0.5, 0.5, 6.2 and 2.5 ml/24 hr, respectively.

5 Transpiration and amino acid accumulation 1277 Table 1 Transpiration of leaf disci and accumulation of radioactivity due to a-ctminoisobutyric acid- ll C after treatment with BAP and FC -r «. t Transpiration Radioactivity T-,* --* Transpiration Radioactivity Treatment (mg H,6/cm'/hr) (dpm) Treatment (mg Hs,6/cm'/hr) (dpm) H a O 3,10±0.59* 1360±205' H,O- -vaseline 1.86±0.18' 445±63* BAP 4.95±0 f ±184 BAP+vaseline l,91± ±71 FC 7,12±0, ±321 FC+vaseline 1.83± ±67 ' Standard deviation was indicated. ABA alone was observed in some experiments repeated. This suggests that the stomata of discs floated on water in the dark close almost completely and that the transpiration of water-treated discs is mainly dut to cuticlar transpiration. ABA inhibited both cytokinin-stimulated transpiration and amino acid accumulation. The dose-response curve of ABA on the cytokinin-stimulated transpiration was quite similar to that on the cytokinin-stimulated accumulation. After 2 hr pretreatment with 2 X lo" 6 M BAP and 2 X 10~ 7 M FC in the dark, the leaf discs were smeared with thin layer of Vaseline to cover the leaf surface. The vaseline treatment greatly lowered both die transpiration and the accumulation regardless of pretreatment (Table 1). Mothes and Engelbrecht (3, 12, 13) showed the accumulation of radioactivity due to a-aminoisobutyric acid- 14 C at the cytokinin-treated site of leaves. If the amino acid accumulation in die leaves is caused by die stimulation of die cytokinininduced transpiration, FC, a stimulator of transpiration (4, 9, 14, 15, 19, 20), also should accumulate radioactivity due to labelled amino acid at the FC-treated site. Furthermore, smearing vaseline on die leaf surface should inhibit amino acid accumulation at die vaseline-treated site. Petioles of Brassica leaf were dipped into the solution of a-aminoisobutyric acid- 14 C, to observe the accumulation of radioactivity in die FC-, BAP-, and vaseline-treated portions (Fig. 4). The radioactivity due to labelled amino acid was accumulated at both FC- and BAP-treated sites. Furthermore, die amino acid accumulation by FC was greater than diat by BAP. This may reflect die difference in die rate of transpiration due to FC and BAP, since FC stimulate transpiration more dian BAP. Aldiough a large amount of radioactivity was seen in die FC-treated site, some radioactivity was seen near die veins of FC-non-treated sites. This suggests that FC was transported through the vascular system, and die transported FC accumulated amino acid in the new locus. Vaseline-treated portions hardly accumulated any radioactivity due to amino acid. All tiiese results strongly support die idea that die growdi-regulator-stimulated transpiration results in die amino acid accumulation at die regulator-treated site. Discussion Cytokinin-stimulated transpiration has been shown in detached Hordeum (2, 6-8, 10), Avena (1, 7, 8), Lolium (7), Nicotiana (5, 11), Lycopersicon (15), Pharbitis, Brassica, and Puerria (5). A previous paper (5) describes the importance of the

6 1278 S. Kuraishi and F. I«hikawa stomatal opening in cytokinin-induced chlorophyll retention, since chlorophyll retention was seen only when cytokinins stimulated transpiration. FC, a fungal metabolic product, causes the stimulation of stomatal openings in bean (17-19), Avena (18), Commelina (16), Corntis (20), Lycopersicon, Prunus and even mosses (6). Thus, treatment with either cytokinins or FC induced stomatal opening, resulting in the stimulation of transpiration. Since the transpiration of the discs was measured in the dark, stomata of water-treated discs should be closed. This has been proved by the experiment showing that ABA, which induces stomatal closure (2, 11, 15, 16) had almost no effect on the transpiration of discs in the dark (Fig. 3). Thus, cytokinins and FC treatments induce opening of the closed stomata in the dark. The stimulation of transpiration of leaves or leaf discs in the dark can bring about a passive uptake of amino acid- l4 C from the medium. Leaves treated with cytokinins and FC increased transpiration (uptake) of water by 5 and 12.4 times, respectively, compared to the water-treated leaf (Fig. 4, legend). Water taken up by the leaf also should contain radioactivity due to amino acid- 14 C. Thus, it is not necessary to assume active uptake of amino acid for the mode of action of amino acid accumulation in the growth regulator-treated portion. Present results and a previous paper (5) showed a beautiful parallelism between stomatal opening and some physiological effects mediated by FC or cytokinins. Moreover, FC retarded the chlorophyll retention of Brassica leaf discs (unpublished data). Thus, the importance of the stomatal opening in the growth regulatormediated amino acid accumulation and chlorophyll retention should be emphasized. Although all the physiological effects caused by cytokinins cannot be attributed only to cytokinin-stimulated transpiration, the importance of the stimulation of transpiration in the cytokinin-induced physiological effects of leaf cannot be underestimated. The authors wish to express their thanks to Mrs Rumiko Yoshino for her technical assistance. References ( 1) Chowdhurry, K. A. and G. M. Buth: Explanation for the stomatal response of excised leaves to Irinetin. Nahtri 227: (1970). ( 2) Cooper, M. J., J. Digby and P. J. Cooper: Effects of plant hormones on the stomata of barley: A study on the interaction between abtcisic acid and kinetin. Planta 105: (1972). ( 3) Engclbrecht, L.: BeitrSge zum Problem der Akkumulation von Aminosauren in Blattzrllen. Flora 150: (1961). ( 4) Graniti, A.: The role of toxins in the pathogenesii of infections by Fusicoccum arnygdali Del. on almond and peach. In Host-Parasite Relations in Plant Pathology. Edited by Z. Kirady and G. Ubrizsy. p Research Institute for Plant Protection, Budapest, ( 5) Kuraishi, S.: Ineffectiveness of cytokinin-induced chlorophyll retention in hypostomatous leaf discs. Plant & Cell Physiol. 17: (1976). (6*) Livne, A. and Y. Vaadia: Stimulation of transpiration rate in barley leaves by kinetin and gibbereuic add. Physiol. Plant. 18: (1965). (7) Luke, H.H. and T. E. Freeman: Rapid bioassay for phytolrinins based on transpiration of excised oat leaves. Nature 215: (1967).

7 Transpiration and amino acid accumulation 1279 ( 8) Luke, H. H. and T. E. Freeman: Stimulation of transpiration by cytokinini. ibid. 217: (1968). ( 9) Marre, E., P. Lado, F. Rasi-Caldogno, R. Colombo and M. I. de Michelis: Evidence for the coupling of proton extrusion to K + uptake in pea internode segments treated in fusicoccin or auxin. Plant So. Letters 3: (1974). (10) Meidner, H.: The effect of kinetin on itomatal opening and the rate of intake of carbon dioxide in mature primary leaves of barley. J. Exp. Bet. 18: (1967). (//) Mizrahi, Y., A, Blumenfeld and A. E. Richmond: Abscisic acid and transpiration in leaves in relation to osmotic stress. Plant PhysvA. 46: (1970). (12) Mothes, K.: The role of kinetin in plant regulation. In Rigulatews Natttrels de la Croissant* Vigitale. p Gifs/Yvette, (13) Mothes, K. and L. Engelbrecht: Kinetin-induced directed transport of substances in excised leaves in the dark. Phytochem. 1: (1961). (14) Squire, G. R. and T. A. Mansfield: Studies on the mechanism of action of fusicoccin, the fungal toxin that induces wilting, and its interaction with abscisic acid. Planta 105: (1972). (15) Tal, M., D. Imber and C. Itai: Abnormal stomatal behavior and hormonal imbalance in fiaaa, a wilty mutant of tomato. Plant Pkysiol. 46: (1970). (75) Tucker, D. J. and T. A. Mansfield: A simple bioassay for detecting "Antitranspirant" activity of naturally occurring compounds such as abscisic acid. Planta 98: (1971). (17) Turner, N. C: K+ Uptake of guard cells stimulated by fusicoccin. Naturt 235: (1972). (18) Turner, N. C.: Stomatal behavior of Avena saliva treated with two phytotoxins, victorin and fusicoccin. Amer. J. Bot. 59: (1972). (19) Turner, N. C.: Action of fusicoccin on the potassium balance of guard cells ofphaseoiw uuigalis. Amtr. J. Bot. 60: (1973). (20) Turner, N. C. and A. Graniti: Fusicoccin: a fungal toxin that opens stoma. Nature 223: (1969).