Interaction of UV-Radiation and IAA During Growth of Seedlings and Hypocotyl Segments of Sunflower

]. Plant Physiol. Vol. 146. pp. 295-302 (1995}

Interaction of UV-Radiation and IM During Growth of Seedlings and Hypocotyl Segments of Sunflower JuRGEN

Ros and MANFRED TEVINI

University of Karlsruhe, Botanical Institute II, Kaiserstr. 12, D-76128 Karlsruhe, Germany Received September 14, 1994 ·Accepted November 20, 1994

Summary

Stem growth of sunflower seedlings (Helianthus annuus, c.v. Polstar L.) was increasingly reduced by UV-B radiation with shorter wavelengths (Schott cut-off filters: WG 360-280) at a constant low white light irradiance. The reduction in stem elongation measured with linear voltage transformers was observed after 10 h of enhanced UV-B irradiation (WG 305) and already after 5 h, when the seedlings were additionally irradiated with lateral shortwave UV-B (WG 305 hi.). Stem elongation of UV-B irradiated sunflower seedlings was not irreversibly reduced as demonstrated by changing UV-B irradiation conditions. UV-B irradiation (WG 305) had no negative influence on stem elongation when the hypocotyls were covered by a non-UV-B transmitting plastic film, indicating that the hypocotyl is most susceptible for the inhibiting effect of UV-B radiation on stem elongation. Elongation growth of isolated hypocotyl segments (HSEG-test) at simultaneous UV-B irradiation was inhibited both in water and IAA solution, dependent on wavelength in the UV-B range. In IAA solutions UV-B preirradiated for 1-48h under a WG 305 filter, elongation growth of segments decreased with preirradiation time. It was assumed that a destruction of IAA and/or a formation of growth inhibiting IAA photoproducts in the IAA solution are reasons for the observed growth reduction. Therefore, IAA-photooxidation kinetics were studied, the IAA photoproducts separated by HPLC and identified as 3-Hydroxymethyloxindole, Indole-3-aldehyde, 3-Methyleneoxindole (3-M), 3-Methyloxindole and Indole. The application of these compounds in the HSEG-test showed that only 3-M inhibited elongation growth. Furthermore, the in vivo IAA concentration of UV-B irradiated sunflower seedlings under WG 305 was reduced by 51% compared with that of seedlings grown under WG 360. On the basis of these results the «IAA destruction» seems to be a potent mechanism for the growth inhibition of UV-B irradiated sunflower seedlings grown at low white light irradiances.

Key words: UV-radiation, elongation growth, /ndole-3-acetic acid, Helianthus annuus. Abbreviations: IAA = Indole-3-acetic acid; HSEG = hypocotyl segment elongation growth; S.E. stem elongation; S.E.R. = stem elongation rate; PAR = photosynthetic active radiation; LVT = linear voltage transformers; UV-A = 400-320nm; UV-B = 320-280nm; UV-C = 200-280nm; HPLC = high pressure liquid chromatography; A. demin = Aqua demineralisata. Introduction

Due to stratospheric ozone depletion (Gleason et al., 1993) increased UV-B radiation has been measured in Antarctica and even in the Northern hemisphere (Kerr and McElroy, 1993; Blumthaler et al., 1994). The biological consequences of increased UV-B radiation are not sufficiently known but © 1995 by Gustav Fischer Verlag, Stuttgart

the research indicates a high damaging potential of shortwave UV-B to sensitive terrestrial (Tevini, 1994) and aquatic organisms (Hader, 1993). Research on terrestrial plants often dealt with the effect of UV-B radiation on plant growth. In many plant species reduced stem growth had been found (Tevini et al., 1981; Tevini, 1994; Caldwell and Flint, 1994). However, neither the growth kinetics nor the action mecha-

296

JURGEN Ros and MANFRED TEVINI

nisms of UV-B radiation on growth and cell elongation had been studied in detail. Auxins, and among them Indole-3-acetic acid (IAA), often strongly stimulate elongation growth of isolated stem segments of different plant species. Fukuyama and Moyed (1964) found that the growth regulator IAA is photooxidized in vitro by white light of high intensity to different riboflavin-catalyzed IAA-photooxidation products, which inhibited growth of bacteria, tomato root tips and the germination of pea seeds. Hager and Schmidt ( 1968 a) postulated that phototropic growth responses are caused by a lateral IAA-photooxidation at the illuminated part of the plant and consequently by an asymmetric distribution of IAA. Despite the fact that this hypothesis is being discussed controversially, IAA may somehow be involved in phototropism (Firn, 1994). In contradiction to phototropism it is quite clear that IAA is involved in geotropism of maize coleoptiles (Bandurski et al., 1990), where an asymmetric distribution of free and esterified IAA was demonstrated after a gravity stimulus in maize seedlings. Irradiation of barley (Tevini and Iwanzik, 1980), rye and oat seedlings (Braun, 1990) with high UVirradiances partially resulted in a loss of their negative geotropical growth response. Consequently, it was assumed that the loss of the geotropical plant growth reactions might be due to a UV-dependent degradation of IAA. This assumption is supported by the fact that IAA can be destroyed in vitro by UV-radiation due to the absorption in the UV-range between 270 and 300 nm, which also points to a possible in vivo photodestruction. Furthermore, UV-B radiation has been shown to lower the auxin concentration in fronds of Spirodela oligorhiza (Witztum et al., 1978) and it was speculated that reductions in plant height are also caused by a lower auxin content in different plant species (Tevini and Iwanzik, 1980; Kulandaivelu et al., 1989; Ziska et al., 1993), since several studies have directly related endogenous auxin of elongating tissues to growth (Bandurski, 1989; Ortuno et al., 1990). Yang et al. (1993) reported that exogenously applied IAA strongly promoted stem elongation in intact lightgrown seedlings of dwarf and tall pea plants. Since dwarf plants, to which IAA was applied, failed to reach the growth rate of tall plants, it was discussed that gibberellins and auxins regulate stem growth together. Gibberellins, however, absorb mainly in the shortwave UV-C (A.max. 254 nm) and are ~ot as susceptible to a UV-B induced photooxidation as auxms. The objective of the present investigation was firstly to measure the effects of enhanced UV-B radiation on elongation growth kinetics of sunflower seedlings highly resoluted with linear voltage transformers, and secondly to prove the hypothesis that growth inhibition of sunflower seedlings by UV-B radiation is correlated to photooxidation of IAA. Materials and Methods

Plant material and growth conditions

Sunflower seeds (Helianthus annuus cv. Polstar) were germinated in plastic pots containing TKS 1 (Floratorf, Oldenburg, Germany) at continuous low white light (WL; Philipps TL 40/29; PAR(4oo-700nm): 50J.Lmolm- 2 sec- 1) at 24°C for 2days. Thereafter,

Table 1: Photon irradiances [J.Lmol m- 2 s- 1] (a), irradiances [mW m- 2] (b) and biologically weighted irradiances [mW m- 2] according to the «PLANT>> weighting function (Caldwell, 1971) normalized at 300 nm in different spectral regions at plant height under different cut-off filters. (c). Under WG 360, no biological effective UV-B radiation was present. WG360

UV-B

a. 280-320 nm b.c. UV-A a. 0.68 320-400 nm b. 219.78 PAR 45.83 a. 400-700 nm b. 9,964.0

WG320

WG305

0.27 102.45 2.31 3.99 1379.40 50.72 11,011.0

2.40 931.33 341.98 4.62 1609.90 51.47 11,175.0

WG 305 hi. 3.85 1500.0 806.50 4.73 1650.20 50.37 10,937.0

WG280 5.60 2190.60 1454.97 4.91 1717.10 50.85 11,041.0

the seedlings continued growth either under white light up to a seedling height of 4 to 5 em or under a light source consisting of two UV-B (Philipps TL 40/12) and three WL tubes for 4 more days. The pots with the seedlings were placed in boxes made either from UVtransmitting or non-UV-B transmitting plexiglass (PG 2458, 209; Ri:ihm, Darmstadt, Germany) open at the top. The top of the plexiglas boxes was covered by different cut-off filters (WG 360, 320, 305, 280; Schott, Wiesbaden, Germany), which filtered UV radiation coming from above. The transmission properties of plexiglass boxes were adjusted to those of the cut-off filters by covering the plexiglass boxes with either UV or non-UV-B transmitting plastic films, so that UV-B radiation of nearly the same fluence rate reached the seedlings from the top and the side. The irradiation condition whereby UV-B radiation from the top was filtered by WG 305 and from the side by only the plexiglass with a transmission down to 280 mm was designated as WG 305 hi. (high). Spectral energy distributions were measured with a double monochromator spectroradiometer, Optronic model 742 (Optronic, Orlando, USA). Irradiation conditions are listed in Table 1.

Growth parameters and growth-kinetic measurements

Hypocotyl length, area of the cotyledons, and fresh and dry weights were determined in 6-day-old sunflower seedlings, which were irradiated for 4days at 5 different UV-B irradiances (Table 1). The area of the cotyledons was measured by an area meter (Li-Cor: Li-3000, Lincoln, USA) and hypocotyllength by a ruler with an accuracy of 0.2 mm. The kinetics of the hypocotyl growth of sunflower seedlings was measured with linear voltage transformers (LVT's; TWK-Electronics, Dusseldorf, Germany; according to Green and Cummins, 1974) in a self-made measuring system. The seedlings were linked to the transducer by a polyfilamentous thread and a metal hook. Growth data are presented as cumulative whole stem elongation [S.E.: em] and stem elongation rates [S.E.R.: mm/ h]. For studying the possible targets of UV-radiation different parts of the intact seedlings (cotyledons, apex, hypocotyl) were either covered with mylar foil (non-UV-B transmitting) or removed in some experiments.

Hypocotyl segment elongation growth test (HSEG-test)

Two em long hypocotyl segments from sunflower seedlings of equal height grown for 6 days under continuous white light (WL) were excised 2-3 mm below the node of the cotyledons. The segments were transferred to different solutions and incubated for 24 h either at simultaneous UV-B irradiation or in the dark.

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Interaction of UV-Radiation and IAA During Growth of Sunflowers Table 2: Growth parameters (hypocotyl length, cotyledons area, fresh and dry weights) of 6-day-old sunflower seedlings grown 4 days un-

der white light and UV-B tubes filtered by different cut-off filters (WG 360-280). Values followed by a different letter are significandy different at the 5% level (ANOVA). hypocotyl length [em] cotyledons area [cm2] fresh weight [g] dry weight [mg]

WG360

WG320

WG305

WG 305 hi.

WG280

6.20 a ±0.12 2.64 a ±0.15 6.13 a ±0.15 340 a ±7.00

6.10 a ±0.15 2.56 a, b ±0.11 6.11 a, b ±0.11 347 a ±6.80

4.90 b ±0.17 2.34 b ±0.11 5.98 b ±0.11 342 a ±7.50

4.26 c ±0.11 2.03 c ±0.14 5.51c ±0.14 343 a ±7.20

3.04 d ±0.11 1.85 d ±0.13 4.25 d ±0.13 319 b ±6.90

Extraction and separation ofIAA and IAA -photoproducts IAA and IAA-photoproducts were separated by HPLC (Hewlett Packard: model HP 1084 B) and detected by a diode array detector at 280 nm (HP 1040 A). The separation was carried out on a Nucleosil RP 18 column with a linear gradient elution starting with 30% B (methanol) and 70% A (0.01 M acetate buffer, pH 4.66) to 99% methanol within 20 min at a flow rate of 1mLImin. The IAAphotoproducts 3-Hydroxymethyloxindole (3-0H) and 3-Methyleneoxindole (3-M) were collected and photometrically quantified using the extinction coefficients given by Fukuyama and Moyed (1964). Quantitative extraction of IAA and IAA-photoproducts from UV-B irradiated sunflower seedlings reported by Rademacher (1978) was optimized. Five ~tL of [14C}IAA (specific activity 9250 Bq) were added as internal standard. Recovery of IAA ranged between 70-80 %, whereas recovery rates for non-radioactive IAA reported by Rademacher (1978) accounted for about 60 %.

Statistics The results represent means and standard deviations except for the hypocotyl growth curves, where a characteristic growth curve was chosen. The mathematical relations of the growth rate were calculated by polynomial regression. The quality of this relation is expressed by the correlation coefficient (r). According to the collection and the distribution of the data, different statistical tests were selected: for statistical calculations of two normally distributed samples the two-sample F-test and the two-sample T-test were used. For the comparison of more than two samples the analyses of variances (ANOVA) and the Fisher's LSD were applied.

Results

General growth parameters and growth characteristics

The growth parameters hypocotyl length, cotyledons area, and fresh and dry weights of 6-day-old sunflower seedlings grown under white light and UV-B lamps filtered by different cut-off filters of the series WG 360-280 for 4 days were increasingly reduced with shorter wavelengths of the filters and thus with increasing UV-B irradiances (Table 2). Highest UV-B irradiance transmitted through a WG 280 filter reduced the hypocotyl length by approximately 50% compared with lower irradiances at longer wavelengths (WG 320 or WG 360), the cotyledons area and the fresh weight by approximately 70 %. Dry weight was only reduced by 8 %. Further experiments on the growth kinetics of sunflower seedlings grown in plexiglass boxes for 40 h were continued only under filters WG 360, WG 305 and WG 305 hi. and are shown in Fig. 1. The cumulative stem elongation [S.E.] of UV-B irradiated seedlings (WG 305) was reduced by about 22% after 40 h compared with the control plants without UV-B (WG 360; Fig. 1 a). The stem elongation rate [S.E.R.; mm/h] of the control plants was about 10 mm/h for the whole period of time (Fig. 1 b) whereas the S.E.R. of UV-irradiated seedlings (WG 305) stayed at 8 mm/h for 30 h and then declined to 6 mm/h after 40 h. S.E.-curves started to divide after 10 h of irradiation. S.E. of seedlings, which were additionally irradiated with lateral short-wave UV-B (WG 305 hi.), was reduced by about 57% after 40 h compared with the control plants (WG 360; Fig. 1 a). Stem elongation rate of the control plants was about 10 mm/h for 40 h (Fig. 1 b), while the S.E.R. of the UV-irradiated seedlings (WG 305 hi.) was about 6 mm/h in the first 30 hand decreased to 1.5 mm/h after 40 h. S.E.-curves started to divide after 5 h of irradiation. The reversibility of the UV-induced growth reduction was demonstrated by changing the filters and consequently the UV-B irradiance received by the seedlings. S.E.R. of seedlings grown for 10 h either under WG 360 or WG 305 was about 14 and 10 mm/h, respectively, and accounted for a 28% reduction in stem elongation in that experiment (Fig. 2 a). After changing the filters, stem elongation rate of the seedlings initially grown without UV-B decreased while

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Fig.1: Cumulative stem elongation (S.E.; a) and stem elongation rate (S.E.R.; b) of sunflower seedlings grown under WG 360, WG 305 and WG 305 hi. for 40 h. Percentages of S.E. of UV-B irradiated seedlings compared with the control plant after 40 h (a) and the correlation coefficients (r) for the S.E.R. data (b) are also given in the graphs.

JORGEN Ros and MANFRED TEVINI

298

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Fig. 2: Stem elongation rate (S.E.R.) of sunflower seedlings grown under WG 360, WG 305 (a) and WG 360, WG 305 hi. (b) for 10 h. Subsequently, the filters and the equivalent plastic boxes were changed (indicated by an arrow). The correlation coefficients (r) for the S.E.R.data are also given in the graphs. r.p.: reversion point.

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Fig. 3: Cumulative stem elongation (S.E.) of sunflower seedlings grown under WG 360 and WG 305 for 40 h. Different parts of the seedlings were either covered by a non-UV-B transmitting plastic film (Ja hypocotyh Jb cotyledons, Jc apex) or excised (cotyledons 3 b, apex 3d). Percentage of S.E. of UV-B irradiated seedlings compared with the control plants after 40 h is also given in the graphs.

S.E.R. of the seedlings initially grown with UV-B stayed at nearly the same level of about 11 mm/h until the end of the experiment. Both S.E.R. curves crossed 12 h (22 h after time 0) after changing the irradiation conditions (Fig. 2 a). This point was called «reversion point». The S.E.R. of seedlings grown for 10 h either under WG 360 or WG 305 hi. was about 15 mm and 8 mm, respectively, and accounted for a 46 % reduction in stem elongation (Fig. 2 b). The S.E.R. of

the seedlings initially grown without UV-B decreased continuously to 3 mm/h at 40 h, while the seedlings initially grown with UV-B recovered 8-10 h after changing the irradiation and reached a S.E.R. of 8 mm/h, which was maintained until the end of the experiment (Fig. 2 b). S.E.R.-curves crossed 20 h after changing the irradiation conditions. The parts of the seedlings mainly responsible for the perception of UV-B radiation were identified either by covering dif-

Interaction of UV-Radiation and IAA During Growth of Sunflowers

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Fig. 4: Length [em] of hypocotyl segments (2 em long) simultaneously irradiated (WG 360-280 filters) and incubated for 24 h in A. demin. (4a) or in 10J,LM IAA (4b). Length [em] of hypocotyl segments incubated for 24h in IAA preirradiated for 1,2,4,8,16,24,48h (4c) and in IAA-photoproducts (IND: indole, OH: 3-Hydroxymethyloxindole, CHO: Indole-3-aldehyde, MET: 3-Methyloxindole, 3M: 3-Methyleneoxindole, Ad: Aqua demin.) in the dark. Values followed by a different letter are significantly different at the 5% level (ANOVA). ferent organs with plastic films not transmitting UV-B or by excising these organs. The seedlings with a covered hypocotyl reached nearly 100% of the hypocotyllength of the UV-B free control {a), while seedlings with covered cotyledons reached about 80% {b), and those with covered apices 73 % of the control (c). The hypocotyllength was reduced by about 50% (b) and 68% (d) after excising the apex or the cotyledons (Fig. 3).

Hypocotyl segment elongation growth (HSEG} Using the hypocotyl segment elongation growth test (HSEG-test) it was demonstrated that the growth inhibition also occurs in excised UV-B irradiated hypocotyl segments and that interactions of UV-B radiation and the growth regulator IAA exist. Segments increased in length by 40% when incubated for 24 h in A. demin. at simultaneous irradiation with UV-B free light (WG 360), whereas length increases were lower when the segments were irradiated at shorter wavelengths (WG 305, 280; Fig. 4 a). The elongation of segments incubated for 24 h in IAA-solution at simultaneous UV-B irradiation (Fig. 4 b) was reduced with shorter filter wavelengths. The increase of hypocotyl length under UV-B free conditions (WG 360) amounted to 65% and was consequently higher than in A. demin., whereas under the highest UV-B irradiance (WG 280) the increase reached only 30 %, similar to that in A. demin. It was concluded that the wavelength dependent inhibi-

tion of hypocotyl segment elongation could be due to a photodestruction of IAA in the incubation medium and!or the formation of IAA photoproducts, which possibly inhibit hypocotyl growth. In IAA solutions preirradiated for 1, 2, 4, 8, 16, 24 and 48 h under a WG 305 filter, hypocotyllength was significantly lower at preirradiation times longer than 8 h (Fig. 4 c). It was assumed that the IAA concentration was too low and! or that IAA photoproducts inhibited hypocotyl elongation after 8 h of UV-B irradiation, when their concentration has reached a certain level. To prove this assumption IAA solution was preirradiated for 1, 2, 24 and 48 h under a WG 305 filter, and aliquots analyzed by HPLC. The content of IAA decreased immediately after the onset of UV-B irradiation and accounted for 72% after 1 h and 43% after 2 h of the original IAA concentration. In pure IAA solutions without any photoproducts (7.2 and 4.3J.1M IAA), in which the IAA concentration corresponded exactly to the concentration appearing after 1 hand 2 h of UV-B preirradiation time, HSEG was not reduced compared with the control in pure IAA (10 11M) and 1 or 2 h preirradiated IAA solutions (Fig. 5 a). In lower concentrated (0.65 and 0.32J.1M) IAA solutions, identical to those appearing after 24 and 48 h UV-B irradiation, HSEG was greater than in A. demin., but lower when incubated in 24 or 48 h preirradiated IAA solutions (Fig. 5 b). The conclusion that a reduced IAA concentration together with growth-inhibiting IAA photoproducts must be responsible for the inhibition of hypocotyl segment

300

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Ros and MANFRED TEVINI

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Fig. 6: a) HPLC-chromatogram of UV-B irradiated IAA (100 J.LM; 1 h under a WG 305 filter). b) Photooxidation of IAA (100 J.LM) and formation of IAA-photoproducts under a WG 305 filter for 16h. 3-Hydroxymethyloxindole (time 5.41: 3-0H), Indole-3-acetic acid (7.51; IAA), Indole-3-aldehyde (9.58; CHO), 3-Methyleneoxindole (11.02; 3-M), Indole (12.02) and 3-Methyloxindole (13.87; 3-MET).

growth in UV-B preirradiated IAA solutions was confirmed by the characterization of the IAA photoproducts. The analysis of the IAA photoproducts in UV-B preirradiated IAA solution by HPLC resulted in five substances (Fig. 6 a): 3-Hydroxymethyloxindole (3-0H), Indole-3-aldehyde (CHO), 3-Methyleneoxindole (3-M), 3-Methyloxindole (3-MET) and Indole (IND). The kinetics of IAA photooxidation showed that the IAA concentration decreased during the entire irradiation period. In the first few hours 3-0H and 3-CHO appeared in higher amounts, whereas 3-Met and Indole were generated at lower rates. 3-M was detected after 1 h of irradiation, but increased continuously with time of irradiation. Therefore, it was assumed that 3-M could be a possible growth inhibitor (Fig. 6 b). The HSEG-test in different IAA photoproducts confirmed that only 3-M inhibited the hypocotyl elongation compared with the water control (Fig. 4d). The IAA content of 6-day-old sunflower seedlings grown with (WG 305) or without UV-B was 59 ng and 116 ng FW-1, which means a loss of 51% (data not shown).

Discussion

The inhibiting effect of UV-B radiation on the growth of seedlings has often been reported (Barnes et al., 1988; Tevini and Teramura, 1989; Tevini, 1994). Furthermore, a fluence response relationship was demonstrated for leaf area and hypocotyllength of cucumber seedlings that were grown in growth chambers under enhanced artificial UV-B radiation (Tevini, 1993). However, the growth kinetics of UV-B irradiated seedlings have not been studied in detail, especially not those measured with LVT's, which continuously record stem elongation as shown for blue light responses (Cosgrove and Green, 1981). Interactions between UV radiation and growth regulators had been earlier discussed as a reason for reduced growth under enhanced UV-B (Tevini and lwanzik, 1980; Kulandaivelu et al., 1989). One objective of this study was to get more information about growth mechanisms under enhanced UV-B irradiation. White light intensity was kept low in this study, because photorepair mechanisms, which can lead to an amelio-

Interaction of UV-Radiation and IAA During Growth of Sunflowers

301

ration of the UV-B effect by high levels of PAR and UV-A/ catalysts for the formation of IAA photoproducts. Furtherblue light, should be minimized (Langer and W ellmann, more, Hager and Schmidt (1968 b) have shown that the elon1990; Pang and Hays, 1991). Plants generally are much more gation growth of isolated maize coleoptiles is inhibited by sensitive to UV-B at low levels of PAR as well as to UV-A 3-M. The Cholodny-Went hypothesis, which explains asymand blue light (Mirecki and Teramura, 1984; Cen and Born- metric growth in phototropism by a displacement of IAA after unilateral irradiation, was traced back to the formation man, 1990; Caldwell et al., 1994). Under these low white light conditions stem elongation of of 3-M on the illuminated part of the stem. Moore (1989) sunflower seedlings was reduced after 10 h of UV-B irradia- emphasized the inhibitory effect of 3-M on SH-group carrytion (WG 305) compared with the control plants without ing enzymes. Consequently, 3-M could inhibit the SH-group UV-B (WG 360). However, seedlings additionally irradiated carrying H+ -plasmalemma ATP-ase, which should play an with lateral shortwave UV-B radiation (WG 305 hi.), already important role in cell elongation despite of a controversial exhibited lower stem elongation after 5 h, which resulted in debate (Kraus et al., 1987; Hager et al., 1991; Rayle and Clea greater S.E. reduction at the end of the experiment (Fig. land, 1992; Schopfer, 1993). However, it can not be excluded 1 a). These results emphasize an important role of shortwave by our results that UV-B radiation acts directly on the H+UV-B radiation (WG 305 hi.) directed laterally to the hypo- ATPase, which was shown for ATPase in rose cells (Murphy, 1993). cotyl. Lower UV-B irradiances (WG 305) were less effective. Furthermore, the in vivo IAA concentration of sunflower It is generally assumed that in dicotyledonous plants IAA is mainly produced in the cotyledons and transported basi- seedlings UV-B irradiated for 4 days under a WG 305 filter petally to the hypocotyl, where it causes cell elongation was significantly reduced by 51% and 3-M formation also (Moore, 1989). Covering of the hypocotyl by a non-UV-B was measurable compared with the control (WG 360; data transmitting plastic film totally prevented the UV-B induced not shown). In our view, the inhibition of elongation growth in seedreduction of hypocotyl growth and again established the importance of the hypocotyl in controlling cell elongation. lings and hypocotyl segments of sunflower could be exCovering of the cotyledons and the apex had minor impor- plained firstly, by the reduction of the endogenous and/or tance. Nevertheless, synthesis of IAA in cotyledons seems to the exogenously applied IAA concentration and, secondly, be necessary for elongation growth. Removal of the cotyle- by the formation of growth inhibiting IAA photoproducts, dons resulted in an inhibition of stem elongation by 68 %, especially 3-M. Therefore, the action of UV-B via IAA-phowhich was also recently described for white light grown sun- tooxidation and formation of 3-M could be one of the possible models, by which UV-B radiation attenuates growth of flower seedlings (Kutschera, 1992). From all the results it seems possible that UV-B radiation sunflower seedlings. On the basis of the presented results the «IAA destrucinteracted with cell elongation mechanisms and IAA in the hypocotyl. In de-etiolated cucumber seedlings a single tion» seems to be a potent mechanism for the growth inhibi6-8h exposure to UV-B reduced the hypocotyl elongation tion of UV-irradiated sunflower seedlings grown at low rate by 50% (Ballare et al., 1991). However, this inhibition white light intensities. It seems reasonable that auxins could was mainly perceived by the cotyledons and is probably be the possible «photoreceptor» for UV-B radiation in terms caused through a specific UV-B photoreceptor, whereas a of regulating elongation growth. covering of hypocotyl and the apex has a small effect in preventing UV-B induced growth inhibition. Phytochrome and Acknowledgements UV-A/blue light receptors may not be involved, since a muThis work was supported by Bundesministerium fiir Forschung tant deficient in stable phytochrome also shows UV-B deund Technologic (BMFT), Germany. We would like to thank Ulli pendent hypocotyl reduction (Ballare et al., 1991). Wolfand E. Heene for excellent experimental and technical assistance. It is well known that white light has a destructive effect on the growth regulator IAA as shown in vitro (Fukuyama and Moyed, 1964) or during phototropic growth (Hager and References Schmidt, 1968 a). Therefore, it was assumed that UV-B radiation could act even better in photodestruction of IAA due to its high energy. The hypocotyl segment growth tests showed BA!LARE, C. L., P. W. BARNES, and R. E. KENDRICK: Photomorphogenetic effects of UV-B radiation on hypocotyl elongation in that UV-B induced inhibition of hypocotyl growth occurred wild type and stable-phytochrome-deficient mutant seedlings of also in excised hypocotyl segments. HSEG-test in A. demin., cucumber. Physiol. Plant. 83, 651-658 (1991}. IAA and preirradiated IAA indicated that the reduction of BANDURKSI, R. S.: Auxin transport and metabolism: the mechanism the hypocotyl segment growth was probably caused by the of tropic curvatures. Giornale botanico italiano 123, 321-335 (1989}. UV-dependent destruction of IAA and a formation of IAA photoproducts, which reduced stem elongation. The identi- BANDURKSI, R. s., A. SCHULZE, M. DESROSIERS, P. jENSEN, D. REINECKE, and B. EPEL: Voltage gated channels as transducers of envification of the IAA photoproducts in UV-B preirradiated ronmental stimuli. In: MoRRE, D. j. and W. F. Boss (eds.}: InosiIAA-solutions by HPLC resulted in five substances: 3-Hytol metabolism in plants, pp. 289-300. Wiley-Liss Inc. (1990}. droxymethyloxindole (3-0H), Indole-3-aldehyde, 3-MethylBARNES, P. W., P. W. joRDAN, W. G. GoLD, S. D. FLINT, and M. M. eneoxindole (3-M), 3-Methyloxindole and Indole. Hager and CALDWELL: Competition, morphology and canopy structure in Schmidt (1968 a) have already shown that IAA-solution wheat (Triticum aestivum L.) and wild oat (Avena fatua L.) excould be destroyed by strong white light and flavin as cataposed to enhanced ultraviolet-B radiation. Funct. Eco. 2, 319lyst to 3-0H and 3-M. UV-B radiation, however, needs no 330 (1988}.

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