Phototropism in seedlings of sunflower, Helianthus annuus L.

In this thesis the phototropic bending of hypocotyls of sunflower seedlings, Helianthus annuus L., is investigated.Chapter 1 gives the reasons for this project. Although phototropism has been studied extensively over the past 100 years, the understanding of the mechanism is far from clear. During this century two main hypotheses were developed, namely the theory of BLAAUW (1915), explaining phototropism as an effect of growth inhibition by light, and the CHOLODNY-WENT theory (1927, 1928), in which the lateral distribution and basipetal transport of the growth-promoting substance, auxin, are involved. Especially the latter received the attention of many investigators, probably because it deals with the first phytohormone discovered. The research on this subject has been mainly concentrated on the properties of auxin and the way in which it influences the pattern of curvature in etiolated coleoptiles. Much experimentation has been done with the exogenous application of diffusate from coleoptile tips or IAA, the auxin, to etiolated monocotyledonous seedlings, without measuring the endogenous levels of this growth substance in the plants themselves. In 1975 BRUINSMA et al., investigating the levels of IAA, in illuminated and shaded halves of phototropically stimulated, green sunflower seedlings, showed that this hormone is equally distributed between both halves. This discovery, and the fact that the evidence for both hypotheses mentioned above is still inconclusive, led to the present investigation.As experimental material sunflower seedlings, Helianthus annuus L., were chosen. Chapter 2 lists the material and methods used for the experiments described in the subsequent Chapters.Chapter 3 describes the influence of other processes on phototropism. It is shown that geotropism acts strongly on the deflected hypocotyl. Only the first hour after the beginning of the phototropic response can be considered to show pure phototropism. Because the delay in the onset of the response, the maximal degree of curvature is determined for each individual seedling during the first 2 hours of phototropic stimulation. The influences of the age of the seedling and length of the hypocotyl were studied as well. Seedlings of 4 to 5 days old and 40 to 60 mm long bend as uniformly as possible, and were used in the experiments described in this thesis. Careful manipulation of the seedlings did not influence the response.Chapter 4 deals with the influence of the other organs of the green seedling on the bending of the hypocotyl. It is concluded that neither the cotyledons and the shoot tip, nor the roots are essential for the response, although a stress effect due to the removal of these organs may cause a delay. The epidermis of the hypocotyl is indirectly involved; the peripheral layers are not necessary for the perception of the unilateral light, but probably control the growth, without which curvature cannot occur.In Chapter 5 the results of treatments with different wavelengths are shown. Unilateral blue light is the main active region, although the response caused by blue light is always slightly less than when caused by unilateral white light. In contrast to seedlings grown in white light, etiolated ones are not phototropically sensitive. Pretreatment of dark-grown seedlings with white or blue light renders them able to curve, and only illumination of the hypocotyl is necessary. The role of the cotyledons, that cannot be removed during this treatment without decreasing the bending, is probably to supply the substrates essential for the growth of the hypocotyl. Pretreatment with darkness of seedlings grown in white light makes them phototropically unresponsive. Again, only the hypocotyl needs this treatment and this indicates that the mechanism for phototropism is located within the hypocotyl. Green seedlings pretreated with red light only show poor curvature, but the growth rate of the seedlings is not related to the bending capacity.Chapter 6 presents the results of the effect of pretreatment with far-red irradiation on the phototropic response. It is shown that far red, more than a treatment in darkness, decreases the curvature in seedlings grown in white light and that the response is a low-energy reaction (L.E.R.). Red light did not affect the lack of bending of etiolated plants, but prolonged red illumination could reverse the effect of far-red irradiance in green seedlings. It is concluded that the perception of the far-red irradiance is located in both the hypocotyl and the cotyledons. Again, no correlation between the growth rate and the curvature of plants pretreated with far red can be detected.The involvement of hormones in the phototropic response is studied in Chapter 7. IAA is not unilaterally distributed in curving parts of the seedlings that were 30 to 45 min phototropically stimulated. No IAA could be detected in diffusates from the seedlings, and this absence is not due to destruction of IAA at the cut surface. However, a growth-inhibiting activity was found in the diffusates, both from hypocotyls and from cotyledons, and this activity was not asymmetrically distributed in bending plants.The role of the growth inhibitor, xanthoxin, was investigated. Experiments are described to show that xanthoxin is not contaminated with ABA after the extraction procedure, and that a bioassay can be properly used for the determination of xanthoxin. In straight hypocotyls xanthoxin is equally distributed, but in curving and curved ones more inhibitor can be extracted from the illuminated side than from the shaded one. From seedlings pretreated with white or blue light, which are phototropically sensitive, a higher amount of xanthoxin can be extracted than from etiolated, phototropically unresponsive, plants. In seedlings pretreated with red light, which show only a poor bending, a variable amount of xanthoxin was detected. The results of experiments in which xanthoxin was exogenously applied to seedlings were inconclusive.Gibberellic acid, GA 3 , applied to the seedlings pretreated with red light 1 hour before the onset of the phototropic experiment, increases the curvature, whereas GA 3 applied to plants grown in white light has no effect. The growth rate of the treated plants does not differ from that of the control group. Seedlings treated daily with various concentrations of the growth retardant CCC show different lengths after four days. Their phototropic curvature, however, is the same, again demonstrating that the extent of curvature is not related to the elongation growth.In the General Discussion (Chapter 8) the CHOLODNY-WENT theory is rejected as an explanation of phototropism in the sunflower seedling. Instead the theory of BLAAUW is modified by ascribing the phototropic reaction to a growth inhibition at the irradiated side, caused by light-induced relative accumulation of xanthoxin at that side. This may account for a curvature independent of the growth rate regulated by auxin and gibberellin.