New Phytol. (1982) 90, fill 618 PHOTOMORPHOGENESIS IN IMP A TIENS PAR VIFLORA AND OTHER PLANT SPECIES UNDER SIMULATED NATURAL CANOPY RADIATIONS Department BY G. C. WHITELAM* AND C. B. JOHNSON of Botany, Plant Science Laboratories, University of Reading, Whlteknights, Reading RG6 2AS, U.K. (Accepted 19 October 1981) SUMMARY Nitrate reductase activity in the leaves of Si/tapis alba L. and Chenopodium album L. was found to be related to the photoequilibrium of phytochrome established under simulated natural canopy radiation (fluorescent white light with added far-red light) of a relatively high photon Huence rate. Under simulated canopy radiation of low photon Huence rate, more characteristic of 'shade' light, the degree of responsiveness of S. alba nitrate reductase has been seen to be reduced. However, in the leaves of Impatiens parviflora L., a species frequently occurring in dense canopy shade, nitrate reductase activity showed a rapid and marked response to changes in phytochrome photoequilibrium with the low-fluence rate sources. Under these same conditions the elongation growth of /. parviflora internodes also showed a marked and rapid response to changes in the phytochrome system, rather than displaying a reduced response as has been proposed for shade tolerating species. INTRODUCTION It has been proposed that a central function of phytochrome, the red/far-red (R/FR) photoreversible plant photoreceptor, is the detection of mutual shading and the subsequent redirection of development of plants in the natural environment (e.g. Morgan and Smith, 1976, 1978, 1979; Holmes and Smith, 1977). This suggestion has been supported by numerous observations that changes in light quality in the R and FR regions of the visible spectrum, characteristic of those resulting from the attenuation of polychromatic light by a vegetation canopy, effect major alterations in the growth and development of a number of plant species (e.g. McLaren and Smith, 1978; Morgan and Smith, 1979; Morgan, O'Brien and Smith, 1980). The responses reported by Smith and his co-workers have been observed under simulated natural radiation whereby increased canopy shade is mimicked by the addition of increased amounts of supplementary FR light to a constant source of white fluorescent light, such that the R: P"R ratio is significantly decreased, resulting in a decrease in the photoequilibrium of phytochrome (Holmes and Smith, 1977). The photosynthetically active radiation (PAR) provided by these sources is uncharacteristic of shade light, however, being typically in the region of 130 /*mol m ' ^ s~^. Consequently, very high fluence rates of FR light (up to as much as 500 //mol m"'' s~'; even more atypical of shade light) are required in Present address: Department of Biological Sciences, University of Dundee, Dundee DDl 4HN, U.K. Abbreviations: PAR, photosynthetically active radiation; R, red light; FR, far-red light; (p,,, phytochrome photoequilibrium, estimated from the ratio of the photon Huence rates at 6fiO:73O nni; (^j^, phytochrome photoequilibrium (= [Pi,]/[Pr] + [Ptr]) measured in etiolated mung bean hypocotyl hooks. 0028-646X/82/040611 +08 $03.00/0 1982 The New Phytologist 21 ANI'9O
6i2 G. C. WHITELAM AND C. B. JOHNSON order to reduce phytochrome photoequilibria to values which are characteristic of those obtaining in shade light. Nevertheless, using these sources many growth responses have been found to be related to (j)^. Furthermore, Morgan and Smith (1979) have proposed that a systematic relationship exists between responsiveness to reduced (j)^ and species habitat based upon the observation that shade-tolerant woodland species show little or no response to a decrease in R:F'R ratio. Firm conclusions regarding the reduced responsiveness of woodland species are difficult to draw, however, because of the considerably lower growth rates of such species compared with those from more open habitats. We have recently described a novel approach to the design of polychromatic light sources which simulate canopy light (Wall and Johnson, 1981). The light provided by these sources is significantly depleted in the blue and R regions of the spectrum, a characteristic of shade light, and is of only 13/^mol m"^ s~i PAR, again characteristic of woodland shade on a sunny day. For this reason, significantly lower fluence rates of FR light (up to 50 //mol m"'^ s^*) are required to reduce phytochrome photoequilibria to values typical of dense shade. Employing these sources Wall and Johnson (1981) have observed relationships between (j) and elongation growth of the mustard seedling, similar to those reported by Smith's group. However, the low PAR provided by these sources poses problems when responses such as nitrate reductase activity are observed in such shade-intolerant species as 5. alba (Johnson and Whitelam, 1982). The 13 /imox m"^ s^i provided by the sources is likely to be below the light compensation point for photosynthesis and hence the capacity of the plant to respond to changes in the phytochrome system may be limited by the supply of photosynthetic products. Hence, these sources are unsuitable for long-term experimentation with shade-intolerant species. This problem may be expected to be particularly serious with respect to the photocontrol of nitrate reductase since this enzyme requires a source of photosynthetically-derived reducing power for its catalytic activity (Beevers and Hageman, 1972). For these reasons we have investigated the responses of Impatiens parviflora to simulated canopy light at low fluences of PAR. Although /. parviflora is a shade-tolerant woodland species per se and is frequently found growing under dense canopies, it is nevertheless of extremely wide distribution and it does retain the capacity for a high growth rate. There is a considerable body of published work pertaining to the effects of quantitative change in the radiation environment on Impatiens, involving both artificial controlled environments (e.g. Hughes, 1965) and under neutral screens in the field (e.g. Evans and Hughes, 1961) but only relatively little analysis of the effects of light quality on its growth and development (see Young, 1975 for a review). Thus it seemed appropriate to extend these observations with an examination of the effects of shade light quality on this remarkable woodland annual. The seedlings used in this investigation were collected from a densely shaded habitat where PAR was typically below 20 fimol m^ s~i; nevertheless the seedlings' responses were found to be more characteristic of those of species from more open habitats as defined by Morgan and Smith (1979). We report here some studies of the responses of /. parvifiora to added FR light and have compared them with those of Chenopodium album, an arable weed, and Sinapis alba, a vigorous shade-avoider, under simulated canopy radiations with high fluence rates.
Photomorphogenesis under natural canopy 613 MATERIALS AND METHODS Plant material and growth conditions Uniform seedlings oi Impatiens parviflora L., at the first leaf pair stage, were collected from dense shade cast by Aesculus hippocastum in the 'Wilderness' at Reading University campus. Seedlings were transplanted to pots of peat and maintained in a controlled environment growth room prior to use. Mature seedlings of Chenopodium alhutn L. were obtained by germinating seeds, originally collected from Field 12 on the Experimental Farm, Sutton Bonington, Leics., in sandwich boxes on moist blotting paper. After 14 days seedlings were transplanted to pots containing a potting compost (John Innes, No. 1.) The seedlings were grown up until 28 days old before treatment for a further 14 days. Light-grown Sinapis alba L. plants were obtained by germinating seeds (Agrow, Freiburg- Ebnet), which had previously been imbibed for 6 h with distilled water, on the surface of moist peat in 5 cm pots. Seedlings were grown up under controlled conditions for 14 days prior to treatment for 2 days. Light sources The broad band light sources of high PAR (130//mol m"^ s"') were those described by McLaren and Smith (1978) and the broad band sources of low PAR (13 fimol m~^ s"') have been described in detail by Wall and Johnson (1981). Assay of nitrate reductase Nitrate reductase was determined either by the in situ method described by Whitelam and Johnson (1980) or by the in vitro method also previously described (Whitelann, Johnson and Smith, 1979). In each case, qualitatively similar results were obtained if the alternative assay was used. The results are typically the mean of six replicates, the bar lengths being equivalent to twice the standard error. Phytochrofne photoequilibria The values given were either estimated from the R:FR photon fluence rate ratios at 660 and 730 nm (cf. Morgan and Smith, 1976) and designated ^5^, or were based on nneasurements of the phytochrome photoequilibria obtained in etiolated mung bean hooks irradiated at 0 C {ipm)- Because of selective screening by chlorophyll they are not necessarily an accurate reflection of the values obtaining in the green plant tissues but are systematically related to these (see Morgan and Smith, 1978). RESULTS AND DISCUSSION The growth responses of a range of plant species to simulated shade light of a high fluence rate have been well documented (e.g. Morgan and Smith, 1978, 1979). A general feature of the responses, reported by Smith and co-workers, is an apparent linear relationship between elongation growth rate and phytochrome photoequilibrium in the range (j)^ 0-20 to ^p 0'70. Similar results are often obtained also with nitrate reductase: the data in Figure 1 for C. album and in Figure 2 for S. alba are typical of those obtained in the high fluence rate sources employed by Smith's group. In both cases the relationship approaches linearity, as for the growth responses, and as observed for nitrate reductase activity in light-grown cauliflower curd (Whitelam, Johnson and Smith, 1979). Indeed, a relationship between photoequilibrium and nitrate reductase activity with greater enzyme activities at
G. C. WHITELAM AND C. B. JOHNSON 0-7 Fig. 1. The relationship between estimated phytochrome photoequilibrium {(^,.) and nitrate reductase activity, assayed in vitro, from the third leaves of 42-day-old Chenopodium album seedlings. The seedlings were grown in a standard pre-treatment controlled environment for 28 days before treatment. PAR during the treatment was 130//mol m"'' s~'. 0-7 Fig. 2. The relationship between estimated phytochrome photoequilibrium ((ij.) and nitrate reductase activity, assayed in vitro from the second leaves of 16-day-old Sinapis alba seedlings (PAR = 130/ymol m^'s-'). The seedlings were grown in a standard pre-treatment controlled environment for 12 days before treatment. higher photoequilibria seems to be a characteristic feature in many i\x\\ de-etiolated light-grown plants under these conditions. Under simulated shade light quality and low fluence rate, Wall and Johnsoi (1981) have shown that the growth responses of S. alba are broadly comparabl with those observed under higher fluence rates. However, we find that at lo\^, fluence rates the responsiveness of nitrate reductase activity from 5. alba leaves appears to be reduced as compared with the data in Figure 2, for the higher fluenc rate sources (Fig. 3). It is possible that a limitation of the capacity to respond is the result of the reduced rate of photosynthesis under the low fluence rate sources When seedlings of /. parviflora, a shade tolerating species, at the second leaf
Photomorphogenesis under natural canopy 615 0-1 Fig. 3. The relationship between phytochrome photoequilibrium (^i,,,) and nitrate roductase activity, assayed in situ, from the second leaf of 16-day-old light-grown Siiia/iis alba seedlings. Added FR light was provided by an Oriel RG N9 colour glass filter: PAR was 13 /;mol m"'^ s~'. Seedlings were grown for 14 days in pre-treatment controlled conditions prior to treatment. Fig. 4. The relationship between phytochrome photoequilibrium (^i,,,) and the linear growth, over 72 h, of the first internode of Impaliensparviflora seedlings: PAR was 13/;mol m""- s~'. Added FR light was provided by an Oriel RG 715 colour glass filter. pair stage are treated under simulated shade light and low fluence rate, there is a marked and rapid photomodulation of elongation growth. It can be seen from Figure 4 that after only 3 days of treatment there is a ninefold increase in elongation of the first internode when sufficient FR light is added to reduced phytochrome photoequilibrium to <j) 010. In Figure 5 a typical batch of such seedlings is shown following 3 days treatment. The increased elongation of the internodes is the most dramatic of the observed responses but a number of other light quality effects are apparent. For example, petiolar elongation and the angle at which the
6i6 G. C. WHITELAM AND C. B. JOHNSON Fig. 5. Impatiens parviflora seedlings treated for 3 days under polychromatic radiations of equal PAR (13 /tmol m~^ s~'), but with diflerent amounts of added FR light, establishing different phytochrome photoequilibria. The phytochrome photoequilibria (^6,,,) were, from left to right, 010, 0-25, 0-40, 0-55 and 0-67. 180 fresti wt) 160 en 140 / o' 1 120 100 / 1ie activ Nitrate 80 60 40 20 0-2 0-3 0-4 0-5 0-6 0-7 I'ig. 6. The relationship between phytochrome photoequilibrium (96,,,) and nitrate reductase activity, assayed in situ, from the third leaf of Impatiens parviflora seedlings: PAR was 13 /ymol m~^ s"'. The seedlings, which were collected from a densely shaded habitat, were kept in a controlled environment growth room prior to treatment for 3 days.
Photoniorphogenesis under natural canopy 617 petiole is held are also modulated by added FR light. The extent and rapidity of this growth response is as great as the responses of many shade-intolerent species from more open habitats as reported by Morgan and Smith (1979) employing higher fluence rate sources. A similarly marked and rapid response to shade light quality is observed with nitrate reductase activity is determined in the third leaf of /. parviflora seedlings after 3 days of treatment (Fig. 6). The results obtained represent the greatest degree of responsiveness that we have yet observed between nitrate reductase activity and photoequilibrium in the range (j)^ 0-10 to ^i,,, 0-67, there being a fivefold difference in enzyme activity as compared with a 30 to 40% difference observed in C. album and S. alba (Figs 1 and 2). The relationship obtained in Figure 6 is not the characteristically linear correlation as previously observed. One reason for this might be that differential attenuation of the fluorescent and the FR light within the leaf results in a shifting of photoequilibria to lower values when the leaf is irradiated with such mixtures (the (j) = 0-67 value is obtained with the PAR source alone and no added FR). The ability of seedlings of I. parviflora to display such marked responses to added FR light is possibly a consequence of their ability to tolerate the very reduced fluence rates of PAR often found in their natural habitats. Despite the low fluence rates the seedlings maintain the capacity for rapid growth rate, and can effect modulations in nitrate reductase activity as a result of changes in the phytochrome systenn. It may be suggested, therefore, that the lack of capacity to respond quickly to added FR light, for example the reduced responsiveness of S. alba leaf-nitrate reductase under low fluences and the apparent reduced responsiveness of some woodland species to added FR light is due either to a limitation in the availability of some photosynthetically derived substrate(s) or to an inherently low growth rate. Care should therefore be taken when categorizing plant species on the basis of their responsiveness to lowered photoequilibrium. A species able to tolerate dense shade, such as /. parviflora, has been found to be as responsive to added FR light as any 'open-habitat' species. ACKNOWLEDGEMENT This Avork was supported by an N.E.R.C. studentship to G.C.W. We thank Professor H. Smith for the provision of some of the light sources used here. REFERENCES BEEVERS, L. & HAGEMAN, R. H. (1972). The role of light in nitrate metabolism in higher plants. Photophysiology, 7, 85-113. EVANS, G. C. & HUGHES, A. P. (1961). Plant growth and the aerial environment. L Effect of artificial shading on Impatiens parviflora. New Phytotngist, 60, 150-180. HOLMES, M. G. & SMITH, II. (1977). The function of phytochrome in the natural environment. IV. Light quality and plant development. Pholochcmistry and Photobiology, 25, 551-557. HUGHES, A. P. (1965). Plant growth and the aerial environment. IX. A synopsis of the autecology of/;«/)o/;>hs parviflora. New Pliytotogisl, 64, 399-413. JOHNSON, C. B. & WHITEI.AM, G. C. (1982). Phytochrome action in light-grown plants: the control of nitrate reduction as a model response. Photochemistry and Photohiotogy, 35, 251-254. MCLAKEN, J. S. & SMITH, H. (1978) The function of phytochrome in the natural environment. VI. The growth and development of Rumex ohtensifotius under simulated canopy light environments. Ptant, Celt and EriTtirowfient, 1, 61-67.
6i8 G.C.WHITELAMANDC. B.JOHNSON MORGAN, D. C, O'BKIEN, T. & SMITH, H. (1980). Rapid photomodulation of stem extension in light-grown Sinapis alba L. Planta, 150, 95 101. MORGAN, D. C. & SMITH, H. (1976). Linear relationship hetween phytochrome photoequilibrium and growth in plants under simulated natural radiation. Nature, 262, 210-212. MORGAN, D. C. & SMITH, H. (1978). The relationship between phytochrome photoequilibrium and development in light-grown Chenopodium album. Planta, 142, 187 193. MORGAN, D. C. & SMITH, H.( 1979). A systematic relationship between phytochrome-controued development and species habitat, for plants grown in simulated natural radiation. Planta, 145, 253-258. WALL, J. K. & JOHNSON, C. B. (1981). The influence of light quality and light quantity in the control of extension growth in light-grown Sinapis alba L. Planta, 153, 101-108. WHITELAM, G. C. & JOHNSON, C. B. (1980). Phytoehrome control of nitrate reductase activity and anthocyanin synthesis in light-brown Sinapis alba (L.). Differential responses of cotyledons and hypocotyls. New Phytologist, 85, 475-482. WHITELAM, G. C, JOHNSON, C. B. & SMITH, H. (1979). The control by phytochrome of nitrate reductase in the curd of light-grown cauliflower. Photochemistry and Photobiologv, 30, 589-594. YOUNG, J. E. (1975). Effects of the spectral composition of light sources on the growth of a higher plant In: Light as an Ecological Factor II, Symposia of the British Ecological Society, vol. 16, pp. 135 160 Blackwell, Oxford.