Alex C. McCormac

A Test of the Adaptive Plasticity Hypothesis Using Transgenic and Mutant Plants Disabled in Phytochrome-Mediated Elongation Responses to Neighbors

Many plants display characteristic phytochrome-mediated stem elongation responses to crowding and vegetation shade, commonly referred to as the shade avoidance syndrome. We tested the hypothesis that this elongation is a form of adaptive plasticity by comparing the relative performance at high and low density of wild-type plants and transgenic and mutant plants in which the shade avoidance response was disabled. Transgenic tobacco plants in which elongation in response to neighbors was blocked by expression of the oat PHYA gene had decreased relative fitness when grown in competition with elongated wild-type plants. In contrast, constitutively elongated Brassica ein mutant plants, deficient in light-stable phytochrome, had lower fitness relative to nonelongated wild type at low density than in competition with elongated wild type at high density. The observation that phytochrome-mediated elongation is advantageous in dense stands, but disadvantageous for uncrowded plants, indicates that a response to foliage shade allows plants to develop an appropriate morphology for the level of competition they experience. This observation supports the adaptive plasticity hypothesis for this ecologically important trait.

Photoresponses of transgenic tobacco plants expressing an oat phytochrome gene

The physiological responses of transgenic tobacco (Nicotiana tabacum L.) plants that express high levels of an introduced oat (Avena sativa L.) phytochrome (phyA) gene to various light treatments are compared with those of wild-type (WT) plants. Seeds, etiolated seedlings, and light-grown plants from a homozygous transgenic tobacco line (9A4) constructed by Keller et al. (EMBO J, 8, 1005–1012, 1989) were treated with red (R), far-red (FR), or white light (WL) with or without supplemental FR light, revealing major perturbations of the normal photobiological responses. White light stimulated germination of both WT and transgenic seed, but addition of FR to the WL treatment suppressed germination. In the WT, all fluence rates tested inhibited germination, but in the transgenics, reduction effluence rate partially relieved germination from the FR-mediated inhibition. It is suggested that the higher absolute levels of the FR-absorbing form of phytochrome (Pfr) in the irradiated transgenics, compared to the WT, may be responsible for the reduced FR-mediated inhibition of germination in the former. Hypocotyl extension of dark-grown seedlings of both WT and transgenic lines was inhibited by continuous R or FR irradiation, typical of the high-irradiance response (HIR). After 2 d of de-etiolation in WL, the WT seedlings had lost the FR-mediated inhibition of hypocotyl extension, whereas it was retained in the transgenics. The FR-mediated inhibition of hypocotyl extension in the transgenic seedlings after de-etiolation may reflect the persistence of an, FR-HIR response mediated by the overexpressed oat PhyA phytochrome. Light-grown WT seedlings exhibited typical shade-avoidance responses when treated with WL supplemented with high levels of FR radiation. Internode and petiole extension rates were markedly increased, and the chlorophyll a∶b ratio decreased, in the low-R: FR treatment. The transgenics, however, showed no increases in extension growth under low-R: FR treatments, and at low fluence rates both internode and petiole extension rates were significantly decreased by low R ∶ FR. Interpretation of these data is difficult. The depression of the chlorophyll a∶b ratio by low R ∶ FR was identical in WT and transgenic plants, indicating that not all shade-avoidance responses of light-grown plants were disrupted by the over-expression of the introduced oat phyA gene. The results are discussed in relation to the proposal that different members of the phytochrome family may have different physiological roles.

Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.

PHOTORESPONSES OF Arabidopsis SEEDLINGS EXPRESSING AN INTRODUCED OAT phyA cDNA: PERSISTENCE OF ETIOLATED PLANT TYPE RESPONSES IN LIGHT‐GROWN PLANTS

The photocontrol of hypocotyl elongation has been studied in etiolated and light‐grown wild type (WT) Arabidopsis thaliana (L. Heynh) seedlings, and in two homozygous isogenic lines that have been transformed with the oat phy A gene coding sequence under the control of the cauliflower mosaic virus (CaMV) 35S promoter. For etiolated seedlings the inhibition of hypocotyl elongation by continuous broad band far‐red light (FR) is saturated at much lower photon fluence rates in the transgenic seedlings compared with WT seedlings. Furthermore, whereas de‐etiolation of WT seedlings leads to loss of responsiveness of the hypocotyls to prolonged FR, de‐etiolated transgenic seedlings continue to show a pronounced FR‐mediated inhibition of elongation. This may reflect the persistence of a FR‐high irradiance response (HIR) mediated by the introduced oat phytochrome A. Although the hypocotyls of light‐grown transgenic seedlings display a qualitatively normal end‐of‐day FR growth promotion, such seedlings display an aberrant shade‐avoidance response to reduced red:far‐red ratio (R:FR). These results are discussed in relation to the proposal that the constitutive expression of phytochrome A leads to the persistence of photoresponse modes normally restricted to etiolated plants.

Contrasting Responses of Etiolated and Light-Adapted Seedlings to Red: Far-Red Ratio: A Comparison of Wild Type, Mutant and Transgenic Plants has Revealed Differential Functions of Members of the Phytochrome family

Summary The photosensory roles of the light-labile phytochrome A and light-stable phytochrome B in etiolated and light-adapted plants were studied using wild type (WT) seedlings, and seedlings of photomorphogenic mutant and transgenic plants. Under continuous white light (WL) red (R): far-red (FR) ratio = 6.8), hypocotyl elongation growth of etiolated seedlings of WT mustard, Arabidopsis , tomato and tobacco was strongly inhibited, and anthocyanin synthesis was stimulated in mustard and tomato. During the initial period following transfer from darkness, supplementation of WL with far-red radiation (WL + FR) (R:FR = 0.18) caused increased levels of growth inhibition in mustard, Arabidopsis and tobacco and higher levels of anthocyanin synthesis in mustard and tomato. Subsequent to a period of light adaptation (which varied in length according to species) elongation growth rate became accelerated and total levels of anthocyanin decreased under WL + FR relative to WL alone. This transition in photomorphogenic sensitivity to FR was variously impaired in the phytochrome B-deficient mutant of Arabidopsis ( hy3 ), the phytochrome A-deficient mutant of tomato ( aurea ) and transgenic lines of tobacco and tomato expressing an introduced oat- phy A-gene. Overall growth acceleration as stimulated by exogenous gibberellin A 3 did not alter the growth response to FR mediated by the heterologous phytochrome A of the transgenic tobacco. The data are consistent with differential roles for phytochrome A and B in photomorphogenic sensitivity to the R : FR ratio of the light environment; namely phytochrome A operates via the FR-mediated high irradiance reaction, and the light-stable phytochromes, including phytochrome B, regulate response according to the photoequilibrium established by the R : FR ratio.

Photoregulation of germination in seed of transgenic lines of tobacco and Arabidopsis which express an introduced cDNA encoding phytochrome A or phytochrome B

Photoinduction and photoinhibition of germination in seed from a homozygous tobacco (Nicotiana tabacum L.) line containing an introduced oat phyA cDNA (encoding phytochrome A) is compared with that of isogenic wild-type (WT) tobacco. Under continuous irradiation by a light source with a low red∶far-red (R∶FR) ratio the transgenic tobacco seed appeared to be less susceptible to photoinhibition of germination compared with WT seed. However, induction of germination following a short pulse by R (666 nm) was not enhanced in the genotype transformed by oat phyA cDNA compared with the WT; neither did germination of the transgenic tobacco seed show an increased sensitivity to saturating pulses of light of longer wavelengths (666–730 nm). In seeds of transgenic Arabidopsis thaliana (L.) Heynh. which contained an introduced phytochrome-B-encoding cDNA, levels of dark germination were enhanced, consistent with mediation of response by phytochrome B-Pfr. The germination behaviour of Arabidopsis genotypes wich contained an introduced cDNA encoding phytochrome A, however, did not significantly differ from that of the WT.

Do the Members of the Phytochrome Family have Different Roles? Physiological Evidence from Wild-Type, Mutant and Transgenic Plants

The realisation that phytochrome represents a family of photoreceptors, encoded by at least three, and probably more, nuclear genes (Sharrock and Quail, 1989), has led to the proposal that the members of the family may have differential roles in the regulation of development and metabolism (see Smith and Whitelam, 1990, for review). In principle, this idea simplifies our understanding of the complexity of photomorphogenesis, by providing a potential resolution of the many conflicts in the physiological data. Investigations over several decades have shown that a number of different response modes, separable on classical photobiological grounds, can be incontrovertibly attributed to phytochrome, but this multiplicity of roles has been extremely difficult to reconcile with the classical concept of phytochrome as a single photoreceptor. Such conflicts may be traced back at least two decades, to the proposal by Hillman (1967) that there must be at least two populations of phytochrome, one that is ‘active’ but present in low concentration, and one that is ‘bulk’, i.e., present in high concentration and responsible for the spectrophotometric observations but not active in the physiological responses. It is, therefore, tempting to speculate that different phytochrome-mediated response modes may be mediated by different phytochromes. This article considers this idea and some of the evidence that currently appears to support it. We conclude that the hypothesis is at least sustainable on present evidence, but the attractiveness of the concept must be weighed in balance with the implications it has for the central dogmas of photomorphogenesis.