R.E. Kendrick

The aurea mutant of tomato is deficient in spectrophotometrically and immunochemically detectable phytochrome.

The aurea locus mutant (auw) of tomato contains less than 5% of the level of phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. Immunoblot analysis using antibodies directed against etiolated-oat phytochrome demonstrates that crude extracts of etiolated mutant tissue are deficient in a major immunodetectable protein (116 kDa) normally present in the parent wild type. Analyses of wild-type tissue extracts strongly indicate that the 116-kDa protein is phytochrome by showing that this protein: a) is degraded more rapidly in vitro after a brief far-red irradiation than after a brief red irradiation (Vierstra RD, Quail PH, Planta 156: 158–165, 1982); b) contains a covalently bound chromophore as detected by Zn-chromophore fluorescence on nitrocellulose blots; and c) has an apparent molecular mass comparable to phytochrome from other species on size exclusion chromatography under non-denaturing conditions. The demonstration that the aurea mutant is deficient in this 116-kDa phytochrome indicates that the lack of spectrally detectable phytochrome in this mutant is the result of a lesion which affects the abundance of the phytochrome molecule as opposed to its spectral integrity.

Immunochemically detectable phytochrome is present at normal levels but is photochemically nonfunctional in the hy 1 and hy 2 long hypocotyl mutants of Arabidopsis.

The hy 1 and hy 2 long hypocotyl mutants of Arabidopsis thaliana contain less than 20% (the detection limit) of the phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. In contrast, spectral measurements for the hy 3, hy 4, and hy 5 long hypocotyl mutants indicate that they each contain levels of phytochrome equivalent to the wild-type parent. Immunoblot analysis using a monoclonal antibody directed against the chromophore-bearing region of etiolated-oat phytochrome demonstrates that extracts of all mutant and wild-type Arabidopsis tissues, prepared by extraction of proteins into hot SDS-containing buffer, have identical levels of one major immunodetectable protein (116 kDa). An assay involving controlled in vitro proteolysis, known to produce distinctive fragmentation patterns for Pr and Pfr (Vierstra RD, Quail PH, Planta 156: 158–165, 1982), indicates that the 116 kDa polypeptide from the wild-type parent represents Arabidopsis phytochrome. The 116 kDa protein from either hy 3, hy 4, or hy 5 displays the same fragmentation pattern found for the wild type. Together with the spectral data, these results indicate that the mutant phenotype of these variants does not involve lesions in the polypeptide sequence that lead to gross conformational aberrations, and suggest that the genetic lesions may affect steps in the transduction chain downstream of the photoreceptor. In contrast, this same analysis for hy 1 and hy 2 has revealed that the 116 kDa protein from either of these mutants is not degraded differently in response to the different wavelengths of irradiation given in vitro. Moreover, whereas immunoblot analysis of tissue extracts from light-grown wild-type seedlings show that the 116 kDa phytochrome protein level is greatly reduced relative to dark-grown tissue as expected, similar extracts of light-grown hy 1 and hy 2 seedlings contain the 116 kDa polypeptide in amounts equivalent to those of dark-grown tissue. Combined, these data indicate that the hy 1 and hy 2 mutants both produce normal levels of immunochemically detectable phytochrome that is photochemically nonfunctional.

A Temporarily Red Light-Insensitive Mutant of Tomato Lacks a Light-Stable, B-Like Phytochrome

We have selected four recessive mutants in tomato (Lycopersicon esculentum Mill.) that, under continuous red light (R), have long hypocotyls and small cotyledons compared to wild type (WT), a phenotype typical of phytochrome B (phyB) mutants of other species. These mutants, which are allelic, are only insensitive to R during the first 2 days upon transition from darkness to R, and therefore we propose the gene symbol tri (temporarily red light insensitive). White light-grown mutant plants have a more elongated growth habit than that of the WT. An immunochemically and spectrophotometrically detectable phyB-like polypeptide detectable in the WT is absent or below detection limits in the tri1 mutant. In contrast to the absence of an elongation growth response to far-red light (FR) given at the end of the daily photoperiod (EODFR) in all phyB-deficient mutants so far characterized, the tri1 mutant responds to EODFR treatment. The tri1 mutant also shows a strong response to supplementary daytime far-red light. We propose that the phyB-like phytochrome deficient in the tri mutants plays a major role during de-etiolation and that other light-stable phytochromes can regulate the EODFR and shade-avoidance responses in tomato.

High pigment mutants of tomato exhibit high sensitivity for phytochrome action

Summary Anthocyanin synthesis and hypocotyl growth have been studied in tomato ( Lycopersicon esculentum Mill.) seedlings of high pigment ( hp ) mutants, aurea ( au ) mutants deficient in the labile type of phytochrome, an auhp double mutant and wild types. The phytochrome controlled [red light (RL)/far-red light reversible] anthocyanin synthesis occurring in a 24 h dark period after a 12 h pretreatment with RL or blue light (BL) is similar in the hp mutant, whereas in the case of the wild type, pretreatment with BL is more effective than RL. When grown under continuous broad-band UV-A, BL and RL for 5 d the au and auhp double mutants only accumulate low levels of anthocyanin compared to the wild type and the hp mutant. Under these conditions the hp mutant accumulates the highest levels of anthocyanin, but the relative effectiveness of RL and BL in the hp mutant and the wild type is reversed (BL being more effective than RL in wild type, whereas in the hp mutant RL is more effective than BL). These results suggest that the hp mutation enables maximal anthocyanin synthesis to be achieved without activation of the BL photoreceptor. When grown for 7 d in a regime of 14 h white light/10 h darkness the activation of anthocyanin synthesis is reduced in the au and auhp mutants compared to the wild type and the hp mutant. After the same treatment, the hypocotyls of hp -mutant seedlings of each cultivar are similar in length to those of their corresponding wild types and those of the au and auhp mutants are longer. However, after 5 d continuous low fluence rate (3 μ-tmol m- 2 s- 1 ) UV-A, BL and RL, the hypocotyl growth of the hp mutant is inhibited more than the wild type. The auhp double mutant shows a small but significantly higher anthocyanin accumulation than the au mutant, as well as increased inhibition of hypocotyl growth. It is proposed that the hp mutation increases the sensitivity to the labile phytochrome pool.

Photophysiology of a Tomato Mutant Deficient in Labile Phytochrome

Summary Photomorphogenetic responses have been studied in a tomato (Lycopersicon esculentum Mill.) aurea (au) mutant which has yellow-green leaves and elongated hypocotyls when grown in white light. Whereas etiolated tissue of the au mutant contains

Photomorphogenetic Responses of a Long Hypocotyl Mutant of Cucumis sativus L.

Summary Photomorphogenetic responses have been studied in a Cucumis sativus mutant (lh) which has long hypocotyls in white light (WL) compared to its wild type. Seeds and dark-grown etiolated seedlings of the lh mutant and wild type contain similar levels of spectrophotometrically detectable phytochrome. In continuous WL after 20 d there is a six-fold difference in hypocotyl length as a result of differences in cell elongation since the hypocotyls were estimated to be composed of similar numbers of cells. Although de-etiolation was retarded in the lh mutant in red light (RL), RL/far-red light reversibility of hypocotyl elongation and cotyledon expansion was shown. However, de-etiolated seedlings (seedlings pre-irradiated with 8 h WL) showed no inhibition of hypocotyl growth or promotion of cotyledon expansion in response to RL. Covering of one cotyledon of de-etiolated wildtype seedlings resulted in a marked curvature of the hypocotyl in RL given from above; the lh mutant did not exhibit this simulated phototropism. Little difference in responsiveness of wild type and lh mutant to UV-A and blue light (BL) was observed. It is provisionally concluded that this lh mutant lacks phytochrome function in light-grown plants perhaps due to the absence or lack of function of light stable phytochrome. The usefulness of the lh mutant for understanding the role of phytochrome and the BL-photoreceptor in controlling photomorphogenesis in light-grown plants is discussed.

Phytochrome control of anthocyanin biosynthesis in tomato seedlings : Analysis using photomorphogenic mutants

Anthocyanin biosynthesis has been studied in hypocotyls and whole seedlings of tomato (Lycoperskon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. In white light (WL)/dark (D) cycles the fri1 mutant, deficient in phytochrome A (phyA), shows an enhancement of anthocyanin accumulation, whereas the tri1 mutant, deficient in phytochrome Bl (phyBl) has a WT level of anthocyanin. Under pulses of red light (R) or R followed by far‐red light (FR) given every 4 h, phyA is responsible for the non‐R/FR reversible response, whereas phyBl is partially responsible for the R/FR reversible response. From R and blue light (B) pretreatment studies, B is most effective in increasing phytochrome responsiveness, whereas under R itself it appears to be dependent on the presence of phyBl. Anthocyanin biosynthesis during a 24 h period of monochromatic irradiation at different flu‐ence rates of 4 day‐old D‐grown seedlings has been studied. At 660 nm the fluence rate‐response relationships for induction of anthocyanin in the WT are similar, yet complex, showing a low fluence rate response (LFRR) and a fluence rate‐dependent high irradiance response (HIR). The high‐pigment‐1 (hp‐1) mutant exhibits a strong amplification of both the LFRR and HIR. The fri1 mutant lacks the LFRR while retaining a normal HIR. In contrast, a transgenic tomato line overexpressing the oat PHYA3 gene shows a dramatic amplification of the LFRR. The tri1 mutant, retains the LFRR but lacks the HIR, whereas the fri1, tri1 double mutant lacks both components. Only an LFRR is seen at 729 nm in WT; however, an appreciable HIR is observed at 704 nm, which is retained in the tri1 mutant and is absent in the fri1 mutant, indicating the labile phyA pool regulates this response component.

Phytochrome, gibberellins, and hypocotyl growth : a study using the cucumber (Cucumis sativus L.) long hypocotyl mutant

The possible involvement of gibberellins (GAs) in the regulation of hypocotyl elongation by phytochrome was examined. Under white light the tall long hypocotyl (lh) cucumber (Cucumis sativus L.) mutant, deficient in a type B-like phytochrome, shows an increased “responsiveness” (defined as response capability) to applied GA4 (the main endogenous active GA) compared to the wild type. Supplementing far-red irradiation results in a similar increase in responsiveness in the wild type. Experiments involving application of the precursor GA9 and of an inhibitor of GA4 inactivation suggest that both the GA4 activation and inactivation steps are phytochrome independent. Endogenous GA levels of whole seedlings were analyzed by combined gas chromatography-mass spectrometry using deuterated internal standards. The levels of GA4 (and those of GA34, the inactivated GA4) were lower in the lh mutant under low-irradiance fluorescent light compared with the wild type, similar to wild type under higher irradiance light during the initial hypocotyl extension phase, and higher during the phase of sustained growth, in which extension involved an increase in the number of cells in the upper region. In all cases, growth of the lh mutant was more rapid than that of the wild type. It is proposed that GA4 and phytochrome control cell elongation primarily through separate mechanisms that interact at a step close to the terminal response.

Response of light-grown wild-type and long hypocotyl mutant cucumber plants to end-of-day far-red light.

Abstract— A long‐hypocotyl mutant (lh) of cucumber (Cucumis sativus L.) has been studied which has previously been shown to lack phytochrome control of growth in de‐etiolated seedlings and thought to be modified with respect to the light‐stable type of phytochrome. We have analyzed the response of lh mutant and isogenic wild‐type (WT) plants to daily treatment with end‐of‐day far‐red light (EODFR). Only the WT responded to this treatment resulting in a large increase in internode length; an increase in petiole length; changes in leaf development (increased area, decreased thickness and reduction in indentation); redistribution of dry matter from leaf blades to stem; increased apical dominance and promotion of tendril formation. There were only small or no significant effects on chlorophyll and total carotenoid content, chlorophyll alb ratio, soluble protein levels and net photosyn‐thetic rates. The lh mutant failed to respond to EODFR treatment, and had the appearance of a shade‐avoiding plant growing in extreme shade. The lh mutant appears to completely lack the phytochrome responses attributable to the type of phytochrome that is active in shade detection. A discussion of the possible roles of the stable and labile types of phytochrome in light grown plants follows.

Response of light-grown wild-type and aurea-mutant tomato plants to end-of-day far-red light.

Abstract The effect of end-of-day (EOD) far-red (FR) light treatment on tomato (Lycopersicon esculentum Mill.) plants of the wild type (WT) and an aurea (au) mutant (which is deficient in the spectrophotometrically detectable light-labile phytochrome pool in etiolated seedlings) was studied. Both the WT and au mutant exhibit a quantitatively similar EOD-FR response (i.e. stimulation of elongation growth) indicating that phytochrome is functional in light-grown seedlings of the au mutant. However, no dramatic effects of EOD-FR light on leaf elongation, chlorophyll, carotenoid and soluble protein levels were observed in either the WT or au mutant. The au mutant contained less chlorophyll and more soluble protein per unit fresh weight than the WT. Although practically no anthocyanin could be detected in leaves of the au mutant, the WT showed a tenfold reduction in anthocyanin in response to EOD-FR light. The lower productivity of the au mutant compared with the WT results from its reduced leaf surface area, since its maximum rate of photosynthesis or photosynthetic efficiency appears to be slightly higher than that of the WT. Phytochrome was extracted and partially purified from light-grown plants of the WT and au mutant and was quantified by spectrophotometry. On a fresh weight basis the phytochrome content of au-mutant plants was 66% of that of WT plants. It is proposed that the light-labile phytochrome pool regulates the synthesis of the photosynthetic apparatus in light-grown plants and that the light-stable phytochrome pool functions in the EOD-FR elongation response, the latter pool being present and functional in the au mutant.