Stem extension-growth responses to blue light require Pfr in tomato seedlings but are not reduced by the low phytochrome levels of the aurea mutant

The effects of blue light (B) on stem extension growth were investigated in wild-type (WT) and aurea (au ) mutant seedlings of tomato. The au mutant has reduced phytochrome levels. Etiolated seedlings were grown under background red light (R) or far-red light (FR) with or without B. Hypocotyl growth was inhibited by B added to R but not by B added to FR, both in WT and au seedlings. The levels of B and/or R reaching the stem of fully de-etiolated seedlings grown in a glasshouse were reduced by means of collars around it. Both in WT and au -mutant seedlings the responses to B were larger at high than at low R/FR quantum ratios. In etiolated and light-grown au seedlings, changing the levels of phytochrome-absorbable radiation did not cause the same effect as changing B levels, indicating the action of specific BL/UV-A photoreceptor(s) (BAP). The responses to B are reduced by the low calculated levels of Pfr established by light treatments but not by the low levels of phytochrome present in the au mutant. The au mutant appears to be deficient in a phytochrome pool that is not essential for the interdependent co-action observed between phytochrome and BAP in the control of stem extension growth in tomato.     

Light-dependent,but phytochrome-independent,translational control of the accumulation of the P700 chlorophyll-a protein of photosystem I in barley (Hordeum vulgare L.)

This work reports on the regulation of synthesis of the P700 chlorophyll-a apoprotein of photosystem I in barley. The mRNA for the P700 apoprotein is almost exclusively confined to the plastid membrane-bound polysomes. However, the mRNA for the 32-kDa herbicide-binding protein of photosystem II is found in both the soluble and membrane-bound polysomes.The mRNA for the P700 apoprotein is found in similar amounts in dark-grown and light-grown wild-type as well as mutant xantha-l⁸¹ barley. The latter mutant is deficient in chlorophyll biosynthesis. However, while wild-type leaves accumulate the P700 chlorophyll-a protein only in the light, mutant leaves never accumulate the P700 apoprotein.A more sensitive approach was taken using isolated plastids to study P700 apoprotein synthesis. Etioplasts did not synthesize detectable P700 apoprotein even when the etioplasts were exposed to light. However, only a 1-min exposure of leaves to light was necessary to induce P700 apoprotein synthesis by isolated plastids.Phytochrome involvement in controlling P700 apoprotein synthesis was tested by using red/farred light treatment of leaves. These treatments showed no far-red reversibility of red-induced P700-apoprotein synthesis in isolated plastids even after 3 h of darkness after the light treatments. From these data we conclude that the accumulation of P700 apopootein is not under the control of phytochrome and that the light induction of P700 apoprotein is most likely mediated through the protochlorophyllide/chlorophyllide system. This control, however, may also involve cytoplasmic signals as the synthesis of the P700 apoprotein is not turned on in illuminated etioplasts.     

Metabolic Adaptations of Plant Respiration to Nutritional Phosphate Deprivation

Shoots of the lazy-2 (lz-2) gravitropic mutant of tomato (Lycopersicon esculentum Mill.) have a normal gravitropic response when grown in the dark, but grow downward in response to gravity when grown in the light. Experiments were undertaken to investigate the nature of the light induction of the downward growth of lz-2 shoots. Red light was effective at causing downward growth of hypocotyls of lz-2 seedlings, whereas treatment with blue light did not alter the dark-grown (wild-type) gravity response. Downward growth of lz-2 seedlings is greatest 16 h after a 1-h red light irradiation, after which the seedlings begin to revert to the dark-grown phenotype. lz-2 seedlings irradiated with a far-red light pulse immediately after a red light pulse exhibited no downward growth. However, continuous red or far-red light both resulted in downward growth of lz-2 seedlings. Thus, the light induction of downward growth of lz-2 appears to involve the photoreceptor phytochrome. Fluence-response experiments indicate that the induction of downward growth of lz-2 by red light is a low-fluence phytochrome response, with a possible high-irradiance response component.     

Altered Phytochrome Regulation of Greening in an aurea Mutant of Tomato

A brief pulse of red light accelerates chlorophyll accumulation upon subsequent transfer of dark-grown tomato (Lycopersicon esculentum) seedlings to continuous white light. Such potentiation of greening was compared in wild type and an aurea mutant W616. This mutant has been the subject of recent studies of phytochrome phototransduction; its dark-grown seedlings are deficient in phytochrome, and light-grown plants have yellow-green leaves. The rate of greening was slower in the mutant, but the extent (relative to the dark control) of potentiation by the red pulse was similar to that in the wild type. In the wild type, the fluence-response curve for potentiation of greening indicates substantial components in the VLF (very low fluence) and LF (low fluence) ranges. Far-red light could only partially reverse the effect of red. In the aurea mutant, only red light in the LF range was effective, and the effect of red was completely reversed by far-red light. When grown in total darkness, aurea seedlings are also deficient in photoconvertible PChl(ide). Upon transfer to white light, the aurea mutant was defective in both the abundance and light regulation of the light-harvesting chlorophyll a/b binding polypeptide(s) [LHC(II)]. The results are consistent with the VLF response in greening being mediated by phytochrome. Furthermore, the data support the hypothesis that light modulates LHC(II) levels through its control of the synthesis of both chlorophyll and its LHC(II) apoproteins. Some, but not all, aspects of the aurea phenotype can be accounted for by the deficiency in photoreception by phytochrome.     

Genetic analysis of the roles of phytochromes A and B1 in the reversed gravitropic response of the lz-2 tomato mutant

The lz-2 mutation in tomato ( Lycopersicon esculentum ) causes conditional reversal of shoot gravitropism by light. This response is mediated by phytochrome. To further elicit the mechanism by which phytochrome regulates the lz-2 phenotype, phytochrome-deficient lz-2 plants were generated. Introduction of au alleles, which severely block chromophore biosynthesis, eliminated the reversal of hypocotyl gravitropism in continuous red and far-red light. The fri ¹ and tri ¹ alleles were introduced to specifically deplete phytochromes A and B1, respectively. In dark-grown seedlings, phytochrome A was necessary for response to high-irradiance far-red light, a complete response to low fluence red light, and also mediated the effects of blue light in a far-red reversible manner. Loss of phytochrome B1 alone did not significantly affect the behaviour of lz-2 plants under any light treatment tested. However, dark-grown lz-2 plants lacking both phytochrome A and B1 exhibited reduced responses to continuous red and were less responsive to low fluence red light and high fluence blue light than plants that were deficient for phytochrome A alone. In high light, full spectrum greenhouse conditions, lz-2 plants grew downward regardless of the phytochrome deficiency. These results indicate that phytochromes A and B1 play significant roles in mediating the lz-2 phenotype and that at least one additional phytochrome is involved in reversing shoot gravitropism in this mutant.     

Mutations of Arabidopsis in potential transduction and response components of the phototropic signaling pathway.

Four genetic loci were recently identified by mutations that affect phototropism in Arabidopsis thaliana (L.) Heyhn. seedlings. It was hypothesized that one of these loci, NPH1, encodes the apoprotein for a phototropic photoreceptor. All of the alleles at the other three mutant loci (nph2, nph3, and nph4) contained wild-type levels of the putative NPH1 protein and exhibited normal blue-light-dependent phosphorylation of the NPH1 protein. This indicated that the NPH2, NPH3, and NPH4 proteins likely function downstream of NPH1 photoactivation. We show here that, although the nph2, nph3, and nph4 mutants are all altered with respect to their phototropic responses, only the nph4 mutants are also altered in their gravitropic responsiveness. Thus, NPH2 and NPH3 appear to act as signal carriers in a phototropism-specific pathway, whereas NPH4 is required for both phototropism and gravitropism and thus may function directly in the differential growth response. Despite their altered phototropic responses in blue and green light as etiolated seedlings, the nph2 and nph4 mutants exhibited less dramatic mutant phenotypes as de-etiolated seedlings and when etiolated seedlings were irradiated with unilateral ultraviolet-A (UV-A) light. Examination of the phototropic responses of a mutant deficient in biologically active phytochromes, hy1-100, indicated that phytochrome transformation by UV-A light mediates an increase in phototropic responsiveness, accounting for the greater phototropic curvature of the nph2 and nph4 mutants to UV-A light than to blue light.     

Identification of photo-inactive phytochrome A in etiolated seedlings and photo-active phytochrome B in green leaves of the aurea mutant of tomato

The contents of spectrophotometrically measurable phytochrome A (PhyA) and phytochrome B (PhyB) and the corresponding immunochemically detectable apoproteins (PHYA and PHYB) were examined in dark- and light-grown tissues of the aurea mutant of tomato and its wild-type (WT). The amount of PHYA in etiolated aurea seedlings was found to be about 20% of that in the WT; this PHYA showed no photoreversible changes in absorbance, no downregulation of the level of PHYA in light-grown seedlings, and no differential proteolysis of Pr and Pfr species in vitro which was seen in the case of the WT. By contrast, the amount of PHYB in aurea seedlings was not significantly different from that in WT seedlings. Phytochrome isolated from green leaves of the aurea mutant and purified by ion-exchange chromatography showed a red/far-red reversible spectral change, and its elution profile during chromatography was essentially similar to that of PHYB. The results indicate that aurea is a mutant that is deficient in photoactive PhyA at the etiolated stage, when it contains a spectrally inactive PHYA. However, the mutant contains spectrally active PhyB in its green tissue as does the WT.     

Photophysiology of the Elongated Internode (ein) Mutant of Brassica rapa: ein Mutant Lacks a Detectable Phytochrome B-Like Polypeptide

Several phytochrome-controlled processes have been examined in etiolated and light-grown seedlings of a normal genotype and the elongated internode (ein/ein) mutant of rapid-cycling Brassica rapa. Although etiolated ein seedlings displayed normal sensitivity to prolonged far-red light with respect to inhibition of hypocotyl elongation, expansion of cotyledons, and synthesis of anthocyanin, they displayed reduced sensitivity to prolonged red light for all three of these deetiolation responses. In contrast to normal seedlings, light-grown ein seedlings did not show a growth promotion in response to end-of-day far-red irradiation. Additionally, whereas the first internode of light-grown normal seedlings showed a marked increase in elongation in response to reduced ratio of red to far-red light, ein seedlings showed only a small elongation response. When blots of protein extracts from etiolated and light-treated ein and normal seedlings were probed with monoclonal antibody to phytochrome A, an immunostaining band at about 120 kD was observed for both extracts. The immunostaining intensity of this band was substantially reduced for extracts of light-treated normal and ein seedlings. A mixture of three monoclonal antibodies directed against phytochrome B from Arabidopsis thaliana immunostained a band at about 120 kD for extracts of etiolated and light-treated normal seedlings. This band was undetectable in extracts of ein seedlings. We propose that ein is a photoreceptor mutant that is deficient in a light-stable phytochrome B-like species.     

Phytochrome A enhances the promotion of hypocotyl growth caused by reductions in levels of phytochrome B in its far-red-light-absorbing form in light-grown Arabidopsis thaliana.

We sought to determine if phytochrome B (phyB)-mediated responses to the red light (R)/far-red light (FR) ratio are affected by phytochrome A (phyA) activity in light-grown seedlings of Arabidopsis thaliana. Pulses of FR delayed into the dark period were less effective than end-of-day (EOD) FR in promoting hypocotyl growth over a given period in darkness. White light minus blue light interposed instead of darkness between the end of the white-light photoperiod and the FR pulse was sufficient to maintain responsivity to the decrease in phyB in FR-light-absorbing form in wild-type (WT) seedlings, but not in the phyA mutant. Compared with EOD R, hourly R+FR pulses provided throughout the night caused a stronger promotion of stem growth than a single EOD R+FR pulse in WT Arabidopsis, cucumber, mustard, sunflower, tobacco, and tomato, but not in phyA Arabidopsis or in the aurea mutant of tomato. WT seedlings of Arabidopsis responded to a range of high EOD R/FR ratios, whereas the phyA mutant required stronger reductions in the EOD R/FR ratio. In sunlight, phyA seedlings of Arabidopsis showed no response to the "early warning" signals of neighboring vegetation, and hypocotyl-growth promotion occurred at higher plant densities than in the WT. Thus, under a series of light conditions, the sensitivity or responsivity to reductions in the R/FR ratio were larger in WT than in phyA seedlings. A product of phyA is therefore proposed to enhance the hypocotyl-growth response to decreases in phyB in FR-light-absorbing form in light grown seedlings.     

Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception

Transgenic tomato [Lycopersicon esculentum (=Solanum lycopersicum)] lines overexpressing tomato PHYA, PHYB1, or PHYB2, under control of the constitutive double-35S promoter from cauliflower mosaic virus (CaMV) have been generated to test the level of saturation in individual phytochrome-signalling pathways in tomato. Western blot analysis confirmed the elevated phytochrome protein levels in dark-grown seedlings of the respective PHY overexpressing (PHYOE) lines. Exposure to 4 h of red light resulted in a decrease in phytochrome A protein level in the PHYAOE lines, indicating that the chromophore availability is not limiting for assembly into holoprotein and that the excess of phytochrome A protein is also targeted for light-regulated destruction. The elongation and anthocyanin accumulation responses of plants grown under white light, red light, far-red light, and end-of-day far-red light were used for characterization of selected PHYOE lines. In addition, the anthocyanin accumulation response to different fluence rates of red light of 4-d-old dark-grown seedlings was studied. The elevated levels of phyA in the PHYAOE lines had little effect on seedling and adult plant phenotype. Both PHYAOE in the phyA mutant background and PHYB2OE in the double-mutant background rescued the mutant phenotype, proving that expression of the transgene results in biologically active phytochrome. The PHYB1OE lines showed mild effects on the inhibition of stem elongation and anthocyanin accumulation and little or no effect on the red light high irradiance response. By contrast, the PHYB2OE lines showed a strong inhibition of elongation, enhancement of anthocyanin accumulation, and a strong amplification of the red light high irradiance response.     

Effect of White and Red Light on the Pigment Content and Functional Activity in Pine Chloroplasts

The pigment content and rates of primary photosynthetic reactions were determined in chloroplasts of 14-day-old pine (Pinus silvestris L.) seedlings grown in light and darkness. In addition, the functional activities were investigated in chloroplasts from dark-grown seedlings exposed to white, red ( = 670 nm), and red + far-red ( = 748 nm) light. Dark-grown seedlings were capable of performing the Hill reaction, noncyclic photophosphorylation, and phenazine methosulfate–supported photophosphorylation, although the reaction rates in chloroplasts from dark-grown plants were considerably lower than in preparations from light-grown plants. Light treatment of dark-grown seedlings rapidly activated the photoreduction of ferricyanide and photophosphorylation, while the additional accumulation of green pigments started only after a lag period of two hours. Preirradiation of dark-grown seedlings with red light stimulated the formation of pigments, especially chlorophyll b, as well as the functional activity of chloroplasts. When far-red light was applied after red-light exposure, the processes examined were inhibited. It is concluded that accumulation of the light-harvesting complex and functional activities of chloroplasts at the photosystem II level in pine seedlings are controlled by the phytochrome.     

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

The aurea locus mutant (au ^w) 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.     

Translation and stability of proteins encoded by the plastid psbA and psbB genes are regulated by a nuclear gene during light-induced chloroplast development in barley

We have characterized a nuclear mutant of barley, viridis-115, lacking photosystem II (PSII) activity and compared it to wild-type seedlings during light-induced chloroplast development. Chloroplasts isolated from wild-type and viridis-115 seedlings illuminated for 1 h synthesized similar polypeptides and had similar protein composition. After 16 h of illumination, however, mutant plastids exhibited reduced ability to radiolabel D1, CP47, and several low Mr membrane polypeptides, and by 72 h, synthesis of these proteins was undetectable. Immunoblot analysis showed that plastids of dark-grown wild-type barley lacked several PSII proteins (D1, D2, CP47, and CP43) and that 16 h of illumination resulted in the accumulation of these polypeptides. In contrast, these polypeptides did not accumulate in illuminated viridis-115 seedlings, although mutant plastids accumulated two PSII proteins that participate in oxygen evolution, oxygen-evolving enhancers 1 and 3. Northern analysis showed that the levels of psbA and psbB mRNA in mutant plastids were equal to or greater than levels in wild-type plastids throughout the developmental period examined here. These results indicate that the nuclear mutation present in viridis-115 affects the translation and stability of the chloroplast-encoded D1 and CP47 polypeptides and that its influence is expressed after the onset of light-induced chloroplast development.     

Light-stimulated cotyledon expansion in the blu3 and hy4 mutants of Arabidopsis thaliana.

Cotyledon expansion in response to blue light was compared for wild-type Arabidopsis thaliana (L.) Heynh. and the mutants blu3 and hy4, which show reduced inhibition of hypocotyl growth in blue light. White, blue, and red light stimulated cotyledon expansion in both intact and excised cotyledons of wild-type seedlings (ecotypes No-0, WS, Co-0, La-er). Cotyledons on intact blu3 and hy4 seedlings did not grow as well as those on the wild type in response to blue light, but pretreatment of blu3 seedlings with low fluence rates of red light increased their responsiveness to blue light. Excision of cotyledons alleviated the mutant phenotype so that both mutant and wild-type cotyledons grew equally well in blue light. The loss of the mutant cotyledon phenotype upon excision indicates that the blu3 and hy4 lesions affect cotyledon expansion indirectly via a whole-plant response to light. Furthermore, the ability of excised, mutant cotyledons to grow normally in blue light shows that this growth response to blue light is mediated by a photosystem other than the ones impaired by the blu3 and hy4 lesions.     

Photocontrol of the Accumulation of Plastid Polypeptides during Greening of Tomato Cotyledons : Potentiation by a Pulse of Red Light

A pulse of red light acting through phytochrome accelerates the formation of chlorophyll upon subsequent transfer of dark-grown seedlings to continuous white light. Specific antibodies were used to follow the accumulation of representative subunits of the major photosynthetic complexes during greening of seedlings of tomato (Lycopersicon esculentum). The time course for accumulation of the various subunits was compared in seedlings that received a red light pulse 4 h prior to transfer to continuous white light and parallel controls that did not receive a red light pulse. The light-harvesting chlorophyll-binding proteins of photosystem II (LHC II), the 33-kD extrinsic polypeptide of the oxygen-evolving complex (OEC1), and subunit II of photosystem I (psaD gene product) all increased in the light, and did so much faster in seedlings that received the inductive red light pulse. The red light pulse had no significant effect on the abundance of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), nor on several plastid-encoded polypeptides: the large subunit of Rubisco, the β subunit of the CF₁ complex of plastid ATPase, and the 43- and 47-kD subunits of photosystem II (CP43, CP47). Subunits I (cytochrome b₆f) and III (Rieske Fe-S protein) of the cytochrome b₆f complex showed a small or no increase as a result of the red pulse. The potentiation of greening by a pulse of red light, therefore, is not expressed uniformly in the abundance of all the photosynthetic complexes and their subunits.