Phytochrome Regulation of Greening in Pisum: Chlorophyll Accumulation and Abundance of mRNA for the Light-Harvesting Chlorophyll a/b Binding Proteins

A brief pulse of red light eliminates or reduces the lag in chlorophyll accumulation that occurs when dark-grown pea seedlings are transferred to continuous white light. The red light pulse also induces the accumulation of specific mRNAs. We compared time courses, escape from reversal by far-red light, and fluence-response behavior for induction of mRNA for the light-harvesting chlorophyll a/b binding proteins (Cab mRNA) with those for induction of rapid chlorophyll accumulation in seedlings of Pisum sativum cv Alaska. In both cases the time courses of low fluence and very low fluence responses diverged from each other in a similar fashion: the low fluence responses continued to increase for at least 24 hours, while the very low fluence responses reached saturation by 8 to 16 hours. Both responses escaped from reversibility by far-red slowly, approaching the red control level after 16 hours. The fluence-response curve for the Cab mRNA increase, on the other hand, showed threshold and saturation at fluences 10-fold lower than threshold and saturation values for the greening response. Therefore, the level of Cab mRNA, as measured by the presence of sequences hybridizing to a cDNA probe, does not limit the rate of chlorophyll accumulation after transfer of pea seedlings to white light. The Cab mRNA level in the buds of seedlings grown under continuous red light remained high even when the red fluence rate was too low to allow significant greening. In this case also, abundance of Cab mRNA cannot be what limits chlorophyll accumulation.     

Photobiology of phytochrome-mediated growth responses in sections of stem tissue from etiolated oats and corn

The far-red reversibility of the phytochrome-controlled stimulation of elongation of coleoptile sections by low fluence red light has been characterized in subapical coleoptile sections from dark-grown Avena sativa L., cv Lodi seedlings. The fluence dependence of the far-red reversal was the same whether or not the very low fluence response is also expressed. The capacity of far-red light to reverse the red light-induced response began to decline if the far-red light was given more than 90 minutes after the red irradiation. Escape was complete if the far red irradiation was given more than 240 minutes after the red irradiation. Sections consisting of both mesocotyl and coleoptile tissue from dark-grown Avena seedlings were found to have physiological regulation of the very low fluence response by indole 3-acetic acid and low external pH similar to that seen for sections consisting entirely of coleoptile tissue. The fluence-dependence of the red light-induced inhibition of mesocotyl elongation was studied in mesocotyl sections from dark grown Zea mays L. hybrid T-929 seedlings. Ten micromolar indole 3-acetic acid stimulates the control elongation of the sections, while at the same time increasing the sensitivity of the tissue for the light-induced inhibition of growth by a factor of 100.     

Ethylene is not involved in the blue light-induced growth inhibition of red light-grown peas

Although the growth of intact plants is inhibited by irradiation with blue light, the growth rate of isolated stem segments is largely unaffected by blue light. We hypothesized that this loss of responsiveness was a result of ethylene production as part of the wounding response. However, we found no interaction between ethylene- and blue light-induced growth inhibition in dark- or red light-grown seedlings of pea (Pisum sativum L.). Inhibition of growth begins in dark-grown seedlings exposed to blue light within 3 min of the onset of blue light, as was known for red light-grown seedlings. By contrast, ethylene-induced inhibition of growth occurs only after a lag of 20 to 30 min or more (dark-grown seedlings) or 60 min (red light-grown seedlings). Also, the inhibition response of red light-grown seedlings is the same whether ethylene is present from the onset of continuous blue-light treatment or not. Finally the spatial distribution of inhibition following blue light was different from that following ethylene treatment.     

Light-controlled Stem Elongation in Pea Seedlings Grown under Varied Light Conditions

There appears to be an orderly transition from one photosensitive state to another in light-controlled stem elongation in the garden pea, Pisum sativum L. cv. Alaska. Stem elongation in dark-grown plants is known to be phytochrome-controlled. However, seedlings are insensitive to phytochrome after a red light pretreatment. An action spectrum for inhibition in these seedlings has peaks at 440 and 620 nm. Protochlorophyll is suggested as the photoreceptor. If these red pretreated seedlings are further exposed to white light, sensitivity to 440 to 620 nm light is lost. Promotion by blue-green light is the only photoresponse shown by these seedlings. Inhibition of completely white light-grown seedlings required simultaneous exposure to high intensity blue light and 600 nm light.     

Phytochrome-mediated cellular photomorphogenesis

Red light-induced cell elongation and division in intact, etiolated oat (Avena sativa cv Lodi) seedlings have been assessed. The middle of coleoptile was especially responsive in the very low fluence range whereas the region immediately below the coleoptile tip and the two regions just above the coleoptilar node were more responsive than the entire organ in the low fluence range. These responses in the coleoptile are both the result of an increase in cell elongation. Coleoptile cell division is slightly inhibited in the very low and slightly stimulated by red light in the low fluence range.

The one-sixth of the mesocotyl closest to the node is more suppressed in its growth than is any other region in the very low fluence range. However, the low fluence response involved the entire mesocotyl equally. In the apical one-sixth of the mesocotyl, a strong suppression of cell division and a weak suppression of cell elongation occurs. In the lower five regions of the mesocotyl, red light in both fluence ranges suppresses only cell elongation. Apparently, the difference between red light-induced oat growth stimulation and suppression primarily involves differences in the response of the cell elongation process.

     

Light regulation of protein synthesis factor EF-G in pea chloroplasts

The activity of pea chloroplast elongation factor G (EF-G), a nuclear-coded protein required for the elongation cycle of chloroplast protein synthesis, is regulated in response to light. In pea seedlings germinated and grown under continuous white or red light, EF-G specific activity reaches a maximum between days 10 to 15, and then decreases. EF-G activity is almost undetectable in extracts from dark-grown seedlings. When 13-day dark-grown pea seedlings are transferred to light, EF-G specific activity reaches a higher value after 2 to 3 days than observed in seedlings grown under continuous light. The small and large subunits of ribulose bisphosphate carboxylase continue to accumulate after EF-G specific activity has reached maximum levels. Cytoplasmically synthesized components of the chloroplast protein synthetic apparatus, such as EF-G, may help coordinate cytoplasmic and nuclear events with chloroplast gene expression during light-induced chloroplast differentiation.     

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.     

Changes in Nucleic Acids in Phytochrome-dependent Elongation of the Alaska Pea Epicotyl

Red light, which produces the physiologically active form of phytochrome (Pfr), inhibited epicotyl elongation in intact dark-grown Alaska pea seedlings. This red light response was detectable 3 hours after the light treatment and became pronounced after 5 hours. The growth inhibition was completely reversed by far red light applied immediately after the red or by pretreatment of the seedlings with the plant hormone gibberellin A₃.     

Phytochrome-mediated phototropism in de-etiolated seedlings : occurrence and ecological significance

Phototropic responses to broadband far red (FR) radiation were investigated in fully de-etiolated seedlings of a long-hypocotyl mutant (lh) of cucumber (Cucumis sativus L.), which is deficient in phytochrome-B, and its near isogenic wild type (WT). Continuous unilateral FR light provided against a background of white light induced negative curvatures (i.e. bending away from the FR light source) in hypocotyls of WT seedlings. This response was fluence-rate dependent and was absent in the lh mutant, even at very high fluence rates of FR. The phototropic effect of FR light on WT seedlings was triggered in the hypocotyls and occurred over a range of fluence rates in which FR was very effective in promoting hypocotyl elongation. FR light had no effect on elongation of lh-mutant hypocotyls. Seedlings grown in the field showed negative phototropic responses to the proximity of neighboring plants that absorbed blue (B) and red light and back-reflected FR radiation. The bending response was significantly larger in WT than in lh seedlings. Responses of WT and lh seedlings to lateral B light were very similar; however, elimination of the lateral B light gradients created by the proximity of plant neighbors abolished the negative curvature only in the case of lh seedlings. More than 40% of the total hypocotyl curvature induced in WT seedlings by the presence of neighboring plants was present after equilibrating the fluence rates of B light received by opposite sides of the hypocotyl. These results suggest that: (a) phytochrome functions as a phototropic sensor in de-etiolated plants, and (b) in patchy canopy environments, young seedlings actively project new leaves into light gaps via stem bending responses elicited by the B-absorbing photoreceptor(s) and phytochrome.     

Red light enhancement of the phototropic response of etiolated pea stems

In the subapical third internode of 7-day-old etiolated pea seedlings, the magnitude of phototropic curvature in response to continuous unilateral blue illumination is increased when seedlings are pre-exposed to brief red light. The effect of red light on blue light-induced phototropism becomes manifest maximally 4 or more hours after red illumination, and closely parallels the promotive action of red light on the elongation of the subapical cells. Ethylene inhibits phototropic curvature by an inhibitory action on cell elongation without affecting the lateral transport of auxin. Pretreatment of seedlings with gibberellic acid causes increased phototropic curvature, but experiments using ¹⁴C-gibberellic acid indicate that gibberellic acid itself is not laterally transported under phototropic stimuli. Neither red light nor gibberellic acid treatment has any promotive effect on blue light-induced lateral transport of ³H-indoleacetic acid. Under conditions where phototropic curvature is increased by red light treatment, low concentrations of indoleacetic acid applied in lanolin paste to the apical cut end of the seedling cause an increased elongation response in subapical tissue. This could explain increased phototropic curvature caused by red light treatment.     

The involvement of indole-3-acetic acid in the control of stem elongation in dark- and light-grown pea (Pisum sativum) seedlings

We investigated the role of auxin on stem elongation in pea (Pisum sativum L.) grown for 10d in continuous darkness or under low-irradiance blue, red, far red and white light. The third internode of treated seedlings was peeled and the tissues (epidermis and cortex+central cylinder) were separately analyzed for the concentration of free and conjugated indole-3-acetic acid (IAA). Under red, far red and white light internode elongation was linearly related with the free IAA content of all internode tissues, suggesting that phytochrome-dependent inhibition of stem growth may be mediated by a decrease of free IAA levels in pea seedlings. The correlation between IAA and internode elongation, however, did not hold for blue light-grown seedlings. The hypothesis that the growth response under low-irradiance blue light might be correlated with the lack of phytochrome B signalling and changes in gibberellin metabolism is discussed in view of current knowledge on hormonal control of stem growth.     

Blue-light-mediated shade avoidance requires combined auxin and brassinosteroid action in Arabidopsis seedlings

Plant growth in dense vegetation can be strongly affected by competition for light between neighbours. These neighbours can not only be detected through phytochrome-mediated perception of a reduced red:far-red ratio, but also through altered blue light fluence rates. A reduction in blue light (low blue) induces a set of phenotypic traits, such as shoot elongation, to consolidate light capture; these are called shade avoidance responses. Here we show that both auxin and brassinosteroids (BR) play an important role in the regulation of enhanced hypocotyl elongation of Arabidopsis seedlings in response to blue light depletion. Only when both hormones are experimentally blocked simultaneously, using mutants and chemical inhibitors, will the response be fully inhibited. Upon exposure to low blue several members of the cell wall modifying XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH) protein family are regulated as well. Interestingly, auxin and BR each regulate a subset of these XTHs, by which they could regulate cell elongation. We hypothesize that auxin and BR regulate specific XTH genes in a non-redundant and non-synergistic manner during low-blue-induced shade avoidance responses of Arabidopsis seedlings, which explains why both hormones are required for an intact low-blue response.     

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m⁻² s⁻¹ of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.     

Comparison of fluence-response relationships of phototropism in light- and dark-grown buckwheat

Fluence-response relationships of phototropism in light- and dark-grown buckwheat (Fagopyrum esculentum Moench.) were compared using systematically varied fluence rates and irradiation times of unilateral monochromatic blue light. Etiolated seedlings respond to most fluence rates in a tri-phasic manner. Phase one differs from classic first positive in that reciprocity is not observed and the peak occurs at a wide variety of fluences, often orders of magnitude less than those characteristic of first positive. Light-grown plants display this pattern only when stimulated by low fluence rates. Phase three is an ascending arm directly related to irradiance time and is comparable to classic second positive. Phase two is a nearly indifferent zone separating phases one and three. At the lowest fluence rates, the maximal observed curvature is greater for dark-grown than for light-grown plants and the former curve more in response to short (2-second) exposures than do the latter. At the highest fluence rates, the maximal observed curvature is much greater for light-grown than for dark-grown seedlings, particularly at irradiation times of 2 to 3 minutes or more. Tropic curvatures correlate positively with increasing fluence rate up to some inflection range, above which the relationship becomes negative. This inflection range is approximately two orders of magnitude higher for light-grown plants.     

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.