Regulation of phytochrome A mRNA abundance in parsley seedlings and cell-suspension cultures

A cDNA clone encoding the apoprotein of a parsley phytochrome was isolated and classified as parsley PHYA phytochrome, on the basis of a sequence homology comparison with all available phytochrome sequences. Red light pulses led to a phytochrome-dependent down-regulation of PHYA mRNA abundance in etiolated parsley seedlings to a level of 10–20% compared with the dark control. The PHYA mRNA abundance in a parsley cell suspension culture was also down-regulated by light pulses. Transient expression assays in parsley protoplasts showed light regulation of a chimeric pea PHYA promoter uidA-gene construct.     

Neighbour signals perceived by phytochrome B increase thermotolerance in Arabidopsis

Due to the preeminence of reductionist approaches, understanding of plant responses to combined stresses is limited. We speculated that light‐quality signals of neighbouring vegetation might increase susceptibility to heat shocks because shade reduces tissue temperature and hence the likeness of heat shocks. In contrast, plants of Arabidopsis thaliana grown under low‐red/far‐red ratios typical of shade were less damaged by heat stress than plants grown under simulated sunlight. Neighbour signals reduce the activity of phytochrome B (phyB), increasing the abundance of PHYTOCHROME‐INTERACTING FACTORS (PIFs). The phyB mutant showed high tolerance to heat stress even under simulated sunlight, and a pif multiple mutant showed low tolerance under simulated shade. phyB and red/far‐red ratio had no effects on seedlings acclimated with nonstressful warm temperatures before the heat shock. The phyB mutant showed reduced expression of several fatty acid desaturase (FAD) genes and less proportion of fully unsaturated fatty acids and electrolyte leakage of membranes exposed to heat shocks. Red‐light‐activated phyB also reduced thermotolerance of dark‐grown seedlings but not via changes in FADs expression and membrane stability. We propose that the reduced photosynthetic capacity linked to thermotolerant membranes would be less costly under shade, where the light input limits photosynthesis.     

Fluorescence and photochemical properties of phytochromes in wild-type wheat and a transgenic line overexpressing an oat phytochrome A (PHYA) gene: functional implications

Etiolated seedlings of wild‐type wheat and a transgenic line overexpressing an oat PHYA gene were investigated by the use of in situ low‐temperature fluorescence spectroscopy. The red‐absorbing phytochrome form, Pr, was characterized by (1) fluorescence emission spectrum; (2) total phytochrome content, and (3) by the extent of the Pr → lumi‐R photoconversion at low temperature (γ₁), and of the Pr → Pfr photoconversion at ambient temperature (γ₂) as derived from emission data. All the characteristics were shown to be variable and to depend on (1) organ and tissue used; (2) seedling age; (3) transgenic wheat modification, and (4) continuous far‐red irradiation of seedlings during their growth. These variations were interpreted in terms of the existence in wheat seedlings of the two phenomenological Pr types: (a), Pr′– major longer wavelength (687/673 nm, emission/absorption maxima) variable and light‐labile with γ₁ ≈ 0·5; and (b), Pr′′– minor, shorter wavelength (682/668 nm), relatively constant with its concentration not changing significantly with the increase of total phytochrome content in tissues and light‐stable with γ₁ ≤ 0·05–0·1. Overexpression of oat phyA increases primarily the content of Pr′ suggesting that it is comprised of phyA (phyA′) whereas Pr′′ is believed to consist of the minor phyA fraction (phyA′′) and phyB. The transgenic wheat line has been demonstrated to have a modified phenotype – the appearance of the far‐red high irradiance reaction (FR‐HIR) (Shlumukov et al. Plant, Cell and Environment 24, 703–712). The increased content of phyA′ in the transgenic line, whereas the total [phyA′′ + phyB] remains the same as in the wild type, indicates that the phyA′ pool is primarily responsible for the observed modification of the phenotype and suggests that even in wild‐type plants the phyA′ component of the phyA pool may mediate the FR‐HIR.     

Red And far‐red regulation of filament movement correlates with the expression of phytochrome and FHY1 genes in Spirogyra varians (Zygnematales,Streptophyta)1

Spirogyra filaments show unique photomovement that differs in response to blue, red, and far‐red light. Phototropins involved in the blue‐light movement have been characterized together with downstream signaling components, but the photoreceptors and mechanical effectors of red‐ and far‐red light movement are not yet characterized. The filaments of Spirogyra varians slowly bent and aggregated to form a tangled mass in red light. In far‐red light, the filaments unbent, stretched rapidly, and separated from each other. Mannitol and/or sorbitol treatment significantly inhibited this far‐red light movement suggesting that turgor pressure is the driving force of this movement. The bending and aggregating movements of filaments in red light were not affected by osmotic change. Three phytochrome homologues isolated from S. varians showed unique phylogenetic characteristics. Two canonical phytochromes, named SvPHY1 and SvPHY2, and a noncanonical phytochrome named SvPHYX2. SvPHY1 is the first PHY1 family phytochrome reported in zygnematalean algae. The gene involved in the transport of phytochromes into the nucleus was characterized, and its expression in response to red and far‐red light was measured using quantitative PCR. Our results suggest that the phytochromes and the genes involved in the transport system into the nucleus are well conserved in S. varians.     

Establishment of far‐red high irradiance responses in wheat through transgenic expression of an oat phytochrome A gene

A transgenic wheat line over‐expressing an oat phytochrome A gene under the control of the constitutive maize ubiquitin promoter was generated using a biolistic particle delivery system from immature wheat embryos. The resulting line showed increased levels of total phytochrome A protein in both dark‐grown and light‐grown plants. When grown under continuous far‐red light, seedlings of this line showed additional inhibition of the coleoptile extension in comparison with wild‐type seedlings. Unlike the response of wild‐type seedlings to continuous far‐red, this additional inhibition was dependent on fluence rate and was not observed under half‐hourly pulses of far‐red delivering the same total fluence as the continuous irradiation treatment. These observations suggest that increase in phytochrome A levels in wheat leads to the establishment of a far‐red high irradiation reaction in this monocotyledonous plant. Exposure to continuous red light caused a similar inhibition of coleoptile extension in both the wild types and the transgenic seedlings. When wild‐type seedlings were grown under continuous far‐red, their coleoptiles remained completely colourless and first leaves remained tightly rolled. In contrast, transgenic seedlings grown in the same conditions produced significant levels of anthocyanins in their coleoptiles and their first leaves became unrolled. Taken together, our data suggest that the increased levels of phytochrome A in wheat can change the type of response of some developmental processes to light signals, leading to the generation of a high irradiance reaction which is otherwise absent in the wild types under the conditions used.     

A role for ABCB19‐mediated polar auxin transport in seedling photomorphogenesis mediated by cryptochrome 1 and phytochrome B

During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochrome 1 (cry1) and phytochrome B (phyB) photosensory pathways, respectively. How these photosensory pathways integrate with growth control mechanisms to achieve the appropriate degree of stem elongation was investigated by combining cry1 and phyB photoreceptor mutations with genetic manipulations of a multidrug resistance‐like membrane protein known as ABCB19 that influenced auxin distribution within the plant, as evidenced by a combination of reporter gene assays and direct auxin measurements. Auxin signaling and ABCB19 protein levels, hypocotyl growth rates, and apical hook opening were measured in mutant and wild‐type seedlings exposed to a range of red and blue light conditions. Ectopic/overexpression of ABCB19 (B19OE) greatly increased auxin in the hypocotyl, which reduced the sensitivity of hypocotyl growth specifically to blue light in long‐term assays and red light in high‐resolution, short‐term assays. Loss of ABCB19 partially suppressed the cry1 hypocotyl growth phenotype in blue light. Hypocotyl growth of B19OE seedlings in red light was very similar to phyB mutants. Altered auxin distribution in B19OE seedlings also affected the opening of the apical hook. The cry1 and phyB photoreceptor mutations both increased ABCB19 protein levels at the plasma membrane, as measured by confocal microscopy. The B19OE plant proved to be a useful tool for determining aspects of the mechanism by which light, acting through cry1 or phyB, influences the auxin transport process to control hypocotyl growth during de‐etiolation.     

Control of hypocotyl elongation in Arabidopsis thaliana by photoreceptor interaction

In order to test the interaction of different phytochromes and blue-light receptors, etiolated seedlings of wild-type Arabidopsis thaliana (L.) Heynh., a phytochrome (phy) B-overexpressor line (ABO), and the photoreceptor mutants phyA-201, phyB-5, hy4-2.23n, fha-1, phyA-201/phyB-5, and phyA-201/hy4-2.23n were exposed to red and far-red light pulses after various preirradiations. The responsiveness to the inductive red pulses is primarily mediated by phyB which is rather stable in its far-red-absorbing form as demonstrated by a very slow loss of reversibility. Without preirradiation the red pulses had an impact on hypocotyl elongation only in PHYA mutants but not in the wild type. This indicates a suppression of phyB function by the presence of phyA. Preirradiation with either far-red or blue light resulted in an inhibition of hypocotyl elongation by red pulses in the wild type. Responsiveness amplification by far-red light is mediated by phyA and disappears slowly in the dark. The extent of responsiveness amplification by blue light was identical in the wild type and in the absence of phyA, or the cryptochromes cryl (hy4-2.23n) or cry2 (fha-1). Therefore, we conclude that stimulation of phyB by blue light preirradiation is either mediated by an additional still-unidentified blue-light-absorbing pigment or that phyA, cry1 and cry2 substitute for each other completely. Both blue and red preirradiation established responsiveness to red pulses in phyA-201/phyB-5 double mutants. These results demonstrate that inhibition of hypocotyl elongation by red pulses is not only mediated by phyB but also by a phytochrome(s) other than phyA and phyB. Received: 21 July 1998 / Accepted: 7 December 1998     

Rice sucrose transporter1 (OsSUT1) up‐regulation in xylem parenchyma is caused by aphid feeding on rice leaf blade vascular bundles

The role of the sucrose transporter OsSUT1 in assimilate retrieval via the xylem, as a result of damage to and leakage from punctured phloem was examined after rusty plum aphid (Hysteroneura setariae, Thomas) infestation on leaves from 3‐week‐old rice (Oryza sativa L. cv Nipponbare) plants. Leaves were examined over a 1‐ to 10‐day infestation time course, using a combination of gene expression and β‐glucuronidase (GUS) reporter gene analyses. qPCR and Western blot analyses revealed differential expression of OsSUT1 during aphid infestation. Wide‐field fluorescence microscopy was used to confirm the expression of OsSUT1‐promoter::GUS reporter gene in vascular parenchyma associated with xylem elements, as well as in companion cells associated with phloem sieve tubes of large, intermediate and small vascular bundles within the leaf blade, in regions where the aphids had settled and were feeding. Of great interest was up‐regulation of OsSUT1 expression associated with the xylem parenchyma cells, abutting the metaxylem vessels, which confirmed that OsSUT1 was not only involved in loading of sugars into the phloem under normal physiological conditions, but was apparently involved in the retrieval of sucrose leaked into the xylem conduits, which occurred as a direct result of aphid feeding, probing and puncturing of vascular bundles. The up‐regulation of OsSUT1 in xylem vascular parenchyma thus provides evidence in support of the location within the xylem parenchyma cells of an efficient mechanism to ensure sucrose recovery after loss to the apoplast (xylem) after aphid‐related feeding damage and its transfer back to the symplast (phloem) in O. sativa leaves.     

Light-regulated overexpression of an Arabidopsis phytochrome A gene in rice alters plant architecture and increases grain yield

The phytochromes are a family of red/far-red light absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in the light environment. We report here the overexpression of Arabidopsis thaliana PHYTOCHROME A (PHYA) gene in a commercially important indica rice variety (Oryza sativa L. Pusa Basmati-1). The expression of the transgene was driven by the light-regulated and tissue-specific rice rbcS promoter. Several independent homozygous sixth generation (T₅) transgenic lines were characterized and shown to accumulate relatively high levels of PHYA protein in the light. Under both far-red and red light, PHYA-overexpressing lines showed inhibition of the coleoptile extension in comparison to non-transgenic seedlings. Furthermore, compared with non-transgenic rice plants, mature transgenic plants showed significant reduction in plant height, internode length and internode diameter (including differences in cell size and number), and produced an increased number of panicles per plant. Under greenhouse conditions, rice grain yield was 6–21% higher in three PHYA-overexpressing lines than in non-transgenic plants. These results demonstrate the potential of manipulating light signal-transduction pathways to minimize the problems of lodging in basmati/aromatic rice and to enhance grain productivity.     

Overexpression of rice phytochrome A in tobacco seedlings modifies responsiveness but not competence to phytochrome

To analyse the control of rice phytochrome A (phyA) overexpression (wild type or variously mutated) on gene regulation, transgenic tobacco lines overexpressing various rice phyA constructs were crossed with transgenic tobacco lines containing mustard Lhcb1 or Chs1 promoters fused to the uidA reporter gene (-glucuronidase). It was demonstrated that the temporal pattern of competence to respond to phytochrome was not altered by rice phyA overexpression. Also, overexpression of rice phyA did not change the spatial pattern of gene expression. The responsiveness to red and far-red light, on the other hand, depended on the type of overexpressed rice phyA in a structure-function relation: the serine-to-alanine mutant mediated an enhanced response both under continuous red and far-red light, whereas the N-terminal deletion mutant showed a dominant negative effect under continuous far-red light and even after red light pulses. However, the effectiveness of rice phyA overexpression depended on the promoter construct and the developmental stage of the seedlings. The Lhcb1 promoter also conferred -glucuronidase activity in etiolated seedlings. This dark expression could be decreased by a long-wavelength farred light pulse given early in development (24 h after sowing), indicating that this phenomenon is under the control of stable types of phytochrome.Abbreviations Chs1 chalcone synthase - GUS -glucuronidase - Lhcb1 type 1 light-harvesting chlorophyll a/b-binding protein - NTD N-terminal deletion mutant of rice phyA - phyA phytochrome A - phyB phytochrome B - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - RW rice wild-type phyA - S/A serine-to-alanine mutant of rice phyA - XAN wild-type tobacco cv. Xanthi We thank N.-H. Chua (Rockefeller Univ., New York, USA) and J. Stockhaus (Heinrich-Heine-Universität, Düsseldorf, Germany) for providing seeds from tobacco lines overexpressing the diverse rice phyA proteins. The work was supported by a grant from the Human Frontier Science Program and a grant from Deutsche Forschungsgemeinschaft (SFB 388). K.E. is a recipient of a Landesgraduierten-förderung fellowship     

Regulation of promoter activity of ferredoxin-dependent glutamate synthase

The GLU1 promoter for Fd-glutamate synthase (Fd-GOGAT, EC 1.4.1.7) of Arabidopsis thaliana (ecotype Columbia) confers the expression of the β-glucuronidase (GUS) reporter gene on transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) transformed with the GLU1 promoter-GUS construct. Histochemical analysis reveals that GUS expression is associated with mesophyll and vascular tissue of 14-d-old tobacco seedlings. Red light substitutes for white light and induces a 2-fold increase in the GUS expression associated with mesophyll, veins and vascular tissue. Sucrose also serves as a signal to induce GUS expression in mesophyll and veins of cotyledons. Mature leaves, adapted to the dark for 3 d, conserves the red light- and white light-dependent inductions of GUS activity, while GUS expression is repressed by white light in roots. The mesophyll-located expression of the GLU1 promoter suggests that Fd-glutamate synthase has a function in the photorespiratory ammonium cycling and primary ammonium assimilation. The distinct location of GLU1 promoter expression in the vascular tissue supports the view that Fd-glutamate synthase synthesises glutamate for intracellular transport of glutamine and glutamate.     

The serine-rich N-terminal region of <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochrome A is required for protein stability

Deletion or substitution of the serine-rich N-terminal stretch of grass phytochrome A (phyA) has repeatedly been shown to yield a hyperactive photoreceptor when expressed under the control of a constitutive promoter in transgenic tobacco or Arabidopsis seedlings retaining their native phyA. These observations have lead to the proposal that the serine-rich region is involved in negative regulation of phyA signaling. To re-evaluate this conclusion in a more physiological context we produced transgenic Arabidopsis seedlings of the phyA-null background expressing Arabidopsis PHYA deleted in the sequence corresponding to amino acids 6–12, under the control of the native PHYA promoter. Compared to the transgenic seedlings expressing wild-type phyA, the seedlings bearing the mutated phyA showed normal responses to pulses of far-red (FR) light and impaired responses to continuous FR light. In yeast two-hybrid experiments, deleted phyA interacted normally with FHY1 and FHL, which are required for phyA accumulation in the nucleus. Immunoblot analysis showed reduced stability of deleted phyA under continuous red or FR light. The reduced physiological activity can therefore be accounted for by the enhanced destruction of the mutated phyA. These findings do not support the involvement of the serine-rich region in negative regulation but they are consistent with a recent report suggesting that phyA turnover is regulated by phosphorylation. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Different Phototransduction Kinetics of Phytochrome A and Phytochrome B in Arabidopsis thaliana

The kinetics of phototransduction of phytochrome A (phyA) and phytochrome B (phyB) were compared in etiolated Arabidopsis thaliana seedlings. The responses of hypocotyl growth, cotyledon unfolding, and expression of a light-harvesting chlorophyll a/b-binding protein of the photosystem II gene promoter fused to the coding region of β-glucuronidase (used as a reporter enzyme) were mediated by phyA under continuous far-red light (FR) and by phyB under continuous red light (R). The seedlings were exposed hourly either to n min of FR followed by 60 minus n min in darkness or to n min of R, 3 min of FR (to back-convert phyB to its inactive form), and 57 minus n min of darkness. For the three processes investigated here, the kinetics of phototransduction of phyB were faster than that of phyA. For instance, 15 min R h⁻¹ (terminated with a FR pulse) were almost as effective as continuous R, whereas 15 min of FR h⁻¹ caused less than 30% of the effect of continuous FR. This difference is interpreted in terms of divergence of signal transduction pathways downstream from phyA and phyB.