Phytochrome-Deficient hy1 and hy2 Long Hypocotyl Mutants of Arabidopsis Are Defective in Phytochrome Chromophore Biosynthesis

The hy1 and hy2 long hypocotyl mutants of Arabidopsis contain normal levels of immunochemically detectable phytochrome A, but the molecule is photochemically nonfunctional. We have investigated the biochemical basis for this lack of function. When the hy1 and hy2 mutants were grown in white light on a medium containing biliverdin IX[alpha], a direct precursor to phytochromobilin, the phytochrome chromophore, the seedlings developed with a morphological phenotype indistinguishable from the light-grown wild-type control. Restoration of a light-grown phenotype in the hy1 mutant was also accomplished by using phycocyanobilin, a tetrapyrrole analog of phytochromobilin. Spectrophotometric and immunochemical analyses of the rescued hy1 and hy2 mutants demonstrated that they possessed wild-type levels of photochemically functional phytochrome that displayed light-induced conformational changes in the holoprotein indistinguishable from the wild type. Moreover, phytochrome A levels declined in vivo in response to white light in rescued hy1 and hy2 seedlings, indicative of biliverdin-dependent formation of photochemically functional phytochrome A that was then subject to normal selective turnover in the far-red-light-absorbing form. Combined, these data suggest that the hy1 and hy2 mutants are inhibited in chromophore biosynthesis at steps prior to the formation of biliverdin IX[alpha], thus potentially causing a global functional deficiency in all members of the phytochrome photoreceptor family.     

Blue-light promotion of flowering is absent in hy4 mutants of Arabidopsis

Loss of a blue-light photoreceptor in the hy4 mutants of Arabidopsis thaliana (L.) Heynh substantially delayed flowering (>100 d to flower vs. 40–50 d), especially with blue light exposure from lamps lacking much red (R) and/or far-red (FR) light. Red night breaks were promotory but flowering was still later for the hy4-101 mutant. However, with exposure to light from FR-rich lamps, flowering of all mutants was early and no different from the wild type. Thus, flowering of Arabidopsis involves a blue-light photoreceptor and other, often more effective photoreceptors. The latter may involve phytochrome photoresponses to R and FR, but with little or no phytochrome response to blue wavelengths.Abbreviations HIR high irradiance response - FR far-red - R red - WT wild type     

Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl

A majority of the cells in the Arabidopsis hypocotyl undergo endoreduplication. The number of endocycles in this organ is partially controlled by light. Up to two cycles occur in light-grown hypocotyls, whereas in the dark about 30% of the cells go through a third cycle. Is the inhibition of the third endocycle in the light an indirect result of the reduced cell size in the light-grown hypocotyl, or is it under independent light control? To address this question, the authors examined the temporal and spacial patterns of endoreduplication in light- or dark-grown plants and report here on the following observations: (i) during germination two endocycles take place prior to any significant cell expansion; (ii) in the dark the third cycle is completed very early during cell growth; and (iii) a mutation that dramatically reduces cell size does not interfere with the third endocycle. The authors then used mutants to study the way light controls the third endocycle and found that the third endocycle is completely suppressed in far red light through the action of phytochrome A and, to a lesser extent, in red light by phytochrome B. Furthermore, no 16C nuclei were observed in dark-grown constitutive photomorphogenic 1 seedlings. And, finally the hypocotyl of the cryptochrome mutant, hy4, grown in blue light was about three times longer than that of the wild-type without a significant difference in ploidy levels. Together, the results support the view that the inhibition of the third endocycle in light-grown hypocotyls is not the consequence of a simple feed-back mechanism coupling the number of cycles to the cell volume, but an integral part of the phytochrome-controlled photomorphogenic program.     

Transgene-mediated auxin overproduction in Arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin-resistant mutants

Transgenic Arabidopsis thaliana plants constitutively expressing Agrobacterium tumefaciens tryptophan monooxygenase (iaaM) were obtained and characterized. Arabidopsis plants expressing iaaM have up to 4-fold higher levels of free indole-3-acetic acid (IAA) and display increased hypocotyl elongation in the light. This result clearly demonstrates that excess endogenous auxin can promote cell elongation in a whole plant. Interactions of the auxin-overproducing transgenic plants with the phytochrome-deficient hy6-1 and auxin-resistant axrl-3 mutations were also studied. The effects of auxin overproduction on hypocotyl elongation were not additive to the effects of phytochrome deficiency in the hy6-1 mutant, indicating that excess auxin does not counteract factors that limit hypocotyl elongation in hy6-1 seedlings. Auxin-overproducing seedlings are also qualitatively indistinguishable from wild-type controls in their response to red, far-red, and blue light treatments, demonstrating that the effect of excess auxin on hypocotyl elongation is independent of red and blue light-mediated effects. All phenotypic effects of iaaM-mediated auxin overproduction (i.e. increased hypocotyl elongation in the light, severe rosette leaf epinasty, and increased apical dominance) are suppressed by the auxin-resistant axr1-3 mutation. The axr1-3 mutation apparently blocks auxin signal transduction since it does not reduce auxin levels when combined with the auxin-overproducing transgene.     

Avena sativa L. contains three phytochromes,only one of which is abundant in etiolated tissue

Phytochrome from leaves of light-grown oat (Avena sativa L. cv. Garry) plants is characterized with newly generated monoclonal antibodies (MAbs) directed to it. The results indicate that there are at least two phytochromes in green oat leaves, each of which differs from the phytochrome that is most abundant in etiolated oat tissue. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with reference to 124-kilodalton (kDa) phytochrome from etiolated oats, the two phytochromes from green oats have monomer sizes of 123 of 125 kDa. Immunoblot analysis of SDS, sample buffer extracts of lyophilized, green oat leaves indicates that neither the 125-kDa nor the 123-kDa polypeptide is a degradation product arising after tissue homogenization. Of the two, the 123-kDa phytochrome appears to be the predominant species in light-grown oat leaves. During SDS-PAGE in the presence of 1 mM Zn²⁺, 123-kDa phytochrome undergoes a mobility shift corresponding to an apparent mass increase of 2 kDa. In contrast, the electrophoretic mobility of 125-kDa phytochrome is unaffected by added Zn²⁺. Some MAbs that recognize 123-kDa phytochrome fail to recognize 125-kDa phytochrome and vice versa, indicating that these two phytochromes are not only immunochemically distinct from 124-kDa phytochrome, but also from each other. It is evident, therefore, that there are at least three phytochromes in an oat plant: 124-kDa phytochrome, which is most abundant in etiolated tissue, plus 123-and 125-kDa phytochromes, which predominate in light-grown tissue.Abbreviations Da Dalton - HA hydroxyapatite - MAb monoclonal antibody - PAb polyclonal antibody preparation - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). We thank Dr. Alan Jones, Department of Biology, University of North Carolina, Chapel Hill, USA, for kindly providing rabbit antiserum 4032, and Mrs. Donna Tucker and Mrs. Danielle Neal for their technical assistance.     

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.     

NADPH : protochlorophyllide oxidoreductases in white pine (Pines strobes) and loblolly pine (P. taeda)

Summary NADPH : protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide in higher plants. Cloned cDNAs encoding two distinct pchlide reductases were isolated from a gt11 library constructed from poly(A)⁺ RNA prepared from the cotyledons of dark-grown white pine (Pines strobes) seedlings and a nuclear gene (lpcr) analogous to one of these cDNAs has been characterized from loblolly pine (P. taeda). The pine gene encodes an approximately 43 kDa precursor polypeptide consisting of a 334-amino acid mature protein and a 66-amino acid transit peptide. The deduced primary structures for the pine proteins are highly homologous to those reported from monocots and dicots. The coding portion of the pine lpcr gene is interrupted by four introns. The placement of these introns within the pine lpcr gene is identical to that observed in pea (Pisum sativum), suggesting conservation in gene organization between dicot and gymnosperm species. Western blot analysis using polyclonal antiserum against oat pchlide reductase detected in extracts of dark-grown pine cotyledons a single immunoreactive protein, which declined in abundance during a 48 h period of illumination with white light. Cotyledons of dark-grown seedlings were also found to accumulate high levels of pchlide reductase mRNA; however, little or no change in the steady-state levels of mRNA encoding pchlide reductase was observed in these tissues following illumination. Stem tissue of dark-grown seedlings did not contain significant levels of pchlide reductase mRNA, whereas stems of light-grown plants of the same age accumulated substantial amounts of the message. These results suggest that light and the developmental age of the tissue affect regulation of lpcr expression in pine.     

Gibberellins control Arabidopsis hypocotyl growth via regulation of cellular elongation

The gibberellins (GAs) are endogenous regulators of plant growth. Experiments are described here that test the hypothesis that GA regulates hypocotyl growth by altering the extent of hypocotyl cell elongation. These experiments use GA-deficient and altered GA-response mutants of Arabidopsis thaliana (L.) Heyhn. It is shown that GA regulates elongation, in both light- and dark-grown hypocotyls, by influencing the rate and final extent of cellular elongation. However, light- and dark-grown hypocotyls exhibit markedly different GA dose-response relationships. The length of dark-grown hypocotyls is relatively unaffected by exogenous GA, whilst light-grown hypocotyl length is significantly increased by exogenous GA. Further analysis suggests that GA control of hypocotyl length is close to saturation in dark-grown hypocotyls, but not in light grown hypocotyls. The results show that a large range of possible hypocotyl lengths is achieved via dose-dependent GA-regulated alterations in the degree of elongation of individual hypocotyl cells.Key words: Arabidopsis, cell elongation, gibberellin (GA), GA mutants, hypocotyl.      

Far-red light-insensitive, phytochrome A-deficient mutants of tomato

We have selected two recessive mutants of tomato with slightly longer hypocotyls than the wild type, one under low fluence rate (3 mol/m²/s) red light (R) and the other under low fluence rate blue light. These two mutants were shown to be allelic and further analysis revealed that hypocotyl growth was totally insensitive to far-red light (FR). We propose the gene symbol fri (far-red light insensitive) for this locus and have mapped it on chromosome 10. Immunochemically detectable phytochrome A polypeptide is essentially absent in the fri mutants as is the bulk spectrophotometrically detectable labile phytochrome pool in etiolated seedlings. A phytochrome B-like polypeptide is present in normal amounts and a small stable phytochrome pool can be readily detected by spectrophotometry in the fri mutants. Inhibition of hypocotyl growth by a R pulse given every 4 h is quantitatively similar in the fri mutants and wild type and the effect is to a large extent reversible if R pulses are followed immediately by a FR pulse. After 7 days in darkness, both fri mutants and the wild type become green on transfer to white light, but after 7 days in FR, the wild-type seedlings that have expanded their cotyledons lose their capacity to green in white light, while the fri mutants de-etiolate. Adult plants of the fri mutants show retarded growth and are prone to wilting, but exhibit a normal elongation response to FR given at the end of the daily photoperiod. The inhibition of seed germination by continuous FR exhibited by the wild type is normal in the fri mutants. It is proposed that these fri mutants are putative phytochrome A mutants which have normal pools of other phytochromes.     

Nicotiana plumbaginifolia hlg mutants have a mutation in a PHYB-type phytochrome gene: they have elongated hypocotyls in red light, but are not elongated as adult plants

Two new allelic mutants of Nicotiana plumbaginifolia have been isolated which display a hypocotyl which is long (hlg) when seedlings are grown in continuous white light (W). This can be accounted for by the decreased response to red light (R) of the hypocotyl elongation rate in these mutants. Responses to other wavelengths are unaffected in the mutants. When grown in white light, mature hlg mutants are not elongated with respect to the wild-type; they also bolt and flower later. The shade-avoidance responses to red/far red ratio (R:FR) are intact in these mutants. Both mutants are deficient in a phyB-like polypeptide that is immunodetectable in the wild-type; both have wild-type levels of a phyA-like polypeptide. These alleles are inherited in a partially dominant manner, and correspond to single-base missense mutations in a gene highly homologous to N. tabacum PHYB, which codes for a phytochrome B-type photoreceptor. One allele, hlg-1, has an introduced amino acid substitution; this may define a residue essential for phytochrome protein stability. The other allele, hlg-2, has a stop codon introduced C-terminal to the chromophore binding domain. As these phyB mutants are unaffected in shade-avoidance responses, but deficient in perception of R, it is concluded that the phyB absent in these mutants is responsible for R perception in the N. plumbaginifolia seedling, but is not a R:FR sensor in light-grown plants.     

Extension-growth responses and expression of flavonoid biosynthesis genes in the Arabidopsis hy4 mutant

The hy4 mutant of Arabidopsis thaliana(L.) Heynh. was previously shown to be impaired in the suppression of hypocotyl extension specifically by blue light. We report here that hy4 is altered in a range of blue-light-mediated extension-growth responses in various organs in seedlings and mature plants: it shows greater length of bolted stems, increased petiole extension and increased leaf width and area in blue light compared to the wild type. The hy4 mutant shows decreased cotyledon expansion in both red and blue light compared to the wild type. Anthocyanin formation and the expression of several flavonoid biosynthesis genes is stimulated by blue light in the wild type but to a much lower extent in hy4. The results indicate that the HY4 gene product is concerned with the perception of blue light in a range of extension-growth and gene-expression responses in Arabidopsis.Abbreviations DFR dihydroflavonol reductase - CHS chalcone synthase - CHI chalcone isomerase We thank the UK Agricultural and Food Research Council for supporting this work through the award of a research grant to G.I.J. We are grateful to Robert Brown for excellent technical assistance and Drs B.W. Shirley (Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, USA), C.D. Silflow (Department of Genetics and Cell Biology, University of Minnesota, St. Paul, USA) and I.E. Somssich (Department of Biochemistry, Max-Planck-Institut, Köln, Germany) for providing plasmid DNA.     

Photomorphogenic development of the Arabidopsisshy2–1D mutation and its interaction with phytochromes in darkness

We previously reported a photomorphogenic mutation of Arabidopsis thaliana, shy2–1D, as a dominant suppressor of a hy2 mutation. Here, we report that shy2–1D confers various photo-responsive phenotypes in darkness and the dark phenotypes of the mutant are affected by phytochrome deficiency. Dark-grown seedlings of the mutant developed several photomorphogenic characteristics such as short hypocotyls, cotyledon expansion and opening, and partial differentiation of plastids. When grown further in darkness, the mutant plant underwent most of the developmental stages of a light-grown wild-type plant, including development of foliar leaves, an inflorescence stem with cauline leaves, and floral organs. In addition, two light-inducible genes, the nuclear-encoded CAB and the plastid-encoded PSBA genes, were highly expressed in the dark-grown mutant seedlings. Furthermore, reduced gravitropism, a phytochrome-modulated response, was observed in the mutant hypocotyl in darkness. Thus, shy2–1D is one of the most pleiotropic photomorphogenic mutations identified so far. The results indicate that SHY2 may be a key component regulating photomorphogenesis in Arabidopsis. Surprisingly, double mutants of the shy2–1D mutant with the phytochrome-deficient mutants hy2, hy3 (phyB-1) and fre1–1 (phyA-201) showed reduced photomorphogenic response in darkness with a longer hypocotyl, a longer inflorescence stem, and a lower level expression of the CAB gene than the shy2–1D single mutant. These results showed that phytochromes function in darkness in the shy2–1D mutant background. The implications of these results are discussed.     

Phytochrome control of plastid mRNA in mustard (Sinapis alba L.)

The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with ³²P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.Abbreviations LS large subunit - RuBP ribulose-1,5-bisphosphate - ptDNA plastid DNA     

Effect of overexpression of Arabidopsis Damaged DNA-binding protein 1A on De-etiolated 1

In Arabidopsis thaliana, de-etiolated 1 mutants (det1) grown in the dark resemble light-grown wild-type seedlings. Arabidopsis DET1 encodes a 62 kD protein, which is a negative regulator of light signaling. UV-damaged DNA-binding protein 1 (DDB1) was initially identified due to its role in human DNA damage repair. Arabidopsis has two DDB1 homologs: DDB1A and DDB1B. DDB1A mutation enhances det1 mutant phenotypes. In this study, we generated Arabidopsis lines that overexpress DDB1A-3HA in wild-type, det1, as well as Myc-DET1 or GFP-DET1 rescued genetic backgrounds. DDB1A-3HA overexpression resulted in decreased apical hook formation in wild-type dark-grown seedlings, and enhanced det1 small rosette and early flowering time phenotypes. In the Myc-DET1 background, DDB1A-3HA overexpression resulted in decreased rescue of dark- and light-grown hypocotyl length, light-grown anthocyanin and chlorophyll levels, adult height and stem number phenotypes. This result is consistent with the decreased levels of Myc-DET1 protein detected in the DDB1A-3HA overexpression line. The GFP-DET1 DDB1A-3HA double overexpression line exhibited increased rescue of dark and light-grown hypocotyl length and light-grown chlorophyll level phenotypes relative to GFP-DET1 alone, despite the fact that GFP-DET1 protein also decreased in the double overexpression line. In addition, increased DET1 resulted in decreased DDB1A-3HA levels due to proteasomal degradation. Overall, DDB1A-3HA overexpression affected phenotypes in a variety of DET1 backgrounds, reduced epitope-tagged DET1 levels, and, correlatively, in general dampened the rescue of det1 mutants by the DET1–DDB1A complex.     

Tagetitoxin affects plastid development in seedling leaves of wheat

Ultrastructural and biochemical approaches were used to investigate the mode of action of tagetitoxin, a nonhost-specific phytotoxin produced by Pseudomonas syringae pv. tagetis (Hellmers) Young, Dye and Wilkie, which causes chlorosis in developing — but not mature — leaves. Tagetitoxin has no effect on the growth rate or morphology of developing leaves of wheat (Triticum aestivum L.) seedlings. Its cytological effects are limited to plastid aberrations; in both light-and dark-grown leaves treated with toxin, internal plastid membranes fail to develop normally and plastid ribosomes are absent, whereas mitochondrial and cytoplasmic ribosomes are unaffected. The activity of a plastid stromal enzyme, ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39), which is co-coded by nuclear and chloroplast genes, is markedly lower in extracts of both light-and dark-grown toxin-treated leaves, whereas the activity of another stromal enzyme, NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-G-3P-DH, EC 1.2.1.13), which is coded only by the nuclear genome, is significantly lower in extracts of light-grown, but not of dark-grown, treated leaves. The mitochondrial enzymes fumarase (EC 4.2.1.2) and cytochrome-c oxidase (EC 1.9.3.1) are unaffected by toxin in dark-grown leaves, but fumarase activity is reduced in light-grown ones. Four peroxisomal enzyme activities are lowered by toxin treatment in both light- and dark-grown leaves. Light- and dark-grown, toxintreated leaves contain about 50% and 75%, respectively, of the total protein of untreated leaves. There are threefold and twofold increases in free amino acids in light-grown and dark-grown treated leaves, respectively. In general, the effects of tagetitoxin are more extensive and exaggerated in light-grown than in dark-grown leaves. We conclude that tagetitoxin interferes primarily with a light-independent aspect of chloroplast-specific metabolism which is important in plastid biogenesis.Abbreviations NADP-G-3-DH NADP-glyceraldehyde-3-phosphate dehydrogenase - PLB prolamellar body - RuBP-Case ribulose-1,5-bisphosphate carboxylase - SADH shikimic acid dehydrogenase     

Different Roles for Phytochrome in Etiolated and Green Plants Deduced from Characterization of Arabidopsis thaliana Mutants

We have isolated a new complementation group of Arabidopsis thaliana long hypocotyl mutant (hy6) and have characterized a variety of light-regulated phenomena in hy6 and other previously isolated A. thaliana hy mutants. Among six complementation groups that define the HY phenotype in A. thaliana, three (hy1, hy2, and hy6) had significantly lowered levels of photoreversibly detectable phytochrome, although near wild-type levels of the phytochrome apoprotein were present in all three mutants. When photoregulation of chlorophyll a/b binding protein (cab) gene expression was examined, results obtained depended dramatically on the light regime employed. Using the red/far-red photoreversibility assay on etiolated plants, the accumulation of cab mRNAs was considerably less in the phytochrome-deficient mutants than in wild-type A. thaliana seedlings. When grown in high-fluence rate white light, however, the mutants accumulated wild-type levels of cab mRNAs and other mRNAs thought to be regulated by phytochrome. An examination of the light-grown phenotypes of the phytochrome-deficient mutants, using biochemical, molecular, and morphological techniques, revealed that the mutants displayed incomplete chloroplast and leaf development under conditions where wild-type chloroplasts developed normally. Thus, although phytochrome may play a role in gene expression in etiolated plants, a primary role for phytochrome in green plants is likely to be in modulating the amount of chloroplast development, rather than triggering the initiation of events (e.g., gene expression) associated with chloroplast development.     

Physiological Roles of Brassinosteroids in Early Growth of <Emphasis Type="Italic">Arabidopsis:</Emphasis> Brassinosteroids Have a Synergistic Relationship with Gibberellin as well as Auxin in Light-Grown Hypocotyl Elongation

We examined the physiological effects of brassinosteroids (BRs) on early growth of Arabidopsis. Brassinazole (Brz), a BR biosynthesis inhibitor, was used to elucidate the significance of endogenous BRs. It inhibited growth of roots, hypocotyls, and cotyledonous leaf blades dose-dependently and independent of light conditions. This fact suggests that endogenous BRs are necessary for normal growth of individual organs of Arabidopsis in both photomorphogenetic and skotomorphogenetic programs. Exogenous brassinolide (BL) promoted hypocotyl elongation remarkably in light-grown seedlings. Cytological observation disclosed that BL-induced hypocotyl elongation was achieved through cell enlargement rather than cell division. Furthermore, a serial experiment with hormone inhibitors showed that BL induced hypocotyl elongation not through gibberellin and auxin actions. However, a synergistic relationship of BL with gibberellin A₃ (GA₃) and indole-3-acetic acid (IAA) was observed on elongation growth in light-grown hypocotyls, even though gibberellins have been reported to be additive to BR action in other plants. Taken together, our results show that BRs play an important role in the juvenile growth of Arabidopsis; moreover, BRs act on light-grown hypocotyl elongation independent of, but cooperatively with, gibberellins and auxin.     

Co-action between phytochrome B and HY4 in Arabidopsis thaliana

A combination of physiological and genetic approaches was used to investigate whether phytochromes and blue light (BL) photoreceptors act in a fully independent manner during photomorphogenesis of Arabidopsis thaliana (L.) Heynh. Wild-type seedlings and phyA, phyBand hy4 mutants were daily exposed to 3 h BL terminated with either a red light (R) or a far-red light (FR) pulse. In wild-type and phyA-mutant seedlings, BL followed by an R pulse inhibited hypocotyl growth and promoted cotyledon unfolding. The effects of BL were reduced if exposure to BL was followed by an FR pulse driving phytochrome to the R-absorbing form (Pr). In the wild type, the effects of R versus FR pulses were small in seedlings not exposed to BL. Thus, maximal responses depended on the presence of both BL and the FR-absorbing form of phytochrome (Pfr) in the subsequent dark period. Impaired responses to BL and to R versus FR pulses were observed in phyB and hy4 mutants. Simultaneous irradiation with orange light indicated that BL, perceived by specific BL photoreceptors (i.e. not by phytochromes), required phytochrome B to display a full effect. These results indicate interdependent co-action between phytochrome B and BL photoreceptors, particularly the HY4 gene product. No synergism between phytochrome A (activated by continuous or pulsed FR) and BL photoreceptors was observed.Abbreviations BL blue light - D darkness - FR far-redlight - FRc continuous FR - Pfr FR-absorbing form of phytochrome - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - WT wild type We thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands), Professor J. Chory (Salk Institute, Calif., USA) and the Arabidopsis Biological Resource Center (Ohio State University, Ohio, USA) for their kind provision of the original seed batches. This work was financially supported by CONICET, Universidad de Buenos Aires (AG 040) and Fundación Antorchas (A-12830/1 0000/9)     

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain Columbia, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and napin (1.7 S) seed-storage proteins of Brassica napus. The 12 S protein from Arabidopsis was resolved by SDS-PAGE into two groups of subunits with approximate relative molecular weights of 22–23 kDa (kilodalton) and 30–34 kDa. These polypeptides accumulated late in embryo development, disappeared early in germination, and were not detected in other vegetative or reproductive tissues. Accumulation of the 12 S proteins in aborted seeds from nine embryo-lethal mutants with different patterns of abnormal development was studied to determine the extent of cellular differentiation in arrested embryos from each mutant line. Abundant 12 S proteins were found in arrested embryos from two mutants with late lethal phases, but not in seven other mutants with lethal phases ranging from the globular to the cotyledon stages of embryo development. These results indicate that the accumulation of seed-storage proteins in wild-type embryos of Arabidopsis is closely tied to morphogenetic changes that occur during embryo development. Embryo-lethal mutants may therefore be useful in future studies on the developmental regulation of storage-protein synthesis.Abbreviations kDa kilodalton - M_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate