Article

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

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Abstract

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.

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... 10 Analyses of phytochrome chromophore-deficient mutants have demonstrated that phytochromes are important for maintaining chloroplast number and development as these mutants have reduced numbers of chloroplasts, reduced chlorophyll levels, increased chl a/chl b ratios, and reduced granal stacking in plastids. [14][15][16][17] Specific aspects of photosynthesis are also impaired in phytochrome synthesis mutants. High light-grown (400 µmol m −2 s −1 ) phytochrome A mutants (phyA-201) exhibit low levels of NPQ 18 and hy1 mutants (hy1-1), which are defective in phytochromobilin chromophore biosynthesis, also show low levels of NPQ, 18,19 suggesting that phytochromes play either direct or indirect roles in regulating NPQ to allow plants to cope with excess absorbed light energy in high light conditions. ...
... This impact of mesophyll-specific phytochromes on these processes may be in part through the association of phytochrome with regulating chlorophyll levels 45 and the quantitative input of phytochromes into the accumulation of Lhcb proteins. 16 Additionally, reduced chlorophyll levels have been previously correlated with disruptions in thylakoid architecture, including grana formation and stacking. 14−17 We also noted reduced grana and disturbed stacking, especially at early developmental stages for leaves lacking phytochromes compared to WT (Supplemental Figure 1). ...
... The association of trimeric LHCII with chlorophyll was intact in lines constitutively expressing BVR (35S::pBVR3) or weakly expressing BVR in the mesophyll (CAB3::pBVR3) compared to WT. Whereas phytochromes have been previously associated with accumulation of Lhcb proteins, 16 these data suggested that the pool of mesophyllspecific phytochromes is required for accumulation of Lhcb1 and may impact assembly of LHCII antenna complexes that are necessary for WT levels of NPQ. 49 ...
Article
Phytochromes regulate light-dependent plastid development and plant growth and development. Prior analyses demonstrated that phytochromes regulate expression of Sigma factor 2 (SIG2), which is involved in plastid transcription and coordinates expression of plastid‐ and nuclear‐encoded genes involved in plastid development, as well as plant growth and development. Mutation of SIG2 impacts distinct aspects of photosynthesis, resulting in elevated levels of cyclic electron flow and nonphotochemical quenching (NPQ). As we initially identified SIG2 expression as misregulated in a line lacking phytochromes in mesophyll tissues (i.e., CAB3::pBVR lines), here we report on an investigation of whether photosynthetic parameters such as NPQ are also disrupted in CAB3::pBVR lines. We determined that a specific parameter of NPQ, i.e., energy-dependent quenching (qE) which is a rapidly induced photoprotective mechanism that dissipates stressful absorption of excess light energy during photosynthesis, is disrupted when mesophyll phytochromes are significantly depleted. The observed reduction in NPQ levels in strong CAB3::pBVR lines is associated with a reduction in the accumulation of Lhcb1 proteins and assembly or stability of light-harvesting complexes (LHCs), especially trimeric LHC. These results implicate mesophyll-localized phytochromes in a specific aspect of phytochrome-mediated NPQ, likely through regulation of chlorophyll synthesis and accumulation and the associated impacts on chlorophyll–protein complexes. This role is distinct from the impact of mesophyll phytochrome-dependent control of SIG2 and associated NPQ regulation.
... It is of crucial importance in the biosynthetic pathway of PFB, a cofactor necessary for the formation of photoconvertible phytochromes and thus for proper light sensing (Parks and Quail, 1991;Rockwell et al., 2006). Light signaling deficiency, together with heme accumulation, also affects chlorophyll biosynthesis in hy1 (Chory et al., 1989;Terry and Kendrick, 1999). As indicated by the name that we originally used to designate the mutant line (lph), hy1-7 exhibits marked photomorphogenic defects, a phenotype that is consistent with the previously described role of HY1 in light perception (Davis et al., 1999;Muramoto et al., 1999). ...
... In addition to attenuated light signaling, other developmental aberrations were reported in the heme oxygenase-deficient plants. Defects in chlorophyll biosynthesis and chloroplast development due to nonfunctional phytochrome signaling in hy1 mutants were described (Chory et al., 1989). Furthermore, chlorophyll biosynthesis in hy1 plants is also inhibited via a negative feedback loop mediated by heme accumulation (Terry and Kendrick, 1999). ...
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In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The crosstalk between cytokinin response and light is known for a long time. However, the molecular mechanism underlying the interactionbetween light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL (LPH) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT-1 (CKI1), encoding the constitutively active sensor histidine kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE 1 (HY1) which encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertiblephytochromes. Our analysis confirmed the light-dependent regulation oftheCKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors PHYTOCHROME INTERACTING FACTOR 3 (PIF3) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). Changes in CKI1 expression observed in lph/hy1-7 and phy mutants correlatewithmisregulation of MSP signaling, changedcytokinin sensitivity and developmental aberrations,previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development.
... The transcriptional networks of the PIF proteins can overlap; however, specific regulatory functions exist: for example, PIF1 has been described as functioning in germination and chlorophyll biosynthesis, and PIF3 in the greening of seedlings (Huq et al. 2004; Monte et al. 2004). Indeed, phyB mutants are pale green throughout the entire plant cycle due to impaired chloroplast development (Chory et al. 1989). In the absence of functional PhyB, the PIF transcription factors are not degraded and thus continue to repress transcription of the essential genes required for chloroplast development and function (Chory et al. 1989; Stephenson et al. 2009). ...
... Indeed, phyB mutants are pale green throughout the entire plant cycle due to impaired chloroplast development (Chory et al. 1989). In the absence of functional PhyB, the PIF transcription factors are not degraded and thus continue to repress transcription of the essential genes required for chloroplast development and function (Chory et al. 1989; Stephenson et al. 2009). Loss of PhyB function not only impairs chloroplast development but also affects other processes such as shade avoidance, water use efficiency and flowering time (Reed et al. 1993; Boccalandro et al. 2009). ...
Article
Plant development is regulated by external and internal factors such as light and chloroplast development. A revertant of the Arabidopsis thaliana (L.) Heyhn. chloroplast biogenesis mutant snowy cotyledon 3 (sco3-1) was isolated partially recovering the impaired chloroplast phenotype. The mutation was identified in the Phytochrome B (PhyB) gene and is a result of an amino acid change within the PAS repeat domain required for light-induced nuclear localisation. An independent phyB-9 mutation was crossed into sco3-1 mutants, resulting in the same partial reversion of sco3-1. Further analysis demonstrated that SCO3 and PhyB influence the greening process of seedlings and rosette leaves, embryogenesis, rosette formation and flowering. Interestingly, the functions of these proteins are interwoven in various ways, suggesting a complex genetic interaction. Whole-transcriptome profiling of sco3-1phyB-9 indicated that a completely distinct set of genes was differentially regulated in the double mutant compared with the single sco3-1 or phyB-9 mutants. Thus, we hypothesise that PhyB and SCO3 genetically suppress each other in plant and chloroplast development.
... These screens have identified the phytochrome apoprotein and chromophore biosynthetic genes (HY1, HY2, PHYA, PHYB, and PHYD), cryptochrome genes (HY4; for review, see Fankhauser and Chory, 1997), and downstream elements involved in the control of elongation or flowering, including ELF3 (Zagotta et al., 1996), FHY1 and FHY3 (Whitelam et al., 1993), HY5 (Oyama et al., 1997), PEF1 (Ahmad and Cashmore, 1996), and RED1 (Wagner et al., 1997). With the exception of phyB and the chromophore mutants hy1 and hy2, none of the other mutants appears to be defective in phytochrome-regulated gene expression or chloroplast development (Chory et al., 1989a;Reed et al., 1994). ...
... Figure 7 shows the effect of a single pulse of red light on the derepression of CAB gene expression: a 14-fold increase in levels of total CAB mRNAs in the wild type. As reported previously and as shown in Figure 7, CAB accumulation was slightly reduced in phyB and about 80% reduced in hy1 (Chory et al., 1989a;Reed et al., 1994). In these mutants, the dark basal levels of CAB mRNA accumulation were similar to the wild type. ...
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We searched for new components that are involved in the positive regulation of nuclear gene expression by light by extending a screen for Arabidopsis cue(chlorophyll a/b-binding [CAB] protein-underexpressed) mutants (H.-M. Li, K. Culligan, R.A. Dixon, J. Chory [1995] Plant Cell 7: 1599–1610). cue mutants display reduced expression of the CAB3 gene, which encodes light-harvesting chlorophyll protein, the main chloroplast antenna. The new mutants can be divided into (a) phytochrome-deficient mutants (hy1and phyB), (b) virescent or delayed-greening mutants (cue3, cue6, and cue8), and (c) uniformly pale mutants (cue4 andcue9). For each of the mutants, the reduction inCAB expression correlates with the visible phenotype, defective chloroplast development, and reduced abundance of the light-harvesting chlorophyll protein. Levels of protochlorophyllide oxidoreductase (POR) were reduced to varying degrees in etiolated mutant seedlings. In the dark, whereas the virescent mutants displayed reduced CAB expression and the lowest levels of POR protein, the other mutants expressed CAB and accumulated POR at near wild-type levels. All of the mutants, with the exception ofcue6, were compromised in their ability to derepressCAB expression in response to phytochrome activation. Based on these results, we propose that the previously postulated plastid-derived signal is closely involved in the pathway through which phytochrome regulates the expression of nuclear genes encoding plastid proteins.
... The extract was measured at wavelengths of both 645 and 663 nm with a Lambda EZ201 spectrophotometer (Perkin Elmer). Chlorophyll a, chlorophyll b, and total chlorophyll contents were calculated using MACKINNEY'S (1941) specific absorption coefficients as reported by CHORY et al. (1989). chlorophyll a (mg/g)=12.72(A663)-2.59(A645)×V/1000×W ...
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This study was performed to genetical and morphological investigation of a novel chlorophyll deficiency gene in tobacco leaf. One low chlorophyll content (LCC) variety (Urumieh 2) and high chlorophyll content (HCC) variety (Burley Ree 103) from the Burley type was crossed and the F2 generation was grown on the field. One hundred plants were selected, contained low and high chlorophyll content. These plants were sampled and DNA was extracted. Sixty RAPD primers were tested on parents, LCC and HCC Bulks based on Bulk Segregant Analysis (BSA). Chi-square test confirmed the monogenic segregation. Regresion analysis showed that there was strong relationship between greenness degree and chlorophyll contents. Four primers (OPE17, OPC09, OPB08 and OPR02) showed polymorphism and after the test on 97 samples from the F2 generation two markers were selected (OPB08-1050 and OPC09-1900). That showed 15.9 and 10.8 CM distance from chlorophyll locus respectively
... This is important because several lightregulatory mechanisms essential for photosynthetic efficiency and adaptation occur in mature leaves (Melis, 1991; Aro et al., 1993). In addition, the action spectrum for various light-regulated processes (Fluhr et al., 1986; Cosgrove, 1994; Mohr, 1994) and the types of phytochromes that predominate in tissues (Chory et al., 1989; Quail, 1994) change during leaf development. The types of RNA polymerases predominating in plastids also change during leaf development , from a nuclear-encoded T7 phage-type in immature plastids to a plastid-encoded Escherichia coli-like RNA polymerase (PEP) in mature chloroplasts (Igloi and Kö ssel, 1992; Iratni et al., 1994; Allison et al., 1996; Hajdukiewicz et al., 1997). ...
Article
We characterized the photobiology of light-activated chloroplast transcription and transcript abundance in mature primary leaves by using the following two systems: transplastomic promoter-reporter gene fusions in tobacco (Nicotiana tabacum), and phytochrome (phyA, phyB, and hy2) and cryptochrome (cry1) mutants of Arabidopsis. In both dicots, blue light and UV-A radiation were the major signals that activated total chloroplast and psbA, rbcL, and 16S rrntranscription. In contrast, transcription activities in plants exposed to red and far-red light were 30% to 85% less than in blue light/UV-A, depending on the gene and plant species. Total chloroplast,psbA, and 16S rrn transcription were 60% to 80% less in the Arabidopsis phyA mutant exposed to blue light/UV-A relative to wild type, thus definitively linking phyA signaling to these photoresponses. To our knowledge, the major role of phyA in mediating the blue light/UV-A photoresponses is a new function for phyA in chloroplast biogenesis at this stage of leaf development. AlthoughrbcL expression in plants exposed to UV-A was 50% less in the phyA mutant relative to wild type, blue light-induced rbcL expression was not significantly affected in the phyA, phyB, andcry1 mutants. However, rbcL expression in blue light was 60% less in the phytochrome chromophore mutant,hy2, relative to wild type, indicating that another phytochrome species (phyC, D, or E) was involved in blue light-inducedrbcL transcription. Therefore, at least two different phytochromes, as well as phytochrome-independent photosensory pathways, mediated blue light/UV-A-induced transcription of chloroplast genes in mature leaves.
... It is interesting that sunl mutants share morphological phenotypes with phyB mutants. phyB mutants flower early and accumulate less chlorophyll (Chory et al., 1989a), and both phyB and sunl mutants have long hypocotyls in Wc (Koornneef et al., 1980; results not shown). ...
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The expression of the Arabidopsis plastocyanin (PC) gene is developmentally controlled and regulated by light. During seedling development, PC gene expression is transiently induced, and this induction can be repressed by sucrose. In transgenic seedlings carrying a PC promoter-luciferase fusion gene, the luciferase-induced in vivo luminescence was similarly repressed by sucrose. From a mutagenized population of such transgenic seedlings, we selected for mutant seedlings that displayed a high luminescence leve1 when grown on a medium with 3% sucrose. This screening of mutants resulted in the isolation of several sucrose-uncoupled (sun) mutants showing reduced repression of luminescence by sucrose. Analysis of the sun mutants revealed that the accumulation of PC and chlorophyll a/b binding protein (CAB) mRNA was also sucrose uncoupled, although the extent of uncoupling varied. The effect of sucrose on far-red light high-irradiance responses was studied in wild-type, sunl, sun6, and sun7 seedlings. In wild-type seedlings , sucrose repressed the far-red light-induced cotyledon opening and inhibition of hypocotyl elongation. sun7 seedlings showed reduced repression of these responses. Sucrose also repressed the far-red light-induced block of greening in wild-type seedlings, and both sun6 and sun7 were affected in this response. The results provide evidence for a close interaction between sucrose and light signaling pathways. Moreover, the sun6 and sun7 mutants genetically identify separate branches of phytochrome A-dependent signal transduction pathways.
... The analysis of transgenic plants either overexpressing or repressing by antisense methods these specific genes may ultimately be required before the situation is resolved. However, properly characterized monogenic photoreceptor mutants have proved very useful to this end (Adamse et al., 1988a(Adamse et al., , 1988bChory et al., 1989;L6pez-Juez et al., 1990aL6pez-Juez et al., , 1990b; for reviews, see Furuya, 1989;Kendrick and Nagatani, 1991). ...
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A novel cDNA sequence homologous to a phytochrome B (phyB) gene that was isolated in a library from tobacco tissue has been used in an Escherichia coli expression system to raise anti-phytochrome B (anti-PHYB) polypeptide-specific monoclonal antibodies. The specificity of these antibodies has been tested by cross-reactivity against purified pea light-labile type 1 and light-stable type 2 phytochromes, with some antibodies reacting with the type 2 and none with the type 1 phytochromes. One such antibody, monoclonal mAT1, has been employed to analyze the phytochrome molecular species present in a photomorphogenic long hypocotyl (lh) mutant of cucumber. The results indicated that the mutant contains wild-type levels of the light-labile type 1 phytochrome polypeptide (PHYA), which has an apparent molecular mass of approximately 120 kD, but shows less than 1% (detection limit) of a light-stable polypeptide recognized by mAT1 in wild-type seedlings. This protein, not detectable in the lh mutant, has the properties of light-stable type 2 phytochrome, has an apparent molecular mass of 116 to 117 kD, and remains at constant levels under continuous low-fluence-rate red light. Therefore, we conclude that the lh mutant lacks at least one type 2 phytochrome-like polypeptide, most probably a phyB gene product. The correlation between the lack of this protein and the deficiency or absence of physiological responses to a light-stable phytochrome species in this mutant helps to identify the physiological roles played by the products of different subfamilies within the phytochrome gene family.
... The authors suggested that the increased resistance results from higher Chl content or enhanced stomatal conductance due to superproduction of PhyB, which is the primary RL-sensor in green higher plants playing a dominant role in comparison to other phytochromes. The A. thaliana hy3 mutant has an elongated hypocotyl when grown in white light and is paler in color compared to WT (Chory et al. 1989). According to the available data, hy3 is severely defective in the PhyB apoprotein and, hence, in PhyB (Somers et al. 1991). ...
Article
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The effect of UV-A radiation (365 nm) and the protective effect of preillumination with red light (RL, 664 nm, 10 min) or with a combination of red and far-red light (FRL, 727 nm, 10 min) on the activity of the PSII as well as the expression levels of selected genes, especially encoding chloroplast proteins (sAPX, tAPX, CAB1, and D1), were studied in leaves of the 26-d-old hy3 mutant of Arabidopsis thaliana, which is deficient in the phytochrome B apoprotein. The effects were compared with corresponding effects observed in the hy2 mutant of A. thaliana, which is deficient in the phytochrome chromophore. Illumination with UV-A decreased the photosynthetic pigment content and the maximum photochemical quantum yield of PSII (Fv/Fm) and the effective quantum yield of PSII (ФPSII). The reduction of the Fv/Fm ratio and the ФPSII was more pronounced in the mutants as compared to wild-type plants (WT). The preillumination of the leaves with RL caused a significant reduction in the inhibitory effect of UV-radiation on the PSII activity in the WT plants, but it caused only a small decrease in the hy3 mutant. The preillumination of leaves with RL and FRL combination compensated the protective effect of RL on the UV-induced decrease of the fluorescence parameters in the WT. Such reversibility is typical for involvement of red/far-red reversible phytochromes at low intensity light. The results suggest an important role of red/far-red reversible phytochromes (phytochrome B) in the resistance of the PSII to UV-A radiation caused by changes in contents of either carotenoids or other UV-absorbing pigments probably through biosynthesis of these pigments. The data also demonstrated that phytochrome B and other phytochromes can affect the PSII stress-resistance by the fast regulation of the expression of genes encoding antioxidant enzymes and transcription factors at the step of gene transcription.
... Additionally, the phytochrome-deficient mutants of tomato aurea (au) and yellow-green-2 (yg-2) show inhibition of Pchlide synthesis, which in turn, causes loss of the POR protein in these mutants [47]. Under white light, au and yg-2 mature plants present a pale phenotype due to lower chlorophyll levels and reduced chloroplast development [48] [49]. Because the prolamellar body is the main site of accumulation of Pchlide and POR complexes, studies aimed to characterize this protein and its substrate in the new mutant YELP presented here will be required because we found plastids with prolamellar body-like structures despite the yellow cells were always grown in the light. ...
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In this work, we present an ultrastructural and physiological description of a novel chlorophyll-deficient, yellow cell line of the grass Bouteloua gracilis that develops etioplast-like plastids in presence of light (YELP). These mutant cells were compared to the parental, wild-type, highly chlo-rophyllous cells from which they were isolated. Growth analysis, based on fresh and dry weights, indicated that YELP accumulates biomass at a slower rate than the parental, green cells. Besides, YELP developed very low levels of photosynthetic pigments, reaching only 9.3% and 38.4% of chlorophyll a and chlorophyll b, respectively, developed by the wild-type cells. Likewise, the accessory pigments, carotenes and xanthophylls, were only synthesized at 8.0% and 5.4%, respectively , of the levels reached by the green cells. Electron microscopy revealed remarkable differences in plastid ultrastructure between the wild-type and mutant cells. Plastids of YELP were heterogeneous and smaller than those found in wild-type cells. YELP plastids were abnormal with poorly developed membrane systems that prevented the accumulation of chlorophyll and accessory pigments in the mutant cell line. We expect this novel, mutant cell line will provide new tools for studying plastid development and differentiation.
... Several studies have reported that blue light resulted in higher Chl contents than red light (Saebo et al., 1995;Kim et al., 2004;Lee et al., 2007;Poudel et al., 2008), which was inconsistent with our result. No significant difference between treatment R and treatment B on the Chla, Chlb, Chl(a + b) contents and Chl density was observed in this experiment, which probably was due to phytochrome participating in modulating the amount of chloroplast development and inducing chlorophyll accumulation in green plants in response to red light (Chory et al., 1989). In addition to functioning as accessory pigments for photosynthesis, Car is also important for protecting photosynthetic organisms from the destructive photooxidations (Nobel, 2010). ...
Article
In many parts of Asia, balloon flower (Platycodon grandiflorum (Jacq.) A. DC., P. grandiflorum) is much valued because of its ornamental and medicinal attributes. Although previous studies have described techniques of plant regeneration and culture, the influence of different spectra has not been documented. We investigated the effect of light spectra on leaf morphology, anatomy and chemical composition using cultured nodal explants grown under light-emitting diodes (LEDs) at a photosynthetic photon flux density of 50 μmol m−2 s−1. Treatments were: monochromatic blue light (B), 75% blue + 25% red light (BR31), 50% blue + 50% red light (BR11), 25% blue + 75% red light (BR13) and monochromatic red light (R). Fluorescent white lamps (FL) were used as a control. Treatment B induced larger leaf area, leaf thickness and dry mass of whole plant and higher leaf number than treatment R or FL. Plantlets from treatment R had the highest specific leaf mass, soluble sugar and sucrose content. Chlorophyll and L-ascorbic acid content was higher in leaves grown under LED variants than in those grown under FL. The indoleacetic acid content in B- and BR31-treated leaves was higher than that in the R-treated leaves. In addition, B and BR31 induced large and nearly rectangular palisade parenchyma cells, while the palisade parenchyma cells were elliptical in shape and small in the FL, BR11 and BR13 treatments, or irregular in the R treatment. Leaves from the R and BR11 treatments had the highest stomata frequency, moreover, FL-, B- and BR31-treated leaves possessed normal elliptical stomata. Treatment B induced the highest ETR, ΦPSII, and qp, significantly higher than treatment R or FL. Our data revealed that light spectrum strongly affected the morphology and growth index of P. grandiflorum plantlets cultured in vitro.
... Additional studies demonstrated that both blue light-responsive cryptochrome and red/far-red light-responsive phytochrome photoreceptors perceive light and stimulate plastid development (Thum et al., 2001), and phytochromes are involved in regulating chloroplast gene transcription in mature leaves (Chun et al., 2001). Consistent with these observations, phytochrome-deficient mutants, including phyB (Reed et al., 1994) and chromophore-deficient hy1 and hy2 mutants (Chory et al., 1989), exhibit defects in chloroplast development and/or differentiation. However, insights into the identity and functions of specific photoreceptor-dependent effectors that impact chloroplast development and function are limited. ...
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Light perception by photoreceptors impacts plastid transcription, development, and differentiation. This photoreceptor-dependent activity suggests a mechanism for photoregulation of gene expression in the nucleus and plastid that serves to coordinate expression of critical genes of these two organelles. This coordinate expression is required for proper stoichiometric accumulation of components needed for assembly of plastids, photosynthetic light-harvesting complexes and components such as phytochromes. Chloroplast-targeted sigma factors, which function together with the plastid-encoded RNA polymerase to regulate expression of plastid-encoded genes, and nuclear-encoded plastid development factors, such as GLK1 and GLK2, are targets of phytochrome regulation. Such phytochrome-dependent functions are hypothesized to allow light-dependent regulation, and feasibly tuning, of plastid components and function in response to changes in the external environment, which directly affects photosynthesis and the potential for light-induced damage. When the size and protein composition of the light-harvesting complexes are not tuned to the external environment, imbalances in electron transport can impact the cellular redox state and cause cellular damage. We show that phytochromes specifically regulate the expression of multiple factors that function to modulate plastid transcription and, thus, provide a paradigm for coordinate expression of the nuclear and plastid genomes in response to changes in external light conditions. As phytochromes respond to changes in the prevalent wavelengths of light and light intensity, we propose that specific phytochrome-dependent molecular mechanisms are used during light-dependent signaling between the nucleus and chloroplast during photomorphogenesis to coordinate chloroplast development with plant developmental stage and the external environment.
... Since the 1980s, molecular genetic techniques relying on the model species Arabidopsis thaliana (Arabidopsis) have successfully provided great benefits to the field of plant photobiology. For example, the inhibition of hypocotyl elongation under light had been assumed to be controlled by three types of photoreceptors, including blue light receptors, UV-B receptors, red/far-red light-absorbing phytochromes, and screening to identify these photoreceptors on the basis of this phenotype is simple and sophisticated (18)(19)(20). In 1980, several Arabidopsis mutants that exhibit long hypocotyls under white light were identified (18). ...
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Blue light affects many aspects of plant growth and development throughout the plant lifecycle. Plant cryptochromes (CRYs) are UV-A/blue light photoreceptors that play pivotal roles in regulating blue light-mediated physiological responses via the regulated expression of more than one thousand genes. Photoactivated CRYs regulate transcription via two distinct mechanisms: indirect promotion of the activity of transcription factors by inactivation of the COP1/SPA E3 ligase complex or direct activation or inactivation of at least two sets of basic helix-loop-helix transcription factor families by physical interaction. Hence, CRYs govern intricate mechanisms that modulate activities of transcription factors to regulate multiple aspects of blue light-responsive photomorphogenesis. Here, we review recent progress in dissecting the pathways of CRY signaling and discuss accumulating evidence that shows how CRYs regulate broad physiological responses to blue light. This article is protected by copyright. All rights reserved.
... The Plant Cell as hy3 ( phyB ), have contributed to studies of the physiological roles of phytochrome (Nagatani et al., 1993; Reed et al., 1994; Shinomura et al., 1994, 1996). The hy1 and hy2 mutants do not respond to R and FR light, although the phytochrome apoprotein is synthesized normally (Chory et al., 1989). These mutants are probably deficient in phytochrome chromophore biosynthesis, because feeding experiments with biliverdin restore the wild-type phenotype (Parks and Quail, 1991). ...
Article
The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.
... Faure et al. [79] found that the EAM 8 mutant affects circadian clock function by regulating both PRR and the Ppd-H1 (Photoperriod-H1) gene. In Arabidopsis thaliana, the lhy1 (long hypocotyl 1) mutant [84] showed a similar period of circadian oscillation when related with their wild-type (WD) under the long photoperiod of light, but has reduced circadian oscillation under continuous red light, and this knowledge helps us to establish that one or more phytochrome species are present in Arabidopsis thaliana to control or entrain the circadian clock [20]. A (lhy-4) mutant of Arabidopsis thaliana [85] lacked the capability to prevent elongation of hypocotyl in bluelight radiation, but showed normal phytochrome-controlled inhibition in far-red photoperiod. ...
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Many mortal organisms on this planet have developed the potential to merge all internal as well as external environmental cues to regulate various processes running inside organisms and in turn make them adaptive to the environment through the circadian clock. This moving rotator controls processes like activation of hormonal, metabolic, or defense pathways, initiation of flowering at an accurate period, and developmental processes in plants to ensure their stability in the environment. All these processes that are under the control of this rotating wheel can be changed either by external environmental factors or by an unpredictable phenomenon called mutation that can be generated by either physical mutagens, chemical mutagens, or by internal genetic interruption during metabolic processes, which alters normal functionality of organisms like innate immune responses, entrainment of the clock, biomass reduction, chlorophyll formation, and hormonal signaling, despite its fewer positive roles in plants like changing plant type, loss of vernalization treatment to make them survivable in different latitudes, and defense responses during stress. In addition, with mutation, overexpression of gene components sometimes supresses mutation effect and promote normal circadian genes abundance in the cell, while sometimes it affects circadian functionality by generating arrhythmicity and shows that not only mutation but overexpression also effects normal functional activities of plant. Therefore, this review mainly summarizes the role of each circadian clock genes in regulating rhythmicity, and shows that how circadian outputs are controlled by mutations as well as overexpression phenomenon. Graphical abstract
... PHYA protein accumulates to a high level in etiolated seedlings and acts primarily as a far-red sensor at the stages of early seedling development (Parks and Quail, 1993), whereas PHYB is the predominant red sensor (Reed et al., 1993;Franklin et al., 2003). Hypocotyl 1 (HY1) encodes a plastid heme oxygenase necessary for PHY chromophore biosynthesis, and deletion of HY1 leads to lowered levels of photoreversible PHY A, B, C, D, E, thus reduced sensitivity to far-red and red light (Chory et al., 1989;Muramoto et al., 1999). ...
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Primary metabolism is closely linked to plant productivity and quality. Thus, a better understanding of the regulation of primary metabolism by photoreceptors has profound implications for agricultural practices and management. This study aims at identifying the role of light signaling in the regulation of primary metabolism, with an emphasis on starch. We first screened seven cryptochromes and phytochromes mutants for starch phenotype. The phyAB mutant showed impairment in starch accumulation while its biomass, chlorophyll fluorescence parameters, and leaf anatomy were unaffected, this deficiency being present over the whole vegetative growth period. Mutation of plastidial nucleoside diphosphate kinase-2 (NDPK2), acting downstream of phytochromes, also caused a deficit in starch accumulation. Besides, the glucose-1-phosphate adenylyltransferase small subunit (APS1) was down-regulated in phyAB. Those results suggest that PHYAB affect starch accumulation through NDPK2 and APS1. Then, we determined changes in starch and primary metabolites in single phyA, single phyB, double phyAB grown in light conditions differing in light intensity and/or light spectral content. PHYA is involved in starch accumulation in all the examined light conditions, whereas PHYB only exhibits a role under low light intensity (44 ± 1 μmol m-2 s-1) or low R:FR (11.8 ± 0.6). PCA analysis of the metabolic profiles in the mutants and wild type (WT) suggested that PHYB acts as a major regulator of the leaf metabolic status in response to light intensity. Overall, we propose that PHYA and PHYB signaling play essential roles in the control of primary metabolism in Arabidopsis leaves in response to light.
... Although etiolated seedlings also develop under far-red light, the plastid structures differ from the etioplasts developed in darkness ( Figure 8B). Light signaling contributes to plastid development via complex pathways (Chory et al. 1989). Plastid biogenesis in the Arabidopsis mutant cop1 is suppressed in darkness (Deng et al. 1992;Sperling et al. 1998). ...
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Since its domestication from wild rice thousands of years ago, rice has been cultivated largely through transplantation. During transplantation from the nursery to the paddy field, rice seedlings experience transplantation shock which affects their physiology and production. However, the mechanisms underlying transplantation shock and rice adaptation to this shock are largely unknown. Here, we isolated a transplant-sensitive chloroplast-deficient (tsc1) rice mutant that produces albino leaves after transplantation. Blocking light from reaching the juvenile leaves and leaf primordia caused chloroplast deficiencies in transplanted tsc1 seedlings. TSC1 encodes a noncanonical ATP-binding cassette (ABC) transporter homologous to AtNAP14 and of cyanobacterial origin. We found that TSC1 controls plastid development in rice under dark conditions, and functions independently of light signaling. However, light rescued the tsc1 mutant phenotype in a spectrum-independent manner. TSC1 was upregulated following transplantation, and modulated the iron and copper levels, thereby regulating prolamellar body formation during the early P4 stage of leaf development. Therefore, TSC1 is indispensable for plastid development in the absence of light, and contributes to adaptation to transplantation shock. Our study provides insight into the regulation of plastid development and establishes a framework for improving recovery from transplantation shock in rice.
... Of the five GUN loci identified through the original genetic screen (Susek et al., 1993), GUN2 (encoding heme oxygenase 1) and GUN3 (encoding phytochromobilin synthase) are also known as ELONGATED HYPOCOTYL 1 (HY1) and HY2, respectively (Figure 1). Because the defects in the gun2 and gun3 mutants affect the biosynthesis of phytochromobilin, the chromophore of the red and far-red light photoreceptors phytochromes (Bae and Choi, 2008;Li et al., 2011), both hy1 and hy2 were initially identified as key components of light signaling pathways (Chory et al., 1989;Parks and Quail, 1991). Their later identification as gun mutants indicated a close link between light and retrograde signaling (Ruckle et al., 2012;Larkin, 2014). ...
Article
Communication between cellular compartments is vital for development and environmental adaptation. Signals emanating from organelles, so-called retrograde signals, coordinate nuclear gene expression with the developmental stage and/or the functional status of the organelle. Plastids (best known in their green photosynthesizing differentiated form, the chloroplasts) are the primary energy-producing compartment of plant cells, and the site for the biosynthesis of many metabolites, including fatty acids, amino acids, nucleotides, isoprenoids, tetrapyrroles, vitamins, and phytohormone precursors. Signals derived from plastids regulate the accumulation of a large set of nucleus-encoded proteins, many of which localize to plastids. A set of mutants defective in retrograde signaling (genomes uncoupled, or gun) was isolated over 25 years ago. While most GUN genes act in tetrapyrrole biosynthesis, resolving the molecular function of GUN1, the proposed integrator of multiple retrograde signals, has turned out to be particularly challenging. Based on its amino acid sequence, GUN1 was initially predicted to be a plastid-localized nucleic acid-binding protein. Only recently, mechanistic information on the function of GUN1 has been obtained, pointing to a role in plastid protein homeostasis. This review article summarizes our current understanding of GUN-related retrograde signaling and provides a critical appraisal of the various proposed roles for GUNs and their respective pathways.
... However, Kendrick (1996, 1999) recommended in their report that inhibition of Pchlide synthesis is not connected to the inhibition of phytochrome. Some reports strongly support this opinion in which it was revealed that pale yellow-green phenotype had shown during their growth in several phytochrome mutant plants (Chory et al. 1989;Weller et al. 1996) (Table 16.1). ...
Chapter
Heme oxygenase 1 (HO1) is a well-known protector against oxidative stress in an animal system. In plant system, recently it’s also well explored against different stresses. Heme oxygenase (HO) scavenges free heme released from heme proteins with the production of ferrous iron (Fe²⁺), biliverdin-IXα (BV-IXα), and carbon monoxide (CO). HO illustrates functional diversity and engages in several functions. It regulates positively in antioxidant defense mechanism against various types of abiotic stress. Among all forms of HO, HO1 has been well explored against abiotic stresses as metal stress, salt stress, and drought stress; however, the differing roles of the other three HOs are still less identified. HO1 defects lead to reduced chlorophyll content. It delays gibberellin-triggered programmed cell death of aleurone layer due to drought stress by interacting with nitric oxide. HO1 along with nitric oxide and iron shows the long-distance signaling response to salt stress. Overall, the study helps to explore highlights on the recent studies related to plant HO research involving its antioxidant role against abiotic stresses.
... Hypocotyl phenotypes have been used to screen for mutants showing abnormal responses to light, and the genes discovered through these studies have had a great impact on the field of plant physiology. Arabidopsis thaliana (hereafter Arabidopsis) hy mutants were initially characterized on the basis of their long hypocotyl phenotype in the light (Chory et al. 1989). Later research showed that hy3 mutants are deficient in the photoreceptor phytochrome B, while hy5 affects a transcription factor that functions in light signaling, and hy1 and hy2 both affect phytochrome chromophore biosynthesis (Kohchi et al. 2001). ...
... BV, as a product derived from HO-catalyzed oxidative cleavage of heme, is the precursor of PΦB [25,72]. In Arabidopsis, the mutation of HO1 causes the hy1 mutant to do not respond to red and far-red light [73], and be deficient in BV and subsequent PΦB synthesis [27]. Another HO-deficient mutant of pea (pcd1) was also shown to be unable to convert heme to BV and have severely impaired ability in PΦB biosynthesis [74]. ...
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Light is one of the most important environmental factors that affect many aspects of plant growth, including chlorophyll (Chl) synthesis and flowering time. Here, we identified a rice mutant, yellow leaf and early flowering (ye1), and characterized the gene YE1 by using a map-based cloning method. YE1 encodes a heme oxygenase, which is localized to the chloroplasts. YE1 is expressed in various green tissues, especially in leaves, with a diurnal-rhythmic expression pattern, and its transcripts is also induced by light during leaf-greening. The mutant displays decreased Chl contents with less and disorderly thylakoid lamellar layers in chloroplasts, which reduced the photosynthesis rate. The early flowering phenotype of ye1 was not photoperiod-sensitive. Furthermore, the expression levels of Chl biosynthetic genes were downregulated in ye1 seedlings during de-etiolation responses to light. We also found that rhythmic expression patterns of genes involved in photoperiodic flowering were altered in the mutant. Based on these results, we infer that YE1 plays an important role in light-dependent Chl biogenesis as well as photoperiodic flowering pathway in rice.
... The extract was measured at wavelengths of both 645 and 663 nm with T80 UV/VIS Spectrophotometer (PG Instrument Ltd.). Chlorophyll a, chlorophyll b and total chlorophyll contents were calculated using MacKinney's (1941) specific absorption coefficients as described by Chory et al. (1989), in which chlorophyll a = 12.72 (A 663 ) -2.59 (A 645 ), chlorophyll b = 22.88 (A 645 ) -4.68 (A 6663 ), and total chlorophyll = 20.29 (A 645 ) + 8.02 (A 663 ). The total chlorophyll per gram tissue was converted with the formula of, total chlorophyll (mg/g) = chlorophyll (mg/L) X 0.001 (L) / fresh weight (g). ...
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The uppermost leaf below the panicle is the flag leaf that provides the most important source of photosynthetic energy during reproduction and grain filling, thereby has great impact in panicle development and grain yield in rice. In the present investigation in order to explore the relationship between grain yield and flag leaf parameters, yield composition, length and width of the flag leaf, and panicle length were measured in some rice cultivars. Statistical analysis indicated that flag leaf length was positively correlated with panicle length for the studied cultivars demonstrating higher grain yield. Chlorophyll measurement indicated that flag leaf contained more chlorophyll than penultimate leaf. Yield of all the cultivars upon excision of flag leaf was also compared. Removal of flag leaf led to a decline in the seed-setting rate which eventually reduced the grain yield. Besides this, variable pollen viability was also noticed in the different cultivars. mvivsk: av‡bi kx‡li wb‡Pi me©cO_g cvZvwU‡K d¬vM cvZv ejv nq hv av‡bivbv c~Y©Zvi mgq mv‡jvKms‡k−kw³ Drcv`‡b g~Lf~wgKv cvjb K‡i| av‡bi kx‡li MVb I av‡bi dj‡b d¬vM cvZvi f~wgKv Abb¨| GB M‡elYvq av‡bi djb I d¬vM cvZvi gaKvi mcK© †`Lvi RbwKQy av‡bi Rv‡Z dj‡bi cOfveK, d¬vM cvZvi ˆ`N©¨ I cO¯' Ges av‡bi kx‡li ˆ`‡N©¨i cwigvc Kiv nq| M‡elYv djvd‡j †`Lv hvq, mKj Rv‡Zi †¶‡IB d¬vM cvZvi ˆ`N©¨ I kx‡li ˆ`‡N©¨i mcK© abvZiK| d¬vM cvZv I Zvi wb‡Pi cvZvi †K¬v‡ivwd‡ji gvIv cwigvc K‡i †`Lv hvq Zyjbvg~jKfv‡e d¬vM cvZvq †K¬v‡ivwd‡ji cwigvY †ewk| wKQy av‡bi d¬vM cvZv †K‡U w`‡q Zvi djb cwigvc K‡i d¬vM cvZv bv KvUv av‡bi Zyjbvq Kg cvIqv †M‡Q| GQvovI av‡bi wewfbœ Rv‡Zi †¶‡I civM‡iYyi mgZvq wfbœZv cwijwZ nq|
... Loss of phyB results in longer hypocotyl than the wild type under white or red light, and this visible phenotype has been considered useful to assess phyB activity (Chory et al., 1989). No significant differences in hypocotyl length were observed between phyB-9 OG and phyB-9 BC under either white light or red monochromatic light, indicating that the bnen mutation does not affect this well-studied trait of phyB, at least in the fluence rate examined (Fig. 1, H and I). ...
... Altered levels of chloroplast retrograde signal(s), functioning in either chloroplast biogenesis or operation, ultimately contribute to changes in plant growth and development. For example, Arabidopsis mutants over-accumulating plastid signals such as heme (Chory et al., 1989;Woodson et al., 2011;Espinas et al., 2012), methylerythritol cyclodiphosphate (MEcPP) (Gil et al., 2005;Xiao et al., 2012;Bjornson et al., 2017), dihydroxyacetone phosphate (DHAP) (Chen and Thelen, 2010;Vogel et al., 2014), or 3 -phosphoadenosine 5 -phosphate (PAP) (Rossel et al., 2006;Wilson et al., 2009;Estavillo et al., 2011) are smaller in rosette size with varying extents of altered rosette morphology. As some of these retrograde signals such as MEcPP (Xiao et al., 2012) and PAP accumulate upon excess light exposure, perhaps these chloroplast retrograde signaling mutants could be used to dissect if and how chloroplast retrograde, light and hormonal signaling crosstalk with one another to impact on plant morphogenesis. ...
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Plant growth and development are dependent on chloroplast development and function. Constitutive high level accumulation of a chloroplast stress signal, 3′-phosphoadenosine-5′-phosphate (PAP), confers drought tolerance to plants, but slow downs and alters plant growth and development. PAP, a by-product of sulfur metabolism, is maintained at very low levels by the SAL1 phosphatase during vegetative growth of Arabidopsis and accumulates in rosettes during drought and excess light. Eight independent forward genetic screens in Arabidopsis identified SAL1 as the regulator of multiple phenotypes related to stress responses, hormonal signaling and/or perception. In this perspective article, we collate all the sal1 phenotypes published in the past two decades, and distill the different pathways affected. Our meta-analysis of publicly available sal1 microarray data coupled to preliminary hormonal treatment and profiling results on sal1 indicate that homeostasis and responses to multiple hormones in sal1 are altered during rosette growth, suggesting a potential connection between SAL1-PAP stress retrograde pathway and hormonal signaling. We propose the SAL1-PAP pathway as a case study for integrating chloroplast retrograde signaling, light signaling and hormonal signaling in plant growth and morphogenesis.
... Brief red light treatment induces CABP expression with a characteristic waveform consisting of a transient acute response peaking 2 to 4 hr after the light pulse, followed by severa1 cycles of a circadian rhythm with a period of 30 hr or more (Millar et al., 1992a;Kay, 1993;Anderson and Kay, 1997). The red light induction of CAB2 expression is attenuated by subsequent farred illumination, demonstrating that phytochrome mediates this response (Chory et al., 1989;Kay, 1993). ...
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The fundamental notion that phytochrome controls plant development through differential regulation of gene expression (Mohr, 1966) is now well supported by direct experimental evidence (Benfey and Chua, 1989; Gilmartin et al., 1990; Kuhlemeier et al., 1987; Nagy et al., 1988; Tobin and Silverthorne, 1985). However, the molecular mechanism by which the photoreceptor transduces its regulatory signal to genes under its control remains unknown. For some time we have approached this question by simultaneously studying the properties of the photoreceptor molecule and the negative autoregulation of phytochrome (phy) genes as a paradigm of phytochrome-regulated gene expression (Colbert, 1988; Lissemore and Quail, 1988; Quail et al., 1987b, 1990). Recent molecular-genetic studies have revealed that phytochrome is encoded by a small family of divergent and differentially regulated genes (Dehesh et al., 1990b; Sharrock and Quail, 1989); have shown that overexpression of the photoreceptor in heterologous, transgenic plants provides a system for directed mutational analysis of functional regions of the polypeptide (Boylan and Quail, 1989; Kay et al., 1989c; Keller et al., 1989); and have begun to provide insight into the cis-regulatory elements and trans-acting factors involved in phy gene transcription (Brace et al., 1989, 1990; Dehesh et al., 1990a).
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Lesion mimic mutant (LMM) plants display spontaneous necrotic lesions on their leaves without any pathogenic infection. Specific rice LMMs designated as spotted leaf (spl) including spl1, spl3, spl4, spl5 and spl6 are genetically known as lesion resembling disease (lrd) mutant. The inheritance patterns in the and progenies of these mutants are controlled by recessive genetic factors. Lesion development in the rice LMMs were controlled by both development stages and environmental factors. The rice LMMs exhibited higher numbers of spots under temperature than those under . Contents of chlorophyll were drastically reduced at 60 days old LMM leaves when the spot formation was severe. The levels of endogenous hydrogen peroxide were highest at 45 days old mutants but reduced at 60 days old. Transcription levels of stress related genes including thioredoxin peroxidase and protein disulfide isomerase were reduced in spotted leaves than those of non spotted leaves. It could be suggested that scavenging system against reactive oxygen species induced by either stresses or innate metabolisms may not work properly in the rice LMMs. As these rice LMMs autonomously expressed clear lesions of lrd phenotype without pathogen infection, it could be useful to understand stresses responses in plants.
Article
Photon fluence rate-response curves at different wavelengths were generated for the light-induced inhibition of hypocotyl elongation in seedlings of wildtype and photomorphogenic mutants of Arabidopsis thaliana. (L.) Heynh. Treatment of wild-type seedlings with continuous low-fluence-rate light (< 1.0 μmol photons · m(-2) · s(-1)) induced some inhibition of hypocotyl elongation at all wavelengths tested, with maximum inhibition in blue light. At higher fluence rates, inhibition reached a maximum of 70-80% in UV-A, blue, and far-red light. Fluence rate-response curves for seedlings of blu1, a blue light-response mutant, showed a specific reduction in their response to blue light, but their response to UV-A, red, and far-red light was similar to that in wild-type seedlings. In contrast, the phytochromedeficient mutant hy6 showed a loss of response to lowfluence-rate light at all wavelengths, as well as to highfluence-rate far-red light. However, hy6 seedlings retained sensitivity to high-fluence-rate blue and UV-A light. The data support the conclusion that blue-lightand phytochrome-dependent photosensory systems regulate hypocotyl elongation independently and in an additive manner. Furthermore, hypocotyl inhibition in wild-type, blul, hy6 and blul-hy6 double mutants was indistinguishable in UV-A light, whereas marked differences were observed at other wavelengths, indicating the involvement of a third photosensory system with an absorption maximum in the UV-A.
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Plastid genes are transcribed by two different types of RNA polymerases: A plastid-encoded eubacterial-type RNA polymerase (PEP), and nuclear-encoded phage-type RNA polymerases (NEP). To investigate the spatio-temporal expression of PEP, we tagged its α-subunit with a hemagglutinin epitope (HA). Transplastomic tobacco plants were generated and analyzed for the distribution of the tagged polymerase in plastid subfractions, and associated genes were identified under different light conditions. RpoA:HA was detected as early as in 3 days old seedlings and was constitutively expressed in green tissue over 60 days of plant development. We found that the tagged polymerase subunit was preferably associated with the plastid membranes, and less abundant in the soluble stroma fraction. Attachment of RpoA:HA to the membrane fraction during early seedling development was independent of DNA, whereas at later stages of development, DNA seems to facilitate connection of the polymerase to membranes. To survey PEP-dependent transcription units, we probed for nucleic acids enriched in RpoA:HA precipitates using a tobacco chloroplast whole genome tiling array. The most strongly co-enriched DNA fragments represent photosynthesis genes (e.g. psbA, psbC, psbD, psbB, rbcL) known to be driven by PEP promoters, while NEP-dependent genes were less abundant in RpoA:HA precipitates. Additionally, we could demonstrate that the association of PEP with photosynthesis-related genes was reduced during the dark period, indicating that plastome-wide PEP-DNA association is a light-dependent process. This article is protected by copyright. All rights reserved.
Article
A new, elongated mutant of garden pea (Pisum sativum L.) is described, and shown to be conferred by a recessive allele of a new gene, sln. At the seedling stage, the mutant resembles the previously described slender type (genotype la cry s ), possessing markedly longer basal internodes than the wild-type. Furthermore, as for la cry s plants, application of gibberellin (GA)-biosynthesis inhibitors to the dry seeds (before sowing) did not markedly affect internode length in the mutant. However, the inheritance of the new slender phenotype is unusual, since in crosses between sln and Sln plants the mutant phenotype is absent in the F2 generation, reappearing in the F3. Young shoots possessing the new slender phenotype (sln) contained much higher levels of GA1, GA8, GA20 and GA29 than did wild-type shoots. Mature, near-dry seeds from slender plants contained very high levels of GA20, marginally more GA29, and very little (if any) GA29-catabolite, compared with seeds harvested from wild-type (Sln-) plants. It is suggested that sln may impair the catabolism of GA20 in maturing seeds. As a result, GA20 accumulates and on germination may move into the seedling where it is converted to GA1, promoting elongation growth. A model is proposed to explain the inheritance of the sln phenotype and its physiological implications are discussed. The new sln slender mutation has a different mode of action from the established la cry s slender gene combination.
Article
The committed step in the biosynthesis of the phytochrome chromophore phytochromobilin involves the oxidative cleavage of heme by a heme oxygenase (HO) to form biliverdin IXα. Through positional cloning of the photomorphogenic mutant hy1, the Arabidopsis HO (designated AtHO1) responsible for much of phytochromobilin synthesis recently was identified. Using theAtHO1 sequence, we identified families ofHO genes in a number of plants that cluster into two subfamilies (HO1- and HO2-like). The tomato (Lycopersicon esculentum) yg-2 andNicotiana plumbaginifolia pew1 photomorphogenic mutants are defective in specific HO genes. Phenotypic analysis of a T-DNA insertion mutant of Arabidopsis HO2 revealed that the second HO subfamily also contributes to phytochromobilin synthesis. Homozygous ho2-1 plants show decreased chlorophyll accumulation, reduced growth rate, accelerated flowering time, and reduced de-etiolation. A mixture of apo- and holo-phyA was detected in etiolated ho2-1 seedlings, suggesting that phytochromobilin is limiting in this mutant, even in the presence of functional AtHO1. The patterns of ArabidopsisHO1 and HO2 expression suggest that the products of both genes overlap temporally and spatially. Taken together, the family of HOs is important for phytochrome-mediated development in a number of plants and that each family member may uniquely contribute to the phytochromobilin pool needed to assemble holo-phytochromes.
Chapter
Phytochrome action has been demonstrated to affect the transcription of a number of different genes in many different species (see reviews by Tobin and Silverthorne, 1985; Kuhlemeier et al., 1987). The effect may be either a positive or negative one. Other light receptors, circadian rhythms, and tissue type have also been shown to have effects on the transcription of phytochrome regulated genes. Particular short “light responsive elements” that can interact with protein factors have been identified upstream of a number of rbcS and cab genes encoding, respectively, the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the major apoproteins of the photosystem II light-harvesting chlorophyll a/b-protein complex (LHCII). The evidence suggests these sequences play an important role in the overall response to light/dark conditions (reviewed in Silverthorne and Tobin, 1987; Benfey and Chua, 1989). There is also evidence that phytochrome action can alter RNA levels by effects on additional, post-transcriptional processes (Colbert, 1988; Thompson, 1988; Elliott et al., 1989), as well as influence many other processes, such as membrane permeability, that may not involve altered gene expression (Kendrick and Kronenberg, 1986). Although the phytochrome chromoprotein has itself been the subject of biochemical studies for many years, to date there is no clear understanding of the chain of events by which the phototransformation of phytochrome leads to specific transcriptional changes.
Chapter
The realisation that phytochrome represents a family of photoreceptors, encoded by at least three, and probably more, nuclear genes (Sharrock and Quail, 1989), has led to the proposal that the members of the family may have differential roles in the regulation of development and metabolism (see Smith and Whitelam, 1990, for review). In principle, this idea simplifies our understanding of the complexity of photomorphogenesis, by providing a potential resolution of the many conflicts in the physiological data. Investigations over several decades have shown that a number of different response modes, separable on classical photobiological grounds, can be incontrovertibly attributed to phytochrome, but this multiplicity of roles has been extremely difficult to reconcile with the classical concept of phytochrome as a single photoreceptor. Such conflicts may be traced back at least two decades, to the proposal by Hillman (1967) that there must be at least two populations of phytochrome, one that is ‘active’ but present in low concentration, and one that is ‘bulk’, i.e., present in high concentration and responsible for the spectrophotometric observations but not active in the physiological responses. It is, therefore, tempting to speculate that different phytochrome-mediated response modes may be mediated by different phytochromes. This article considers this idea and some of the evidence that currently appears to support it. We conclude that the hypothesis is at least sustainable on present evidence, but the attractiveness of the concept must be weighed in balance with the implications it has for the central dogmas of photomorphogenesis.
Chapter
Since plants are sedentary organisms, they are much more at the mercy of their environment than are animals. It is therefore not surprising that environmental factors, such as light and temperature, have a profound effect on the growth and development of plants. In fact, the greater regulatory importance of external factors is one of the ways in which plant development differs fundamentally from that of animals (Goldberg, 1988). Environmental stimuli frequently act as cues to initiate developmental transitions in plants, a good example being the induction of leaf expansion and chloroplast biogenesis when plants are first exposed to light. Indeed light probably has a more extensive regulatory influence on plant development than any other environmental factor. In addition to detecting the presence or absence of light, plants are able to detect subtle changes in spectral quality and light intensity (Smith, 1982), and they can measure the length of the photoperiod, which is used by many species to initiate flowering (Vince-Prue, 1983). These responses are mediated by photoreceptors that are quite distinct from those that are used to harvest light energy in photosynthesis. Some examples of light-mediated responses in plants are given in Table 1.1.
Chapter
Light affects nearly all aspects of plant life and development, from seed germination to flowering. The most well-known phenomenon, photosynthesis, involves absorption of light in the visible spectrum by chlorophylls and accessory pigments and utilization of that energy to split water and generate ATP and NADPH for the reduction of carbon from CO2. Other phenomena include growth or nongrowth movements in response to directional or diffuse light, measurement of the duration of light or darkness in a 24-h cycle, and a diversity of responses including seed germination, seedling development, and perception of neighboring vegetation, which are broadly included under the term photomorphogenesis.. Light is used as a signal for responses, not as an energy source to fix carbon. The energy requirement for most of these signals is not sufficiently high; in fact, in some cases, it is vanishingly small. Several hormones, particularly gibberellins, indole acetic acid, brassinosteroids, and cytokinins, interact with phytochromes to regulate several processes. Phytochrome signaling interacts with gibberellin signaling in seed germination, de-etiolation, stem elongation, and flowering response. It seems that while the activated phytochrome enhances the synthesis of biologically active 3β-hydroxylated gibberellins in seed germination and apical bud growth and leaf expansion during de-etiolation, it downregulates GA synthesis or sensitivity in stem elongation.
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The purpose of this project was to further our understanding of shelf life in babyleaf spinach and to assess how well a range of indicators could be used to predict shelf life with a view to developing screening tests for plant breeders and field tests for growers.
Chapter
The ferrdoxin gene system. Recently we identified a cDNA clone encoding ferredoxin I and showed that the level of the corresponding transcript in pea seedlings is increased by white light (Sagar et al., 1988a, b) and by red light acting through the phytochrome system (Dobres et al., 1987; Kaufman et al., 1985, 1986). The light responses of this gene — which we have designated Fed-1 — differ strikingly from those of RbcS and Cab which have so far served as the main models for light responsive genes, providing us with an opportunity to significantly broaden our understanding of light regulatory mechanisms.
Chapter
In developing leaves, photosynthetically competent chloroplasts arise from small, undifferentiated proplastids that are present in meristematic cells. This process, called greening, involves the coordinate regulation of many nuclear and chloroplast genes (Mullet, 1988). The cues for the initiation of this developmental program are both extrinsic and intrinsic.
Chapter
In this review, we discuss recent progress in the understanding of various elements of signal transduction cascades activated by blue light. We consider two responses in particular: inhibition of stem growth and phototropism. Included is a discussion of recent work related to the identification of photoreceptors and their chromophores. We also discuss blue light-modulated regulation of gene expression and ion movements. Physiological investigations are included where they appear relevant.
Chapter
During seed germination, mobilization of food reserves stored in the endosperm or the cotyledons, and their subsequent metabolism by the growing embryo axis, leads to the emergence of the radicle and the plumule. As the food materials are exhausted, there is a transition of the embryo from the heterotrophic mode of metabolism to the autotrophic mode and the production of new cellular materials by photosynthesis and other metabolic processes. Within this general framework, the emerging radicle orients its growth through positive gravitropism by extending downward into the soil to form the future root system of the adult plant. At the same time, the plumule acquires the capacity for negative gravitational response as it grows away from the soil into the air and produces the shoot system. These fundamental functional activities are coordinated by the division of cells at the root and shoot tips, cell enlargement and elongation, and differentiation of cells to produce the specialized tissues and organs of the seedling plant. The transition from heterotrophy to autotrophy takes place with the establishment of the root and shoot systems and marks the onset of a new phase of plant growth. As is well known, the duration of this new phase is variable, and in annual plants it is terminated by flowering and fruiting.
Chapter
Leaves are among the most specialized of all plant organs, devoting most of their activity to the production of ribulose-l,5-bisphosphate carboxylase-oxygenase (RuBISCO). This key protein is mentioned here to introduce the chapter on leaves not only because of its importance as a primary enzyme for carbon fixation in the initial reaction of photosynthesis, but also to emphasize that all of the, structural and developmental adaptations of leaves are geared to maximize the production of RuBISCO.
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Tissue culture systems have long been exploited to study the process of organogenesis. In response to externally applied cytokinins, pluripotent cells proliferate into green calli and subsequently regenerate shoots. Conventionally, the cytokinin-induced greening phenotype has been evaluated by counting numbers of green foci or to present photographic evidence of morphological changes. However, because the structure of calli is disorganized and the development of pigmentation takes place gradually from pale white through yellow to green, adequately defining and counting green foci remains difficult. In this study, we employed chlorophyll measurement as an alternative method to statistically assess the greening phenotype in tissue culture material. We found that N,N-dimethylformamide was the most effective solvent for the extraction of chlorophylls from callus tissue and that bead disruption of the structured tissue improved solvent penetration and the consistency of results. The sensitivity of the method facilitated the quantification of chlorophylls in single-cultured root explants and the use of a spectrophotometer increased the efficiency of measuring multiple samples. Our measurements showed that chlorophyll contents from calli of wild-type and altered cytokinin response mutants (cre1; ahk3, or cytokinin hypersensitive 2 (ckh2)/pickle (pkl) were statistically distinguishable, validating the method. Our proposed procedure represents gains in efficiency and precision and leads to more robust standardization than the conventionally used counting of green foci.
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Proteins that contain iron-sulfur clusters play pivotal roles in various metabolic processes, such as photosynthesis and redox metabolism. Among the proteins involved in the biosynthesis of iron-sulfur clusters in plants, the SUFB subunit of the SUFBCD complex appears unique because SUFB has been reported to be involved in chlorophyll metabolism and phytochrome-mediated signaling. To gain insights into the function of the SUFB protein, we analyzed the phenotypes of two SUFB mutants, laf6 and hmc1, and RNAi lines with reduced SUFB expression. When grown in the light, the laf6 and hmc1 mutants and the SUFB RNAi lines accumulated higher levels of the chlorophyll biosynthesis intermediate, Mg-protoporphyrin IX monomethylester (Mg-proto MME) consistent with the impairment of Mg-proto MME cyclase activity. Both SUFC- and SUFD-deficient RNAi lines accumulated the same intermediate suggesting that inhibition of Fe-S cluster synthesis is the primary cause of this impairment. Dark-grown laf6 seedlings also showed an increase in protoporphyrin IX (Proto IX), Mg-proto, Mg-proto MME and 3,8-divinyl protochlorophyllide a (DV-Pchlide) levels, but this was not observed in hmc1 or the SUFB RNAi lines, nor was it complemented by SUFB overexpression. In addition, the long hypocotyl in far-red light phenotype of the laf6 mutant could not be rescued by SUFB overexpression and segregated from the pale-green SUFB-deficient phenotype indicating it is not caused by mutation at the SUFB locus. These results demonstrate that Fe-S cluster biosynthesis is important for chlorophyll biosynthesis, but that the laf6 phenotype is not due to a SUFB mutation. This article is protected by copyright. All rights reserved.
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Characterization of tomato mutants affecting growth, yield and fruit quality traits could provide valuable insights into the underlying biology vis-à-vis source of new alleles for breeding programs. Knowing the presence of useful genes and alleles would help in making decisions on the multiplication of accessions and the maintenance of seed stocks when responding to an expected higher demand for materials. Such information may also help in making decisions on heterogeneous accessions, where only some genotypes may possess useful alleles. Thus, the present study was undertaken to characterize three induced mutants, two parental lines and three hybrids for 9 qualitative and 27 quantitative traits to utilize useful genes in tomato breeding, and to assess genetic variability and character associationship among 27 quantitative characters to identify important selection indices. The variation was observed in the habit of growth, type of blossoms, fruit form and the color of the immature fruit among mutants. A significant effect of mutant genes is recorded in a heterogeneous state to express desirable horticultural qualities in hybrids. Partial effects to excessive dominance have been found to inherit fruit yields and other horticultural qualities. Exploit the hybrid Berika × B Mut-1 appeared as the most promising and could either commercially use for both table and processing purpose after critical evaluation or could be utilized in segregating generation to identify pure lines with desirable horticultural traits. The most important traits for high heritability with high genetic advance are: flower cluster per plant, flower per cluster, fruit per plant, number of locules per fruit, total chlorophyll content of leaf, total chlorophyll content of immature fruits, chlorophyll a content of immature fruits, chlorophyll b content of immature fruits, lycopene content of mature fruits and ascorbic acid content, suggesting selection would be rewarding for improvement of these traits. The top priority should be given to selection based on lycopene content, chlorophyll a content of leaf, ascorbic acid content, fruit number per plant and chlorophyll b content of immature fruit for yield improvement and could be considered while formulating selection indices in the improvement of tomato.
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Arabidopsis thaliana (Arabidopsis), as a model for plant research, is widely used for various aspects of plant science. To provide a more sophisticated and microscopic environment for the germination and growth of Arabidopsis, we report a 384-well type plant array chip in which each Arabidopsis seed is independently seeded in a solid medium. The plant array chip is made of a poly(methyl methacrylate) (PMMA) acrylic material and is assembled with a home-made light gradient module to investigate the light effects that significantly affect the germination and growth of Arabidopsis. The light gradient module was used to observe the growth pattern of seedlings according to the intensity of the white light and to efficiently screen for the influence of the white light. To investigate the response to red light (600 nm), which stimulates seed germination, the light gradient module was also applied to the germination test. As a result, the germination results showed that the plant array chip can be used to simultaneously screen wild type seeds and phytochrome B mutant seeds on a single array chip according to the eight red light intensities.
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Ineffective alfalfa nodules formed by Rhizobium meliloti nif::Tn5 mutants were examined by light and electron microscopy. R. meliloti nifH::Tn5 mutants formed nodules that were similar in structure to wild-type nodules except that nifH- bacteroids accumulated a compact, electron-dense body. In contrast to nodules induced by wild type and nifH mutants, nifDK- R. meliloti mutants induced nodules which contained numerous starch grains and prematurely senescent bacteroids. In addition, meristematic activity in nifDK- nodules ceased significantly earlier than in nifH- nodules. All mutant nodules exhibited elevated levels of rough endoplasmic reticulum and Golgi membranes compared to wild-type nodule cells. These elevated levels may reflect either a response to nitrogen starvation in the ineffective nodules or an abnormal synthesis and export of nodule-specific proteins during later developmental stages.
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Transcription in lysed barley plastids and Northern slot blot analyses were used to determine the relationship between changes in RNA levels and transcription during plastid development. Transcription in plastids of 4.5-9-day-old dark-grown or illuminated barley seedlings declined up to 10-fold as a function of plant age. Decreased transcription of some plastid genes (rbcL, psaA-psaB) was paralleled by decreased levels of mRNA. In other cases (16SrDNA, psbA) the changes in transcription were not followed by proportional changes in RNA levels indicating that RNA stability is important in establishing the amount of plastid RNA for these genes. Further analysis showed that transcription of the plastid rRNA transcription unit is regulated differently than the transcription of protein coding genes such as psbA or rbcL.
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A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P(700)-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.
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A mutant of Arabidopsis thaliana with reduced content of C(18:3) and C(16:3) fatty acids in membrane lipids exhibited a 45% reduction in the cross-sectional area of chloroplasts and had a decrease of similar magnitude in the amount of chloroplast lamellar membranes. The reduction in chloroplast size was partially compensated by a 45% increase in the number of chloroplasts per cell in the mutant. When expressed on a chlorophyll basis the rates of CO(2)-fixation and photosynthetic electron transport were not affected by these changes. Fluorescence polarization measurements indicated that the fluidity of the thylakoid membranes was not significantly altered by the mutation. Similarly, on the basis of temperature-induced fluorescence yield enhancement measurements, there was no significant effect on the thermal stability of chlorophyll-protein complexes in the mutant. These observations suggest that the high content of trienoic fatty acids in chloroplast lipids may be an important factor regulating organelle biogenesis but is not required to support normal levels of the photosynthetic activities associated with the thylakoid membranes.
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G Gardner and HL Gorton (1985 Plant Physiol 77: 540) demonstrated that gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid) inhibits the initial synthesis and resynthesis of spectrophotometrically detectable phytochrome in pea, maize, and oat. We show that the level of immuno-detectable phytochrome in pea is unaffected by the presence of gabaculine at a concentration that reduces spectrophotometrically detectable phytochrome up to 10-fold. This result indicates that gabaculine inhibits chromophore synthesis without affecting phytochrome apoprotein synthesis and that chromophore-less phytochrome is stable in the cell.
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A monoclonal antibody (Pea-25) directed to phytochrome from etiolated peas (Pisum sativum L., cv Alaska) binds to an antigenic domain that has been highly conserved throughout evolution. Antigenic cross-reactivity was evaluated by immunoblotting sodium dodecyl sulfate sample buffer extracts prepared from lyophilized tissue samples or freshly harvested algae. Pea-25 immunostained an approximately 120-kilodalton polypeptide from a variety of etiolated and green plant tissues, including both monocotyledons and dicotyledons. Moreover, Pea-25 immunostained a similarly sized polypeptide from the moss Physcomitrella, and from the algae Mougeotia, Mesotaenium, and Chlamydomonas. Because Pea-25 is directed to phytochrome, and because it stains a polypeptide about the size of oat phytochrome, it is likely that Pea-25 is detecting phytochrome in each case. The conserved domain that is recognized by Pea-25 is on the nonchromophore bearing, carboxyl half of phytochrome from etiolated oats. Identification of this highly conserved antigenic domain creates the potential to expand investigations of phytochrome at a cellular and molecular level to organisms, such as Chlamydomonas, that offer unique experimental advantages.
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Phytochrome action results in a large and rapid increase in the light-harvesting chlorophyll a/b-protein (LHCP) mRNA level in etiolated seedlings of Arabidopsis thaliana: the RNA increase is detectable within 1 hour after 1 minute red illumination, reaches a maximum 30-fold higher than the dark level at ca. 2 hours, and decays back to dark levels by about 8 hours after the brief red illumination. S1 nuclease analysis distinguishes two kinds of mRNAs transcribed from the three members of the LHCP gene family previously characterized for Arabidopsis (LS Leutwiler, EM Meyerowitz, EM Tobin, 1986 Nucleic Acids Res 14: 4051-4064). One of these arises from the AB140 gene, while the other represents the product(s) of the AB165 and/or AB180 gene(s) (AB165/AB180 mRNA). In mature, white light-grown plants, the two kinds of mRNAs are present in nearly equal amounts. In contrast, in etiolated seedlings, 1 minute red light causes a sixfold greater increase in the level of AB140 mRNA than in the level of AB165/AB180 mRNA, although both levels are regulated by phytochrome action. The kinetics of the responses to 1 minute red light are similar for both kinds of transcripts. Additional evidence suggests that this differential expression is developmentally regulated. Because the AB140 gene offers an attractive target for further analysis of phytochrome-regulated gene expression in Arabidopsis, we have further characterized this gene by mapping its 5' and 3' transcript termini.
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Photomorphogenetic responses have been studied in a cucumber (Cucumis sativus L.) mutant (lh), which has long hypocotyls in white light (WL). While etiolated seedlings of this mutant have a similar phytochrome content and control of hypocotyl elongation as wild type, deetiolation is retarded and WL-grown seedlings show reduced phytochrome control. Spectrophotometric measurements exhibit that WL-grown tissues of the lh mutant (flower petals and Norflurazon-bleached leaves) contain 35 to 50% of the phytochrome level in the wild type. We propose that this is a consequence of a lack of light-stable phytochrome, in agreement with our hypothesis proposed on the basis of physiological experiments. The lh mutant lacks an end-of-day far-red light response of hypocotyl elongation. This enables the end-of-day far-red light response, clearly shown by the wild type, to be ascribed to the phytochrome, deficient in the lh mutant. Growth experiments in continuous blue light (BL) and continuous BL + red light (RL) show that when RL is added to BL, hypocotyl growth remains inhibited in the wild type, whereas the lh mutant exhibits significant growth promotion compared to BL alone. It is proposed that the hypocotyls fail to grow long in low fluence rate BL because photosynthesis is insufficient to sustain growth.
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Transformed Arabidopsis thaliana plants have been produced by a modified leaf disk transformation-regeneration method. Leaf pieces from sterilely grown plants were precultured for 2 days and inoculated with an Agrobacterium tumefaciens strain containing an avirulent Ti (tumor-inducing) plasmid with a chimeric gene encoding hygromycin resistance. After cocultivation for 2 days, the leaf pieces were placed on a medium that selects for hygromycin resistance. Shoots regenerated within 3 months and were excised, rooted, and transferred to soil. Transformation was confirmed by opine production, hygromycin resistance, and DNA blot hybridization of both primary transformants and progeny. This process for producing transgenic Arabidopsis plants should enhance the usefulness of the species for experimental biology.
Chapter
Action spectra studies of photomorphogenic responses in plants imply that several photoreceptor molecules probably exist (see Chap. 2, this Vol.). A necessary part of gaining an understanding of the mechanisms of these light-mediated responses in plants is the identification and characterization of the photoreceptor molecules. To date, the only photomorphogenically active molecule to be unequivocally identified and isolated from plants is phytochrome. The detection and isolation of phytochrome (Butler et al. 1959) was made possible by its unique photoreversible absorbance changes in the red and far-red regions of the spectrum which matched action spectra for photoreversible physiological responses in plants (Borthwick et al. 1952). These reversible spectral changes in phytochrome provided the basis for a photometric assay (Chap. 8, this Vol.) and made possible the subsequent purification and characterization of phytochrome.
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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.
Article
Lemna gibba L. G-3 plants grown heterotrophically in the dark with intermittent red light (2 min every 8 h) contain a substantial amount of translatable mRNA encoding the light-harvesting chlorophyll (Chl)a/b-protein. However, very little [(35)S]methionine is incorporated into the apoproteins during a 1-h labeling period in the dark in these plants compared to plants grown in continuous white light. The Chla/b-protein mRNA is found to be associated with functioning polysomes in plants grown in the dark with intermittent red illumination (R plants). The small amounts of the apoproteins which are synthesized by these plants are found in the membrane fraction; neither the mature apoproteins nor their precursor(s) can be detected immunologically in the soluble fraction. The protein does not accumulate in these plants. Pulse-chase experiments with the R plants demonstrate that the newly synthesized apoproteins have a half-life of about 10 h in the dark. This turnover is not sufficient to explain the observed 20-fold difference in [(35)S]methionine incorporation into the apoprotein between white-light-grown and R plants. We therefore suggest that the synthesis of the Chla/b-apoproteins can be regulated by a light-dependent step at the level of translation, and that this regulation occurs after the initiation of translation.
Article
The multigene family encoding the small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase/oxygenase in the crucifer Arabidopsis thaliana has been isolated and the organization and structure of the individual members determined. The family consists of four genes which have been divided into two subfamilies on the basis of linkage and DNA and amino acid sequence similarities. Three of the genes, designated ats1B, ats2B, and ats3B, reside in tandem on an 8 kb stretch of the chromosome. These genes share greater than 95% similarity in DNA sequence and encode polypeptides identical in length and 96.7% similar in amino acid sequence. The fourth gene, ats1A, is at least 10 kb removed from, or completely unlinked to the B subfamily. The B subfamily genes are more similar to each other than to ats1A in nucleotide and amino acid sequence. All four genes are interupted by two introns whose placement within the coding region of the genes is conserved. The introns of the B subfamily genes are similar in length and nucleotide sequence, but show no similarity to the introns of ats1A. Comparison of the DNA sequences within the immediate 5' and 3' flanking sequences among the genes revealed only limited regions of homology. S1 analysis shows that all four genes are expressed.
Article
The relative molecular mass (Mr) of the native phytochrome monomer from etiolated Cucurbita pepo L., Pisum sativum L., Secale cereale L. and Zea mays L. seedlings has been determined using immunoblotting to visualize the chromoprotein in crude extracts subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single phytochrome band is observed for each plant species when the molecule is extracted under conditions previously demonstrated to inhibit the proteolysis of native Avena sativa L. phytochrome. A comparison among plant species indicates that the Mr of native phytochrome is variable: Zea mays=127000; Secale=Avena=124000; Pisum=121000; Cucurbita=120000. The in-vitro phototransformation difference spectrum for native phytochrome from each species is similar to that observed in vivo in each case and is indistinguishable from that described for native Avena phytochrome. The difference minima between the red- and far-red-absorbing forms of the pigment (Pr-Pfr) are all at 730 nm and the spectral change ratios (ΔAr/ΔAfr) are near unity. When incubated in crude extracts, phytochrome from all four species is susceptible to Pr-specific limited proteolysis in a manner qualitatively similar to that observed for Avena phytochrome, albeit with slower rates and with the production of different Mr degradation products. Further examination of the in-vitro proteolysis of Avena phytochrome by endogeneous proteases has identified several additional phytochrome degradation products and permitted construction of a peptide map of the molecule. The results indicate that both the 6000- and 4000-Mr polypeptide segments cleaved by Pr-specific proteolysis are located at the NH2-terminus of the chromoprotein and are adjacent to a 64000-Mr polypeptide that contains the chromophore.
Article
While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.
Article
A microcomputer-controlled dual-wavelength spectrophotometer suitable for automated phytochrome assay is described. The optomechanical unit provides for sequential irradiation of the sample by the two measuring wavelengths with intervening dark intervals and for actinic irradiation to interconvert phytochrome between its two forms. Photomultiplier current is amplified, converted to a digital value and transferred into the computer using a custom-designed IEEE-488 bus interface. The microcomputer calculates mathematically both absorbance and absorbance difference values with dynamic correction for photomultiplier dark current. In addition, the computer controls the operating parameters of the spectrophotometer via a separate interface. These parameters include control of the durations of measuring and actinic irradiation intervals and their sequence. 14 references, 4 figures.
Article
One approach to elucidating the mechanism of phytochrome action is to characterize thoroughly the phytochrome molecule. This approach has added to our understanding of phytochrome action by demonstrating that distinct phytochrome molecules predominate in etiolated and green plants, by allowing determination of the complete amino acid sequences of Avena Sativa L. (oat) and Cucurbita pepo L. (zucchini) phytochrome, by indicating that phytochrome is unlikely to be a membrane spanning protein, and by providing evidence that phytochrome may be a protein kinase. Phytochrome has also proved useful as a model system in which to study the regulation of gene expression in plants. Expression of the phytochrome genes appears to be regulated at the transcriptional, post-transcriptional, and post-translational levels and thus affords an opportunity to investigate the carefully controlled regulation of a gene that has a central role in plant development.
Article
Phytochrome is known to regulate the expression of several major nuclearencoded polypeptides at the level of transcription. Both in vivoin vitro methods are currently being used to determine the step(s) between the light activation of phytochrome and changes in gene expression.
Article
The au w mutant allele of the aurea locus in tomato has previously been shown to cause deficiency for the phytochrome polypeptide (Parks et al. 1987). We have begun to characterize the molecular basis and consequences of this deficiency. Genomic Southern blot analysis indicates that there are at least two and probably more phytochrome polypeptide structural genes in tomato. RNA blot analysis shows that the au w mutant contains normal levels of phytochrome mRNA and in vitro translation of au w poly(A)+ RNA yields a phytochrome apoprotein that is quantitatively and qualitatively indistinguishable on SDS-polyacrylamide gels from that synthesized from wild-type RNA. These results indicate that the phytochrome deficiency in aurea is not the result of lack of expression of phytochrome genes but is more likely due to instability of the phytochrome polypeptide in planta. Possible reasons for such instability are discussed. Analysis of the molecular phenotype of aurea indicates that the phytochrome-mediated increase in the abundance of the mRNA encoding chlorophyll a/b binding protein (cab) is severely restricted in the mutant as compared with wild-type tomato. Thus, the au w strain exhibits defective photoregulation of gene expression consistent with its very reduced level of the phytochrome photoreceptor.
Article
Phytochrome-induced increases in enzyme activities for phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone isomerase (EC 5.5.1.6), and in amounts of the related end products, anthocyanin and the flavonol, quercetin, were measured in cotyledons of mustard (Sinapis alba L.). There was no correlation between the activities of these enzymes and the rate of anthocyanin accumulation; however, some correlation was found with the quercetin accumulation rate. Since anthocyanin and flavonol accumulation is spatially separated in mustard (flavonols in the upper epidermis, anthocyanin in the lower epidermis), it was possible to measure anthocyanin-associated phenylalanine ammonia-lyase independently. This activity correlated well with the accumulation rate for anthocyanin during the first few hours after induction. The phytochrome effect on anthocyanin formation differed from that on quercetin formation: anthocyanin was strongly induced by continuous far-red light and by both continuous red light and red light pulses, whereas quercetin was only effectively induced by continuous far-red light.
Article
Reduced sensitivity to the inhibitory action of white light on hypocotyl elongation was observed in 41 mutants induced in Arabidopsis tbaliana at 5 different loci. Compared to wild type, these mutants show a locus-specific altered inhibition spectrum when grown in light of restricted spectral regions. Spectrophotometrically detectable phytochrome in dark-grown tissue of those mutants (loci by-1 and by-2) in which the inhibitory effect of far red light is almost or completely absent, is either greatly reduced or below the level of detectability. The spectra of the different mutants and their recombinants provide evidence for the presence of more than one photoreceptor pigment for the High Irradiance Reaction and demonstrate the genetic control of light-induced inhibition of hypocotyl growth.
Article
Tomato mutants at the au, yg-2, and yg-6 loci have yellow-green leaves, elongated hypocotyls and reduced anthocyanin content when grown in white light. Mutant auw, in contrast to its wild type, shows little or no effect of light on seed germination, anthocyanin synthesis, and hypocotyl elongation. In addition the chlorophyll content is reduced, the chlorophyll a/b ratio is increased and the stacking of the thylakoids in the chloroplasts is greatly reduced_ Spectrophotometrically determined phytochrome is absent or strongly reduced in its seeds, dark-grown hypocotyls, light-grown leaves, and roots. These results suggest that the phenotype of these mutants is correlated with a reduced phytochrome content.
Article
IN maize, the plant best known genetically, there are no viable chlorophyll b deficient mutants1,2. Röbbelen3 described an Arabidopsis mutant devoid of this pigment which was, however, a lethal one. It has been reported that in barley only chlorophyll a is necessary for viability4. Among algae viable chlorophyll b-less mutants are not infrequent5. Since chlorophyll b is a normal constituent of the photosynthetic apparatus of all angiosperms and its precise role is little understood, viable mutants involved in the development of this pigment are of interest.
Article
Statistical techniques known as the analysis of variance make it possible for the electron microscopist to plan work in such a way as to obtain quantitative data with the greatest possible economy of effort. This paper explains how to decide how many measurements to make per micrograph, how many micrographs per tissue block, how many blocks per experimentally treated organ, and how many organs per experimental treatment group. It is emphasized, that the early application of sound statistical principles to experimental design in morphology can result in sometimes astonishing savings of labor. While this brief article has attempted to instruct the reader on how pilot data can be used to maximize ones efficiency of effort, the soundest course for most investigators would be to consult a professional statistician to aid them in planning meaningful application of statistical principles to their research.
Article
The biosynthesis of ribulose bisphosphate carboxylase (RUBISCO) provides a model system for studying the coordination of nuclear and organelle gene expression, since this abundantly transcribed and expressed chloroplast enzyme is composed of small (SS) and large subunits (LS) encoded by a nuclear multigene family and a single chloroplast gene, respectively. We have tested the possibility that SS mRNA or protein levels affect LS mRNA amounts or LS protein production and accumulation. We find that expression of antisense DNA sequences for the SS in transgenic tobacco plants drastically reduces the accumulation of SS mRNA and SS protein. These changes are accompanied by corresponding reductions of LS protein but not LS mRNA amounts; accumulation of the LS protein appears to be regulated by translational and posttranslational factors. We also find that the transgenic plants display striking variations in growth that are correlated with antisense gene dosage.
Article
We have cloned an Arabidopsis thaliana chalcone synthase (CHS) gene on the basis of cross-hybridization with a Petroselinum hortense CHS cDNA clone. The protein sequence deduced from the A. thaliana CHS DNA sequence is at least 85% homologous to the CHS sequences from P. hortense, Antirrhinum majus, and Petunia hybrida. Southern blot analysis indicated that CHS is a single-copy gene in A. thaliana. High-intensity light treatment of A. thaliana plants for 24 h caused a 50-fold increase in CHS enzyme activity and an accumulation of visibly detectable levels of anthocyanin pigments in the vegetative structures of these plants. A corresponding increase in the steady-state level of CHS mRNA was detected after high-intensity light treatment for the same period of time. The accumulation of CHS mRNA in response to high-intensity light was due, at least in part, to an increased rate of transcription of the CHS gene as demonstrated by nuclear runoff experiments.
Article
The relation between birthday and date of death has so far been studied from two different perspectives: birthdays were either conceived of as emotionally invested deadlines motivating people to ward off their death which causes a 'dip' in death rates before their birthday, or they were considered as stressful events leading to an increase of mortality on or after their birthday. Using a collection of biographies of famous people from the whole world and another of well-known Swiss citizens we tested hypotheses derived from these assumptions. Neither the 'death-dip' hypotheses nor the 'birthday stress' hypothesis was supported by our results.
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Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.
Article
This paper provides additional experimental evidence that biological variation between individuals is likely to be the major factor influencing the overall precision and efficiency of nested sampling schemes for morphometric analysis of thin sections. Four distinct experimental systems (two based on nervous tissue and two on epithelia) have been investigated. Morphometric estimates were obtained from measurements made on micrographs generated by sampling tissues at several levels. Sources of sampling variation were isolated so that the contributions to overall variance made by inter-animal differences could be evaluated. In each case, biological variation was the cardinal component of total observed variance between animals. Relative contributions ranged from 53% to 78%. Examining more animals would be the most efficient way of reducing the variance of the group mean in these sampling designs.
Article
An evaluation is made of the relative efficiency (precision of the final estimate per unit time of measurement on a given set of sections) of different methods for planar analysis aimed at estimating aggregate, overall stereological parameters (such as VV, SV). The methods tested are point-counting with different densities of test points (4 less than or equal to PT less than 900 per picture), semiautomatic computer image analysis with MOP and automatic image analysis with Quantimet, for obtaining VV and SV estimates. One biological sample as well as three synthetic model structures with known coefficients of variation between sections are used. The standard error of an estimate is mainly determined by the coefficient of variation between sampling units (= sections in the present paper) so that measuring each sample unit with a very high precision is not necessary. Automatic image analysis and point-counting with a 100-point grid were the most efficient methods for reducing the relative standard errors of the VV and SV estimates to equivalent levels in the synthetic models. Using a 64-point grid was as precise, and about 11 times faster than using a tracing device for obtaining the estimate of VV in the biological sample.
Article
The aim of the sampling design for stereology is to obtain the maximal amount of quantitative structural information at a given total cost or effort. Principles of such optimal designs are discussed and methods for generating them are illustrated by a biological example. In general, the variation between different individuals--the biological variation--is the major determinant of overall efficiency, whereas the variation between single microscopic features is unimportant. It follows that the expenditure of time and/or money in order to increase the precision of the individual measurements is irrational in almost all studies where the emphasis is on the biological results.
Article
1. When etiolated pea seedlings were exposed to continuous light for 24 h and then returned to darkness, 38% of the chlorophyll a, 74% of the chlorophyll b and 84% of the light-harvesting chlorophyll a/b protein that had accumulated under illumination proved to be unstable in darkness. The unstable chlorophyll displayed a half-life of about 90 min. In contrast, alpha and beta subunits of the chloroplast coupling factor and the large and small subunits of ribulose 1,5-biphosphate carboxylase continued to accumulate in darkness, although at a slower rate than in plants maintained under light. 2. Short-term labelling in vivo with L-[35S]methionine showed that leaves continued to synthesize the light-harvesting protein and the small subunit of ribulose 1,5-biphosphate carboxylase for up to 48 h after transfer of plants from light and darkness. However, after long-term labelling (16 h), the light-harvesting chlorophyll a/b protein was found to be labelled to high specific activity only in illuminated leaves. 3. I conclude that the light-harvesting chlorophyll a/b protein is subject to turnover after transfer of plants from light to darkness. The site of breakdown appears to be the photosynthetic membrane. I suggest that turnover of the protein is part of the normal physiological mechanism for co-ordinating the accumulation of the pigment and protein components of the light-harvesting chlorophyll a/b complex.