Light-grown Plants Research Articles

Effect of chilling and acclimation on the activity of glutamine synthetase isoforms in maize seedlings

Effects of chilling and acclimation on the activity of cytosolic (GS1) and plastidic (GS2) isoforms of glutamine synthetase (E.C. 6.3.1.2) were studied in chilling-sensitive and acclimation-responsive maize inbred G50. Glutamine synthetase activity in mesocotyls and roots of chilled (7 d/4?C) and rewarmed (1 d/27?C) etiolated plants was "1/3 that of controls. In coleoptiles+leaves of light-grown plants, GS1 was reduced to 75%, and GS2 to 50%. Acclimation (3 d/14?C) increased GS activity and alleviated the effects of chilling. Exposure to H2O2 or menadione also reduced GS activity. Since chilling causes oxidative stress in maize, acclimation probably preserves GS activity by protecting GS from oxidative inactivation. .

The PsbQ Protein Is Required in Arabidopsis for Photosystem II Assembly/Stability and Photoautotrophy under Low Light Conditions

RNA interference was used to simultaneously suppress the expression of the two genes that encode the PsbQ proteins of Photosystem II (PS II) in Arabidopsis thaliana, psbQ-1 (At4g21280) and psbQ-2 (At4g05180). Two independent PsbQ-deficient plant lines were examined. These plant lines produced little detectable PsbQ protein. Under normal growth light conditions, the wild type and mutant plants were visually indistinguishable. Additionally, analysis of steady state oxygen evolution rates and chlorophyll fluorescence characteristics indicated little alteration of photosynthetic capacity in the mutant plants. No loss of other PS II proteins was evident. Interestingly, flash oxygen yield analysis performed on thylakoid membranes isolated from the mutant and wild type plants indicated that the oxygen-evolving complex was quite unstable in the mutants. Furthermore, the lifetime of the S2 state of the oxygen-evolving complex appeared to be increased in these plants. Incubation of the wild type and mutant plants under low light growth conditions led to a significantly stronger observed phenotype in the mutants. The mutant plants progressively yellowed (after 2 weeks) and eventually died (after 3-4 weeks). The wild type plants exhibited only slight yellowing after 4 weeks under low light conditions. The mutant plants exhibited a large loss of a number of PS II components, including CP47 and the D2 protein, under low light conditions. Additionally, significant alterations of their fluorescence characteristics were observed, including an increased FO and decreased FV, yielding a large loss in PS II quantum efficiency (FV/FM). Analysis of QA- decay kinetics in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea indicated a defect in electron transfer from QA- to QB, whereas experiments performed in the presence of this herbicide indicated that the recombination rate between QA- and the S2 state was strongly retarded. These results indicate that the loss of the PsbQ protein induces significant changes in Photosystem II function, particularly in low light-grown plants, and that the PsbQ protein is required for photoautotrophic growth under low light conditions.

The an1-4736 mutation of anther ear1 in maize alters scotomorphogenesis and the light response

Dwarf mutants have been instrumental in the elucidation of gibberellin (GA) metabolism and of their role on a wide range of plant physiological processes. Based on their response to the application of exogenous GAs, dwarf mutants have been divided into two major groups, GA-responsive and GA-insensitive mutants. Here we report on the characterization of an1-4736, a chemically (EMS) induced allele of an1 that belongs to the GA-responsive group. Striking aspects of this mutant allele are its early developmental time of response to GA 3 administration and its capacity to partially repress scotomorphogenesis. When grown in continuous darkness the mutant shows traits usually associated with light-grown plants, such as leaf unfolding, reduction in seedling elongation and induction of light controlled genes. A partial impairment of mutant growth is also observed upon its transfer to light. Since other light-regulated processes appear normal in the mutant, it might be that the lesion in the GA metabolism specifically affects the light dependent signal pathway controlling plant elongation. These results, suggesting a dual role of GAs, one of promotion of stem elongation in light and a second one of repression of photomorphogenesis in darkness, are indicative of an interaction between GAs and light signals in the modulation of growth.

Positive Role for COP1 in UV-B Responses

Under dark conditions, the E3 ubiquitin ligase COP1 is enriched in the nucleus, where it contributes to the degradation of various transcriptional regulators involved in the photomorphogenic response, including HY5 (ELONGATED HYPOCOTYL 5). Thus, in Arabidopsis , COP1 is a negative regulator of photomorphogenesis. Oravecz et al . report that COP1 has a positive role in plant responses to low-level ultraviolet B (UV-B) irradiation through a mechanism that includes stimulation of expression of the HY5 gene. Microarray analysis and phenotypic analysis of wild-type and hy5-1 mutant plants indicated that HY5 was necessary for the stimulation of the expression of genes involved in tolerance to UV-B. The nonlethal COP1 mutant cop1-4 also exhibited decreased UV-B-mediated stimulation of several genes, including HY5 , and about half of the genes that required COP1 also required HY5. Plants respond to UV-B by stimulation of the phenylpropanoid biosynthetic pathway, which produces "sunscreen" flavonoids. Both hy5-1 and cop1-4 plants were deficient in this response to UV-B; however, plants with another COP1 mutant allele, cop1 eid6 , did not show this deficiency, which suggests that the structural requirements and functions of COP1 in visible light (photomorphogenesis) and UV-B signaling (flavonoid biosynthesis) are different. Exposure of light-grown plants to supplemental UV-B irradiation promoted the nuclear accumulation of COP1 (in just light-grown plants, COP1 is excluded from the nucleus). HY5 is also abundant in the nucleus of light-grown plants exposed to supplemental UV-B irradiation, which is unexpected given that COP1 promotes its degradation under dark conditions when both proteins are present in the nucleus. How COP1 can mediate degradation of HY5 in the dark, yet be required for HY5 to accumulate in response to UV-B irradiation, remains to be determined. A. Oravecz, A. Baumann, Z. Máté, A. Brzezinska, J. Molinier, E. J. Oakeley, E. Ádám, E. Schäfer, F. Nagy, R. Ulm, CONSTITUTIVELY PHOTOMORPHOGENIC1 is required for the UV-B response in Arabidopsis . Plant Cell 18 , 1975-1990 (2006). [Abstract] [Full Text]

Priming abiotic factors for optimal hybrid Cymbidium (Orchidaceae) PLB and callus induction, plantlet formation, and their subsequent cytogenetic stability analysis

Abstract Abiotic factors affect the induction of PLBs and callus in hybrid Cymbidium Twilight Moon ‘Day Light’. The initiation and proliferation of new PLBs and callus could be achieved on NAA and kinetin, supplemented at 0.1 mg l −1 each, respectively, both within 45–60 days. Bacto agar was found to be the most suitable solidifying agent for PLB induction, although a higher shoot fresh weight was obtained on Gelrite; a pH 5.3 was optimal while pH 4.5 caused 100% explant necrosis; coconut water, when supplied at 10–20% (v/v) resulted in a significant increase in the number of PLBs formed per PLB segment (23.1 versus 14.6 in controls) while a massive (almost four-fold) increase in fresh top weight occurred when PLB explants were placed in liquid culture, as a result of hyperhydricity; Fe-EDTA (1 mg l −1 ) and activated charcoal (1 g l −1 ) stimulated total fresh weight and PLB formation in the presence of PGRs; PLB formation decreased but total fresh shoot weight increased with the addition of niacin or myo -inositol, both vitamins. Dark-grown PLB-induced plants were etiolated and had longer internodes and higher fresh weight than light-grown control plants at 45 μmol m −2 s −1 ; at 15 μmol m −2 s −1 shoots were slightly etiolated, fragile, and PLB formation was scarce. RAPD and mtDNA analysis of all resultant PLBs, callus or plants showed them to be genetically identical, with comparable chlorophyll contents. Despite the detection of cytological variation between different plant parts, little variation resulted from abiotic factor treatment.

Quantitative contributions of blue light and PAR to the photocontrol of plant morphogenesis in Trifolium repens (L.)

Shade-avoidance is a major adaptive response of plants, and is usually considered to be controlled by phytochromes through the perception of changes in the red:far red light ratio. However, few studies on the effects of blue light (BL) and of light intensity [photosynthetically active radiation (PAR)] on light-grown plants have been conducted, especially concerning changes in PAR at constant BL. The objective here was to quantify the photocontrol of aerial morphogenesis by BL and PAR. Experiments were conducted varying BL and PAR independently, with three BL levels (4, 38, and 83 micromol m(-2) s(-1)) at constant PAR (300 micromol m(-2) s(-1)) and three PAR levels (338, 705, and 163 micromol m(-2) s(-1)) at constant BL (36 micromol m(-2) s(-1)). Effects on morphogenetic processes were analysed as quantitative modulations of ontogenic trends and response curves were produced. White clover (Trifolium repens L.) was used, as it is a typical shade-avoider displaying the whole syndrome of shade-avoidance in a purely vegetative stage. Morphological responses were strongly controlled by both BL and PAR changes, through antagonist effects on leaf appearance rate and additive effects on petiole elongation. All the other responses appeared to be the indirect consequences of changes in the leaf appearance rates. BL acted as a light signal for plant morphogenesis. However, the PAR control probably implicates two distinct mechanisms, such as a trophic effect and a signal. Both PAR and BL actions involved organ-specific differences, which are central in the control of the shade-avoidance responses.

Identification of Primary Target Genes of Phytochrome Signaling. Early Transcriptional Control during Shade Avoidance Responses in Arabidopsis

The phytochrome (phy) photoreceptors modulate plant development after perception of light. Upon illumination of etiolated seedlings, phys initiate a transcriptional cascade by directly transducing light signals to the promoters of genes encoding regulators of morphogenesis. In light-grown plants, however, little is known about the transcriptional cascade modulated by phys in response to changes in light. The phy entry points in this cascade are completely unknown. We are particularly interested in the shade avoidance syndrome (SAS). Here we describe a subset of six genes whose expression is rapidly modulated by phys during both deetiolation and SAS in Arabidopsis (Arabidopsis thaliana). Using cycloheximide, we provide evidence that four of these phy rapidly regulated (PAR) genes are direct targets of phy signaling during SAS, revealing these genes as upstream components of the transcriptional cascade. Promoter-beta-glucuronidase fusions confirmed that PAR genes are photoregulated at the transcriptional level. Analysis of gene expression in light signal transduction mutants showed that COP1 and DET1 (but not DET2 or HY5) play a role in modulating PAR expression in response to shade in light-grown seedlings. Moreover, genetic analyses showed that one of the genes identified as a direct target of phy signaling was phy-interacting factor 3-like-1 (PIL1). PIL1 has previously been implicated in SAS in response to transient shade, but we show here that it also plays a key role in response to long-term shade. The action of PIL1 was particularly apparent in a phyB background, suggesting an important negative role for PIL1 under dense vegetation canopies.

Differential adaptation of two varieties of common bean to abiotic stress

The photosynthetic characteristics of two contrasting varieties of common bean (Phaseolus vulgaris) have been determined. These varieties, Arroz and Orfeo, differ in their productivity under stress conditions, resistance to drought stress, and have distinctly different stomatal behaviour. When grown under conditions of high irradiance and high temperature, both varieties displayed evidence of photosynthetic acclimation at the chloroplast level-there was an increase in chlorophyll a/b ratio, a decreased content of Lhcb proteins, and an increased xanthophyll cycle pool size. Both varieties also showed reduced chlorophyll content on a leaf area basis and a decrease in leaf area. Both varieties showed an increase in leaf thickness but only Arroz showed the characteristic elongated palisade cells in the high light-grown plants; Orfeo instead had a larger number of smaller, rounded cells. Differences were found in stomatal development: whereas Arroz showed very little change in stomatal density, Orfeo exhibited a large increase, particularly on the upper leaf surface. It is suggested that these differences in leaf cell structure and stomatal density give rise to altered rates of photosynthesis and stomatal conductance. Whereas, Arroz had the same photosynthetic rate in plants grown at both low and high irradiance, Orfeo showed a higher photosynthetic capacity at high irradiance. It is suggested that the higher yield of Orfeo compared with Arroz under stress conditions can be explained, in part, by these cellular differences.

PsbR, a Missing Link in the Assembly of the Oxygen-evolving Complex of Plant Photosystem II

The oxygen-evolving complex of eukaryotic photosystem II (PSII) consists of three extrinsic nuclear-encoded subunits, PsbO (33 kDa), PsbP (23 kDa), and PsbQ (17 kDa). Additionally, the 10-kDa PsbR protein has been found in plant PSII and anticipated to play a role in water oxidation, yet the physiological significance of PsbR has remained obscure. Using the Arabidopsis psbR mutant, we showed that the light-saturated rate of oxygen evolution is strongly reduced in the absence of PsbR, particularly in low light-grown plants. Lack of PsbR also induced a reduction in the content of both the PsbP and the PsbQ proteins, and a near depletion of these proteins was observed under steady state low light conditions. This regulation occurred post-transcriptionally and likely involves a proteolytic degradation of the PsbP and PsbQ proteins in the absence of an assembly partner, proposed to be the PsbR protein. Stable assembly of PsbR in the PSII core complex was, in turn, shown to require a chloroplast-encoded intrinsic low molecular mass PSII subunit PsbJ. Our results provided evidence that PsbR is an important link in the PSII core complex for stable assembly of the oxygen-evolving complex protein PsbP, whereas the effects on the assembly of PsbQ are probably indirect. The physiological role of the PsbR, PsbP, and PsbQ proteins is discussed in light of their peculiar expression in response to growth light conditions.

Isoflavone Levels in Five Soybean (Glycine max) Genotypes Are Altered by Phytochrome-Mediated Light Treatments

The objective of the present study was to determine whether concentrations of different isoflavones (puerarin, genistein, genistin, daidzein, and daidzin) in shoots and roots of five selected soybean genotypes would respond the same or differently to red (650 nm peak transmittance) and far-red (750 nm peak transmittance) light treatments given under controlled environments. Levels of isoflavones (mg g(-1) dry weight biomass) present in seeds, control roots, and shoots and 10 day light-treated seedlings (light, dark, red, and far-red wavelengths) of soybean (Glycine max) were determined by high-performance liquid chromatography analysis in comparison with known isoflavone standards. Seeds of the five soybean genotypes studied consistently stored most of their isoflavones as glucosyl conjugates (e.g., daidzin, genistin, and puerarin). For the five soybean genotypes, isoflavone levels were lower in the seeds as compared with roots plus shoots of control, time zero (first true leaf stage) seedlings. Following 10 days of the respective light treatments, we found that (i) isoflavone levels were enhanced in dark-grown plants over light-grown plants for three of the five genotypes (a new finding) and the reverse occurred for a single genotype (a typical response of legumes) and (ii) generally, far-red end of day (EOD) light treatment enhanced total isoflavone levels in roots plus shoots over red EOD light treatment. Results from the present study show that phytochrome does appear to play a role in regulating isoflavone levels in developing soybean seedlings and that this influence by red/far-red-mediated phytochrome reactions is strongly dependent on the genotypes selected for study.

Conservation and Divergence of Light-Regulated Genome Expression Patterns during Seedling Development in Rice andArabidopsis [W

Genome-wide 70-mer oligonucleotide microarrays of rice (Oryza sativa) and Arabidopsis thaliana were used to profile genome expression changes during light-regulated seedling development. We estimate that the expression of approximately 20% of the genome in both rice and Arabidopsis seedlings is regulated by white light. Qualitatively similar expression profiles from seedlings grown under different light qualities were observed in both species; however, a quantitatively weaker effect on genome expression was observed in rice. Most metabolic pathways exhibited qualitatively similar light regulation in both species with a few species-specific differences. Global comparison of expression profiles between rice and Arabidopsis reciprocal best-matched gene pairs revealed a higher correlation of genome expression patterns in constant light than in darkness, suggesting that the genome expression profile of photomorphogenesis is more conserved. Transcription factor gene expression under constant light exposure was poorly conserved between the two species, implying a faster-evolving rate of transcription factor gene expression in light-grown plants. Organ-specific expression profiles during seedling photomorphogenesis provide genome-level evidence for divergent light effects in different higher plant organs. Finally, overrepresentation of specific promoter motifs in root- and leaf-specific light-regulated genes in both species suggests that these cis-elements are important for gene expression responses to light.

ArabidopsisNAPandPIRRegulate Actin-Based Cell Morphogenesis and Multiple Developmental Processes

The actin cytoskeleton mediates cellular processes through the dynamic regulation of the time, location, and extent of actin polymerization. Actin polymerization is controlled by several types of evolutionarily conserved proteins, including those comprising the ARP2/3 complex. In animal cells ARP2/3 activity is regulated by WAVE complexes that contain WAVE/SCAR proteins, PIR121, Nap125, and other proteins. The activity of the WAVE complex is regulated by Rho-GTPase-mediated signaling that leads to ARP2/3 activation by WAVE/SCAR proteins. We describe in this report Arabidopsis (Arabidopsis thaliana) genes encoding Nap and PIR proteins. Light-grown Atnap-1 and Atpir-1 mutant plants displayed altered leaf, inflorescence, silique, and seed set phenotypes. Dark-grown Atnap-1 and Atpir-1 seedlings also exhibited longer roots, enhanced skotomorphogenesis and Glc responses, and shorter thicker hypocotyls than those of wild type, showing that AtNAP and AtPIR participate in a variety of growth and developmental processes. Mutations in AtNAP and AtPIR caused cell morphology defects in cotyledon pavement cells and trichomes seen in mutants in ARP2/3 subunits and in plants expressing constitutively active Rop2 GTPase. The patterns and levels of actin polymerization observed in Atnap-1 and Atpir-1 mutant trichome cells and epidermal pavement cell morphology is consistent with Arabidopsis NAP and PIR proteins forming a WAVE complex that activates ARP2/3 activity. The multiple growth and developmental phenotypes of Atnap and Atpir mutants reveals these proteins are also required for a wider variety of cellular functions in addition to regulating trichome cell growth.

Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP-glucose:thiohydroximate S-glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low Km values for phenylacetothiohydroximic acid (approximately 6 microm) and UDP-glucose (approximately 50 microm) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss-of-function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light-grown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole-3-acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis.

Decrement and amplification of slow wave potentials during their propagation in Helianthus annuus L. shoots.

Slow wave potentials (SWPs) are transitory depolarizations occurring in response to treatments that result in a pressure increase in the xylem conduits (P(x)). Here SWPs are induced by excision of the root under water in 40- to 50-cm-tall light-grown sunflower plants in order to determine the effective signal range to a naturally sized pressure signal. The induced slow wave depolarization appears to move up the stem while it is progressively decremented (i.e. the amplitude decreases with increasing distance from the point of excision) with a rate that appears to rise acropetally from 2.5 to 5.5% cm(-1). The decline of the SWP signal, in both amplitude and range, could be experimentally increased (i) when root excision was carried out in air and (ii) when the transpiration of the sunflower shoot was minimized by a preceding removal or coating of the leaves. A further decline of the SW signal was expected to occur when leaves were included in the measured path. However, when the most distant apical electrode was attached to an upper leaf, it showed a considerably larger depolarization than a neighboring stem position. This apparent amplification of the SWP signal is not confined to the leaf blade but includes the petiole as well. The amplification disappeared (i) when the illumination level was lowered to room light, (ii) when the blade was excised either completely or along the remaining midvein and (iii) when the intact leaf blade was submersed in water. These treatments reduce the SWP at the petiole to a small fraction of the signal in the opposite control leaf and specify bright illumination and blade-mediated transpiration as prerequisites of a signal increase that is confined to young, expanding leaves.

Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors

Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.

Endogenous hormonal levels and growth of dark-incubated shoots of Catasetum fimbriatum

Apical shoots and lateral buds of the epiphytic orchid Catasetum fimbriatum give rise to rootless etiolated stolons, when cultured in the presence of light and then transferred to the dark. The stolons are characterized by fast and continuous apical longitudinal growth. Measurements of four endogenous cytokinin, indole-3-acetic acid (IAA) and abscisic acid (ABA) levels in etiolated shoots and light-grown plants were low. However, after transfer of green plants to the dark, cytokinin levels increased 3- and 7-fold by 10 and 30 days of incubation, respectively. IAA levels also increased significantly, but the increase was not as great as for cytokinins. A similar trend was observed in the roots. A close relationship seems to exist between both cytokinin accumulation and the formation of etiolated stolons. Variations in ABA levels were practically inconspicuous. The presence of paclobutrazol in the medium, a potent inhibitor of gibberellin synthesis, strongly inhibited etiolated and non-etiolated longitudinal shoot growth, although no apparent effect was observed on apical meristem activity.

Phytochrome regulation of pea phototropin

Type 1 phototropin, one of the blue light receptors responsible for phototropism, is encoded in peas by at least two genes, PsPHOT1A and PsPHOT1B (formerly PsPK4 and PsPK5), both of which are more similar to Arabidopsis PHOT1 than to Arabidopsis PHOT2. We show here that PsPHOT1B encodes a full-length phototropin, whose expression pattern suggests that Psphot1b is the predominant phot1-type phototropin in etiolated seedlings. The gene encoding the other type 1 phototropin, PsPHOT1A, is expressed at low levels, with its highest levels in the leaves and stems of more mature, light-grown plants. Studies with phyA, phyB and the phyAphyB double mutants show that phyA and phyB have partially redundant roles in the reduction of PsPHOT1B expression under red light.

Analysis of Combinatorial Loss-of-Function Mutants in the Arabidopsis Ethylene Receptors Reveals That the ers1 etr1 Double Mutant Has Severe Developmental Defects That Are EIN2 Dependent

Ethylene responses in Arabidopsis are controlled by the ETR receptor family. The receptors function as negative regulators of downstream signal transduction components and fall into two distinct subfamilies based on sequence similarity. To clarify the levels of functional redundancy between receptor isoforms, combinatorial mutant lines were generated that included the newly isolated ers1-2 allele. Based on the etiolated seedling growth response, all mutant combinations tested exhibited some constitutive ethylene responsiveness but also remained responsive to exogenous ethylene, indicating that all five receptor isoforms can contribute to signaling and no one receptor subtype is essential. On the other hand, light-grown seedlings and adult ers1 etr1 double mutants exhibited severe phenotypes such as miniature rosette size, delayed flowering, and sterility, revealing a distinct role for subfamily I receptors in light-grown plants. Introduction of an ein2 loss-of-function mutation into the ers1 etr1 double mutant line resulted in plants that phenocopy ein2 single mutants, indicating that all phenotypes observed in the ers1 etr1 double mutant are EIN2 dependent.

Misregulation of tetrapyrrole biosynthesis in transgenic tobacco seedlings expressing mammalian biliverdin reductase.

Previous studies have established that the expression of mammalian biliverdin IXalpha reductase (BVR) in transgenic tobacco (Nicotiana tabacum cv. Maryland Mammoth) resulted in the loss of photoregulatory activity of all phytochromes together with a pronounced chlorophyll deficiency. This study was undertaken to assess the contribution of BVR-mediated alteration of tetrapyrrole metabolism to the observed phenotypes of BVR transgenic plants. BVR expression in dark-grown plants led to the reduced accumulation of protochlorophyllide and transcripts for the two committed enzymes for 5-aminolevulinic acid (ALA) synthesis despite the marked increased capacity for synthesis of ALA. Together with the observation that Mg-porphyrin accumulation in dark-grown seedlings treated with an iron chelator was unaffected by BVR expression, these results indicate that BVR diverts tetrapyrrole metabolism toward heme synthesis while also reducing heme levels to de-repress ALA synthesis. By contrast with dark-grown seedlings, light-grown BVR plants showed a marked inhibition of ALA synthesis compared with wild-type plants - a result that was correlated with the disappearance of the CHL I subunit of Mg-chelatase and an increase in heme oxygenase protein levels. As transcript levels of all tetrapyrrole biosynthetic genes tested were not strongly affected by BVR expression, these results implicate misregulated tetrapyrrole metabolism to be a major mechanism for BVR-dependent inhibition of chlorophyll biosynthesis in light-grown plants.

The acclimative response of the main light-harvesting chlorophyll a/ b–protein complex of photosystem II (LHCII) to elevated irradiances at the level of trimeric subunits

The changes in structural organization of the major light-harvesting chlorophyll a/ b–protein complex of photosystem II (LHC II) at the level of trimeric subcomplexes were studied in spinach plants grown under low light conditions (50 μmol quanta m −2 s −1) and then acclimated to elevated irradiances. By monitoring photochemical quenching of fluorescence yield ( q P), photosystem II (PS II) functional status was assessed in leaves of plants acclimated to a range of elevated irradiances. Three separate acclimative irradiances were selected for the experiments, reflecting: limiting light conditions (150 μmol quanta m −2 s −1), near to the inflexion point on the irradiance curve conditions (300 μmol quanta m −2 s −1) and an excessive light, causing a moderate stress in the form of down regulation of PS II (450 μmol quanta m −2 s −1). An immunoblot analysis showed that there was a clear decline in an abundance on chlorophyll basis of Lhcb1-3 apoproteins as an acclimative irradiance increased from 50 to 450 μmol quanta m −2 s −1, with Lhcb1 decreasing to a lesser extent than Lhcb2 and Lhcb3 (only at excessive irradiance). When analyzed by non-denaturing isoelectric focusing BBY membrane fragments (PSII α-enriched, stacked thylakoid membranes) isolated from low light-grown plants were resolved into nine fractions, seven of which (labelled 3–9) were established by us previously [Jackowski and Pielucha, J. Photochem. Photobiol. B: Biol. 64 (2001) 45] to be LHC II subcomplexes representing mixed populations of closely similar trimers, comprising permutations of Lhcb1 and Lhcb2 (subcomplexes 3–7) or Lhcb1-3 (subcomplexes 8 and 9). A heterogeneity with regard to accumulation behaviour of LHC II subcomplexes in response to elevated irradiances was revealed. The subcomplexes 5 and 6 were accumulating at similar level, regardless of the light irradiance experienced. Another group consisting of the subcomplexes 3 and 4 (the most basic ones) showed a progressive increase in relative abundance with increasing an irradiance intensity whereas the subcomplexes 7–9 (the most acidic ones) exhibited a progressive decline in their relative abundance during an acclimation of spinach plants to elevated irradiances thus they may collectively represent an elevated irradiance-responsive subunit of LHCII.