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

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

Response of antioxidant enzymes to transfer from elevated carbon dioxide to air and ozone fumigation, in the leaves and roots of wild‐type and a catalase‐deficient mutant of barley

A catalase-deficient mutant (RPr 79/4) and the wild-type (cv. Maris Mink) barley (Hordeum vulgare L.) counterpart, were grown for 3 weeks in high CO₂ (0.7%) and then transferred to air and ozone (120 nl 1^?1) in the light and shade for a period of 4 days. Leaves and roots were analysed for catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and glutathione reductase (GR, EC 1.6.4.2) activities. CAT activity in the leaves of the RPr 79/4 catalase-deficient mutant was around 5-10% of that determined in Maris Mink, but in the roots, both genotypes contained approximately the same levels of activity. CAT activity in Maris Mink increased in the leaves after transferring plants from 0.7% CO₂ to air or ozone, reaching a maximum of 5-fold, after 4 days in shade and ozone. For the catalase-deficient mutant, only small increases in CAT activity were observed in light/air and light/ozone treatments. In the roots, CAT activity decreased consistently in both genotypes, after plants were transferred from 0.7% CO₂. The total soluble SOD activity in the leaves and roots of both genotypes increased after plants were transferred from 0.7% CO₂. The analysis of SOD isolated from leaves following non-denaturing PAGE, revealed the presence of up to eight SOD isoenzymes classified as Mn-SOD or Cu/Zn-SODs; Fe-SOD was not detected. Significant changes in Mn- and Cu/Zn-SOD isoenzymes were observed; however, they could not account for the increase in total SOD activity. In leaves, GR activity also increased in Maris Mink and RPr 79/4, following transfer from 0.7% CO₂; however, no constant pattern could be established, while in roots, GR activity was reduced after 4 days of the treatments. The data suggest that elevated CO₂ decreases oxidative stress in barley leaves and that soluble CAT and SOD activities increased rapidly after plants were transferred from elevated CO₂, irrespective of the treatment (light, shade, air or ozone).     

The mapping of phytochrome genes and photomorphogenic mutants of tomato

The map positions of five previously described phytochrome genes have been determined in tomato (Lycopersicon esculentum Mill.) The position of the yg-2 gene on chromosome 12 has been confirmed and the classical map revised. The position of the phytochrome A (phy A)-deficient fri mutants has been refined by revising the classical map of chromosome 10. The position of the PhyA gene is indistinguishable from that of the fri locus. The putative phyB1-deficient tri mutants were mapped by classical and RFLP analysis to chromosome 1. The PhyB1 gene, as predicted, was located at the same position. Several mutants with the high pigment (hp) phenotype, which exaggerates phytochrome responses, have been reported. Allelism tests confirmed that the hp-2 mutant is not allelic to other previously described hp (proposed here to be called hp-1) mutants and a second stronger hp-2 allele (hp-2 ^j ) was identified. The hp-2 gene was mapped to the classical, as well as the RFLP, map of chromosome 1. Received: 24 May 1996 / Accepted: 14 June 1996     

Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase

A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS₂) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS₁). A sequence identicalto an N-terminal sequence determined from a purified preparation of the mature GS₂ polypeptide (NH₂-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46–61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS₂ subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS₂-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS₂ mRNA, and an anti-GS antiserum to assay for GS₂ protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS₂ mRNA; class II, which had normal or increased levels of GS₂ mRNA but very little GS₂ protein; and class III, which had significant amounts of GS₂ protein but little or no GS₂ activity.     

The aurea mutant of tomato is deficient in spectrophotometrically and immunochemically detectable phytochrome

The aurea locus mutant (au ^w) of tomato contains less than 5% of the level of phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. Immunoblot analysis using antibodies directed against etiolated-oat phytochrome demonstrates that crude extracts of etiolated mutant tissue are deficient in a major immunodetectable protein (116 kDa) normally present in the parent wild type. Analyses of wild-type tissue extracts strongly indicate that the 116-kDa protein is phytochrome by showing that this protein: a) is degraded more rapidly in vitro after a brief far-red irradiation than after a brief red irradiation (Vierstra RD, Quail PH, Planta 156: 158–165, 1982); b) contains a covalently bound chromophore as detected by Zn-chromophore fluorescence on nitrocellulose blots; and c) has an apparent molecular mass comparable to phytochrome from other species on size exclusion chromatography under non-denaturing conditions. The demonstration that the aurea mutant is deficient in this 116-kDa phytochrome indicates that the lack of spectrally detectable phytochrome in this mutant is the result of a lesion which affects the abundance of the phytochrome molecule as opposed to its spectral integrity.     

Molecular analysis of the phytochrome deficiency in an aurea mutant of tomato

Summary 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.     

Plasma membrane depolarization‐activated calcium channels, stimulated by microtubule‐depolymerizing drugs in wild‐type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half‐life in ton 2 mutant cells affected in the organization of cortical microtubules

Depolarization-activated plasma membrane calcium channels have been suggested to play prominent roles in signal perception and transduction processes during growth and development of higher plants. The existence of such channels has recently been established in higher plant cells. However, patch–clamp experiments have shown that their activity is very low and decreases very rapidly after the establishment of the whole-cell configuration, due most probably to protein–protein interactions involving microtubules. The present study takes advantage of the existence of Arabidopsis thaliana mutants referred to as ton 2 mutants reported to be affected in their microtubule organization, to address the physiological relevance of such a hypothesis based on a pharmacological approach. Patch–clamp studies showed that depolarization-activated calcium channel activities in ton 2 protoplasts were 10-fold higher and their relative half-life three-times longer than in wild-type protoplasts. In addition, oryzalin and colchicine, which disrupt the microtubule organization, stimulated and stabilized calcium channel activities in wild-type but remained ineffective on ton 2 protoplasts. However, although the microtubules appeared important in the regulation of calcium channels in A. thaliana, immunocytological staining of tubulin demonstrated that there was no visible difference in the general organization of microtubule networks or in the amount of microtubules bound to the plasma membrane in ton 2 and wild-type protoplasts. It is suggested that the down-regulation of calcium channels implicating microtubules involves additional component(s) corresponding probably to gene product(s) defective in ton 2 mutant cells.     

The tomato photomorphogenetic mutant, aurea, is deficient in phytochromobilin synthase for phytochrome chromophore biosynthesis

The aurea mutants of tomato have been widely used as phytochrome-deficient mutants for photomorphogenetic and photobiological studies. By expressed sequence tag (EST)-based screening of sequence databases, we found a tomato gene that encodes a protein homologous to Arabidopsis HY2 for phytochromobilin synthase catalyzing the last step of phytochrome chromophore biosynthesis. The tomato protein expressed in Escherichia coli showed phytochromobilin synthase activity. The corresponding loci in all aurea mutants tested have nucleotide substitutions, deletions or DNA rearrangements. These results indicate that aurea is a mutant of phytochromobilin synthase in tomato. We also discuss a phylogenetic analysis of phytochromobilin synthases in the bilin reductase family.     

Physiological and behavioural differences of hatchery and wild‐reared steelhead Oncorhynchus mykiss smolts of the same genetic origin

In many parts of the world release of hatchery‐reared smolts has long been used to mitigate for the deleterious effects of habitat loss and overfishing on salmonid populations. Of increasing concern is whether this may cause harm by spreading non‐native stocks and potentially releasing incompetent smolts. The objective of this study was to determine if smolt physiology and behavior of juveniles produced from a recently founded native broodstock differ from their wild (naturally‐reared) counterparts. In the fall of 2002 and 2003 juvenile wild steelhead were captured, PIT tagged, and returned ( n  = 1360 in 2002 and n  = 2708 in 2003) to Abernathy Creek. In winter of 2003 and 2004 hatchery‐reared fish were PIT tagged and later released ( n  = 1100 in 2003 and n  = 1400 in 2004) into Abernathy Creek. Gill biopsies were collected from wild and hatchery fish throughout the rearing and out‐migration season. The timing and speed of outmigration was assessed using two stationary PIT tag antennas (92–97% efficient). Hatchery migrants in 2003 were larger, had significantly lower gill Na⁺, K⁺‐ATPase activities, and migrated slower than wild fish. Results from the 2004 migratory season will also be presented. This study shows that hatchery rearing can result in smolts which are physiologically and behaviourally different from genetically similar wild fish. Whether these differences are critical enough to affect the rate of adult returns will be determined in future years.     

The terminal O‐acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A‐dependent acyl transfer

The gene encoding acetyl CoA:deacetylvindoline 4-O-acetyltransferase (DAT) (EC 2.3.1.107) which catalyzes the last step in vindoline biosynthesis was isolated and characterized. The genomic clone encoded a 50 kDa polypeptide containing the sequences of nine tryptic fragments derived from the purified DAT heterodimer. However, cleavage of DAT protein to yield a heterodimer appears to be an artifact of the protein purification procedure, since the size of the protein (50 kDa) cross-reacting with anti-DAT antibody in seedlings and in leaves of various ages also corresponds to the size of the active recombinant enzyme. Studies with the intact plant and with developing seedlings showed that induction of DAT mRNA, protein accumulation and enzyme activity occurred preferentially in vindoline producing tissues such as leaves and cotyledons of light-treated etiolated seedlings. The ORF of DAT showed significant sequence identity to 19 other plant genes, whose biochemical functions were mostly unknown. The Mr of ≈ 50 kDa, a HXXXDG triad, and a DFGWGKP consensus sequence are highly conserved among the 20 plant genes and these criteria may be useful to identify this type of acyltransferase. The involvement of some of these genes in epicuticular wax biosynthesis, fruit-ripening and in benzoyltransfer reactions indicates that the plant kingdom contains a superfamily of multifunctional acyltransferases which operate by a reaction mechanism related to the ancient chloramphenicol O-acetyltransferase and dihydrolipoyl acetyltransferase class of enzymes.     

Individual‐level analysis of pre‐ and post first‐feed growth and development in Atlantic salmon

Atlantic salmon Salmo salar were followed from egg to smoltification using genetic analysis to identify individuals and to link observations from pre‐ and post first feeding. Egg size and hatch timing significantly influenced alevin size at first feed but neither egg size, hatch timing or alevin sizes were correlated to size, condition factor or smolt status post first feed. In a hatchery environment the potential advantage gained by early hatching, larger alevin does not persist after first feeding. Different physiological and genetic complexes appear to influence growth in these two distinct phases of the Atlantic salmon's life‐cycle.     

Analysis of protochlorophyllide reaccumulation in the phytochrome chromophore-deficient aurea and yg-2 mutants of tomato by in vivo fluorescence spectroscopy

The aurea and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum) are unable to synthesize the phytochrome chromophore from heme resulting in a block of this branch of the tetrapyrrole pathway. We have previously shown that these mutants also exhibit an inhibition of protochlorophyllide (Pchlide) synthesis and it has been hypothesised that this is due to feedback inhibition by heme on the synthesis of 5-aminolevulinic acid (ALA). In this study we have investigated Pchlide reaccumulation in cotyledons from etiolated wild-type (WT), aurea and yg-2 seedlings using low-temperature fluorescence spectroscopy. WT cotyledons showed two characteristic Pchlide emission maxima at 630 nm (F630) and 655 nm (F655) respectively, while the aurea and yg-2 mutants contained only phototransformable Pchlide F655. Following a white-light flash to WT cotyledons, reaccumulation of phototransformable Pchlide F655 in the first 30 min was absolutely dependent on the presence of Pchlide F630 before the flash. Reaccumulation of Pchlide F630 was not apparent until at least 2 h after the phototransformation. In contrast, Pchlide F630 never accumulated in aurea cotyledons. The relative rates of both Pchlide F655 and total Pchlide synthesis were approximately twice as high in WT compared to aurea. Measurement of ALA synthesis capacity during this period showed that the reduced rate of Pchlide reaccumulation in aurea was due to an inhibition at this step of the pathway. In addition, feeding of ALA resulted in a substantial and equal increase of non-phototransformable Pchlide in both WT and aurea indicating that aurea cotyledons are capable of accumulating high levels of Pchlide that is not associated to the active site of NADPH:Pchlide oxidoreductase (POR). The implications of these results for the mechanism of inhibition of Pchlide synthesis in phytochrome chromophore-deficient mutants and the role of non-phototransformable Pchlide F630 during plastid development are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vivo measurement of phytochrome in tomato fruit

Presence of phytochrome in two kinds of tomatoes (Lycopersicon esculentum Mill.), the yellow lutescent strain and cherry tomatoes (L. esculentum Mill. var. cerasiformecv. Red Cherry), was established by measuring the absorption difference spectra of the whole fruit after irradiation with red and with far red light. Phytochrome content was determined in yellow lutescent tomatoes and decreased gradually during the ripening period.     

Population structure of lake‐type and river‐type sockeye salmon in transboundary rivers of northern British Columbia

The population structure of 'lake‐type' and 'river‐type' sockeye salmon Oncorhynchus nerka , primarily in transboundary rivers in northern British Columbia, was examined with a survey of microsatellite variation. Variation at 14 microsatellite loci was surveyed from c . 3000 lake‐type and 3200 river‐type sockeye salmon from 47 populations in six river drainages in British Columbia. The mean F _ST for the 14 microsatellite loci and 47 populations was 0·068, and 0·034 over all river‐type populations. River‐type sockeye salmon were more genetically diverse than lake‐type sockeye salmon, with expected heterozygosity of river‐type sockeye salmon 0·72 and with an average 12·7 alleles observed per locus, whereas expected heterozygosity of lake‐type sockeye salmon was 0·65 with and average 10·5 alleles observed per locus. River drainage of origin was a significant unit of population structure. There was clear evidence of genetic differentiation among river‐type populations of sockeye salmon from different drainages over a broad geographic range in British Columbia.     

Isolation and characterization of rice phytochrome A mutants

To elucidate phytochrome A (phyA) function in rice, we screened a large population of retrotransposon (Tos17) insertional mutants by polymerase chain reaction and isolated three independent phyA mutant lines. Sequencing of the Tos17 insertion sites confirmed that the Tos17s interrupted exons of PHYA genes in these mutant lines. Moreover, the phyA polypeptides were not immunochemically detectable in these phyA mutants. The seedlings of phyA mutants grown in continuous far-red light showed essentially the same phenotype as dark-grown seedlings, indicating the insensitivity of phyA mutants to far-red light. The etiolated seedlings of phyA mutants also were insensitive to a pulse of far-red light or very low fluence red light. In contrast, phyA mutants were morphologically indistinguishable from wild type under continuous red light. Therefore, rice phyA controls photomorphogenesis in two distinct modes of photoperception--far-red light-dependent high irradiance response and very low fluence response--and such function seems to be unique and restricted to the deetiolation process. Interestingly, continuous far-red light induced the expression of CAB and RBCS genes in rice phyA seedlings, suggesting the existence of a photoreceptor(s) other than phyA that can perceive continuous far-red light in the etiolated seedlings.