Barley Mutant Research Articles

Inefficient rpl2 splicing in barley mutants with ribosome-deficient plastids.

Analysis of transcript accumulation and splicing in plastids of four nuclear mutants of barley revealed that the ribosomal protein L2 (rpl2) gene transcripts containing a group II intron remained entirely unspliced, whereas the intron of the ribosomal protein L16 (rpl16) gene (linked with the rpl2 gene in the same operon) was removed in the mutant plastids. Also, the transcripts of other genes containing group II introns (ribosomal protein S16 gene, rps16; NADH dehydrogenase ND2 gene, ndhB; cytochrome f gene, petD; and intron-containing reading frame 170, irf170) and of the tRNA for leucine, trnL (UAA), possessing the only chloroplast group I intron, were found to be spliced. The mutants used in this investigation are considered to be nonallelic; this excludes the possibility that a single nuclear gene is responsible for the impaired splicing of rpl2 transcripts. The mutants, however, have a severe deficiency in chloroplast ribosomes in common; this deficiency is evident from the lack of the essential ribosomal protein L2 and from an extremely low steady state level of plastid rRNAs. From these results, we conclude that a functioning translational apparatus of the organelle is a prerequisite for splicing of the chloroplast rpl2 class II intron but not for splicing of at least five other group II intron-containing transcripts. This provides genetic evidence for a chloroplast DNA-encoded component (e.g., a maturase) involved in the splicing of rpl2 pre-mRNA.

Isolation and characterization of aleurone protoplasts from a malting variety of barley (Hordeum vulgare L.)

A procedure has been developed to isolate protoplasts from mature aleurone layers of the malting variety Alexis and four other barley genotypes. It combines induction of endogenous cell wall degrading enzymes together with use of Onuzuka cellulase R 10 and driselase and results in better yields for two varieties than can be obtained with the huskless variety Himalaya. The viability of the freshly isolated protoplasts is greater than 90% and in spite of the presence of gibberellic acid during isolation procedures, most of the protoplasts are at an early developmental stage, as judged by ultrastructure. Gibberellic acid-induced changes in protoplast structure resemble those reported for Himalaya protoplasts. The protoplasts secrete both α-amylase (EC 3.2.1.1) and (1-3, 1-4)-β-glucanase (EC 3.2.1.73) into the surrounding medium. Transfection studies using a low pI α-amylase promoter to direct chloramphenicol acetyltransferase expression in aleurone protoplasts from Alexis and Himalaya revealed significant differences in their hormone responsiveness. In the absence of hormones, low levels of expression of the reporter enzyme were obtained in Alexis protoplasts, while high levels were characteristic for Himalaya protoplasts. An 8-fold increase in the expression of the reporter gene was induced by supplying the transfected Alexis protoplasts with gibberellin A3, whereas expression in Himalaya protoplasts remained unchanged. When Himalaya protoplasts were isolated from aleurone layers that had not been incubated with GA3 during the initial stages of protoplasting (the classical procedure), the hormone response of the promoter was 2.5-fold. It is thus possible to optimize the aleurone protoplast isolation procedure for different barley genotypes and mutants of interest in studies of transgenic gene expression and hormone induced secretion of proteins from this unique secretory plant tissue.

Chemical Composition and Physical Characteristics of Proanthocyanidin-free and Normal Barley Isotypes

Abstract Proanthocyanidin-free mutant ( ant ) and parent (normal) barley pairs were grown at the Montana Agricultural Experiment Station, Bozeman, Montana, U.S.A., in 1985 and 1986. The pairs included: Triumph, ant 17-148 (cv. Gallant); Moravian III, ant 13-605; Andre, ant 13-587; Robust, ant 17-625; Advance, ant 17-537, normal and ant -, respectively. The ant barleys contained more ( P P P P P P P ant barleys, but tannin contents did not differ between barley types or among pairs. Physical measurements were similar between barley pairs, but the normal barleys had heavier ( P ant barleys and the 1985 crop had heavier ( P ant gene becomes a desirable genetic trait in barley, the chemical components will not be altered so as to reduce the value of the grain for feed significantly.

Amyloplasts and Starch Characterization in High Lysine Barley Mutant

Starch granules of Notch-2 high lysine barley mutant are small, irregular in shape and show a virtual loss of birefringence and a higher gelatinization temperature, compared to parent NP-113. Starch polymer of Notch-2 is qualitatively different from parent, as evidenced by X-ray diffractograms. There is a higher incidence of V-pattern at early development as also at maturity in mutant as compared to parent. Intensities corresponding to B-pattern are present at early development in both parent and mutant. B-pattern persists at maturity in mutant, while it is replaced by A-pattern in parent. Amyloplasts of mutant are modified in contrast to normal, oblong amyloplasts of parent. Modified amyloplasts show loss of grana, increased granulation and lipid droplets. Extensive lipid complexing with starch polymers in mutant is indicated and possible commercial and nutritional potentials speculated.

Light-harvesting chlorophyll a-b complex requirement for regulation of Photosystem II photochemistry by non-photochemical quenching

Recently, it has been suggested (Horton et al. 1992) that aggregation of the light-harvesting a-b complex (LHC II) in vitro reflects the processes which occur in vivo during fluorescence induction and related to the major non-photochemical quenching (qE). Therefore the requirement of this chlorophyll a-b containing protein complex to produce qN was investigated by comparison of two barley mutants either lacking (chlorina f2) or depressed (chlorina(104)) in LHC II to the wild-type and pea leaves submitted to intermittent light (IL) and during their greening in continuous light.It was observed that qN was photoinduced in the absence of LHC II, i.e. in IL grown pea leaves and the barley mutants. Nevertheless, in these leaves qN had no (IL, peas) or little (barley mutants) inhibitory effect on the photochemical efficiency of QA reduction measured by flash dosage response curves of the chlorophyll fluorescence yield increase induced by a single turn-over flashDuring greening in continuous light of IL pea leaves, an inhibitory effect on QA photoreduction associated to qN developed as Photosystem II antenna size increased with LHC II synthesis. Utilizing data from the literature on connectivity between PS II units versus antenna size, the following hypothesis is put forward to explain the results summarized above. qN can occur in the core antenna or Reaction Center of a fraction of PS II units and these units will not exhibit variable fluorescence. Other PS II units are quenched indirectly through PS II-PS II exciton transfer which develops as the proportion of connected PS II units increases through LHC II synthesis.

Cytological and molecular evidence of deletion of ribosomal RNA genes in chromosome 6 of barley (Hordeum vulgare)

The barley chromosomal mutant T-35, in which only one pair of satellite chromosomes is apparent, was analyzed using a range of cytological and molecular techniques. Using conventional Feulgen staining, Giemsa and silver banding, in situ hybridization, and Southern blot analysis, unequivocal cytological and molecular evidence was obtained that T-35 is a homozygous deletion of rRNA genes residing in the nucleolus organizer region (NOR) of chromosome 6. According to the criteria of arm ratio and Giemsa-banding pattern of this chromosome, the deletion involved the whole NOR, one of the breakpoints being localized in the short arm proximally to the NOR-associated heterochromatic band, the other probably in the satellite of the chromosome. As a result of this deletion, an increased activity of the rRNA genes (as indicated by the size of the silver bands) on the other NOR-bearing chromosome (chromosome 7) was observed. The possible reasons for this phenomenon are discussed.

Vir-115 gene product is required to stabilize D1 translation intermediates in chloroplasts

The nuclear gene mutant of barley, vir-115, shows a developmentally induced loss of D1 synthesis that results in inactivation of Photosystem II. Translation in plastids isolated from 1 h illuminated vir-115 seedlings is similar to wild type. In wild-type barley, illumination of plants for 16 to 72 h results in increased radiolabel incorporation into the D1 translation intermediates of 15-24 kDa. In contrast, these D1 translation intermediates were not observed in vir-115 plastids isolated from plants illuminated for 16-72 h. In addition, after 72 h of illumination, radiolabel incorporation into D1 was undetectable in vir-115 plastids. The level and distribution of psbA mRNA in membrane-associated polysomes was similar in wild-type and vir-115 mutant plastids isolated from plants illuminated for 16-72 h. Toeprint analysis showed similar levels of translation initiation complexes on psbA mRNA in vir-115 and wild-type plastids. These results indicate that translation initiation and elongation of D1 is not significantly altered in the mutant plastids. Ribosome pausing on psbA mRNA was observed in wild-type and vir-115 mutant plastids. Therefore, the absence of D1 translation intermediates in mutant plastids is not due to a lack of ribosome pausing on psbA mRNA. Based on these results, it is proposed that vir-115 lacks or contains a modified nuclear-encoded gene product which normally stabilizes the D1 translation intermediates. In wild-type plastids, ribosome pausing and stabilization of D1 translation intermediates is proposed to facilitate assembly of cofactors such as chlorophyll with D1 allowing continued D1 synthesis and accumulation in mature chloroplasts.

Differential Responses of Growth and Nitrate Reductase Activity in Wild Type and NO 2-Tolerant Barley Mutants to Atmospheric NO 2 and Nutrient Nitrate

Summary Strains of NO 2 -tolerant barley ( Hordeum vulgare L.) were selected from NaN 3 -mutated seeds using high levels of atmospheric 3 (5 μLL -1 ). Seedlings from NO 2 -tolerant mutant seeds (Types B1 and W3) were grown alongside wild type seedlings (cvs. Maris Mink and Zephyr) in glasshouses for 10 d with nutrient solution containing either no nitrogen or 5 mM nitrate and some of each were fumigated with 0.5 μLL -I NO 2 for 3d. Exposure of plants to NO 2 for 3d caused a substantial decline in root dry weight and an increase in the levels of nitrate reductase (NR) activity in the leaves of both mutants. Levels of root NR activities were also higher in NO 2 -fumigated plants but this increase could be explained by the decrease in root weights. The entire population of the wild type seedlings and about 5 % of the B 1 plants showed visible symptoms of NO 2 injury in the form of whitening of leaf tips or brown spots on the leaf blades but none was ever found on the W3 mutant. It would appear from these experiments that these NO 2 -tolerant mutants cope with the toxic symptoms of atmospheric NO 2 injury by reducing their root growth and enhancing the levels of their shoot NR activities.

Growth responses of mutants of spring barley to fumigation with SO2 and NO2in combination

SUMMARYThis study was undertaken to evaluate the range of variation in growth responses of sodium azide‐induced M2mutants (stocks SI and S2) of spring barley, Hordeum vulgare. L. cv. Maris Mink, in relation to air pollution tolerance or sensitivity. The preliminary screening experiment with 150 nl l−1concentrations of SO2 and NO2 in combination for 10 d, yielded well‐defined sensitive and tolerant lines in both the stocks. However, the percentage frequency distribution of plants in different injury classes varied and stock S2 showed a better segregation of different lines. In a subsequent experiment, treatment with a mixture of each gas at 100 nl l−lconcentration for 22 d caused a variable degree of growth reduction. As a result of air pollution treatment, most of the growth parameters were significantly (P < 0.001) reduced in the sensitive lines of both the stocks, whereas the tolerant lines showed a marked resistance to the growth inhibitory effect of the pollutants. The magnitude of the difference between the sensitivities of different lines was usually double or more, and the effects were, more pronounced in stock S2 than in stock SI. Root dry weight was more adversely affected than shoot dry weight in sensitive lines of both the stocks. The results indicated that inter‐line differences in growth responses of these mutants were wide ranging, distinct and consistent. In conclusion, this study clearly demonstrates that artificial mutations may cause a markedly broad base of variation in air pollution sensitivity of crop plants which may be used in future plant breeding programmes of pollution tolerant cultivars.

Characterization of a family of chlorophyll-deficient wheat (Triticum) and barley (Hordeum vulgare) mutants with defects in the magnesium-insertion step of chlorophyll biosynthesis.

During thylakoid membrane biogenesis, chlorophyll (Chl) biosynthesis and the accumulation of Chl-binding proteins are tightly linked, light-regulated processes. We have investigated the consequences faced by mutant plants with defects in Chl biosynthesis by studying a series of five homeologous allelic chlorina mutants in wheat (Triticum) and one phenotypically related barley (Hordeum vulgare) mutant that express the same pleiotropic mutant phenotype but to different extents. These mutants accumulate Chl at different rates, with the most severely affected plants having the slowest rate of Chl accumulation. Analysis of precursor pools in the Chl synthesis pathway indicates they have a partial block in Chl synthesis and accumulate protoporphyrin IX (Proto), the last porphyrin compound common to both heme and Chl synthesis. The affected plants with the most severe phenotypes accumulate the most Proto. Chloroplasts isolated from these mutants exhibit a lower activity of the enzyme Mg-chelatase, which catalyzes the first committed step in Chl synthesis. The most severely affected plants exhibit the greatest reduction in Mg-chelatase activity. Heme levels and protoporphyrinogen oxidase activity were the same for mutant and wild-type plants. We suggest that a block in Mg-chelatase activity in these mutants could account for the other traits of their pleiotropic phenotype previously described in the literature.

Ribosome-deficient plastids affect transcription of light-induced nuclear genes: genetic evidence for a plastid-derived signal

Transcription of ten nuclear genes was analysed in the albostrians mutant of barley (Hordeum vulgare L.). The lack of plastid ribosomes in white seedlings of this mutant results in a complex alteration of nuclear gene expression at the transcriptional level. We found a strong reduction in the accumulation of mRNAs transcribed from nuclear genes encoding chloroplast enzymes involved in the Calvin cycle, the chlorophyll a/b binding protein, and the cytosolic enzyme nitrate reductase. In contrast, the levels of transcripts of the genes encoding the cytosolic glycolytic enzymes glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase were slightly enhanced. Accumulation of chalcone synthase mRNA even reaches much higher levels in white than in green leaves. Ribosome-deficient plastids were combined by crossing with a nuclear genotype heterozygous for the albostrians allele. Analysis of transcript levels in F1 plants having the same nuclear genotype and differing only with respect to their content of normally developed chloroplasts versus undifferentiated mutant plastids, provided strong genetic evidence for the plastid being the origin of a signal (chain) involved in regulation of nuclear gene expression. Results of run-on transcription in isolated nuclei demonstrated that the plastid signal acts at the level of transcription; it does not interfere with gene regulation in general. Mechanisms triggering nuclear gene expression in response to light operate in white mutant leaves: the very low levels of mRNAs derived from nuclear genes encoding chloroplast proteins and the strongly enhanced level of chalcone synthase mRNA were both light inducible. Also the negative regulation of leaf thionein gene expression by light is observed in white albostrians seedlings.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly and composition of the chlorophyll a-b light-harvesting complex of barley (Hordeum vulgare L.): Immunochemical analysis of chlorophyll b-less and chlorophyll b-deficient mutants

The chlorina-f2 mutant of barley (Hordeum vulgare L.) contains no chlorophyll b in its light-harvesting antenna, whereas the chlorina-103 mutant contains approximately 10% of the chlorophyll b found in wild-type. The absolute chlorophyll antenna size for Photosystem-II in wild-type, chlorina-103 and chlorina-f2 mutant was 250, 58 and 50 chlorophyll molecules, respectively. The absolute chlorophyll antenna size for Photosystem-I in wild-type, chlorina-103 and chlorina-f2 mutant was 210, 137 and 150 chlorophyll molecules, respoectively. In spite of the smaller PS I antenna size in the chlorina mutants, immunochemical analysis showed the presence of polypeptide components of the LHC-I auxiliary antenna with molecular masses of 25, 19.5 and 19 kDa. The chlorophyll a-b-binding LHC-II auxiliary antenna of PS II contained five polypeptide subunits in wild-type barley, termed a, b, c, d and e, with molecular masses of 30, 28, 27, 24 and 21 kDa, respectively. The polypeptide composition of the LHC-II auxiliary antenna of PS II was found to be identical in the two mutants, with only the 24 kDa subunit d present at an equal copy number per PS II in each of the mutants and in the wild-type barley. This d subunit assembles stably in the thylakoid membrane even in the absence of chlorophyll b and exhibits flexibility in its complement of bound chlorophylls. We suggest that polypeptide subunit d binds most of the chlorophyll associated with the residual PS II antenna in the chlorina mutants and that is proximal to the PS II-core complex.

Editing of the chloroplast rpoB transcript is independent of chloroplast translation and shows different patterns in barley and maize.

Sequence analysis of amplified cDNAs derived from the maize chloroplast rpoB transcript which encodes the beta subunit of a chloroplast specific, DNA dependent RNA polymerase reveals four C-to-U editing sites clustered within 150 nucleotides of the 5' terminal region of the rpoB message. These newly identified editing sites confirm the bias of chloroplast editing for certain codon transitions and for second codon positions which both appear suggestive for an involvement of the translational apparatus in the editing process. This supposition prompted us to investigate editing of the rpoB transcript from ribosome deficient, and hence protein synthesis deficient, plastids of the barley mutant albostrians. In this mutant editing is, however, not impaired at any of the editing sites functional in the barley wild type rpoB transcript. This demonstrates that chloroplast editing is neither linked to nor dependent on the chloroplast translational apparatus. As a further consequence any peptide components required for chloroplast editing must be encoded in the nuclear genome. In spite of strong sequence conservation only three of the four editing sites identified in the maize rpoB transcript are functional in barley. This indicates that sequences surrounding an editing site alone are not sufficient as determinants for the editing process in chloroplasts, but that trans-acting templates carrying the editing information for each individual site may also be required.

Evaluation of the role of State transitions in determining the efficiency of light utilisation for CO2 assimilation in leaves

Wheat leaves were exposed to light treatments that excite preferentially Photosystem I (PS I) or Photosystem II (PS II) and induce State 1 or State 2, respectively. Simultaneous measurements of CO2 assimilation, chlorophyll fluorescence and absorbance at 820 nm were used to estimate the quantum efficiencies of CO2 assimilation and PS II and PS I photochemistry during State transitions. State transitions were found to be associated with changes in the efficiency with which an absorbed photon is transferred to an open PS II reaction centre, but did not correlate with changes in the quantum efficiencies of PS II photochemistry or CO2 assimilation. Studies of the phosphorylation status of the light harvesting chlorophyll protein complex associated with PS II (LHC II) in wheat leaves and using chlorina mutants of barley which are deficient in this complex demonstrate that the changes in the effective antennae size of Photosystem II occurring during State transitions require LHC II and correlate with the phosphorylation status of LHC II. However, such correlations were not found in maize leaves. It is concluded that State transitions in C3 leaves are associated with phosphorylation-induced modifications of the PS II antennae, but these changes do not serve to optimise the use of light absorbed by the leaf for CO2 assimilation.

Sodium azide mutagenesis: preferential generation of A.T-->G.C transitions in the barley Ant18 gene.

The molecular basis for the absence of anthocyanins and proanthocyanidins in four independent sodium azide-induced ant18 mutants of barley was examined by sequencing the gene encoding dihydroflavonol 4-reductase in these mutants. Sodium azide generated 21 base substitutions, which corresponds to 0.17% of the 12,704 nucleotides sequenced. Of the substitutions, 86% were nucleotide transitions, and 14% were transversions. A.T-->G.C base pair transitions were about 3 times more frequent than G.C-->A.T transitions. No deletions or mutation hot spots were found. The absence of dihydroflavonol 4-reductase activity in ant18-159, ant18-162, and ant18-164 plants is caused by missense mutations in the respective genes. By using microprojectile bombardment, a plasmid harboring the wild-type Ant18 gene was introduced into ant18-161 mutant cells and resulted in the development of anthocyanin pigmentation, which demonstrates that the mutation is corrected by expression of the introduced gene. On the other hand, a plasmid derivative with the two ant18-161-specific base transitions at the 5' splice site of intron 3 prevented complementation. It is concluded that the absence of detectable mRNA for dihydroflavonol 4-reductase in ant18-161 cells is due to the mutations in the pre-mRNA splice donor site.

Comparison of chlorophyll fluorescence quenching in leaves of wild-type with a chlorophyll-b-less mutant of barley ( Hordeum vulgare L.)

A pulse amplitude-modulated fluorometric technique was employed to separate photochemical (qP) and non-photochemical (qN) chlorophyll fluorescence quenching in attached leaves of wild-type and a chlorophyll-b-less mutant of barley ( Hordeum vulgare L.). Whereas a significantly higher qN developed in the wild type in the intensity range at which the photosynthetic light response became non-linear, there were virtually no differences in qP until pronounced photoinhibition was apparent. On monitoring the “dark” recovery of the maximum ( F M) and dark level ( F 0) fluorescence yield, three distinct kinetic phases were resolved, which are ascribed to relaxation of high energy state quenching (qE), state 1-state 2 transitions (qT) and quenching due to photoinhibition (qI). The results provide evidence for heterogeneity of qE. The major part of qE (related to a fast-relaxing phase of F M quenching) is strongly reduced in mutant leaves. A fast-relaxing phase of F 0 quenching, readily observed in wild-type leaves under high light, is absent in the mutant under the same conditions. Hence, qE appears to be associated with the photosystem II light-harvesting complex (LHC II). A medium component of “dark” relaxation kinetics was observed in both mutant and wild-type leaves. However, it appears attributable to qT only in the latter at low light. Supported by the finding that there was no difference in xanthophyll pool size (on a chlorophyll a basis) between wild type and mutant, under high light conditions the medium phase may reflect a slower-relaxing portion of qE, probably due to xanthophyll conversion. Under supersaturating light the very slowly relaxing qI component became dominating, affecting mutant leaves to a considerably greater extent. The results stress the key role of LHC II not only as a solar energy collector but also in protecting the photosynthetic apparatus from adverse effects of excess excitation input.

AN ASSESSMENT OF THE MALTING QUALITY OF A MUTANT OF THE BARLEY CV. TROUBADOUR GROWN UNDER SCOTTISH CONDITIONS

The barley mutant, TR49, selected in Spain for rapid germination, and its parent cultivar Troubadour, were grown in Scotland in 1991 and assessed for a range of grain and malt quality characters. Results suggested that both genotypes had a potential dormancy problem, although TR49 showed a slight but significant improvement over Troubadour. TR49 produced very large grain which did not completely modify during malting, but still showed an advantage over its parent cultivar for all malting parameters assessed.

Hot Water Extracts in a Mutant Derived from the Barley Cultivar Troubadour

Abstract The mutant barley genotype TR49 and the cultivar from which it was derived, Troubadour, were grown at four sites throughout Spain in 1989 and 1990. Significant differences due to genotype and environment were detected for a range of malting quality parameters in 1990. Hot water extract was correlated with a number of factors which contribute to the structure of the endosperm or ease of its modification, such as β-glucan content, malt protein and Kolbach index. The levels of α-amylase and β-glucanase, although significantly different between the genotypes, did not give resultant differences in extract. The higher extract of TR49 over Troubadour is most likely to be due to differences in the structure and composition of the endosperm walls which facilitate modification.

Dynamics of Photosystem Stoichiometry Adjustment by Light Quality in Chloroplasts.

Long-term imbalance in light absorption and electron transport by photosystem I (PSI) and photosystem II (PSII) in chloroplasts brings about changes in the composition, structure, and function of thylakoid membranes. The response entails adjustment in the photosystem ratio, which is optimized to help the plant retain a high quantum efficiency of photosynthesis (W.S. Chow, A. Melis, J.M. Anderson [1990] Proc Nat Acad Sci USA 87: 7502-7506). The dynamics of photosystem ratio adjustment were investigated upon the transfer of pea {Pisum sativum} plants from a predominantly PSI-light to a predominantly PSII-light environment and vice versa. The concentration of functional components (primary electron accepting plastoquinone of PSII [QA], P700) and that of constituent proteins were monitored during acclimation by A difference spectrophotometry and immunoblot analysis, respectively. Fully reversible changes in photosystem ratio occurred with a half-time of about 20 h. They involved closely coordinated changes in the concentration of the QA, reaction center protein D1, D2, and the 9-kD apoprotein of the cytochrome b559 for PSII. Similarly, closely coordinated changes in the relative concentration of P700 and reaction center proteins of PSI were observed. The level of chlorophyll b and that of the light-harvesting complex II changed in accordance with the concentration of PSII in the acclimating thylakoids. Overall, adjustments in the photosystem ratio in response to PSI- or PSII-light conditions appeared to be a well-coordinated reaction in the chloroplast. The response was absent in the chlorophyll b-less chlorina f2 mutant of barley (Hordeum vulgare) and in a phycobilisomeless mutant of Agmenellum quadruplicatum, suggesting that photosystem accessory pigments act as the light-quality perception molecules and that PSI and PSII themselves play a role in the signal transduction pathway.

Chlorophyll fluorescence transients in a barley mutant lacking Photosystem I

We have compared the properties of a mutant of barley lacking Photosystem I (viridis-zb (63) ) with the corresponding wild type using modulated fluorescence measurements. The mutant showed two unexpected characteristics. Firstly, there was a slow decline in the fluorescence signal in the light which was dependent on the presence of O2 at concentrations similar to that in air; 2% O2 in N2 had no effect. The observed decline was mainly due to an increase in the non-photochemical quenching. Secondly, in the absence of O2, saturating light pulses caused a pronounced transient decrease in the fluorescence signal; a similar effect could also be observed in wild type plants when neither CO2 nor O2 was present.