Variability in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Small Subunits and Carboxylation Activity in Fern Gametophytes Grown under Different Light Spectra

Two distinct ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU) populations were observed in Pteris vittata gametophytes grown under different illumination conditions. Exposure of the fern gametophytes to continuous red light (R) resulted in Rubisco SSUs that were not recognized by polyclonal antibodies raised against SSUs from spinach. Unlike the R-induced SSUs, blue light (B) induced SSUs were well recognized. This difference in SSU composition also reflected in Rubisco activity. In vitro, B-induced Rubisco exhibits a significantly higher carboxylation activity as compared to the R-induced Rubisco. Approximately a two- to threefold increase in the V_max value of the B-induced carboxylase as compared to the R-induced one was measured. It thus seems very likely that certain domains in the SSU molecule affect enzyme activity.     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS

The effect of nitrogen supply during growth on the contribution of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; EC 4.1.1.39) to the control of photosynthesis was examined in tobacco (Nicotiana tabacum L.). Transgenic plants transformed with antisense rbcS to produce a series of plants with a progressive decrease in the amount of Rubisco were used to allow the calculation of the flux-control coefficient of Rubisco for photosynthesis (C_R). Several points emerged from the data: (i) The strength of Rubisco control of photosynthesis, as measured by C_R, was altered by changes in the short-term environmental conditions. Generally, C_R was increased in conditions of increased irradiance or decreased CO₂. (ii) The amount of Rubisco in wild-type plants was reduced as the nitrogen supply during growth was reduced and this was associated with an increase in C_R. This implied that there was a specific reduction in the amount of Rubisco compared with other components of the photosynthetic machinery. (iii) Plants grown with low nitrogen and which had genetically reduced levels of Rubisco had a higher chlorophyll content and a lower chlorophyll a/b ratio than wild-type plants. This indicated that the nitrogen made available by genetically reducing the amount of Rubisco had been re-allocated to other cellular components including light-harvesting and electron-transport proteins. It is argued that there is a luxury additional investment of nitrogen into Rubisco in tobacco plants grown in high nitrogen, and that Rubisco can also be considered a nitrogen-store, all be it one where the opportunity cost of the nitrogen storage is higher than in a non-functional storage protein (i.e. it allows for a slightly higher water-use efficiency and for photosynthesis to respond to temporarily high irradiance).Abbreviations C_R flux control coefficient of Rubisco for photosynthesis - rbcS gene for the Rubisco small subunit - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase W.P. Quick is grateful to Professor D.T. Clarkson (Department of Agricultural Sciences, University of Bristol, Long Ashton, UK) for pointing out the connection between stomatal conductance and nutrient availability. This work was supported by the Deutsche Forschungsgemeinschaft.     

Antisense RNA inhibition of Rubisco activase expression

Ribulose bisphosphate carboxylase (Rubisco) activase catalyzes the activation of Rubisco in vivo. Activase antisense DNA mutants of tobacco have been generated to explore the control that activase exerts on the photosynthetic process. These mutants have up to 90% reductions in activase protein levels as a consequence of an inhibition of activase mRNA accumulation. It is shown that photosynthesis, measured as the rate of CO₂ exchange (CER), is modestly decreased in plants exposed to high irradiances. The decreases in CER in the transgenic plants are accompanied by corresponding decreases in Rubisco activation, indicating that activase has a direct effect on photosynthetic rates in the antisense plants by influencing the activation state of Rubisco. It is concluded that in high light conditions, control of photosynthesis is largely shared between Rubisco and activase. Plant growth is also impaired in mutant plants that have severe reductions in activase. The inhibition of activase in the antisense plants does not have an impact on the accumulation of Rubisco large subunit or small subunit mRNAs or proteins. This indicates that the concerted expression of the genes for activase (Rca) and Rubisco (rbcL and rbcS) in response to light, developmental factors and circadian controls is not due to feedback regulation of rbcL or rbcS by the amount of activase protein.     

The effects of light on sex determination in gametophytes of the fern <Emphasis Type="Italic">Ceratopteris richardii</Emphasis>

The sexuality of homosporous fern gametophytes is usually determined by antheridiogen, a pheromone that promotes maleness. In this work the effect of photomorphogenically active light on antheridiogen-induced male development was examined for gametophytes of Ceratopteris richardii. Although blue light did not affect sensitivity to Ceratopteris antheridiogen (A_Ce) in wild-type gametophytes, it was found that the gametophytes of the her1 mutant, which are insensitive to A_Ce, developed into males when grown under blue light in the presence of A_Ce. Thus, we conclude that another A_Ce-signal transduction pathway activated by blue light exists latently in the gametophytes of C. richardii. Red light, on the other hand, suppressed male development. Because simultaneous red and blue light-irradiation did not promote male development in the her1 gametophytes, the action of red light seems to dominate that of blue light. The results of experiments with a photomorphogenic mutant also suggested that phytochrome may be involved in the action of red light.     

Ethylene is a modulator of gibberellic acid-induced antheridiogenesis in <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

In fern (Anemia phyllitidis) gametophytes cellulose in the walls of the antheridial zone cells which was organized in clusters and spots was transformed via dispersed form to fibrillar arrangement (layered in oblique and perpendicular array in relation to the transverse direction of cell expansion) during antheridiogenesis induced by gibberellic acid (GA₃) and/or enhanced by 1-aminocyclopropane-1-carboxylic acid (ACC). In the ACC-treated gametophytes, where antheridia were not induced, the cellulose was arranged in the same manner. Aminooxyacetic acid (AOA), which inhibits antheridiogenesis and development of fern gametophytes, produced in the cell walls both random and longitudinal type of organization of cellulose microfibrils, however, in the GA₃/AOA-treated plants the oblique type was also observed. The total numbers of cells with perpendicular and/or oblique type of cellulose microfibrils in the GA₃-, GA₃/ACC-and GA₃/AOA-treated gametophytes corresponded to the average number of antheridia formed. Moreover, it was found that the extracts from the gametophytes treated with GA₃ or with the mixture of GA₃ and ACC contained significantly less soluble sugars but more α-amylase-and endoglucanase-released sugars than the extracts from the gametophytes of the other series. Thin layer chromatography of the samples from the cell wall extracts hydrolyzed by endoglucanase contained xylose and cellobiose which suggested that these sugars built the xyloglucans, hemicellulose polymers responsible for tethering of walls of fern gametophyte cells like in higher plants.     

Biochemical and molecular basis for impairment of photosynthetic potential

Ozone induces reductions in net photosynthesis in a large number of plant species. A primary mechanism by which photosynthesis is reduced is through impact on carbon dioxide fixation. Ozone induces loss in Rubisco activity associated with loss in concentration of the protein. Evidence is presented that ozone may induce oxidative modification of Rubisco leading to subsequent proteolysis. In addition, plants exposed to ozone sustain reduction in rbcS, the mRNA for the small subunit of Rubisco. This loss in rbcS mRNA may lead to a reduced potential for synthesis of the protein. The regulation of O₃-induced loss of Rubisco, and implications of the decline in this protein in relation to accelerated senescence are discussed.     

Utility of rbcS gene as a novel target DNA region for brown algal molecular systematics

The usefulness of molecular phylogenetic studies has increased remarkably as the quantity and quality of available DNA sequences has increased. When compared with the progress that has occurred in angiosperms and animals, there have been relatively few target DNA regions identified for use in taxonomic studies of brown algae. Therefore, in this study, we developed a new set of primers to amplify Rubisco small subunit (rbcS) gene sequences and determined the rbcS gene sequences of various species of brown algae including those belonging to Dictyotales, Ectocarpales, Fucales and Sphacelariales. The level of sequence variations in the rbcS gene varied according to the brown algal lineages. When focusing on the relationship of species within the genus Sargassum, the rbcS gene sequences provided useful information regarding the phylogenetic relationship among sections of the subgenus Bactrophycus. Based on the broad applicability and phylogenetic utility of the rbcS gene, we suggest that the sequence be used as a new target region for the molecular systematics of brown algae.     

Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Brassinosteroids (BRs) are a new group of plant growth substances that promote plant growth and productivity. We showed in this study that improved growth of cucumber (Cucumis sativus) plants after treatment with 24-epibrassinolide (EBR), an active BR, was associated with increased CO₂ assimilation and quantum yield of PSII (Φ_PSII). Treatment of brassinazole (Brz), a specific inhibitor for BR biosynthesis, reduced plant growth and at the same time decreased CO₂ assimilation and Φ_PSII. Thus, the growth-promoting activity of BRs can be, at least partly, attributed to enhanced plant photosynthesis. To understand how BRs enhance photosynthesis, we have analyzed the effects of EBR and Brz on a number of photosynthetic parameters and their affecting factors, including the contents and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Northern and Western blotting demonstrated that EBR upregulated, while Brz downregulated, the expressions of rbcL, rbcS and other photosynthetic genes. In addition, EBR had a positive effect on the activation of Rubisco based on increased maximum Rubisco carboxylation rates (V _c,max), total Rubisco activity and, to a greater extent, initial Rubisco activity. The accumulation patterns of Rubisco activase (RCA) based on immunogold-labeling experiments suggested a role of RCA in BR-regulated activation state of Rubisco. Enhanced expression of genes encoding other Calvin cycle genes after EBR treatment may also play a positive role in RuBP regeneration (J _max), thereby increasing maximum carboxylation rate of Rubisco (V _c,max). Thus, BRs promote photosynthesis and growth by positively regulating synthesis and activation of a variety of photosynthetic enzymes including Rubisco in cucumber.     

Regulation of Protein Synthesis during Photomorphogenesis of Gametophytes of the Fern Onoclea sensibilis

Gametophytes of the fern Onoclea sensibilis grow as filaments in the dark and in red light and become planar in blue light. Pulse-labeling 4-day-old gametophytes with [³⁵S]methionine at different times after transfer to dark, red, and blue light environments revealed higher rates of amino acid uptake and protein synthesis in blue light than in red light or in the dark. Characterization of the extant and newly synthesized soluble proteins by one- and two-dimensional gel electrophoresis showed that the patterns of protein accumulation and synthesis in gametophytes exposed to short periods of red or blue light were qualitatively indistinguishable from those of gametophytes maintained in the dark. However, some striking increases and decreases in the levels of certain polypeptides were noted and these changes were accentuated during continued growth of gametophytes in the different environments. The results show that photomorphogenesis of gametophytes of O. sensibilis is associated with quantitative rather than qualitative changes in the population of mRNAs available for translation.     

Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.)

Silver fern (Pityrogramma calomelanos L.) is a terrestrial or lithophytic herbaceous fern used for ornamental and medicinal purposes. In its farina it produces the cytotoxic and anticancer compound dihydrochalcone. In vitro induction of apospory and apogamy, and direct field establishment of aposporous gametophytes and subsequent sporophyte development has been accomplished. Half-strength Murashige and Skoog (MS) medium with 3.33 μM N⁶-benzyladenine (BA) and 2.32 μM kinetin (Kn) showed earlier development and produced higher numbers of aposporous gametophytes than half-strength MS basal medium. Crozier explants developed higher numbers (mean value 29.2) of gametophytes, but were slower than frond explants (mean value 23.2). The gametophytes originated from the epidermal hairs progressed from uniseriate filamentous to cordate through bi-, tri- and multiseriate and spatulate stage with the development of antheridia. Reduction in the nutrient and sucrose concentrations in the media favoured apogamy. Sucrose-free 1/10 strength MS medium and agar plates developed a mean of 30.4 and 29.9 sporophytes, respectively in the light. The greenhouse-established gametophytes developed sporophytes. The established sporophytes ex vitro showed 95% survival rate. Apogamous sporophytes and the source plant showed the same chromosome numbers (2n=116). The established protocol accomplishes apogamy and apospory in silver fern, and the aposporous gametophytes can be used for genetic transformation and development of transgenic silver fern.     

Ribonucleic Acid and Protein Changes in the Subcellular Components of the Gametophytes of Pteridium aquilinum during Growth in Red and Blue Light

The distribution of RNA and protein in the gametophytes of bracken fern, Pteridium aquilinum is affected by the quality of light in which they are grown. Two-dimensional gametophytes growing in blue light have a greater amount of RNA and protein than one-dimensional protonema growing in red light. Fractions rich in nuclei, chloroplasts, mitochondria, ribosomes and soluble supernatants obtained from blue-light grown gametophytes by differential centrifugation contain greater amounts of RNA and protein than corresponding fractions of red-light grown plants. Differences in RNA and protein content are detected in some of the fractions within 24 hours after start of the treatment.     

Growth Promotion of Vegetative Gametophytes of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> by Blue Light

Through an acclimation period of 10 days, compared to white light, the maximal net photosynthetic rates were significantly higher for gametophytes of Undaria pinnatifida cultivated under blue light (400–500 nm), and were lower under red light (600–700 nm). Chlorophyll c and the carotenoid content of gametophytes were similar under blue light and red light but were much lower under white light. The growth rate of female gametophytes under blue light was higher than that under other lights, and the growth rate of male gametophytes showed little variation with respect to blue and white light. Male and female gametophytes were mixed together to form sporophytes under white, blue and red light. After approximately 5 days, 50% gametophytes became fertile under blue and white light, but remained vegetative under red light after 10 days.     

Do fern gametophytes have the capacity for irradiance acclimation?

Ferns present two alternant generations: sporophyte and gametophyte. In the present work we address the question of whether fern gametophytes have the potential to acclimate to different irradiances as vascular plants do. We studied the gametophytes of three different fern species belonging to the Aspleniaceae family with different ecological requirements (Asplenium trichomanes, Asplenium scoloprendrium and Ceterach officinarum). Fern spores were germinated and the gametophytes cultivated under photon flux density (PFD) of 10, 50 or 100 μmol m⁻² s⁻¹. From the early stages of spore germination (the formation of the 5-celled germinal filament), photosynthetic apparatus acclimates showing the typical patterns of photochemical responses to high or low PFD. In agreement with the photochemical pattern of acclimation, higher contents of xanthophyll cycle pigments and α-tocopherol was observed in plants grown under high PFD. The α/β-carotene ratio, used as indicator of the acclimation of the photosynthetic apparatus, also sustained the initial hypothesis except for A. trichomanes. We conclude that fern gametophytes display a complete array of photosynthetic and photoprotective traits that allow an effective acclimation to PFD.     

Influence of ozone on transgenic tobacco plants expressing reduced quantities of Rubisco

RbcS-antisense transformed tobacco plants (Nicotiana tabacum cv. Petit Havana) expressing reduced quantities of Rubisco protein were used to examine the role of Rubisco quantity in determining ozone (O₃) sensitivity. Transformed and wild-type plants were exposed to O₃ in the greenhouse and in the field. Stomatal conductance, net photosynthesis and Rubisco protein quantity were measured at various times. Antisense-transformed genotypes responded to O₃ by exhibiting rapid, severe foliar necrosis. The wild-type plants responded more slowly, exhibiting limited injury. Decreases in stomatal conductance, net photosynthesis or Rubisco quantity in plants exposed to O₃ were not observed in asymptomatic leaves. Total biomass was lower for the transformed genotypes and decreased in both genotypes after exposure to O₃. Shoot–root ratio and specific leaf area were higher in the transformed genotypes and increased in both genotypes with exposure to O₃. Measurements of intercellular airspace demonstrated the presence of larger intercellular spaces in the transformed plants. The indirect effects of the rbcS antisense transformation, including morphological changes in the leaf, probably rendered the transformed plants more sensitive to the oxidant. The decreased quantity of Rubisco is not thought to be directly related to increased O₃ sensitivity in the transformed plants.     

Decreased ribulose-1,5-bisphosphate carboxylase/oxygenase in transgenic tobacco transformed with 'antisense'rbcS.VIII. Impact on photosynthesis and growth in tobacco growing under extreme high irradiance and high temperature

Wild-type and antisense rbcS tobacco (Nicotiana tabacum) plants were grown in a glasshouse in midsummer in Portugal with an irradiance of 1500–2000 μmol m⁻²s⁻¹ and daytime temperatures of 30–35 °C. The Rubisco content of the transformants was lower by 35, 80 and over 90% than that of the wild-type. Gas exchange was measured over three separate days. There was a near-linear relation between Rubisco content and photosynthetic rate during the period of high irradiance, allowing a flux control coefficient of 0.83–0.89 to be estimated. The relation deviated slightly from linearity, because the internal CO₂ concentration (c;) was higher in the transformants than in the wild-type (190 and 275 μmol mol⁻¹ in plants with 35 and 80% less Rubisco, respectively, compared with 175 μmol mol⁻¹ for wild-type), compensating to some extent for the decreased Rubisco content. This increase in c_i occurred because the stomatal conductance (g) remained unaltered or was even higher in plants with decreased Rubisco, despite the lower rate of CO₂ assimilation. As a consequence, water use efficiency declined. The decreased rate of photosynthesis was not accompanied by a stoichiometric decrease in apparent growth rate. These results are discussed in relation to earlier studies of the plant set in growth cabinets. It is concluded that tobacco can adjust over a wide range of growth conditions to avoid a onesided limitation by Rubisco, but that in extreme environmental conditions this capacity to adapt is exhausted.     

The drelationship between CO2-assimilation rate,Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO₂-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r₁ progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO₂ supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO₂-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO₂-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO₂-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P 2-carboxyarabinitol-1-phosphate - P_ip_a intercellular, ambient partial pressure of CO₂ - PGA 3-phospho-glycerate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SSU small subunit of Rubisco     

Negative phototropism in young gametophytes of the fern Schizaea pusilla

Schizaea pusilla is a rare fern that occurs in acidic bogs and is one of the few fern species that maintains a filamentous gametophyte throughout its development. To expand our knowledge of the physiology of this fern, phototropic responses were examined in young gametophytes. In contrast to germ filaments of other fern species, apical protonemata of young gametophytes are negatively phototropic in continuous white, red and blue light at all fluence rates tested. The expression of phototropic curvature is not limited by time since apical protonemata are also negatively phototropic when they are given brief exposures of light and then placed in the dark. In other lower plant groups such as mosses and some algae, the direction of phototropic curvature can change depending on light quality and intensity, but in young gametophytes of Schizaea, negative phototropic curvature was observed in all conditions studied. Blue light is the most effective in promoting the negative phototropic response in Schizaea.     

RNA and protein metabolism in the particulate fractions of the gametophytes of bracken fern during growth in red and blue light

Summary The metabolism of RNA and protein in the gametophytes of bracken fern (Pteridium aquilinum) is affected by the quality of light in which they are grown. When sporelings were grown as two-dimensional gametophytes in blue light, particulate fractions separated from the sporelings exhibited greater incorporation of uridine-³H and leucine-³H into RNA and protein, respectively, than those from sporelings grown as one-dimensional protonema in red light. After various periods of exposure of gametophytes to red or blue light in the presence of uridine-³H, the nuclei-rich fraction showed the highest specific activity in RNA, and irrespective of incubation time, blue light was more effective than red light. The possibility that enhanced synthesis of RNA in the nucleus in response to blue light is significantly related to the morphological growth pattern of the gametophytes, is discussed.