Maize plastid photogenes: mapping and photoregulation of transcript levels during light-induced development.

Positively photoregulated regions that show increased transcript levels upon illumination of dark-grown seedlings are scattered over approximately 19% of the maize plastid chromosome. Some photogenes, i.e., genes within these regions, are transcribed individually, whereas others that are transcribed as polycistronic mRNAs appear to be functionally organized into operons. Multiple light-induced transcripts are complementary to most photogenes; these mRNAs are not present in equimolar amounts during plastid photomorphogenesis, but particular transcripts predominate at specific stages of development. Most, but not all, photogene RNA pools reach a maximum size (after either 10, 20, or 44 h of illumination) and then fall to approximately preillumination levels. These data and other considerations argue that photogene expression control is fundamentally transcriptional and that there is more than one expression class. Transcripts of the maize plastid gene for the large subunit of ribulose bisphosphate carboxylase reach a maximum by 20 h of illumination; transcripts of the nuclear gene for the small subunit of this enzyme continue to accumulate and fall considerably later. These data suggest that the level of transcription of the latter gene in the nucleus may be regulated by events in the chloroplast.

Loss of endocytic capacity in aging Paramecium. The importance of cytoplasmic organelles.

Aged cells have significantly fewer food vacuoles and ingest fewer bacteria than young cells. Loss of food vacuoles was explained by a decreasing difference in the food vacuole formation and excretion rates; the formation rate declined more rapidly than the excretion rate, approaching equivalence at 160 fissions, when the proportion of cells with no food vacuoles, in the presence of excess food, abruptly increased. A model for cellular aging is presented in which control of organelle numbers and cyclical interactions between the nucleus and cytoplasm may be of critical importance.

Molecular evolution and nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH).

The nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH) are presented. The evolution of these genes among higher plants is characterized by a transition mutation bias of about 2:1 and by rates of synonymous and nonsynonymous substitution which are much lower than similar rates for genes from other sources. This is consistent with the notion that the plastid genome is evolving conservatively in primary sequence. Yet, the mode and tempo of sequence evolution of these and other plastid-encoded coupling factor genes are not the same. In particular, higher rates of nonsynonymous substitution in atpE (the gene for the epsilon subunit of CF1) and higher rates of synonymous substitution in atpH in the dicot vs. monocot lineages of higher plants indicate that these sequences are likely subject to different evolutionary constraints in these two lineages. The 5'- and 3'-transcribed flanking regions of atpA and atpH from maize, wheat and tobacco are conserved in size, but contain few putative regulatory elements which are conserved either in their spatial arrangement or sequence complexity. However, these regions likely contain variable numbers of "species-specific" regulatory elements. The present studies thus suggest that the plastid genome is not a passive participant in an evolutionary process governed by a more rapidly changing, readily adaptive, nuclear compartment, but that novel strategies for the coordinate expression of genes in the plastid genome may arise through rapid evolution of the flanking sequences of these genes.

Age-correlated changes in expression of micronuclear damage and repair in Paramecium tetraurelia.

In Paramecium, age is defined as the number of mitotic divisions which have elapsed since the previous cross-fertilization (conjugation) or self-fertilization (autogamy). As the mitotic interval between fertilizations increases, the percentage of nonviable progeny clones increases. In the current study, resolution of conflicting previous reports on the pattern of increase of death and reduced viability in progeny from aging parent cells is found. Some exautogamous clones exhibit a high mortality at young clonal ages, others show no mortality throughout their life span, but most (73%) show an abrupt increase in the percent death and reduced viability in progeny from cells 50-80 fissions old. Ultraviolet-irradiation-induced micronuclear mutations, repairable by photoreactivation, increased with increased clonal age when monitored by percent death and reduced viability of exautogamous progeny of irradiated cells. Loss of dark repair is considered a contributor to the increased expression of micronuclear mutations with increased clonal age.

Regulation of Photosynthesis during Leaf Development in RbcS Antisense DNA Mutants of Tobacco.

We have previously characterized RbcS antisense DNA mutants of tobacco that have drastic reductions in their content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; S.R. Rodermel, M.S. Abbott, L. Bogorad [1988] Cell 55: 673-681). In this report we examine the impact of Rubisco loss on photosynthesis during tobacco (Nicotiana tabacum) leaf development. Photosynthetic capacities are depressed in the antisense leaves, but the patterns of change in photosynthetic rates during the development of these leaves are similar to those in wild-type plants: after attaining a maximum in young leaves, photosynthetic capacities undergo a prolonged senescence decline in older leaves. The alterations in photosynthetic capacities in both the wild type and mutant are closely correlated with changes in Rubisco activity and content. During wild-type leaf development, Rubisco accumulation is regulated by coordinate changes in RbcS and rbcL transcript accumulation, whereas in the antisense leaves, Rubisco content is a function of RbcS, but not rbcL, transcript abundance. This indicates that large subunit protein production is controlled posttranscriptionally in the mutants. The antisense leaves accumulate near-normal levels of chlorophyll and representative photosynthetic proteins throughout development, suggesting that photosynthetic gene expression is not feedback regulated by Rubisco abundance. Considered together, the data in this paper indicate that leaf developmental programs are generally insensitive to sharp reductions in Rubisco content and emphasize the metabolic plasticity of plant cells in achieving optimal photosynthetic rates.

Photosynthetic Acclimation to Elevated CO2 Occurs in Transformed Tobacco with Decreased Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content.

Inhibition of net carbon assimilation rates during growth at elevated CO2 was studied in transgenic tobacco (Nicotiana tabacum L.) plants containing zero to two copies of antisense DNA sequences to the small subunit polypeptide (rbcS) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). High- and low-Rubisco tobacco plants were obtained from the selfed progeny of the original line 3 transformant (S.R. Rodermel, M.S. Abbott, L. Bogorad [1988] Cell 55: 673-681). Assimilation rates of high- and low-Rubisco tobacco plants increased 22 and 71%, respectively, when transferred from 35- to 70-Pa CO2 chamber air at 900 [mu]mol m-2 s-1 photon flux density. However, CO2-dependent increases of net carbon assimilation rates of high- and low-Rubisco plants virtually disappeared after 9 d of growth in elevated CO2 chamber air. Total above-ground dry matter production of high- and low-Rubisco plants was 28 and 53% greater, respectively, after 9 d of growth at 70 Pa compared with 35 Pa CO2. Most of this dry weight gain was due to increased specific leaf weight. Rubisco activity, Rubisco protein, and total chlorophyll were lower in both high- and low-Rubisco plants grown in enriched compared with ambient CO2 chamber air. Soluble leaf protein also decreased in response to CO2 enrichment in high- but not in low-Rubisco tobacco plants. Decreased Rubisco activities in CO2-adapted high- and low-Rubisco plants were not attributable to changes in activation state of the enzyme. Carbonic anhydrase activities and subunit levels measured with specific antibodies were similar in high- and low-Rubisco tobacco plants and were unchanged by CO2 enrichment. Collectively, these findings suggested that photosynthetic acclimation to enriched CO2 occurred in tobacco plants either with or without transgenically decreased Rubisco levels and also indicated that the down-regulation of Rubisco in CO2-adapted tobacco plants was related to decreased specific activity of this enzyme.

Photosynthesis, Rubisco Activity and Amount, and Their Regulation by Transcription in Senescing Soybean Leaves.

Senescence is a phase of leaf ontogeny marked by declining photosynthetic activity that, in soybean (Glycine max [L.] Merr.), is paralleled by a decline in chloroplast function. Soybean leaves have different patterns of decline in photosynthetic capacity and chloroplast function associated with nodal position and sink activity. The objective of this work was to determine whether leaves from nodes 3 and 6 of soybean, which show these different patterns, are similarly regulated with respect to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity and content and also to ascertain the degree of regulation of Rubisco content by transcription. Leaves from nodes 3 and 6 of field-grown soybean plants were sampled periodically from the time of their unfolding until near death. In situ CO2-exchange rate (CER) increased to a maximal level in both leaves and then declined slowly. For node 3 leaves the decline was progressive, but for node 6 leaves the decline was arrested at about 75% of maximum CER for a period of about 20 d, coincident with the onset of rapid seed growth, before a short period of very rapid decline immediately preceding leaf death. Rubisco activities and Rubisco content were directly correlated with CER in the leaves exhibiting the two different patterns. Rubisco activation ratio was similar for the two leaves and did not change throughout development. The primary regulator of photosynthesis at the physiological level, thus, was the amount of Rubisco protein. Decreases in Rubisco holoenzyme during senescence of both leaves were accompanied by coordinate decreases in the levels of mRNAs for the small and large subunits of Rubisco, suggesting that the decrease in Rubisco enzyme amounts during soybean leaf senescence is due to slower transcription rates and that levels of these mRNAs are coordinately controlled during senescence as they are during chloroplast development. However, plastid DNA template availability and posttranscriptional controls may also influence Rubisco content during senescence of these leaves. We conclude that soybean leaf photosynthesis likely unfolds according to a single developmental program but that modifications can be superimposed upon this program to maximize photosynthetic rates.

Susceptibility to the sugar beet cyst nematode is modulated by ethylene signal transduction in Arabidopsis thaliana.

Previously, we identified Arabidopsis thaliana mutant rhd1-4 as hypersusceptible to the sugar beet cyst nematode Heterodera schachtii. We assessed rhd1-4 as well as two other rhd1 alleles and found that each exhibited, in addition to H. schachtii hypersusceptibility, decreased root length, increased root hair length and density, and deformation of the root epidermal cells compared with wild-type A. thaliana ecotype Columbia (Col-0). Treatment of rhd1-4 and Col-0 with the ethylene inhibitors 2-aminoethoxyvinylglycine and silver nitrate and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid suggests that the rhd1-4 hypersusceptibility and root morphology phenotypes are the result of an increased ethylene response. Assessment of known ethylene mutants further support the finding that ethylene plays a role in mediating A. thaliana susceptibility to H. schachtii because mutants that overproduce ethylene (eto1-1, eto2, and eto3) are hypersusceptible to H. schachtii and mutants that are ethylene-insensitive (etr1-1, ein2-1, ein3-1, eir1-1, and axr2) are less susceptible to H. schachtii. Because the ethylene mutants tested show altered susceptibility and altered root hair density and length, a discrimination between the effects of altered ethylene signal transduction and root hair density on susceptibility was accomplished by analyzing the ttg and gl2 mutants, which produce ectopic root hairs that result in greatly increased root hair densities while maintaining normal ethylene signal transduction. The observed normal susceptibilities to H. schachtii of ttg and g12 indicate that increased root hair density, per se, does not cause hypersusceptibility. Furthermore, the results of nematode attraction assays suggest that the hypersusceptibility of rhd1-4 and the ethylene-overproducing mutant eto3 may be the result of increased attraction of H. schachtii-infective juveniles to root exudates of these plants. Our findings indicate that rhd1 is altered in its ethylene response and that ethylene signal transduction positively influences plant susceptibility to cyst nematodes.

Changes in mRNA abundance within Heterodera schachtii-infected roots of Arabidopsis thaliana.

Gene expression changes in plant roots infected by plant-parasitic cyst nematodes are involved in the formation of nematode feeding sites. We analyzed mRNA abundance changes within roots of Arabidopsis thaliana during the early compatible interaction with Heterodera schachtii, the sugarbeet cyst nematode. Approximately 1,600 root sections, each containing a single parasitic nematode and its feeding site, and 1,600 adjacent, nematode-free root sections were excised from aseptic A. thaliana cultures 3 to 4 days after inoculation with H. schachtii. These tissue samples were termed infected and uninfected, respectively. Preparasitic nematodes were added to the uninfected tissue sample to maintain the nematode to plant tissue proportion. mRNA extracted from these two tissue samples was subjected to differential display analysis. Thirty-six cDNA clones corresponding to mRNA species with different abundance between both tissue samples were isolated. Of these clones, 24 were of A. thaliana origin and 12 were from H. schachtii. Differential display data predicted that the A. thaliana cDNA clones corresponded to 13 transcripts that were more abundant in the infected root sections and 11 transcripts that were more abundant in the uninfected root sections. H. schachtii cDNA clones were predicted to correspond to four transcripts that were more abundant in parasitic nematodes and to eight transcripts that were more abundant in preparasitic nematodes. In situ hybridization experiments confirmed the mRNA abundance changes in A. thaliana roots predicted by the differential display analyses for two A. thaliana clones.

A Screen for Arabidopsis thaliana Mutants with Altered Susceptibility to Heterodera schachtii.

Genetic approaches are a powerful means to elucidate plant-pathogen interactions. An in vitro screening protocol was developed to identify Arabidopsis thaliana mutants with altered susceptibility to Heterodera schachtii, the sugar beet cyst nematode. In an initial screen of approximately 5,200 ethyl methanesulfonate-generated mutant plants, two stable mutations were identified. Both mutant lines were backcrossed and were found to harbor single recessive mutant alleles. Mutant line 2-4-6 shows an approximately two-fold increase in sedentary and developing nematodes, while mutant line 10-5-2 exhibits a significant decrease in susceptibility that manifests itself only after nematodes become sedentary. Analyses of progeny from crosses of lines 2-4-6 and 10-5-2 indicated that the two mutations are not allelic. However, the mutant gene in line 2-4-6 was found to be allelic to the previously identified mutant rhd1 and was termed rhd1-4. The mutant gene in line 10-5-2 was called asc1 for altered susceptibility to cyst nematodes. Our results demonstrate the feasibility of a nematological mutant screen and strengthen A. thaliana and H. schachtii as a model pathosystem.

Regulation of phytoene desaturase expression is independent of leaf pigment content in Arabidopsis thaliana.

Synthesis of carotenoids in higher plants occurs in the plastids, but all of the required enzymes are coded for in the nuclear genome and are post-transcriptionally imported into the plastid compartment. Regulation of the synthesis of the enzymes is poorly understood. The two-step desaturation of phytoene to zeta-carotene, carried out by the enzyme phytoene desaturase (PDS), is one of the earliest steps in the pathway and has been studied in several systems. Previous analyses of phytoene-accumulating tissue suggested that there may be feedback regulation of PDS gene transcription, with higher expression in white tissue. To investigate this regulation further, we examined phytoene-accumulating tissue in Arabidopsis thaliana (L.) Heynh. Two types of phytoene-accumulating tissue were studied: Norflurazon-bleached plants and white sectors from the immutans variegation mutant. Based on competitive RT-PCR measurements of PDS mRNA and immunochemical detection of PDS protein, we determined that there is no significant induction of PDS gene expression specific to white tissue, indicating that PDS expression is independent of the pigment status of the cells. Reasons why our results differ from those in other systems are discussed.

Nuclear-organelle interactions: nuclear antisense gene inhibits ribulose bisphosphate carboxylase enzyme levels in transformed tobacco plants.

The biosynthesis of ribulose bisphosphate carboxylase (RUBISCO) provides a model system for studying the coordination of nuclear and organelle gene expression, since this abundantly transcribed and expressed chloroplast enzyme is composed of small (SS) and large subunits (LS) encoded by a nuclear multigene family and a single chloroplast gene, respectively. We have tested the possibility that SS mRNA or protein levels affect LS mRNA amounts or LS protein production and accumulation. We find that expression of antisense DNA sequences for the SS in transgenic tobacco plants drastically reduces the accumulation of SS mRNA and SS protein. These changes are accompanied by corresponding reductions of LS protein but not LS mRNA amounts; accumulation of the LS protein appears to be regulated by translational and posttranslational factors. We also find that the transgenic plants display striking variations in growth that are correlated with antisense gene dosage.

Single gene for the large subunit of ribulosebisphosphate carboxylase in maize yields two differentially regulated mRNAs.

A second mRNA coding for the large subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] has been found in the plastids of maize leaves. The 5' terminus of this mRNA was shown by S1 nuclease analysis to be 238 nucleotides upstream of the previously described large subunit mRNA [McIntosh, L., Poulsen, C. & Bogorad, L. (1980) Nature (London) 288, 556-560]. The same two mRNAs were produced in a homologous in vitro transcription system using cloned plastid DNA as a template. The ratio of the two mRNAs changes during light-induced plastid development and can be altered in the in vitro system by manipulation of transcription conditions.

The Arabidopsis immutans mutation affects plastid differentiation and the morphogenesis of white and green sectors in variegated plants.

The immutans (im) variegation mutant of Arabidopsis has green and white leaf sectors due to the action of a nuclear recessive gene, IMMUTANS (IM). This gene encodes the IM protein, which is a chloroplast homolog of the mitochondrial alternative oxidase. Because the white sectors of im accumulate the noncolored carotenoid, phytoene, IM likely serves as a redox component in phytoene desaturation. In this paper, we show that IM has a global impact on plant growth and development and is required for the differentiation of multiple plastid types, including chloroplasts, amyloplasts, and etioplasts. IM promoter activity and IM mRNAs are also expressed ubiquitously in Arabidopsis. IM transcript levels correlate with carotenoid accumulation in some, but not all, tissues. This suggests that IM function is not limited to carotenogenesis. Leaf anatomy is radically altered in the green and white sectors of im: Mesophyll cell sizes are dramatically enlarged in the green sectors and palisade cells fail to expand in the white sectors. The green im sectors also have significantly higher than normal rates of O(2) evolution and elevated chlorophyll a/b ratios, typical of those found in "sun" leaves. We conclude that the changes in structure and photosynthetic function of the green leaf sectors are part of an adaptive mechanism that attempts to compensate for a lack of photosynthesis in the white leaf sectors, while maximizing the ability of the plant to avoid photodamage.

copia-like retrotransposons are ubiquitous among plants.

Transposable genetic elements are assumed to be a feature of all eukaryotic genomes. Their identification, however, has largely been haphazard, limited principally to organisms subjected to molecular or genetic scrutiny. We assessed the phylogenetic distribution of copia-like retrotransposons, a class of transposable element that proliferates by reverse transcription, using a polymerase chain reaction assay designed to detect copia-like element reverse transcriptase sequences. copia-like retrotransposons were identified in 64 plant species as well as the photosynthetic protist Volvox carteri. The plant species included representatives from 9 of 10 plant divisions, including bryophytes, lycopods, ferns, gymnosperms, and angiosperms. DNA sequence analysis of 29 cloned PCR products and of a maize retrotransposon cDNA confirmed the identity of these sequences as copia-like reverse transcriptase sequences, thereby demonstrating that this class of retrotransposons is a ubiquitous component of plant genomes.

Co-transcription pattern of an introgressed operon in the maize chloroplast genome comprising four ATP synthase subunit genes and the ribosomal rps2.

Several examples of the introduction of a gene from one gene complex into another (introgression) are found when chloroplast RP gene clusters are compared to those in Escherichia coli or cyanobacteria. Here we describe the transcript pattern of one such cluster from maize (Zea mays) that includes the genes for 4 subunits of the thylakoid ATP synthase (atpI, H, F, A) and the rps2 gene. Twelve transcript species covering the size range from 7,000 to 800 nt were identified in RNA isolated from dark-grown and greening maize seedlings, and several of them were characterized by reverse transcription analysis. A major species of 6,200 nt, with its 5' end at 181 nt upstream of the initiating ATG of rps2, contained the transcripts of all the 5 genes. Two further sets of transcripts having their 5' ends ca. 120 and 50 nt upstream of the initiation codons of the atpI and atpH genes were also identified. Thus, this plastid gene cluster in maize is functionally organized as an operon with additional regulatory features to allow for increased accumulation of mRNAs for the thylakoid components.

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with "antisense" rbcS : I. Impact on photosynthesis in ambient growth conditions.

Experiments were carried out to determine how decreased expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) affects photosynthetic metabolism in ambient growth conditions. In a series of tobacco (Nicotiana tabacum L.) plants containing progressively smaller amounts of Rubisco the rate of photosynthesis was measured under conditions similar to those in which the plants had been grown (310 µmol photons · m(-2) · s(-1), 350 µbar CO2, 22° C). (i) There was only a marginal inhibition (6%) of photosynthesis when Rubisco was decreased to about 60% of the amount in the wildtype. The reduced amount of Rubisco was compensated for by an increase in Rubisco activation (rising from 60 to 100%), with minor contributions from an increase of its substrates (ribulose-1,5-bisphosphate and the internal CO2 concentration) and a decrease of its product (glycerate-3-phosphate). (ii) The decreased amount of Rubisco was accompanied by an increased ATP/ADP ratio that may be causally linked to the increased activation of Rubisco. An increase of highenergy-state chlorophyll fluorescence shows that thylakoid membrane energisation and high-energy-state-dependent energy dissipation at photosystem two had also increased. (iii) A further decrease of Rubisco (in the range of 50-20% of the wildtype level) resulted in a strong and proportional inhibition of CO2 assimilation. This was accompanied by a decrease of fructose-1,6-bisphosphatase activity, coupling-factor 1 (CF1)-ATP-synthase protein, NADP-malate dehydrogenase protein, and chlorophyll. The chlorophyll a/b ratio did not change, and enolase and sucrose-phosphate synthase activity did not decrease. It is argued that other photosynthetic enzymes are also decreased once Rubisco decreases to the point at which it becomes strongly limiting for photosynthesis. (iv) It is proposed that the amount of Rubisco in the wildtype represents a balance between the demands of light, water and nitrogen utilisation. The wildtype overinvests about 15% more protein in Rubisco than is needed to avoid a strict Rubisco limitation of photosynthesis. However, this "excess" Rubisco allows the wildtype to operate with lower thylakoid energisation, and decreased high-energy-state-dependent energy dissipation, hence increasing light-use efficiency by about 6%. It also allows the wildtype to operate with a lower internal CO2 concentration in the leaf and a lower stomatal conductance at a given rate of photosynthesis, so that instantaneous water-use efficiency is marginally (8%) increased.

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with 'antisense' rbcS : II. Flux-control coefficients for photosynthesis in varying light, CO2, and air humidity.

Transgenic tobacco (Nicotiana tabacum L.) plants transformed with 'antisense' rbcS to produce a series of plants with a progressive decrease in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been used to investigate the contribution of Rubsico to the control of photosynthesis at different irradiance, CO2 concentrations and vapour-pressure deficits. Assimilation rates, transpiration, the internal CO2 concentration and chlorophyll fluorescence were measured in each plant. (i) The flux-control coefficient of Rubisco was estimated from the slope of the plot of Rubisco content versus assimilation rate. The flux-control coefficient had a value of 0.8 or more in high irradiance, (1050 µmol·m(-2)·s(-1)), low-vapour pressure deficit (4 mbar) and ambient CO2 (350 µbar). Control was marginal in enhanced CO2 (450 µbar) or low light (310 µmol·m(-2)·s(-1)) and was also decreased at high vapour-pressure deficit (17 mbar). No control was exerted in 5% CO2. (ii) The flux-control coefficients of Rubisco were compared with the fractional demand placed on the calculated available Rubisco capacity. Only a marginal control on photosynthetic flux is exerted by Rubisco until over 50% of the available capacity is being used. Control increases as utilisation rises to 80%, and approaches unity (i.e. strict limitation) when more than 80% of the available capacity is being used. (iii) In low light, plants with reduced Rubisco have very high energy-dependent quenching of chlorophyll fluorescence (qE) and a decreased apparent quantum yield. It is argued that Rubisco still exerts marginal control in these conditions because decreased Rubisco leads to increased thylakoid energisation and high-energy dependent dissipation of light energy, and lower light-harvesting efficiency. (iv) The flux-control coefficient of stomata for photosynthesis was calculated from the flux-control coefficient of Rubisco and the internal CO2 concentration, by applying the connectivity theorem. Control by the stomata varies between zero and about 0.25. It is increased by increased irradiance, decreased CO2 or decreased vapour-pressure deficit. (v) Photosynthetic oscillations in saturating irradiance and CO2 are suppressed in decreased-activity transformants before the steady-state rate of photosynthesis is affected. This provides direct evidence that these oscillations reveal the presence of "excess" Rubisco. (vi) Comparison of the flux-control coefficients of Rubisco with mechanistic models of photosynthesis provides direct support for the reliability of these models in conditions where Rubisco has a flux-control coefficient approach unity (i.e. "limits" photosynthesis), but also indicates that these models are less useful in conditions where control is shared between Rubisco and other components of the photosynthetic apparatus.

Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis.

The immutans (im) variegation mutant of Arabidopsis thaliana contains green- and white-sectored leaves due to the action of a nuclear recessive gene. The mutation is somatically unstable, and the degree of sectoring is influenced by light and temperature. Whereas the cells in the green sectors contain normal chloroplasts, the cells in the white sectors are heteroplastidic and contain non-pigmented plastids that lack organized lamellar structures, as well as small pigmented plastids and/or rare normal chloroplasts. This indicates that the plastids in im white cells are not affected equally by the nuclear mutation and that the expression of immutans is 'plastid autonomous'. In contrast to other variegation mutants with heteroplastidic cells, the defect in im is not maternally inherited. immutans thus represents a novel type of nuclear gene-induced variegation mutant. It has also been found that the white tissues of immutans accumulate phytoene, a non-colored C40 carotenoid intermediate. This suggests that immutans controls, either directly or indirectly, the activity of phytoene desaturase (PDS), the enzyme that converts phytoene to zeta-carotene in higher plants. However, im is not the structural gene for PDS. A secondary effect of carotenoid deficiency, both in immutans and in wild-type plants treated with a herbicide that blocks carotenoid synthesis, is an increase in acid ribonuclease activity in white tissue. It is concluded that the novel variegation generated by the immutans mutation should offer great insight into the complex circuitry that regulates nuclear-organelle interactions.

Recovery of YAC-end sequences through complementation of an Escherichia coli pyrF mutation.

We have developed a genetic means to recover sequences from YAC-ends near the yeast selectable marker URA3. This strategy is based on the ability of URA3 to complement mutations in pyrF, an Escherichia coli gene required for pyrimidine biosynthesis. We have developed an E.coli strain with a non-reverting allele of pyrF that is also suitable for cloning (recA-, hsdR-). We demonstrate the utility of this complementation strategy to obtain right-end clones from three YACs containing Arabidopsis thaliana DNA.