LAM1 is required for dorsoventrality and lateral growth of the leaf blade in Nicotiana.

The role of LAM1 in dorsoventrality and lateral growth of the leaf blade was investigated in the 'bladeless' lam1 mutant of Nicotiana sylvestris and in periclinal chimeras with lam1 and wild-type (N. glauca) cell layers. Mutant lam1 primordia show normal dorsoventrality at emergence, but produce blade tissue that lacks dorsal cell types and fails to expand in the lateral plane. In leaves of a lam1-glauca-glauca (L1-L2-L3) chimera, we observed restoration of dorsal identity in the lam1 upper epidermis, suggesting non-cell-autonomous movement of a dorsalizing factor between cell layers of the blade. A lam1-lam1-glauca chimera generated a leaf blade with lam1 cells in the L1-derived epidermis and the L2-derived upper and lower mesophyll. An in situ lineage analysis revealed that N. glauca cells in the L3-derived middle mesophyll restore palisade differentiation in the adjoining lam1 upper mesophyll. Movement of dorsalizing information appears short-range, however, having no effect on the upper lam1 epidermis in lam1-lam1-glauca. Clusters of lam1 mesophyll in distal or proximal positions show a localized default to radial growth, indicating that the LAM1 function is required for dorsoventrality and lateral growth throughout blade expansion.

LOP1: a gene involved in auxin transport and vascular patterning in Arabidopsis.

We have taken a genetic approach to understanding the mechanisms that control vascular patterning in the leaves of higher plants. Here we present the identification and characterization of the lop1 mutant of Arabidopsis which is defective in basipetal transport of IAA. Mutant leaf midveins show disoriented axial growth, and bifurcation into twin veins that are frequently rotated out of the normal dorsal/ventral axis of the leaf. Mutant plants also display abnormal patterns of cell expansion in the midrib cortex and in the epidermis of the elongation zone of lateral roots. Lateral roots show abnormal curvature during initiation, sometimes encircling the primary root prior to growth in a normal downward direction. Mutant seedlings have normal levels of free IAA, and appear normal in auxin perception, suggesting that transport is the primary lesion. The abnormalities in vascular development, lateral root initiation and patterns of cell expansion observed in the lop] mutant are consistent with a basic disruption in basipetal transport of IAA.

LAM-1 and FAT Genes Control Development of the Leaf Blade in Nicotiana sylvestris.

Leaf primordia of the lam-1 mutant of Nicotiana sylvestris grow normally in length but remain bladeless throughout development. The blade initiation site is established at the normal time and position in lam-1 primordia. Anticlinal divisions proceed normally in the outer L1 and L2 layers, but the inner L3 cells fail to establish the periclinal divisions that normally generate the middle mesophyll core. The lam-1 mutation also blocks formation of blade mesophyll from distal L2 cells. This suggests that LAM-1 controls a common step in initiation of blade tissue from the L2 and L3 lineage of the primordium. Another recessive mutation (fat) was isolated in N. sylvestris that induces abnormal periclinal divisions in the mesophyll during blade initiation and expansion. This generates a blade approximately twice its normal thickness by doubling the number of mesophyll cell layers from four to approximately eight. Presumably, the fat mutation defines a negative regulator involved in repression of periclinal divisions in the blade. The lam-1 fat double mutant shows radial proliferation of mesophyll cells at the blade initiation site. This produces a highly disorganized, club-shaped blade that appears to represent an additive effect of the lam-1 and fat mutations on blade founder cells.

A nuclear mutation blocking initiation of the lamina in leaves of Nicotiana syhestris.

Isolation of a nuclear recessive mutation (lam-1) blocking initiation of the lamina in leaves of Nicotiana sylvestris Speg. et Comes is described. Histological analysis of the mutant apex demonstrates a defect in establishment of meristematic activity along the margins of emerging primordia, the earliest cytological event in lamina formation. Mutant leaves grow to their normal length (about 30 cm) but fail to expand. Transverse sections show that mutant leaves are essentially naked midribs, lacking the characteristic cell types of the wild-type mesophyll. In the absence of lateral expansion, all secondary veins develop along the longitudinal axis, producing abnormal parallel venation. The mutant is defective in phase transition and grows indefinitely as a juvenile vegetative rosette. Exogenous gibberellic acid induces rapid stem elongation and flowering, but does not correct the lamina defect, indicating that juvenile arrest is a pleiotropic consequence of a gibberellin deficiency in bladeless leaves. The calyx, corolla and gynoecium in mutant flowers show defects in lateral development, indicating that the lam-1 gene plays a crucial role in development of floral organs as well as leaves.

Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with o(2)-resistant photosynthesis.

Experiments were conducted with a tobacco (Nicotiana tabacum) mutant with 40 to 50% greater catalase activity than wild type that is associated with a novel form of O(2)-resistant photosynthesis. The apparent K(m) for H(2)O(2) was the same in mutant and wild-type leaf extracts. Tobacco RNAs were hybridized with Nicotiana sylvestris catalase cDNA, and a threefold greater steady-state level of catalase mRNA was found in mutant leaves. Steady-state levels of ribulose-1,5-bisphosphate carboxylase small subunit mRNA were similar in mutant and wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified catalase showed that the protein concentration in the band corresponding to catalase was higher in the mutant than in the wild type. Separation of leaf catalase proteins by isoelectric focusing revealed the presence of five major bands and one minor band of activity. The distribution of the catalase activity among these forms was similar in mutant and wild type, although the total activity was higher in the mutant in all five major bands. The results indicate that the enhanced catalase activity in mutant leaves is caused by an increase in synthesis of catalase protein and that this trait is mediated at the nucleic acid level.

Purification and characterization of an isozyme of catalase with enhanced-peroxidatic activity from leaves of Nicotiana sylvestris.

Two isozymes of catalase (EC 1.11.1.6), one with typically low peroxidatic activity (CAT-1) and the other with enhanced-peroxidatic activity (EP-CAT or CAT-3) have been purified to electrophoretic homogeneity from tobacco (Nicotiana sylvestris) seedlings and antibodies prepared against each. The isozyme proteins showed no immunological cross-reactivity. The subunit Mr was 55,300 +/- 750 for CAT-1 and 53,300 +/- 850 for CAT-3 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the catalatic reaction, the apparent Km values for CAT-1 and CAT-3 were 0.057 and 0.054 M, respectively, and the kcat values were 4.8 x 10(7) and 3.0 x 10(6) min-1, respectively. In the peroxidatic reaction, both have similar apparent Km's for H2O2. The apparent Km values for CAT-3 for the series methyl, ethyl, propyl, butyl, and allyl alcohols were 2.48, 5.6, 38.6, 429, and 16.3 mM, respectively. For CAT-1, the values were 697, 55.8, no detectable reaction with propyl and butyl, and 163 mM, respectively. Neither isozyme utilized dianisidine or guaiacol in the peroxidatic reaction. Catalase activity (CAT-2) which eluted in an intermediate position between CAT-1 and CAT-3 from a chromatofocusing column was composed of only one subunit whose Mr coincided with CAT-1, and only the antibody to CAT-1 reacted with CAT-2 protein. Thus, CAT-2 and CAT-1 appear closely related while CAT-3 is distinctly different.

Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase.

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with [gamma-(32)P]ATP decreased in the presence of Glc-6-P and Glc-1,6-P(2), but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with [gamma-(32)P]ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with [(32)P]Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either [gamma-(32)P]ATP or [(32)P]Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.

Enhanced-peroxidatic activity in specific catalase isozymes of tobacco, barley, and maize.

Separation of catalase isozymes from leaf extracts of three diverse plant species (Nicotiana sylvestris, Zea mays, Hordeum vulgare L.) revealed a distinct isozyme with enhanced peroxidatic activity (30-, 70-, 28-fold over typical catalase, respectively) which constituted 10 to 20% of the total catalase activity. In maize this isozyme is the product of the Cat3 gene, which is expressed only in mesophyll cells (AS Tsaftaris, AM Bosabalidis, JG Scandalios [1983] Proc Natl Acad Sci USA 80: 4455-4459). A mutation in barley reducing levels of peroxisomal catalase (AC Kendall et al. [1983] Planta 159: 505-511) does not reduce the amount of the isozyme with enhanced peroxidatic activity. Similarly, this isozyme is unaffected in dark-grown barley in spite of a 75% decrease in total catalase activity. These results suggest that this catalase isozyme is under separate genetic control in barley. This may also be the case in tobacco where the catalase isozyme with enhanced peroxidatic activity is an immunologically distinct protein (EA Havir, NA McHale [1989] Plant Physiol 89: 952-957).

Regulation of Catalase Activity in Leaves of Nicotiana sylvestris by High CO(2).

The effect of high CO(2) (1% CO(2)/21% O(2)) on the activity of specific forms of catalase (CAT-1, -2, and -3) (EA Havir, NA McHale [1987] Plant Physiol 84: 450-455) in seedling leaves of tobacco (Nicotiana sylvestris, Nicotlana tabacum) was examined. In high CO(2), total catalase activity decreased by 50% in the first 2 days, followed by a more gradual decline in the next 4 days. The loss of total activity resulted primarily from a decrease in CAT-1 catalase. In contrast, the activity of CAT-3 catalase, a form with enhanced peroxidatic activity, increased 3-fold in high CO(2) relative to air controls after 4 days. Short-term exposure to high CO(2) indicated that the 50% loss of total activity occurs in the first 12 hours. Catalase levels increased to normal within 12 hours after seedlings were returned to air. When seedlings were transferred to air after prolonged exposure to high CO(2) (13 days), the levels of CAT-1 catalase were partially restored while CAT-3 remained at its elevated level. Levels of superoxide dismutase activity and those of several peroxisomal enzymes were not affected by high CO(2). Total catalase levels did not decline when seedlings were exposed to atmospheres of 0.04% CO(2)/5% O(2) or 0.04% CO(2)/1% O(2), indicating that regulation of catalase in high CO(2) is not related directly to suppression of photorespiration. Antibodies prepared against CAT-1 catalase from N. tabacum reacted strongly against CAT-1 catalase from both N. sylvestris and N. tabacum but not against CAT-3 catalase from either species. This observation, along with the rapid changes in CAT-1 and the much slower changes in CAT-3 suggest that one form is not directly derived from the other.

Photorespiratory toxicity in autotrophic cell cultures of a mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity.

Procedures were devised for heterotrophic culture and autotrophic establishment of protoplast-derived cell cultures from the sat mutant of Nicotiana sylvestris Speg. et Comes lacking serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) activity. Increasing photon flux rates (dark, 40, 80 µmol quanta·m(-2)·s(-1)) enhanced the growth rate of autotrophic (no sucrose) wild-type (WT) cultures in air and 1% CO2. Mutant cultures showed a similar response to light under conditions suppressing photorespiration (1% CO2), and maintained 65% of WT chlorophyll levels. In normal air, however, sat cultures developed severe photorespiratory toxicity, displaying a negligible rate of growth and rapid loss of chlorophyll to levels below 1% of WT. Low levels of sucrose (0.3%) completely reversed photorespiratory toxicity of the mutant cells in air. Mutant cultures maintained 75% of WT chlorophyll levels in air, displayed light stimulation of growth, and fixed (14)CO2 at rates identical to WT. Autotrophic sat cultures accumulated serine to levels nearly nine-fold above that of WT cultures in air. Serine accumulated to similar levels in mixotrophic (0.3% sucrose) sat cultures in air, but had no deleterious effect on fixation of (14)CO2 or growth, indicating that high levels of serine are not toxic, and that toxicity of the sat mutation probably stems from depletion of intermediates of the Calvin cycle. Autotrophic sat cultures were employed in selection experiments designed to identify spontaneous reversions restoring the capacity for growth in air. From a population of 678 000 sat colonies, 23 plantlets were recovered in which sustained growth in air resulted from reacquisition of SGAT activity. Twenty-two had SGAT levels between 25 and 50% of WT, but one had less than 10% of WT SGAT activity, and eventually developed symptoms typical of the sat mutant. The utility of autotrophic sat cultures for selection of chloroplast mutations diminishing the oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is discussed.

A Starchless Mutant of Nicotiana sylvestris Containing a Modified Plastid Phosphoglucomutase.

A mutant (NS 458) of Nicotiana sylvestris (Spegazzini and Comes) unable to synthesize leaf starch was isolated in the M(2) generation following ethyl methanesulfonate mutagenesis by testing with iodine. Segregation ratios in reciprocal F(2) progenies showed that the starchless phenotype resulted from a recessive mutation in a single nuclear gene. DEAE-agarose chromatography showed that the mutant is grossly deficient in plastid phosphoglucomutase (EC 2.1.5.1) activity. The structure of the enzyme is changed, as evidenced by increased Michaelis constants and by the prolonged activation period (>40 minutes) observed when the enzyme is assayed in triethanolamine buffer rather than imidazole buffer. The activity of the wild-type enzyme with saturating glucose 6-P alone was 7% of the activity when saturating glucose 1,6-P(2) was also present. The results suggest that glucose 1,6-P(2) is both an effector and a dissociable reaction intermediate. The growth rate of mutant and wild-type plants were not significantly different in continuous light and on an 8-hour dark, 16-hour light cycle and the mutants grew normally under greenhouse conditions. The mutant supports growth during diurnal periods of darkness by vacuolar storage of sugars instead of chloroplast storage of starch. The simplification in metabolism achieved by blocking the diversion of plastid fructose-6-P to starch facilitates the induction of oscillations in CO(2) fixation.

A Nuclear Mutation in Nicotiana sylvestris Causing a Thiamine-Reversible Defect in Synthesis of Chloroplast Pigments.

We report the recovery of a nuclear recessive mutation in Nicotiana sylvestris (Spegazzini and Comes) producing a conditional disruption in the pathway for synthesis of chlorophyll a and b and carotenoids which is fully reversible by exogenous thiamine (0.3 micromolar). In the absence of supplemental thiamine, chlorophyll levels declined by 50% after 5 days, and fell to undetectable levels by 11 days. Mitochondrial (KCN sensitive) respiration rates remained normal in albino leaves (80% loss of chlorophyll), suggesting that chlorosis results primarily from a deficiency of thiamine in the chloroplasts. After thiamine removal, mutant plants produced at least 10 albino leaves with a substantial capacity for growth (0-15 centimeters; 70-fold increase in area), demonstrating sustained operation of many cellular functions in spite of chloroplast disruption. Activities of the plastid isozymes of phosphoglucomutase and phosphoglucoisomerase in albino leaves indicated that the decline in pigment synthesis does not result from a general loss of metabolic activity in chloroplast. Plastid pyruvate dehydrogenase from mutant and wild-type plants displayed a similar affinity for thiamine pyrophosphate, showing that chlorosis does not result from an alteration in this enzyme. Growth of albino leaves and ultrastructural evidence for thylakoid membranes in the chloroplasts suggest that a certain level of fatty acid synthesis is maintained after the interruption of pigment synthesis. Since thiamine deprivation is expected to block production of acetyl-coenzyme A from pyruvate by pyruvate dehydrogenase, acetyl-coenzyme A supporting fatty acid synthesis in albino leaves may be derived solely from mitochondrial acetate.

A Mutant of Nicotiana sylvestris Lacking Serine:Glyoxylate Aminotransferase: Substrate Specificity of the Enzyme and Fate of [2-C]Glycolate in Plants with Genetically Altered Enzyme Levels.

The photorespiratory mutant of Nicotiana sylvestris, NS 349, lacking serine:glyoxylate aminotransferase (SGAT) grows in 1% CO(2) but not in normal air (NA McHale, EA Havir, I Zelitch 1988 Theor Appl Genet. In press). Alanine:hydroxypyruvate and asparagine:hydroxypyruvate aminotransferase activities were also lacking in the mutant, and plants heterozygous with respect to SGAT which grow in normal air had 50% of the activities present in homozygous plants. Therefore, all these activities are associated with the same enzyme. On feeding [2-(14)C]glycolate to leaf discs in the light, NS 349 showed reduced incorporation of radioactivity into the neutral and organic acid fractions and increased incorporation into the amino acid fraction, principally into serine. The effect of reducing SGAT by 50% in heterozygous plants produced little change in the metabolism of [2-(14)C]glycolate, showing there is a large excess of this enzyme in wild-type plants.

A mutant of Nicotiana sylvestris deficient in serine glyoxylate aminotransferase activity : Callus induction and photorespiratory toxicity in regenerated plants.

A photorespiration mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity was isolated in the M2 generation following EMS mutagenesis. Mutants showing chlorosis in air and normal growth in 1% CO2 were fed [(14)C]-2-glycolate to examine the distribution of (14)C among photorespiratory intermediates. Mutant strain NS 349 displayed a 9-fold increase in serine accumulation relative to wild-type controls. Enzyme assays revealed an absence of serine: glyoxylate aminotransferase (SGAT) activity in NS 349, whereas other peroxisomal enzymes were recovered at normal levels. Heterozygous siblings of NS 349 segregating air-sensitive M3 progeny in a 3∶1 ratio were shown to contain one half the normal level of SGAT activity, indicating that air sensitivity in NS 349 results from a single nuclear recessive mutation eliminating SGAT activity. Since toxicity of the mutation depends on photorespiratory activity, callus cultures of the mutant were initiated and maintained under conditions suppressing the formation of functional plastids. Plantlets regenerated from mutant callus were shown to retain the SGAT deficiency and conditional lethality in air. The utility of photorespiration mutants of tobacco as vehicles for genetic manipulation of ribulose bisphosphate carboxylase/oxygenase at the somatic cell level is discussed.

Effects of CO(2) and O(2) on Photosynthesis and Growth of Autotrophic Tobacco Callus.

Mean inhibition of net photosynthesis in autotrophic tobacco callus by 21 and 40% O(2) was 30 and 47%, respectively, similar to intact leaves. Increasing CO(2) concentrations (500-2000 microliters per liter) produced a steady decline in percent inhibition at both O(2) concentrations, indicating that O(2) inhibition resulted primarily from photorespiration. Net photosynthetic rate was plotted as a function of CO(2) concentration at 1, 21, and 40% O(2) for calculation of kinetic constants. Values for V(max) were similar at all O(2) concentrations (x = 5.27 mumol CO(2) per gram fresh weight per hour), indicating that O(2) inhibition of net photosynthesis was fully reversible by CO(2). To determine whether CO(2) and O(2) produced similar effects on growth, autotrophic callus was incubated for three weeks in atmospheres of normal air, high CO(2), high O(2) and high CO(2)/high O(2). Growth in high CO(2) was nearly double that in normal air. High O(2) decreased growth significantly relative to air, but growth in high CO(2)/high O(2) was similar to that in air. Lack of CO(2) reversal of growth inhibition by O(2) indicates that prolonged exposure to high O(2) results in toxicity arising from a nonphotorespiratory source. Growth of heterotrophic callus (2% sucrose), however, was not inhibited by 40 or 60% O(2), suggesting that O(2) toxicity in autotrophic callus results primarily from disruption of photosynthetic functions.

Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves.

Leaf extracts of both Nicotiana tabacum and Nicotiana sylvestris contain multiple forms of catalase (H(2)O(2):H(2)O(2) oxidoreductase, EC 1.11.1.6) which are separable at different pH values by chromatofocusing columns. Marked changes in distribution of these catalases occur during seedling development and leaf maturation. The form of catalase eluting first (peak 1) was predominant during early seedling growth and present at all stages of development. Two more acidic forms (peaks 2 and 3) appeared later and comprised 29% of the total activity by 11 days postgermination. Mature leaves of N. tabacum contained peak 1 catalase, but peaks 2 and 3 represented 62% of the total activity. No interconversion of peaks 1, 2, and 3 was detected. The three forms of catalase differed in thermal stability with peak 1 > peak 2 >> peak 3. For N. sylvestris, t((1/2)) at 55 degrees C was 31.5 and 3.0 min for peaks 1 and 3, respectively, and for N. tabacum, t((1/2)) was 41.5 and 3.2 min, respectively. All forms of catalase in tobacco show peroxidatic (measured as ethanol to acetaldehyde conversion) as well as catalatic activities. However, for both Nicotiana species the ratio peroxidatic/catalatic activity is at least 30-fold higher in peak 3 than in peaks 1 and 2. Chromatofocusing of extracts from spinach leaves separated at least four peaks of catalase activity, one of which had a 10-fold higher ratio of peroxidatic/catalatic activity than the others. Short-term growth (5 days) of tobacco seedlings under atmospheric conditions suppressing photorespiration (1% CO(2)/21% O(2)) reduced total catalase activity and caused a decline in peak 1 catalase and a substantial increase in the activity of peaks 2 and 3 relative to air-grown seedlings at the same stage.

Conditions for strict autotrophic culture of tobacco callus.

Organic gelling agents such as agar and agarose provide a heterotrophic substrate for growth of illuminated tobacco callus. When green cells are incubated in CO(2)-free air on a medium lacking sucrose but solidified with 1% agar, an increase in relative dry weight is sustained through two passages. Similar results with different inoculum sources, and with three brands of agar and two forms of agarose, suggest this is a general phenomenon. A fully autotrophic culture system was developed employing polyurethane pads to support cells in a liquid medium lacking sucrose. Growth was negligible in two passages in CO(2)-free air, and increased with each added increment in CO(2) concentration.