Cellular genetic study of a somatic instability in a tobacco mutant: in vitro isolation of valine-resistant spontaneous mutants

Summary A chlorophyll-deficient mutant line of tobacco (Nicotiana tabacum), named tl, displays spontaneously on leaves green, white, and twinned green/white somatic variations at high frequencies (10^–3 to 10^–2). The frequency of cell events leading to the somatic variations has been shown to be closely dependent on the stage of differentiation of cells during plant development. The activity of transposable elements is suspected in the tl genotype, and a study of its mutagenic ability was performed by selecting in vitro new mutant cellular types. The cellular marker chosen was the resistance to toxic doses of valine conferred by a permeation deficiency. A reproducible method allowing efficient selection of valine-resistant mutant clones from haploid tobacco mesophyll protoplast-derived cells was used. In 10 out of 12 experiments, the frequency of spontaneous valine-resistant clones obtained with the wild-type control was null for cell populations tested to the 10⁶. On the other hand, spontaneous valine-resistant clones were repeatedly isolated at variable and sometimes high frequencies (greater than 10^–3) from cell populations of the tl type. Valine resistance of plants regenerated from these clones was transmitted to the progeny as a single monogenic mutation. These results indicate an increased mutagenic ability of the tl genotype, as compared to the wild-type line.     

The rates of shoot and root growth in intact plants of pea mutants in leaf morphology

Isogenic lines of pea (Pisum sativum L.) with the genetically determined changes in leaf morphology, afila (af) and tendril-less (tl), were used to study the relationship between shoot and root growth rates. The time-course of shoot and root growth was followed during the pre-floral period in the intact plants grown under similar conditions. The af mutation produced afila leaves without leaflets, whereas in the case of the tl mutations, tendrils were substituted with leaflets, and acacia-like leaves were developed. Due to the changes in leaf morphology caused by these mutations, pea genotypes differed in leaf area: starting from day 7, the leaf area was lower in the af plants and larger in the tl plants as compared to the wild-type plants. Such divergence was amplified in the course of plant development and reached its maximum immediately before the transition to flowering. Plants of isogenic lines did not notably differ in stem surface areas. In spite of significant difference in total leaf area, the wild type and tl plants did not differ in leaf dry weight. Starting from leaf 9, the af plants lagged behind two leaflet-bearing genotypes (wild type and tl) in leaf dry weight, whereas stem dry weight was similar in the wild type and tl forms and slightly lower in the af plants. Root dry weights were practically similar in the wild type and tl plants until flowering. The reduction of leaf area in the af plants drastically reduced root dry weight. In other words, the latter index was related to the total weight and total area of leaves and stems. The correlation analysis demonstrated an extremely low relationship between leaf and stem area and dry weight and those of roots early in plant development (when plants develop five to seven leaves). Later, immediately before flowering (nine to eleven leaves), root weight was positively related to leaf weight and area; however, stem area and root weight did not correlate. Thus, in three genotypes (wild type, af, and tl), at the end of their vegetative growth phase, leaf and root biomass accumulated in proportion, independently of leaf area expansion.     

\Cytokinin oxidase/dehydrogenase (CKX) activity in wild and ethylene-insensitive mutant eti5 type of Arabidopsis thaliana (L.) Heynh plants and the effect of cytokinin N1-(2-chloro-4-pyridyl)-N2-phenylurea on enzymatic activity and leaf morphology

The specific activity of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) (CKX) was determined in leaves of wild type (wt) and ethylene-insensitive mutant (eti5) of Arabidopsis thaliana (L.) Heynh plants. Comparative studies showed that this mutation has lower basal CKX activity than wt. Application of 4PU-30 (N¹-(2-chloro-4-pyridyl)-N²-phenylurea) resulted in decreased CKX activity in both wt and mutant plants. The treatment increased leaf blade thickness and the volume of chlorophyll-containing cells per unit leaf area in wt but these changes were not observed in the eti5 mutant. The reduction in chlorophyll “a” and “b”, as well as in carotenoids content in the treated wt tissues resulting from altered leaf morphology was not detected in eti5 plants.     

Genetic control of leaf-blade morphogenesis by the <Emphasis Type="Italic">INSECATUS</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis>

To understand the role of INSECATUS (INS) gene in pea, the leaf blades of wild-type, ins mutant and seven other genotypes, constructed by recombining ins with uni-tac, af, tl and mfp gene mutations, were quantitatively compared. The ins was inherited as a recessive mutant allele and expressed its phenotype in proximal leaflets of full size leaf blades. In ins leaflets, the midvein development was arrested in distal domain and a cleft was formed in lamina above this point. There was change in the identity of ins leaflets such that the intercalary interrupted midvein bore a leaf blade. Such adventitious blades in ins, ins tl and ins tl mfp were like the distal segment of respective main leaf blade. The ins phenotype was not seen in ins af and ins af uni-tac genotypes. There was epistasis of uni-tac over ins. The ins, tl and mfp mutations interacted synergistically to produce highly pronounced ins phenotype in the ins tl mfp triple mutant. The role(s) of INS in leaf-blade organogenesis are: positive regulation of vascular patterning in leaflets, repression of UNI activity in leaflet primordia for ectopic growth and in leaf-blade primordium for indeterminate growth of rachis, delimitation of proximal leaflet domain and together with TL and MFP homeostasis for meristematic activity in leaflet primordia. The variant apically bifid shape of the affected ins leaflets demonstrated that the leaflet shape is dependent on the venation pattern.     

ABA content in shoots and roots of pea mutants af and tl as related to their growth and morphogenesis

The comparative study of shoot and root growth was carried out, and the level of ABA therein determined in the mutant af and tl and wild-type isogenic lines of pea. The recessive af mutation transformed the leaflets into tendrils, and the tl mutation transformed the tendrils into leaflets. These mutations did not affect the length and number of internodes. In all plants, the level of ABA in the leaves was 3–10 times greater than in the roots, and in the course of vegetative growth it rose in both organs. An increase in the shoot area of tl mutant did not change the dry weight of underground and above-ground parts; therefore, the ratio shoot/root in the mutant was identical to that in the wild-type plants. The maintenance of shoot dry weight in the tl mutant at the level of wild-type plant while its area considerably increased was accounted for by a decrease in the thickness of the leaflet and stipule blades. The level of ABA in the stipules of mutant plants was greater than in the wild-type plants. A decrease in the shoot area in the af mutant brought about a decline in its dry weight; however, the ratio root/shoot was maintained at the wild-type level due to a reduced accumulation of dry weight by the root. The level of ABA in the roots of the af mutant was twice greater than in the leafy forms. ABA was assumed to participate in the control over the root growth exerted by the shoot. The absence of leaflets in the af plants was partially compensated for by expanding stipules. The level of ABA therein was three times higher than in the plants of wild type and comparable with the level in the leaflets of the tl mutant and in the wild-type plants. The role of ABA in the growth and final size of leaf blades is discussed.     

<Emphasis Type="Italic">mesophyll cell defective1</Emphasis>, a mutation that disrupts leaf mesophyll differentiation in sunflower

Mutants with altered leaf morphology are useful as markers for the study of genetic systems and for probing the leaf differentiation process. One such mutant with deficient greening and altered development of the leaf mesophyll appeared in an inbred line of sunflower (Helianthus annuus L.). The objectives of the present study were to determine the inheritance of the mutant leaf trait and its morphological characterisation. The mutation, named mesophyll cell defective1 (mcd1), has pleiotropic effects and it is inherited as a monogenic recessive. The structure and tissue organization of mcd1 leaves are disrupted. In mcd1 leaves, the mesophyll has prominent intercellular spaces, and palisade and spongy tissues are not properly shaped. The mutant palisade cells also appear to be more vacuolated and with a reduced number of chloroplasts than the wild type leaves of equivalent developmental stage. The lamina thickness of mcd1 leaves is greatly variable and in some areas no mesophyll cells are present between the adaxial and abaxial epidermis. The leaf area of the mcd1 mutant is extremely reduced as well as the stem height. A deficient accumulation of photosynthetic pigments characterizes both cotyledons and leaves of the mutant. In mcd1 leaves, chlorophyll (Chl) fluorescence imaging evidences a spatial heterogeneity of leaf photosynthetic performance. Little black points, which correspond to photosystem II (PSII) maximum efficiency (F_v/F_m) values close to zero, characterize the mcd1 leaves. Similarly, the lightadapted quantum efficiency (Φ_PSII) values show a homogeneous distribution over wild type leaf lamina, while the damaged areas in mcd1 leaves, represented by yellow zones, are prominent. In conclusion, the loss of function of the MCD1 gene in Helianthus annuus is correlated with a variegated leaf phenotype characterized by a localized destruction of mesophyll morphogenesis and defeat of PSII activity.     

Isolation and characterization of a new mutant allele of <Emphasis Type="Italic">brachytic 2</Emphasis> maize gene

More than 40 monogenic dwarfing mutants have been described in maize; however, the majority of these lead to great reductions in grain yield and, consequently, they have not been used to enhance crop yield in germplasm that is sensible to lodging. An exception in terms of commercial value is the maize mutant brachytic 2 (br2). Br2 gene was cloned in 2003, and it encodes for a protein most probably involved in auxin polar transport. We have isolated a new brachytic mutation that is allelic to the br2 locus and denoted this novel mutant as br2–23. Characterization of this mutant revealed that the br2 mutation modified not only the length of the internodes, as previously reported, but the structure of the leaves as well. Br2–23/br2–23 heterozygotes have a useful intermediate phenotype in terms of plant height, ear height and leaf angle, suggesting a possible utilization of this effect in developing new hybrids. This mutant also appears to be an useful tool by which to study the switch points of the complex developmental program determining maize plant height and architecture.     

Estimation of leaf number and leaf area of hydroponic pak-choi plants (<Emphasis Type="Italic">Brassica campestns</Emphasis> ssp,<Emphasis Type="Italic">chinensis</Emphasis>) using growing degree-days

Temperature is a principal environmental factor that directly affects the growth and timing of appearance for crop leaves. To estimate the leaf number and leaf area of ‘Seoul’ pak-choi plants (Brassica campestns ssp.chinensis), we applied the concept of growing degree-days GDD=(T_avg-T_base) × days, where T_avg, T_base and days were the daily average air temperature, base temperature, and days after transplanting, respectively. Leaves that were beginning to unfold with a leaf area ≥1 cm₂ were counted every 2 to 3 d. Linear relationships were found between leaf number and days after transplanting as well as between leaf number and GDD. The rate of appearance and the number of leaves per GDD were 0.542 leaves d^-1 and 0.051 leaves oC^-1 d^-1, respectively. In contrast, the relationship was non-linear between leaf number and leaf area, with the latter being calculated as [(128.9+11.6×GDD-0.03×GDD²)/1+(0.051×GDD+3.5) /13.7)^-3.9] (cm₂oC¹ d^-1). Using model validation, we found that the estimated leaf number and leaf area showed strong agreement with measured values. our results demonstrate the usefulness of modeling to estimate total leaf area and growth from hydroponically grown pak-choi plants.     

Early stages of leaf development in <Emphasis Type="Italic">has</Emphasis> mutant of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The elucidation of molecular mechanisms underlying the leaf development can be facilitated by the detailed anatomical study of leaf development mutants. We present an analysis of leaf anatomy and morphogenesis during early developmental stages in has mutant of Arabidopsis thaliana. The recessive has mutation affects a number of aspects in plant development, including the shape and size of both cotyledons and leaves. The earliest developmental observations suggest almost synchronous growth of the first two leaf primordia of has mutant. No significant disruption of the cell division pattern in the internal tissue is observed at the earliest stages of development, with the major anatomical difference compared to wild type primordia being the untimely maturation of mesophyll tissue cells in has mutant. At the stage of leaf blade formation, structure disruption becomes clearly evident, by irregular arrangement of the cell layers and the lack of polarity in juvenile has leaves. One distinguishing feature of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. Altered has mutant leaf morphology could be at least partially accounted for by the ectopic STM activity that was found at the base of leaf primordia during early stages of leaf development in has plants.     

Effect of enzyme concentrations on protoplast isolation and protoplast culture of <Emphasis Type="Italic">Spathiphyllum</Emphasis> and <Emphasis Type="Italic">Anthurium</Emphasis>

Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 10⁶ protoplasts g⁻¹ and 1 × 10⁵ protoplasts g⁻¹ could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl₂·2H₂O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm⁻¹ alternating current for 60 s and subsequent generation of two pulses of 4500 V cm⁻¹ direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.     

A single-base deletion mutation in <Emphasis Type="Italic">SlIAA9</Emphasis> gene causes tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) <Emphasis Type="Italic">entire</Emphasis> mutant

The entire (e) locus of tomato (Solanum lycopersicum L.) controls leaf morphology. Dominant E and recessive e allele of the locus produce pinnate compound and complex reduced leaves. Previous research had indicated that SlIAA9, an Aux/IAA gene, was involved in tomato leaf morphology. Down-regulation of SlIAA9 gene by antisense transgenic method decreased the leaf complex of tomato and converted tomato compound leaves to simple leaves. The leaf morphology of these transgenic lines was similar with leaf morphology of tomato entire mutant. In this paper, we report that a single-base deletion mutation in the coding region of SlIAA9 gene results in tomato entire mutant phenotypes.     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

<Emphasis Type="Italic">NARROW LEAF 7</Emphasis> controls leaf shape mediated by auxin in rice

Elucidation of the genetic basis of the control of leaf shape could be of use in the manipulation of crop traits, leading to more stable and increased crop production. To improve our understanding of the process controlling leaf shape, we identified a mutant gene in rice that causes a significant decrease in the width of the leaf blade, termed narrow leaf 7 (nal7). This spontaneous mutation of nal7 occurred during the process of developing advanced backcrossed progeny derived from crosses of rice varieties with wild type leaf phenotype. While the mutation resulted in reduced leaf width, no significant morphological changes at the cellular level in leaves were observed, except in bulliform cells. The NAL7 locus encodes a flavin-containing monooxygenase, which displays sequence homology with YUCCA. Inspection of a structural model of NAL7 suggests that the mutation results in an inactive enzyme. The IAA content in the nal7 mutant was altered compared with that of wild type. The nal7 mutant overexpressing NAL7 cDNA exhibited overgrowth and abnormal morphology of the root, which was likely to be due to auxin overproduction. These results indicate that NAL7 is involved in auxin biosynthesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB354301 and AB354302.     

b-D-glucosidase activity in pelargonium and poinsettia leaves infected by <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Pers. ex Fr.

The involvement of β-D-glucosidase activity in grey mould was studied in two ornamental plant species attacked by Botrytis cinerea. β-D-glucosidase activity in the susceptible pelargonium cultivar ‘Shiva’ gradually increased with the disease development in the leaf spots and their surroundings. The endogenic level of the studied hydrolase in the resistant pelargonium ‘Cascade’ was several times higher than in the susceptible cultivar ‘Shiva’ and in principle underwent no changes after inoculation. The postinfection increase in the activity of β-D-glucosidase noted in the leaves of the susceptible poinsettia cultivar ‘Malibu Red’ was evidently weaker in the intensity, but its tendencies were similar to those of the susceptible pelargonium cultivar. In the leaves of the medium-resistant poinsettia ‘Coco White’ the constitutional level of β-D-glucosidase was 2-3-fold higher in that cultivar than in the susceptible cv. ‘Malibu Red’. In attacked leaves of ‘Coco White’, the enzyme activity continued to increase temporarily until the 3^rd h after inoculation. The process of healthy leaf senescence in both species had no significant influence on the change of the studied enzyme activity which was generally low. A high activity of β-D-glucosidase was also observed in the homogenate prepared from mycelium and in the fungal spores.     

Estimation of the levels of radiation-induced <Emphasis Type="Italic">P</Emphasis>-element transposition in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> experimental populations and laboratory strains

When experimental P + M populations were exposed to chronic γ-irradiation (0.31 mGy/h), the highest instability level of the singed-weak (sn _w ) locus was observed in F₃–F₁₀ with a subsequent decrease and stabilization of the mutation rate. The sn ^w mutation rate was within the range of spontaneous variation in conditions of P-M hybrid dysgenesis and irradiation of males of the Harwich laboratory strain with active P elements at 1.61 mGy/h. The instability of the sn ^w locus was significantly higher at lower dose rates (0.23 and 0.31 mGy/h), suggesting a nonlinear dose-effect relationship.     

Genetic analyses of the formation of the serrated margin of leaf blades in Arabidopsis : combination of a mutational analysis of leaf morphogenesis with the characterization of a specific marker gene expressed in hydathodes and stipules

Developmental control of the formation of the serrated margin of leaf blades was investigated. First, the expression was characterized of a marker gene encoding β-glucuronidase in strain #1-35-38, a transgenic strain of Arabidopsis thaliana (L.) Heynh, derived by the use of a previously described transposon-tagging system. In strain #1-35-38, expression of the marker gene was tissue-specific, being restricted to stipules and the toothed margins of laminae. Using this transgenic marker gene, we examined the development of leaf blade margins in Arabidopsis. We compared the pattern of expression of the marker gene in the leaves of the wild-type plant with that in plants carrying the asymmetric leaves1 (as1) mutation, which causes dramatic changes in leaf-blade morphology in Arabidopsis. The as1 mutant showed normal morphology of early leaf primordia. The mutation affected the development of leaf segmentation in Arabidopsis without any change in the number or morphology of cells in laminae. The as1 mutation affected leaf morphology independently of mutations in other genes known to affect leaf morphogenesis, such as the acaulis1 mutation and the angustifolia mutation. Based upon these results, the development of the morphology of leaf margins in Arabidopsis is discussed. Received: 9 January 1997 / Accepted: 24 June 1997     

Light absorption by isolated chloroplasts and leaves: effects of scattering and ‘packing’

Light absorption was quantified in the following systems: isolated chloroplasts and leaves of spinach (Spinacea oleracea L.), a mutant of geranium (Pelargonium zonale L.) widely differing in pigment content, and coleus (Coleus blumei Benth.) at different stages of leaf ontogenesis. For these species and pea (Pisum sativum L.), scattering-compensated absorption spectra of chloroplast suspensions are presented. Comparison of leaf and chloroplast spectra showed considerable changes in the extent of the ‘package’ effect and the lengthening of the effective optical path in a leaf. The difference between leaf and isolated chloroplast absorption could be quantitatively described by adapting Duysens’s treatment of flattening. It was found that the accumulation of chlorophyll in leaves is accompanied by a monotonous enhancement of the package effect. The results are discussed with special reference to the role of light scattering in leaf optics, light utilization in photosynthesis and wavelength-dependent light gradients in a leaf.     

Effects of carbon source and concentration on development of lingonberry (<Emphasis Type="Italic">Vaccinium vitis-idaea</Emphasis> L.) shoots cultivated <Emphasis Type="Italic">in vitro</Emphasis> from nodal explants

Summary Carbohydrate type and concentration and their interactive effects on in vitro shoot proliferation of three lingonberry (Vaccinium vitis-idaea ssp. vitis-idaea L.) cultivars (‘Regal’, ‘Splendor’, and ‘Erntedank’) and two V. vitis-idaea ssp. minus (Lodd) clones (‘NL1’ and ‘NL2’) were studied. Nodal explants were grown in vitro on medium with 2 μM zeatin and either glucose, sorbitol, or sucrose at a concentration of 0, 10, 20, or 30 gl⁻¹. The interactive effects of carbohydrate type and concentration and genotype were important for shoot proliferation. The best response was afforded by sucrose at 20 gl⁻¹ both in terms of explant response and shoot developing potential, although glucose supported shoot growth equally well, and in ‘NL1’ at 10 gl⁻¹ it resulted in better in vitro growth than sucrose. Carbohydrate concentration had little effect on shoot vigor. The genotypes differed in terms of shoots per explant, length, and vigor, leaves per shoot, and callus formation at the base of explants; this was manifested with various types and concentrations of carbohydrate. Changing the positioning of explants on the medium from vertically upright to horizontal increased the shoot and callus size, but decreased shoot height and leaves per shoot. Proliferated shoots were rooted on a peat:perlite (1∶1, v/v) medium and the plantlets were acclimatized and eventually established in the greenhouse.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: implications of the stay-green mutation for photosynthesis, growth and nitrogen nutrition

Mutation of the nuclear gene sid disables chlorophyll degradation during leaf senescence in the pasture grass Festuca pratensis. This study investigated the effect of the mutation on photosynthesis and on leaf and whole plant growth under a range of nitrogen regimes. When plants were cultivated in a static hydroponic system, the chlorophyll content of fourth leaves of the stay-green mutant Bf993 remained virtually unchanged from full expansion to complete senescence, while tissue of the wild-type (cv. Rossa) became completely yellow. The retention of chlorophyll in Bf993 was not associated with maintenance of photosynthetic activity as shown by rates of light-saturated CO₂ fixation and apparent quantum efficiency. Higher levels of total N in senescing leaves of Bf993 than in Rossa indicated reduced nitrogen remobilization in the mutant. When using a range of [NH₄NO₃], dry matter production and tillering Mere lower for Bf993 at all but the highest [NH₄NO³, which was supra-optimal for the wild type. In contrast to the static system, where fluctuations in N supply occurred, growth and [NO₃^?] uptake were similar in mutant and wild type when [NO₃^?] was continuously maintained by a flowing solution culture system. The results are discussed in relation to the role of N supply and the effect of the stay-green mutation on N recycling.