Characterization of the Snowy Cotyledon 1 Mutant of Arabidopsis Thaliana: The Impact of Chloroplast Elongation Factor G on Chloroplast Development and Plant Vitality

During seedling development chloroplast formation marks the transition from heterotrophic to autotrophic growth. The development and activity of chloroplasts may differ in cotyledons that initially serve as a storage organ and true leaves whose primary function is photosynthesis. A genetic screen was used for the identification of genes that affect selectively chloroplast function in cotyledons of Arabidopsis thaliana. Several mutants exhibiting pale cotyledons and green true leaves were isolated and dubbed snowy cotyledon (sco).One of the mutants, sco1, was characterized in more detail. The mutated gene was identified using map-based cloning. The mutant contains a point mutation in a gene encoding the chloroplast elongation factor G, leading to an amino acid exchange within the predicted 70S ribosome-binding domain. The mutation results in a delay in the onset of germination. At this early developmental stage embryos still contain undifferentiated proplastids, whose proper function seems necessary for seed germination. In light-grown sco1 seedlings the greening of cotyledons is severely impaired, whereas the following true leaves develop normally as in wild-type plants. Despite this apparent similarity of chloroplast development in true leaves of mutant and wild-type plants various aspects of mature plant development are also affected by the sco1 mutation such as the onset of flowering, the growth rate, and seed production. The onset of senescence in the mutant and the wild-type plants occurs, however, at the same time, suggesting that in the mutant this particular developmental step does not seem to suffer from reduced protein translation efficiency in chloroplasts.     

Influences of nitrogen load on the growth and photosynthetic responses of <Emphasis Type="Italic">Quercus serrata</Emphasis> seedlings to O<Subscript>3</Subscript>

The objectives of this study were to clarify the influences of nitrogen (N) load on the growth and photosynthetic responses of Quercus serrata seedlings to O₃ and to obtain basic data for evaluating the critical levels of O₃ for protecting Q. serrata forests in Japan. The effects of O₃ and/or N load on growth and photosynthetic activity of Q. serrata seedlings were investigated during the two growing seasons. Two-year-old seedlings were assigned to 12 experimental treatments, which were comprised of the combination of four gas treatments (charcoal-filtered air and three levels of O₃ at 1.0, 1.5 and 2.0 times ambient concentration) and three N treatments (0, 20 and 50 kg ha⁻¹ year⁻¹). During the second growing season, no significant interactive effects of O₃ and N load on the growth and net photosynthetic rate of the seedlings were detected. Threrfore, we concluded that N supply to the soil at ≤50 kg ha⁻¹ year⁻¹ does not significantly influence the growth and photosynthetic responses of Q. serrata seedlings to O₃. Based on the O₃ exposure-response relationships for the whole-plant growth of the seedlings, the critical level of O₃ for Q. serrata was estimated to be approximately 36 nmol mol⁻¹ as the average 15-h O₃ concentration during the one growing season.     

Factors influencing shoot regeneration from cotyledons of tetraploid <Emphasis Type="Italic">Isatis indigotica</Emphasis> fort

Summary An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS) medium was found to be best for promoting shoot regeneration, followed by Gamborg's B₅ and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented with 2.0 mgl⁻¹ (8.9 μM) 6-benzyladenine and 1.0 mgl⁻¹ (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl⁻¹ (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the chinese medicinal plant, <Emphasis Type="Italic">Dendrobium candidum</Emphasis> wall. ex lindl., from axenic nodal segments

Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l⁻¹ HYPONeX, 80 g l⁻¹ potato homogenate and 2 g l⁻¹ activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l⁻¹ NAA and the plantlets were successfully acclimatized in soil.     

Isolation and Characterization of a Xanthophyll Aberrant Mutant of the Green Alga Nannochloropsis oculata

Novel mutants (xan1 and xan2) of the unicellular green alga Nannochloropsis oculata are impaired in xanthophyll biosynthesis, thereby producing aberrant levels of xanthophylls. High-performance liquid chromatography (HPLC) analysis revealed that the xan1 and xan2 mutants have double the violaxanthin (V) content, but have significantly decreased lutein content in their cells compared to the wild type. Furthermore, these mutants contain two to three times more zeaxanthin than the wild type under low light (LL) growth conditions. However, this xanthophyll aberration in N. oculata did not affect the normal growth and the major cellular chemical composition of the xan1 strain. The xanthophyll pool size of the LL-grown mutant was 1.8-fold greater than that of the wild type. Under high light (HL) growth conditions, V content was substantially decreased in both the mutant and wild types because of the epoxidation state of the xanthophylls. Under LL growth conditions, the deepoxidation states of the xanthophyll pool sizes were 0.1 and 1.2 in the wild type and the mutant, respectively. However, the deepoxidation states of the xanthophyll pool sizes were 0.78 in the wild type and 0.87 in the mutant under HL growth conditions. We observed that the level of one of the commercially important xanthophylls, zeaxanthin, was higher in the mutant than in the wild type under all culture conditions. This mutant is discussed in terms of its commercial value and potential utilization by the algal biotechnology industry for the production of zeaxanthin.     

Direct somatic embryogenesis from leaves,cotyledons and hypocotyls of <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis>

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in seabuckthorn (Hippophae rhamnoides L.) was achieved. The influences of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants were studied. The highest frequency of somatic embryos production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg dm⁻³ kinetin and 0.2, 0.5 mg dm⁻³ indole-3-acetic acid. Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55 %. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.     

Photosynthesis,chloroplast ultrastructure,chemical composition and oxidative stress in <Emphasis Type="Italic">Theobroma cacao</Emphasis> hybrids with the lethal gene <Emphasis Type="Italic">Luteus-Pa</Emphasis> mutant

The Theobroma lethal character Luteus-Pa segregates in a 3:1 ratio, expresses in recessive homozygosis, initially inducing leaf chlorosis and finally provoking seedlings death. The objective of this work was to evaluate gas exchange, chlorophyll fluorescence emission, chemical composition and oxidative stress of wild and mutant seedlings resulting from the crosses Pa 30 × Pa 169 and its reciprocal, aiming to elucidate the seedlings death induced by Luteus-Pa. At 15 day after emergence (DAE) differences began to appear between the wild type and mutant. Mutant seedlings showed: (1) lack of photosynthesis and alterations in chloroplast morphology; (2) lower level of three abundant groups of proteins in leaves; (3) decrease in the content of chloroplastidic pigments (4) decrease in peroxidases activities and increase in leaf polyphenol oxidase activity; (5) decrease in carbohydrate and concentration of some nutrients and low dry mass in all plant parts. In leaves of mutant seedlings of both crosses damages occurred in the system responsible for the photochemical phase of photosynthesis. Variations in growth parameters and subsequent seedling death up to 60 DAE were related to exhaustion of cotyledonary reserves, inactive photosynthetic apparatus and oxidative stress.     

Photosynthetic net O<Subscript>2</Subscript> evolution enhancement as a sign of acclimation to phosphorus deficiency in bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) leaves

Primary leaves of bean (Phaseolus vulgaris L.) seedlings cultivated for 14 days in a growth chamber on complete (control) and phosphate deficient (−P) Knop liquid medium were used for measurements. The −P leaves were smaller and showed an increased specific leaf area (SLA). Their inorganic phosphate (P_i) concentration was considerably lowered. They did not show any significant changes in chlorophyll (Chl) (a + b) concentration and in their net CO₂ assimilation rate when it was estimated under the conditions close to those of the seedlings growth. Light response curves of photosynthetic net O₂ evolution (P _NO₂) of the leaves for the irradiation range up to 500 μmol(photon) m⁻² s⁻¹ were determined, using the leaf-disc Clark oxygen electrode. The measurements were taken under high CO₂ concentration of about 1 % and O₂ concentrations of 21 % or lowered to about 3 % at the beginning of measurement. The results obtained at 21 % O₂ and the irradiations close to or higher than those used during the seedlings growth revealed the phosphorus stress suppressive effect on the leaf net O₂ evolution, however, no such effect was observed at lower irradiations. Other estimated parameters of P _NO₂ such as: apparent quantum requirement (QR_A) and light compensation point (LCP) for the control and −P leaves were similar. However, with a high irradiation and lowered O₂ concentration the rate of P _NO₂ for the −P leaves was markedly higher than that for the control, in relation to both the leaf area and leaf fresh mass. This difference also disappeared at low irradiations, but the estimated reduced QR_A values indicate, under those conditions, the increased yield of photosynthetic light reaction, especially in the −P leaves. The presented results confirm the suggestion that during the initial phase of insufficient phosphate feeding the acclimations in the light phase of photosynthesis, both structural and functional appear. They correspond, probably, to the increased energy costs of carbon assimilation under phosphorus stress, e.g. connected with raised difficulties in phosphate uptake and turnover and enhanced photorespiration. Under the experimental conditions especially advantageous for the dark phase of photosynthesis (saturating CO₂ and PAR, low O₂ concentration), those acclimations may be manifested as an enhancement of photosynthetic net O₂ evolution.     

Utilization of <Emphasis Type="Italic">fadA</Emphasis> knockout mutant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> for overproduction of medium chain-length-polyhydroxyalkanoate

The fadBA operon in the fatty acid β-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA). Succinate at 150 mg l⁻¹ stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l⁻¹ with a cell dry weight of 10.3 g l⁻¹.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> constitutive photomorphogenic mutant, <Emphasis Type="Italic">bls1</Emphasis>,displays altered brassinosteroid response and sugar sensitivity

We have isolated an Arabidopsis mutant impaired in light- and brassinosteroid (BR) induced responses, as well as in sugar signalling. The bls1 (brassinosteroid, light and sugar1) mutant displays short hypocotyl, expanded cotyledons, and de-repression of light-regulated genes in young seedlings, and leaf differentiation and silique formation on prolonged growth in dark. In light, the bls1 mutant is dwarf and develops a short root, compact rosette, with reduced trichome number, and exhibits delayed bolting. The activity of the BR inducible TCH4 and auxin inducible SAUR promoters, fused with GUS gene, is also altered in seedlings harbouring bls1 mutant background. In addition, the bls1 mutant is hypersensitive to metabolizable sugars. The short hypocotyl phenotype in dark, short root phenotype in light and sugar hypersensitivity could be rescued with BR application. Moreover, the bls1 mutant also showed higher expression of a BR biosynthetic pathway gene CPD, which is known to be feedback-regulated by BR. Using a genome-wide AFLP mapping strategy, the bls1 mutant has been mapped to a 1.4Mb region of chromosome 5. Since no other mutant with essentially a similar phenotype has been assigned to this region, we suggest that the bls1 mutant defines a novel locus involved in regulating endogenous BR levels, with possible ramifications in integrating light, hormone and sugar signalling.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Enhancing the frequency of somatic embryogenesis following <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of immature cotyledons of soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill.]

Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl⁻¹ hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl⁻¹ hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols for transformation of soybean should be established under both Agrobacterium and selection conditions.     

Strain improvement of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> Rut C-30 for increased cellulase production

The strain of Trichoderma reesei Rut C-30 was subjected to mutation after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG) for 6 h followed by UV irradiation for 15 min. Successive mutants showed enhanced cellulase production, clear hydrolysis zone and rapid growth on Avicel-containing plate. Particularly, the mutant NU-6 showed approximately two-fold increases in activity of both FPA and CMCase in shake flask culture when grown on basal medium containing peptone (1%) and wheat bran (1%). The enzyme production was further optimized using eight different media. When a mixture of lactose and yeast cream was used as cellulase inducer, the mutant NU-6 yielded the highest enzyme and cell production with a FPase activity of 6.2 U ml⁻¹, a CMCase activity of 54.2 U ml⁻¹, a β-glucosidase activity of 0.39 U ml⁻¹, and a fungal biomass of 12.6 mg ml⁻¹. It deserved noting that the mutant NU-6 also secreted large amounts of xylanases (291.3 U ml⁻¹). These results suggested that NU-6 should be an attractive producer for both cellulose and xylanase production.     

EMB1211 is required for normal embryo development and influences chloroplast biogenesis in Arabidopsis

Chloroplast biogenesis is tightly linked with embryogenesis and seedling development. A growing body of work has been done on the molecular mechanisms underlying chloroplast development; however, the molecular components involved in chloroplast biogenesis during embryogenesis remain largely uncharacterized. In this paper, we show that an Arabidopsis mutant carrying a T‐DNA insertion in a gene encoding a multiple membrane occupation and recognition nexus (MORN)‐containing protein exhibits severe defects during embryogenesis, producing abnormal embryos and thereby leading to a lethality of young seedlings. Genetic and microscopic studies reveal that the mutation is allelic to a previously designated Arabidopsis embryo‐defective 1211 mutant (emb1211). The emb1211 +/? mutant plants produce approximately 25% of white‐colored ovules with abnormal embryos since late globular stage when primary chloroplast biogenesis takes place, while the wild‐type plants produce all green ovules. Transmission electron microscopic analysis reveals the absence of normal chloroplast development, both in the mutant embryos and in the mutant seedlings, that contributes to the albinism. The EMB1211 gene is preferentially expressed in developing embryos as revealed in the EMB1211::GUS transgenic plants. Taken together, the data indicate that EMB1211 has an important role during embryogenesis and chloroplast biogenesis in Arabidopsis.     

Plant regeneration from mesophyll-and cell suspension-derived protoplasts of <Emphasis Type="Italic">Dianthus acicularis</Emphasis> and characterization of regenerated plants

Summary Plant regeneration systems from mesophyll- and cell suspension-derived protoplasts were established in Dianthus acicularis (2n=90), a species with resistance to Burkholderia caryophylli (Psedomonas caryophylli). Protoplasts were isolated from both leaves of in vitro-grown plants and cell suspension cultures established from the calluses originated from leaves of in vitro-grown plants. Protoplasts isolated from both sources showed about the same response to the type and concentration of cytokinins, and gave the highest frequencies of cell division and colony formation in 0.1% (w/v) Gelrite^?-solidified Murashige and Skoog (MS) medium supplemented with 0.5M glucose, 1.0mgl⁻¹ (4.53 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5 mg l⁻¹ (2.28 μM) zeatin. Numerous plantlets were regenerated after transfer of the colonies to 0.8% (w/v) agar-solidified half-strength MS medium supplemented with 0.5 mgl⁻¹ (2.28 μM) zeatin. Most plantlets exhibited normal phenotypes, but some showed variations, such as abnormal morphology with reduced chromosome number, precocious flowering, and vigorous growth with a tetraploid chromosome number. Possible mechanisms responsible for the observed somaclonal variation are discussed.     

Somatic embryogenesis of <Emphasis Type="Italic">Gentiana cruciata</Emphasis> (L.): Histological and ultrastructural changes in seedling hypocotyl explant

Summary Structure and ultrastructure changes that occurred during tissue culture of upper explants of hypocotyl (adjacent to cotyledons) of 10-d-old seedlings of Gentiana cruciata were studied. The explants were cultured on Murashige and Skoog induction medium supplemented with 1.0 mg l⁻¹ dicamba +0.1 mg l⁻¹ naphthaleneacetic acid +2.0 mg l⁻¹ benzyladenine +80.0 mg l⁻¹ adenine sulfate. The initial response of the explant and callus formation were ultrastructurally analyzed during the first 11 d of culture. After 6–8 wk, various methods were employed to collect evidence of indirect somatic embryogenesis. After 48 h of culture, the earliest cell response was cell division of epidermis and primary cortex. There were numerous disturbances of karyo- and cytokinesis, leading to formation of multinuclear cells. With time, the divisions ceased, and cortex cells underwent strong expansion, vacuolization and degradation. About the 6th day of culture, callus tissue proliferated and the initial divisions of vascular cylinder cells were observed. Their division appeared normal. Cells originating from that tissue were small, weakly vacuolated, with dense cytoplasm containing active-looking cell organelles. Numerous divisions occurred in the vascular cylinder, which led to its expansion and the formation of embryogenic callus tissue. During the 6–8th wk of culture, in the proximal end of the explant, masses of somatic embryos were formed from outer parts of intensively proliferating tissue.