Male‐sterile maize plants produced by targeted mutagenesis of the cytochrome P450‐like gene (MS26) using a re‐designed I–CreI homing endonuclease

The I–CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double‐strand breaks and enhancing DNA recombination reactions in maize cells. The DNA‐binding properties of this protein were re‐designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single‐chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium‐mediated transformation, the cleavage resulted in mutations at a co‐delivered extra‐chromosomal ms26‐site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26‐site in 5.8% of transgenic T₀ plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of double‐strand break repair generated by non‐homologous end joining. One of 21 mutagenized T₀ plants carried two mutated alleles of the MS26 gene. As expected, the bi‐allelic mutant T₀ plant and the T₁ progeny homozygous for the ms26 mutant alleles were male‐sterile. This paper described the second maize chromosomal locus (liguless‐1 being the first one) mutagenized by a re‐designed I–CreI–based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.     

Targeted mutagenesis in the progeny of maize transgenic plants

We have demonstrated that targeted mutagenesis can be accomplished in maize plants by excision, activation, and subsequent elimination of an endonuclease in the progeny of genetic crosses. The yeast FLP/FRT site-specific recombination system was used to excise and transiently activate the previously integrated yeast I-SceI homing endonuclease in maize zygotes and/or developing embryos. An artificial I-SceI recognition sequence integrated into genomic DNA was analyzed for mutations to indicate the I-SceI endonuclease activity. Targeted mutagenesis of the I-SceI site occurred in about 1% of analyzed F1 plants. Short deletions centered on the I-SceI-produced double-strand break were the predominant genetic lesions observed in the F1 plants. The I-SceI expression cassette was not detected in the mutant F1 plants and their progeny. However, the original mutations were faithfully transmitted to the next generation indicating that the mutations occurred early during the F1 plant development. The procedure offers simultaneous production of double-strand breaks and delivery of DNA template combined with a large number of progeny plants for future gene targeting experiments.     

Photosystem II inhibition by moderate light under low temperature in intact leaves of chilling-sensitive and -tolerant plants

Photosystem II (PSII) activity was examsined in leaves of chilling-sensitive cucumber ( Cucumis sativus L.), tomato ( Lycopersicum esculentum L.), and maize ( Zea mays L.), and in chilling-tolerant barley ( Hordeum vulgare L.) illuminated with moderate white light (300 µmol m⁻² s⁻¹) at 4°C using chlorophyll a fluorescence measurements. PSII activity was inhibited in leaves of all the four plants as suggested by the decline in F _v/ F _m, 1/ F _o − 1/ F _m, and F _v/ F _o values. The changes in initial fluorescence level ( F _o), F _v/ F _m, 1/ F _o − /1/ F _m, and F _v/ F _o ratios indicate a stronger PSII inhibition in cucumber, maize and tomato plants. The kinetics of chlorophyll a fluorescence rise showed complex changes in the magnitudes and rise of O-J, J-I, and I-P phases caused by photoinhibition. The selective suppression of the J-I phase of fluorescence rise kinetics provides evidence for weakened electron donation from the oxidizing side, whereas the accumulation of reduced Q_A suggests damage to the acceptor side of PSII. These findings imply that the process of chilling-induced photoinhibition involves damage to more than one site in the PSII complexes. Furthermore, comparative analyses of the decline in F _v/ F _o and photooxidation of P700 explicitly show that the extent of photoinhibitory damage to PSII and photosystem I is similar in leaves of cucumber plants grown at a low irradiance level.     

Fumonisin B1 production by Fusarium proliferatum strains isolated from Allium fistulosum plants and seeds in Japan

Aims:  To test the fumonisin B₁ - producing ability of Fusarium proliferatum strains isolated from Welsh onion ( Allium fistulosum ) plants and seeds of commercial cultivars in Japan and to examine the applicability of PCR-based assays to discriminate between fumonisin B₁-producing and nonproducing isolates.
Methods and Results:  Fumonisin B₁ levels in 20 Fusarium isolates obtained from Welsh onion plants and seeds of seven commercial cultivars were determined by HPLC. Thirteen of the 20 isolates produced fumonisin B₁. PCR assay with FUM1 gene-specific primers amplified a DNA fragment (700 bp) only from fumonisin-producing isolates.
Conclusions:  Fusarium proliferatum isolates that can produce fumonisin B₁ were often associated with wilted Welsh onion plants and seeds of some commercial cultivars. The PCR assay with FUM1 gene-specific primers has the potential to discriminate between fumonisin B₁-producing and nonproducing isolates.
Significance and Impact of the Study:  This study revealed that F. proliferatum producing fumonisin B₁ is associated with Welsh onion plants and that commercial cultivar seeds may be contaminated with the fungus. PCR amplification of FUM1 gene can be a useful tool for the rapid identification of fumonisin B₁-producing F. proliferatum isolates.     

Laboratory studies on the effects of pollen from Bt-maize on larvae of some butterfly species

Abstract:   Three lepidopteran species were tested to determine their susceptibility against the ingestion of pollen from genetically modified maize plants. To prove the existence of dose–response relations between the applied amount of pollen (Bt-maize) and the damages on the tested larvae, a method was developed which makes it possible to feed caterpillars with defined amounts of pollen. If their food plants were contaminated with pollen of a cultivar of the Bt-176 maize-line Pieris brassicae , Pieris rapae and Plutella xylostella -larvae fed less, grew more slowly and showed a higher mortality than caterpillars of an untreated control group. The 50% lethality (LD₅₀)-values were calculated for P.xylostella (L₄) with 19.2, for P. rapae (L₂) with 39.0 and also for P. brassicae (L₂) with 139.2 pollen of the transgenic maize Pactol CB. Studies with P. brassicae -caterpillars of different larval stages indicated, that older individuals showed a higher tolerance against pollen from Bt-maize than younger ones. It must be stated on the basis of the present studies, that ingestion of non-transgenic maize pollen has neither a positive nor a negative effect on caterpillars. It becomes clear from the information presented here that it is still difficult to make general statements about the endangering of butterflies, arising from cultivation of genetically modified maize lines. Further investigations on this issue are needed. Initially, the LD₅₀-values concerning the larvae of certain butterfly species have to be determined to anticipate the risks, and in addition the distances between habitats with caterpillar host plants and maize fields, and the abundance of these plants have to be considered.     

Does the 13C of foliage‐respired CO2 and biochemical pools reflect the 13C of recently assimilated carbon?

Isotopic labelling experiments were conducted to assess relationships among ¹³C of recently assimilated carbon ( δC _A), foliage respiration ( δC _F), soluble carbohydrate ( δC _SC), leaf waxes ( δC _LW) and bulk organic matter ( δC _OM). Slash pine, sweetgum and maize were grown under ¹³C depleted CO₂ to label biomass and then placed under ambient conditions to monitor the loss of label. In pine and sweetgum, δC _F of labelled plants (∼−44 and −35‰, respectively) rapidly approached control values but remained depleted by ∼4–6‰ after 3–4 months. For these tree species, no or minimal label was lost from δC _SC, δC _LW and δC _OM during the observation periods. δC _F and δC _SC of labelled maize plants rapidly changed and were indistinguishable from controls after 1 month, while δC _LW and δC _OM more slowly approached control values and remained depleted by 2–6‰. Changes in δC _F in slash pine and sweetgum fit a two-pool exponential model, with the fast turnover metabolic pool (∼3–4 d half-life) constituting only 1–2% of the total. In maize, change in δC _F fits a single pool model with a half-life of 6.4 d. The ¹³C of foliage respiration and biochemical pools reflect temporally integrated values of δC _A, with change in isotopic composition dampened by the size of metabolic carbon reserves and turnover rates.     

Delineation of the recombination sites necessary for integration of pathogenicity islands II and III into the Escherichia coli 536 chromosome

In uropathogenic Escherichia coli strain 536, six pathogenicity islands (PAIs) encode key virulence factors. All PAIs except PAI IV₅₃₆ are flanked by direct repeats and four of them encode integrases responsible for their chromosomal excision. To study recombination sites used for the integration by PAI II₅₃₆ and III₅₃₆ integrases, we measured site-specific recombination between the chromosomal integration site attB , and the PAI-specific attachment site attP . We show that PAI III₅₃₆ IntB, but not IntA, mediates PAI III₅₃₆ integration. Studies of integrative recombination sites of both PAIs show that, when using a large cognate attP site (839 bp for PAI II₅₃₆ and 268 bp for PAI III₅₃₆), PAI II₅₃₆ and III₅₃₆ attB sites could be reduced to 16 bp and 20 bp, respectively, without affecting recombination. Further reduction to 14 bp for PAI II₅₃₆ and 13 bp for PAI III₅₃₆ diminished recombination efficiency. Surprisingly, attP sites could also be reduced to 14 bp (PAI II₅₃₆) and 20 bp (PAI III₅₃₆). The integration host factor (IHF) and the DNA-bending HU protein do not influence PAI II₅₃₆ recombination, but IHF enhances PAI-III₅₃₆ excision and negatively affects its integration. These data suggest that PAI intasomes differ from those of lambda and P4 integrase paradigms.     

Elevated CO2 increases photosynthesis, biomass and productivity, and modifies gene expression in sugarcane

Because of the economical relevance of sugarcane and its high potential as a source of biofuel, it is important to understand how this crop will respond to the foreseen increase in atmospheric [CO₂]. The effects of increased [CO₂] on photosynthesis, development and carbohydrate metabolism were studied in sugarcane ( Saccharum ssp.). Plants were grown at ambient (∼370 ppm) and elevated (∼720 ppm) [CO₂] during 50 weeks in open-top chambers. The plants grown under elevated CO₂ showed, at the end of such period, an increase of about 30% in photosynthesis and 17% in height, and accumulated 40% more biomass in comparison with the plants grown at ambient [CO₂]. These plants also had lower stomatal conductance and transpiration rates (−37 and −32%, respectively), and higher water-use efficiency (c.a. 62%). cDNA microarray analyses revealed a differential expression of 35 genes on the leaves (14 repressed and 22 induced) by elevated CO₂. The latter are mainly related to photosynthesis and development. Industrial productivity analysis showed an increase of about 29% in sucrose content. These data suggest that sugarcane crops increase productivity in higher [CO₂], and that this might be related, as previously observed for maize and sorghum, to transient drought stress.     

Susceptibility of various developmental stages of the maize weevil, Sitophilus zeamais Motschulsky (Col., Curculionidae) to methyl iodide in brown rice

Abstract:  The efficacy of methyl iodide (MI) as a fumigant against all developmental stages of the maize weevil, Sitophilus zeamais Motsch. was investigated. Tests were conducted with concentrations of 1.5, 1.8, 2.1, 2.4, 2.7 and 3.0 mg/l, for a 6-h exposure period. Values of LC₅₀, LC₉₅ and LC₉₉ of MI for immatures and adult stages were determined. The present laboratory tests showed that MI was toxic to various life stages of S. zeamais at relatively short exposure periods. At the LC₅₀ and LC₉₅ levels, the most susceptible stage was the egg stage followed by larvae, pupae and adults (1-day mortality). The egg was found to be most susceptible to MI, requiring 0.81 and 2.16 mg/l for 50 and 99% mortality, respectively, while the adult was most tolerant, requiring 2.30 and 3.02 mg/l for 50 and 99% mortality, respectively, based on 1-day mortality count. Pupae were less susceptible to MI than egg and larvae, requiring 1.47 and 3.19 mg/l for 50 and 99% mortality, respectively. Based on the present toxicity tests, MI has the potential for use as a fumigant to control all developmental stages of the maize weevil, S. zeamais .     

A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4-dechlorination agent pyridine-2,6-bis(thiocarboxylic acid)

A spontaneous mutant of Pseudomonas stutzeri strain KC lacked the carbon tetrachloride (CCl₄) transformation ability of wild-type KC. Analysis of restriction digests separated by pulsed-field gel electrophoresis (PFGE) indicated that the mutant strain CTN1 differed from strain KC by deletion of approximately 170 kb of chromosomal DNA. CTN1 did not produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), the agent determined to be responsible for CCl₄ dechlorination in cultures of strain KC. Cosmids from a genomic library of strain KC containing DNA from within the deleted region were identified by hybridization with a 148 kb genomic Spe I fragment absent in strain CTN1. Several cosmids identified in this manner were further screened for complementation of the PDTC biosynthesis-negative (Pdt⁻) phenotype. One cosmid (pT31) complemented the Pdt⁻ phenotype of CTN1 and conferred CCl₄ transformation activity and PDTC production upon other pseudomonads. Southern analysis showed that none of three other P. stutzeri strains representing three genomovars contained DNA that would hybridize with the 25 746 bp insert of pT31. Transposon mutagenesis of pT31 identified open reading frames (ORFs) whose disruption affected the ability to make PDTC in the strain CTN1 background. These data describe the pdt locus of strain KC as residing in a non-essential region of the chromosome subject to spontaneous deletion. The pdt locus is necessary for PDTC biosynthesis in strain KC and is sufficient for PDTC biosynthesis by other pseudomonads but is not a common feature of P. stutzeri strains.     

Control of Aspergillus section Flavi growth and aflatoxin accumulation by plant essential oils

Aims:  The antifungal effect of Pimpinella anisum (anise), Pëumus boldus (boldus), Mentha piperita (peppermint), Origanum vulgare (oregano) and Minthosthachys verticillata (peperina) essential oils against Aspergillus section Flavi (two isolates of Aspergillus parasiticus and two isolates of Aspergillus flavus ) was evaluated in maize meal extract agar at 0·982 and 0·955 water activities, at 25°C.
Methods and Results:  The percentage of germination, germ-tube elongation rate, growth rate and aflatoxin B₁ (AFB₁) accumulation at different essential oils concentrations were evaluated. Anise and boldus essential oils were the most inhibitory at 500 mg kg⁻¹ to all growth parameters of the fungus. These essential oils inhibited the percentage of germination, germ-tube elongation rate and fungal growth. AFB₁ accumulation was completely inhibited by anise, boldus and oregano essential oils. Peperina and peppermint essential oils inhibited AFB₁ production by 85–90% in all concentrations assayed.
Conclusions:  Anise and boldus essential oils could be considered as effective fungitoxicans for Aspergillus section flavi .
Significance and Impact of the Study:  Our results suggest that these phytochemical compounds could be used alone or in conjunction with other substances to control the presence of aflatoxigenic fungi in stored maize.     

Ozone induced emissions of biogenic VOC from tobacco: relationships between ozone uptake and emission of LOX products

Volatile organic compound (VOC) emissions from tobacco ( Nicotiana tabacum L. var. Bel W3) plants exposed to ozone (O₃) were investigated using proton-transfer-reaction mass-spectrometry (PTR-MS) and gas chromatography mass-spectrometry (GC-MS) to find a quantitative reference for plants' responses to O₃ stress. O₃ exposures to illuminated plants induced post-exposure VOC emission bursts. The lag time for the onset of volatile C₆ emissions produced within the octadecanoid pathway was found to be inversely proportional to O₃ uptake, or more precisely, to the O₃ flux density into the plants. In cases of short O₃ pulses of identical duration the total amount of these emitted C₆ VOC was related to the O₃ flux density into the plants, and not to ozone concentrations or dose–response relationships such as AOT 40 values. Approximately one C₆ product was emitted per five O₃ molecules taken up by the plant. A threshold flux density of O₃ inducing emissions of C₆ products was found to be (1.6 ± 0.7) × 10⁻⁸ mol m⁻² s⁻¹.     

Clostridia spore formation during aerobic deterioration of maize and sorghum silages as influenced by Lactobacillus buchneri and Lactobacillus plantarum inoculants

Aims:  The effect of the inoculation of maize and sorghum silages with Lactobacillus plantarum (LP) and Lactobacillus buchneri (LB) on the clostridia spore formation during aerobic deterioration has been studied.
Methods and results:  The crops were ensiled in 30 l jars, without a lactic acid bacteria inoculant (C), and with an LP or LB inocula (theoretical rate of 1 × 10⁶). After 90 days of conservation, the silages were analysed for the chemical and microbiological characteristics and subjected to an aerobic stability test, during which pH, temperature, nitrate, yeast, mould and clostridia spores were measured. Compared to the C and LP silages, yeasts were reduced in the LB silages, resulting in an increased aerobic stability. Clostridia spores, determined by most probable number (MPN) procedure, increased to 6 log₁₀ MPN g⁻¹ in the C and LP maize silages, whereas they reached 3 log₁₀ MPN g⁻¹ in C and LP sorghum silages.
Conclusions:  Clostridia spore count only slightly increased in the LB maize silages after 342 h (2·59 log₁₀ MPN g⁻¹), whereas it did not show any increase in the LB sorghum silages for the whole period of air exposure.
Significance and impact of the study:  The data indicated that clostridia spore outgrowth can take place during silo feedout in aerobic-deteriorated silages and that LB inoculation reduces the risk of clostridia outgrowth after silage opening by increasing the aerobic stability.     

Early life stage toxicity of extracts from the African fish poison plants Tephrosia vogelii Hook. f. and Asystasia vogeliana Benth. on zebrafish embryos

Embryos of Danio rerio are highly susceptible to extracts of the plants Tephrosia vogelii and Asystasia vogeliana . The concentration of the dried extracts at which 50% of the embryos were affected (EC₅₀) after 24 h exposure were 320 and 572 μg l⁻¹, respectively; corresponding 50% mortality (LC₅₀) values after 48 h exposure were 493 and 869 μg l⁻¹. Results indicate that the use of these ichthyotoxic plants might have a severe impact on the survival of fish larvae in the field.     

Transgenic Bt maize and Rhopalosiphum padi (Hom., Aphididae) performance

Abstract.  1. The population abundance and age structure of Rhopalosiphum padi , one of the most common maize aphid species, on transgenic Bt (expressing the Cry1Ab protein) and non-Bt isogenic maize was studied in commercial plots during three crop seasons.
2. A higher density of aphids, particularly alates and young nymphs, occurred in Bt plots at very young maize development stages, corresponding to the settlement period, in the 3 years studied. Possible causes are discussed. After this period, there were no differences between Bt and non-Bt maize.
3. Mortality, development, and reproduction of the offspring of alate forms of R. padi and the offspring of different generations of apterous forms fed with Bt maize were evaluated in the laboratory under controlled conditions.
4. The developmental and pre-reproductive times of the offspring of the first generation of alatae were shorter and the intrinsic rate of natural increase ( r _m ) higher when aphids fed on Bt maize. The opposite occurs with the offspring of the first generation of apterous mothers, which have lower nymphal and adult mortality, shorter developmental and pre-reproductive times, a higher effective fecundity rate, and greater r _m , when fed on non-Bt maize. The differences in aphid development on the two cultivars may be linked to changes in host-plant quality due to pleiotropic effects of the genetic modification.
5. No differences on aphid mortality, developmental and pre-reproductive times, fecundity, and r _m were found between the offspring of apterous aphids maintained on Bt or non-Bt maize for several generations.     

Photosynthesis is improved by exogenous glycinebetaine in salt-stressed maize plants

The effects of exogenous application of glycinebetaine (GB) (10 m M ) on growth, leaf water content, water use efficiency, photosynthetic gas exchange, and photosystem II photochemistry were investigated in maize plants subjected to salt stress (50 and 100 m M NaCl). Salt stress resulted in the decrease in growth and leaf relative water content as well as net photosynthesis and the apparent quantum yield of photosynthesis. Stomatal conductance, evaporation rate, and water use efficiency were decreased in salt-stressed plants. Salt stress also caused a decrease in the actual efficiency of PSII ( Φ _PSII), the efficiency of excitation energy capture by open PSII reaction centers ( F _v'/ F _m'), and the coefficients of photochemical quenching ( q _P) but caused an increase in non-photochemical quenching (NPQ). Salt stress showed no effects on the maximal efficiency of PSII photochemistry ( F _v/ F _m). On the other hand, in salt-stressed plants, GB application improved growth, leaf water content, net photosynthesis, and the apparent quantum yield of photosynthesis. GB application also increased stomatal conductance, leaf evaporation rate, and water use efficiency. In addition, GB application increased Φ _PSII, F _v'/ F _m', and q _P but decreased NPQ. However, GB application showed no effects on F _v/ F _m. These results suggest that photosynthesis was improved by GB application in salt-stressed plants and such an improvement was associated with an improvement in stomatal conductance and the actual PSII efficiency.     

Internode length in Pisum. A new allele at the Lh locus

A new allele at the Lh locus has been identified in Pisum sativum L. and named lhⁱ . This allele results in reduced GA levels in young shoots, and a dwarf phenotype. Gas chromatography-selected ion monitoring (GC-SIM) with dideuterated internal standards has been used to demonstrate a quantitative relationship between the level of endogenous GA₁ and internode length using the three alleles ( Lh, lh and lhⁱ ) at the Lh locus. These results are consistent with previous findings in peas (for alleles at the Le locus) and other species possessing a predominant early 13-hydroxylation pathway for GA biosynthesis and support the role of GA₁ as the major native GA in peas with biological activity in its own right. However, in contrast to alleles at the Le locus, GA₂₀ levels are also reduced in lh and lhⁱ plants. The lhⁱ allele also has possible pleiotropic effects on seed abortion, leading to a reduction in seed yield compared to plants homozygous for the previously characterised Lh or lh alleles.     

Isolation of photorespiratory mutants from Lotus japonicus deficient in glutamine synthetase

A mutagenesis programme using ethyl methanesulphonate (EMS) was carried out on Lotus japonicus (Regel) Larsen cv. Gifu in order to isolate photorespiratory mutants in this model legume. These mutants were able to grow in a CO₂-enriched atmosphere [0.7% (v/v) CO₂] but showed stress symptoms when transferred to air. Among them, three mutants displayed low levels of glutamine synthetase (GS; EC 6.3.1.2) activity in leaves. The mutants accumulated ammonium in leaves upon transfer from 0.7% (v/v) CO₂ to air. F₁ plants of back crosses to wild type were viable in air and F₂ populations segregated 3 : 1 (viable in air : air-sensitive) indicative of a single Mendelian recessive trait. Complementation tests showed that the three mutants obtained were allelic. Chromatography on DEAE-Sephacel used to separate the cytosolic and plastidic GS isoenzymes together with immunological data showed that: (1) mutants were specifically affected in the plastidic GS isoform, and (2) in L. japonicus the plastidic GS isoform eluted at lower ionic strength than the cytosolic isoform, contrary to what happens in most plants. The plastidic GS isoform present in roots of wild type L. japonicus was also absent in roots of the mutants, indicating that this plastidic isoform from roots was encoded by the same gene than the GS isoform expressed in leaf tissue. Viability of mutant plants in high-CO₂ conditions indicates that plastidic GS is not essentially required for primary ammonium assimilation. Nevertheless, mutant plants did not grow as well as wild type plants in high-CO₂ conditions.