Opposite Effects on Spodoptera littoralis Larvae of High Expression Level of a Trypsin Proteinase Inhibitor in Transgenic Plants1

This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI.

Manipulation of Catalase Levels Produces Altered Photosynthesis in Transgenic Tobacco Plants1

Constructs containing the cDNAs encoding the primary leaf catalase in Nicotiana or subunit 1 of cottonseed (Gossypium hirsutum) catalase were introduced in the sense and antisense orientation into the Nicotiana tabacum genome. The N. tabacum leaf cDNA specifically overexpressed CAT-1, the high catalytic form, activity. Antisense constructs reduced leaf catalase specific activities from 0.20 to 0.75 times those of wild type (WT), and overexpression constructs increased catalase specific activities from 1.25 to more than 2.0 times those of WT. The NADH-hydroxypyruvate reductase specific activity in transgenic plants was similar to that in WT. The effect of antisense constructs on photorespiration was studied in transgenic plants by measuring the CO2 compensation point (Γ) at a leaf temperature of 38°C. A significant linear increase was observed in Γ with decreasing catalase (at 50% lower catalase activity Γ increased 39%). There was a significant temperature-dependent linear decrease in Γ in transgenic leaves with elevated catalase compared with WT leaves (at 50% higher catalase Γ decreased 17%). At 29°C, Γ also decreased with increasing catalase in transgenic leaves compared with WT leaves, but the trend was not statistically significant. Rates of dark respiration were the same in WT and transgenic leaves. Thus, photorespiratory losses of CO2 were significantly reduced with increasing catalase activities at 38°C, indicating that the stoichiometry of photorespiratory CO2 formation per glycolate oxidized normally increases at higher temperatures because of enhanced peroxidation.

A change of ploidy can modify epigenetic silencing.

A silent transgene in Arabidopsis thaliana was reactivated in an outcross but not upon selfing of hemizygous plants. This result could only be explained by assuming a genetic difference between the transgene-free gametes of the wild-type and hemizygous transgenic plants, respectively, and led to the discovery of ploidy differences between the parental plants. To investigate whether a change of ploidy by itself can indeed influence gene expression, we performed crosses of diploid or tetraploid plants with a strain containing a single copy of a transgenic resistance gene in an active state. We observed reduced gene expression of the transgene in triploid compared with diploid hybrids. This led to loss of the resistant phenotype at various stages of seedling development in part of the population. The gene inactivation was reversible. Thus, an increased number of chromosomes can result in a new type of epigenetic gene inactivation, creating differences in gene expression patterns. We discuss the possible impact of this finding for genetic diploidization in the light of widespread, naturally occurring polyploidy and polysomaty in plants.

Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light.

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal, transduction required by the CRY1 protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.

Involvement of small GTP-binding proteins in defense signal-transduction pathways of higher plants.

Small GTP-binding proteins play a critical role in the regulation of a range of cellular processes--including growth, differentiation, and intracellular transportation. Previously, we isolated a gene, rgp1, encoding a small GTP-binding protein, by differential screening of a rice cDNA library with probe DNAs from rice tissues treated with or without 5-azacytidine, a powerful inhibitor of DNA methylation. To determine the physiological role of rgp1, the coding region was introduced into tobacco plants. Transformants, with rgp1 in either sense or antisense orientations, showed distinct phenotypic changes with reduced apical dominance, dwarfism, and abnormal flower development. These abnormal phenotypes appeared to be associated with the higher levels of endogenous cytokinins that were 6-fold those of wild-type plants. In addition, the transgenic plants produced salicylic acid and salicylic acid-beta-glucoside in an unusual response to wounding, thus conferring increased resistance to tobacco mosaic virus infection. In normal plants, the wound- and pathogen-induced signal-transduction pathways are considered to function independently. However, the wound induction of salicylic acid in the transgenic plants suggests that expression of rgp1 somehow interfered with the normal signaling pathways and resulted in cross-signaling between these distinct transduction systems. The results imply that the defense signal-transduction system consists of a complicated and finely tuned network of several regulatory factors, including cytokinins, salicylic acid, and small GTP-binding proteins.

Dominância funcional e monitoramento da resistência de Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) a tecnologias Bt no Brasil

Plantas transgênicas que expressam toxinas de Bacillus thuringiensis Berliner (Bt) têm sido amplamente utilizadas para o controle de Spodoptera frugiperda (J. E. Smith) no Brasil. Entretanto, a evolução da resistência é um dos maiores entraves para a continuidade do uso desta tecnologia. Para subsidiar programas de Manejo da Resistência de Insetos (MRI), foram conduzidos estudos para o aprimoramento dos programas de manejo da resistência de S. frugiperda a tecnologias Bt. Foram realizadas estudos para determinar a dominância funcional da resistência de S. frugiperda a tecnologias Bt mediante a avaliação da sobrevivência de larvas neonatas provenientes das linhagens de S. frugiperda resistentes ao milho Herculex® que expressa a proteína Cry1F (HX-R), ao milho YieldGard VT PRO™ que expressa as proteínas Cry1A.105 e Cry2Ab2 (VT-R), ao milho PowerCore™ que expressa as proteínas Cry1A.105, Cry2Ab2 e Cry1F (PW-R), e ao milho Agrisure Viptera™ que expressa a proteína Vip3Aa20 (Vip-R), além da linhagem suscetível (Sus) e de suas respectivas linhagens heterozigotas em diversas tecnologias de milho e algodão Bt. Posteriormente, um método prático para o monitoramento fenotípico da suscetibilidade a diferentes tecnologias de milho e algodão Bt foi testado a partir da avaliação da sobrevivência de larvas neonatas em folhas de plantas Bt em populações de S. frugiperda provenientes dos Estados do Rio Grande do Sul, Paraná, São Paulo, Goiás e Bahia na safra agrícola 2014/15. E por último, a estimativa da frequência de alelos de resistência de S. frugiperda a Vip3Aa20 foi validada pelo método de F1 screen. Em geral, observou-se alta mortalidade dos heterozigotos nas tecnologias Bt testadas, comprovando que a resistência de S. frugiperda a proteínas Bt é funcionalmente recessiva o que suporta a estratégia de refúgio em programas de MRI. Verificou-se também que linhagens resistentes a eventos que expressam proteínas Cry não sobrevivem em tecnologias que expressam proteína Vip. No monitoramento prático da suscetibilidade a tecnologias Bt, sobrevivência larval superior a 70% foi observada para populações de campo do Paraná, Goiás e Bahia no milho Herculex®. Em tecnologias de milho PowerCore™ e YieldGard VT PRO™ houve sobrevivência larval variando de 1,1 a 17,9%. Em contraste, não houve sobreviventes em tecnologias de milho Viptera™. Em algodão WideStrike® que expressa as proteínas Cry1Ac e Cry1F, sobrevivência acima de 41% foi observada para populações de campo de S. frugiperda. A sobrevivência larval em Bollgard II® que expressa as proteínas Cry1Ac e Cry2Ab2 variou de 14 a 40%. No algodão TwinLink® que expressa as proteínas Cry1Ab e Cry2Ae, a sobrevivência larval das populações foi menor que 20%. O método de F1 screen foi eficiente na detecção de alelos de resistência a Vip3Aa20 em populações de S. frugiperda provenientes de diferentes regiões produtoras de milho no Brasil na safra 2014/2015. De 263 isofamílias testadas, foram detectadas três isofamílias positivas oriundas do Paraná, Mato Grosso e Goiás. A frequência de resistência estimada a Vip3Aa20 variou de 0,0140 a 0,0367 nas populações avaliadas, sendo que a frequência total foi de 0,0076. Neste estudo, fornecemos informações para refinar as estratégias de MRI, além de introduzir novas técnicas para monitorar a resistência de S. frugiperda a tecnologias Bt no Brasil.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

Herbicides cross resistance of a multiple resistant short-awn foxtail ( Alopecurus aequalis Sobol.) population in wheat field

Alopecurus aequalis Sobol. is a common grass weed, which has become increasingly troublesome to control in China wheat fields. One A. aequalis population, collected from Anhui Province China, was suspected to be resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl. This study aimed to establish the cross-resistance pattern using the purified subpopulation and explore the potential targetsite and non-target-site based resistance mechanisms. Sequencing results showed that a single nucleotide change of ATT to AAT was present in acetyl-CoA carboxylase (ACCase) gene of the resistant (R) plants, resulting in an Ile2041Asn amino acid substitution. Besides, another single nucleotide change of CCC to CGC was present in acetolactate synthase (ALS) gene of the R plants, resulting in a Pro197Arg amino acid substitution. The homozygous resistant plants were isolated and the seeds were used in whole-plant herbicide bioassays. Compared with the susceptible (S) population, R population displayed high level resistance to fenoxaprop-P-ethyl and mesosulfuronmethyl. Cross resistance patterns showed that the R population was highly resistant to clodinafop-propargyl, moderately resistant to pyroxsulam and flucarbazoncsodium, lowly resistant to pinoxaden, and susceptible to tralkoxydim, sethoxydim, and isoproturon. The pretreatment of piperonyl butoxide reduced the 50% growth reduction (GR50) value of fenoxaprop-P-ethyl, suggesting that target-site resistance and non-target-site resistance mechanisms were both present in fenoxaprop- P-ethyl-resistance of A. aequalis. This is the first report of ACCase Ile2041Asn and ALS Pro197Arg mutation in A. aequalis.

Sci1 Is A Component Of The Auxin-dependent Control Of Cell Proliferation In Arabidopsis Upper Pistil.

To characterize the recently described SCI1 (stigma/style cell cycle inhibitor 1) gene relationship with the auxin pathway, we have taken the advantage of the Arabidopsis model system and its available tools. At first, we have analyzed the At1g79200 T-DNA insertion mutants and constructed various transgenic plants. The loss- and gain-of-function plants displayed cell number alterations in upper pistils that were controlled by the amino-terminal domain of the protein. These data also confirmed that this locus holds the functional homolog (AtSCI1) of the Nicotiana tabacum SCI1 gene. Then, we have provided some evidences the auxin synthesis/signaling pathways are required for downstream proper AtSCI1 control of cell number: (a) its expression is downregulated in yuc2yuc6 and npy1 auxin-deficient mutants, (b) triple (yuc2yuc6sci1) and double (npy1sci1) mutants mimicked the auxin-deficient phenotypes, with no synergistic interactions, and (c) the increased upper pistil phenotype in these last mutants, which is a consequence of an increased cell number, was able to be complemented by AtSCI1 overexpression. Taken together, our data strongly suggests SCI1 as a component of the auxin signaling transduction pathway to control cell proliferation/differentiation in stigma/style, representing a molecular effector of this hormone on pistil development.

Sugarcane genes associated with sucrose content

Background -: Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants. Results -: We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways. Conclusion -: Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.

In vitro organogenesis of Passiflora alata

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO(3) incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA(3) and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.

High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca

Albicidins are a family of phytotoxins and antibiotics which play an important role in the pathogenesis of sugarcane leaf scald disease. The albA gene from Klebsiella oxytoca encodes a protein which inactivates albicidin by heat-reversible binding. Albicidin ligand binding to a recombinant AlbA protein, purified by means of a glutathione S-transferase gene fusion system, is an almost instant and saturable reaction. Kinetic and stoichiometric analysis of the binding reaction indicated the presence of a single high affinity binding site with a dissociation constant of 6.4 x 10(-8) M. The AlbA-albicidin complex is stable from 4 to 40 degrees C, from ph 5 to 9 and in high salt solutions. Treatment with protein denaturants released all bound albicidin. These properties indicate that AlbA may be a useful affinity matrix for selective purification of albicidin antibiotics. AlbA does not bind to p-nitrophenyl butyrate or alpha-naphthyl butyrate, the substrates of the albicidin detoxification enzyme AlbD from Pantoea dispersa. The potential exists to pyramid genes for different mechanisms in transgenic plants to protect plastid DNA replication from inhibition by albicidins.

Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm(2) embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T-1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).

Engineered detoxification confers resistance against a pathogenic bacterium

We generated transgenic sugarcane plants that express an albicidin detoxifying gene (albD), which was cloned from a bacterium that provides biocontrol against leaf scald disease. Plants with albicidin detoxification capacity equivalent to 1-10 ng of AlbD enzyme per mg of leaf protein did not develop chlorotic disease symptoms in inoculated leaves, whereas all untransformed control plants developed severe symptoms. Transgenic lines with high AlbD activity in young stems were also protected against systemic multiplication of the pathogen, which is the precursor to economic disease. We have shown that genetic modification to express a toxin-resistance gene can confer resistance to both disease symptoms and multiplication of a toxigenic pathogen in its host.