Functional Genomic Approaches for Deciphering Plant-Virus Interactions

Plant-virus interactions are very complex in nature and lead to disease and symptom formation by causing various physiological, metabolic and developmental changes in the host plants. These interactions are mainly the outcomes of viral hijacking of host components to complete their infection cycles and of host defensive responses to restrict the viral infections. Viral genomes contain only a small number of genes often encoding for multifunctional proteins, and all are essential in establishing a viral infection. Thus, it is important to understand the specific roles of individual viral genes and their contribution to the viral life cycles. Among the most important viral proteins are the suppressors of RNA silencing (VSRs). These proteins function to suppress host defenses mediated by RNA silencing and can also serve in other functions, e.g. in viral movement, transactivation of host genes, virus replication and protein processing. Thus these proteins are likely to have a significant impact on host physiology and metabolism. In the present study, I have examined the plant-virus interactions and the effects of three different VSRs on host physiology and gene expression levels by microarray analysis of transgenic plants that express these VSR genes. I also studied the gene expression changes related to the expression of the whole genome of Tobacco mosaic virus (TMV) in transgenic tobacco plants. Expression of the VSR genes in the transgenic tobacco plants causes significant changes in the gene expression profiles. HC-Pro gene derived from the Potyvirus Y (PVY) causes alteration of 748 and 332 transcripts, AC2 gene derived from the African cassava mosaic virus (ACMV) causes alteration of 1118 and 251transcripts, and P25 gene derived from the Potyvirus X (PVX) causes alterations of 1355 and 64 transcripts in leaves and flowers, respectively. All three VSRs cause similar up-regulation in defense, hormonally regulated and different stress-related genes and down-regulation in the photosynthesis and starch metabolism related genes. They also induce alterations that are specific to each viral VSR. The phenotype and transcriptome alterations of the HC-Pro expressing transgenic plants are similar to those observed in some Potyvirus-infected plants. The plants show increased protein degradation, which may be due to the HC-Pro cysteine endopeptidase and thioredoxin activities. The AC2-expressing transgenic plants show a similar phenotype and gene expression pattern as HC-Pro-expressing plants, but also alter pathways related to jasmonic acid, ethylene and retrograde signaling. In the P25 expressing transgenic plants, high numbers of genes (total of 1355) were up-regulated in the leaves, compared to a very low number of down-regulated genes (total of 5). Despite of strong induction of the transcripts, only mild growth reduction and no other distinct phenotype was observed in these plants. As an example of whole virus interactions with its host, I also studied gene expression changes caused by Tobacco mosaic virus (TMV) in tobacco host in three different conditions, i.e. in transgenic plants that are first resistant to the virus, and then become susceptible to it and in wild type plants naturally infected with this virus. The microarray analysis revealed up and down-regulation of 1362 and 1422 transcripts in the TMV resistant young transgenic plants, and up and down-regulation of a total of 1150 and 1200 transcripts, respectively, in the older plants, after the resistance break. Natural TMV infections in wild type plants caused up-regulation of 550 transcripts and down-regulation of 480 transcripts. 124 up-regulated and 29 down-regulated transcripts were commonly altered between young and old TMV transgenic plants, and only 6 up-regulated and none of the down-regulated transcripts were commonly altered in all three plants. During the resistant stage, the strong down-regulation in translation-related transcripts (total of 750 genes) was observed. Additionally, transcripts related to the hormones, protein degradation and defense pathways, cell division and stress were distinctly altered. All these alterations may contribute to the TMV resistance in the young transgenic plants, and the resistance may also be related to RNA silencing, despite of the low viral abundance and lack of viral siRNAs or TMV methylation activity in the plants.

Plant species and functional group effects on abiotic and microbial soil properties and plant-soil feedback responses in two grasslands

1 Plant species differ in their capacity to influence soil organic matter, soil nutrient availability and the composition of soil microbial communities. Their influences on soil properties result in net positive or negative feedback effects, which influence plant performance and plant community composition. 2 For two grassland systems, one on a sandy soil in the Netherlands and one on a chalk soil in the United Kingdom, we investigated how individual plant species grown in monocultures changed abiotic and biotic soil conditions. Then, we determined feedback effects of these soils to plants of the same or different species. Feedback effects were analysed at the level of plant species and plant taxonomic groups (grasses vs. forbs). 3 In the sandy soils, plant species differed in their effects on soil chemical properties, in particular potassium levels, but PLFA (phospholipid fatty acid) signatures of the soil microbial community did not differ between plant species. The effects of soil chemical properties were even greater when grasses and forbs were compared, especially because potassium levels were lower in grass monocultures. 4 In the chalk soil, there were no effects of plant species on soil chemical properties, but PLFA profiles differed significantly between soils from different monocultures. PLFA profiles differed between species, rather than between grasses and forbs. 5 In the feedback experiment, all plant species in sandy soils grew less vigorously in soils conditioned by grasses than in soils conditioned by forbs. These effects correlated significantly with soil chemical properties. None of the seven plant species showed significant differences between performance in soil conditioned by the same vs. other plant species. 6 In the chalk soil, Sanguisorba minor and in particular Briza media performed best in soil collected from conspecifics, while Bromus erectus performed best in soil from heterospecifics. There was no distinctive pattern between soils collected from forb and grass monocultures, and plant performance could not be related to soil chemical properties or PLFA signatures. 7 Our study shows that mechanisms of plant-soil feedback can depend on plant species, plant taxonomic (or functional) groups and site-specific differences in abiotic and biotic soil properties. Understanding how plant species can influence their rhizosphere, and how other plant species respond to these changes, will greatly enhance our understanding of the functioning and stability of ecosystems.

Simple measures of climate, soil properties and plant traits predict national scale grassland soil carbon stocks

1. Soil carbon (C) storage is a key ecosystem service. Soil C stocks play a vital role in soil fertility and climate regulation, but the factors that control these stocks at regional and national scales are unknown, particularly when their composition and stability are considered. As a result, their mapping relies on either unreliable proxy measures or laborious direct measurements. 2. Using data from an extensive national survey of English grasslands we show that surface soil (0-7cm) C stocks in size fractions of varying stability can be predicted at both regional and national scales from plant traits and simple measures of soil and climatic conditions. 3. Soil C stocks in the largest pool, of intermediate particle size (50-250 µm), were best explained by mean annual temperature (MAT), soil pH and soil moisture content. The second largest C pool, highly stable physically and biochemically protected particles (0.45-50 µm), was explained by soil pH and the community abundance weighted mean (CWM) leaf nitrogen (N) content, with the highest soil C stocks under N rich vegetation. The C stock in the small active fraction (250-4000 µm) was explained by a wide range of variables: MAT, mean annual precipitation, mean growing season length, soil pH and CWM specific leaf area; stocks were higher under vegetation with thick and/or dense leaves. 4. Testing the models describing these fractions against data from an independent English region indicated moderately strong correlation between predicted and actual values and no systematic bias, with the exception of the active fraction, for which predictions were inaccurate. 5. Synthesis and Applications: Validation indicates that readily available climate, soils and plant survey data can be effective in making local- to landscape-scale (1-100,000 km2) soil C stock predictions. Such predictions are a crucial component of effective management strategies to protect C stocks and enhance soil C sequestration.

Structural and functional leaf traits of two Gochnatia species from distinct growth forms in a sclerophyll forest site in Southeastern Brazil

O gênero Gochnatia é comumente encontrado em diferentes fitofisionomias do Cerrado do Estado de São Paulo, crescendo desde ambientes mais abertos até áreas florestais mais fechadas. Aqui foram comparadas a anatomia foliar e alguns parâmetros ecofisiológicos de duas espécies do gênero Gochnatia, uma arbustiva (Gochnatia barrosii Cabrera) e a outra arbórea (Gochnatia polymorpha (Less.) Cabrera), ambas ocorrendo em área de cerradão na Estação Ecológica de Assis, SP. Encontraram-se diferenças estruturais qualitativas entre as espécies, com G. barrosii apresentando folhas anfiestomáticas, com epiderme unisseriada e G. polymorpha apresentando folhas hipoestomáticas, com epiderme múltipla ou hipoderme, na face adaxial. Além disso, as folhas de G. barrosii apresentaram menores valores para a espessura dos tecidos (com exceção da epiderme na face abaxial) e da folha em relação a G. polymorpha. Foram observadas diferenças na assimilação de CO2 tanto em base de área quanto de massa seca foliar, além de diferenças na área foliar específica, sendo esta maior em G. barrosii. Apesar das folhas de G. barrosii possuírem estrutura bem menos escleromorfa do que as folhas de G. polymorpha, não foram encontradas diferenças na eficiência do uso de água. Os resultados sugerem que espécies de formas distintas de crescimento de um mesmo gênero possuem características foliares diferenciadas para lidar com as variações ambientais a que são submetidas.

Root, shoot and leaf traits of the congeneric Styrax species may explain their distribution patterns in the cerrado sensu lato areas in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

ZmPUMP encodes a fully functional monocot plant uncoupling mitochondrial protein whose affinity to fatty acid is increased with the introduction of a His pair at the second matrix loop

Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins that uncouple respiration from ATP synthesis. In this study, cDNA encoding maize uncoupling protein (ZmPUMP) was expressed in Escherichia coli and recombinant ZmPUMP reconstituted in liposomes. ZmPUMP activity was associated with a linoleic acid (LA)-mediated H+ efflux with Km of 56.36 ± 0.27 μM and Vmax of 66.9 μmol H+ min-1 (mg prot)-1. LA-mediated H+ fluxes were sensitive to ATP inhibition with Ki of 2.61 ± 0.36 mM (at pH 7.2), a value similar to those for dicot UCPs. ZmPUMP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in plant UCPs. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism. © 2006 Elsevier Inc. All rights reserved.

Association of IGF1 and KDM5A polymorphisms with performance, fatness and carcass traits in chickens

Two functional and positional candidate genes were selected in a region of chicken chromosome 1 (GGA1), based on their biological roles, and also where several quantitative trait loci (QTL) have been mapped and associated with performance, fatness and carcass traits in chickens. The insulin-like growth factor 1 (IGF1) gene has been associated with several physiological functions related to growth. The lysine (K)-specific demethylase 5A (KDM5A) gene participates in the epigenetic regulation of genes involved with the cell cycle. Our objective was to find associations of selected single-nucleotide polymorphisms (SNPs) in these genes with performance, fatness and carcass traits in 165 F2 chickens from a resource population. In the IGF1 gene, 17 SNPs were detected, and in the KDM5A gene, nine SNPs were detected. IGF1 SNP c. 47673G > A was associated with body weight and haematocrit percentage, and also with feed intake and percentages of abdominal fat and gizzard genotype × sex interactions. KDM5A SNP c. 34208C > T genotype × sex interaction affected body weight, feed intake, percentages of abdominal fat (p = 0. 0001), carcass, gizzard and haematocrit. A strong association of the diplotype × sex interaction (p < 0. 0001) with abdominal fat was observed, and also associations with body weight, feed intake, percentages of carcass, drums and thighs, gizzard and haematocrit. Our findings suggest that the KDM5A gene might play an important role in the abdominal fat deposition in chickens. The IGF1 and KDM5A genes are strong candidates to explain the QTL mapped in this region of GGA1. © 2012 Institute of Plant Genetics, Polish Academy of Sciences, Poznan.

Development of a thermoeconomic methodology for the optimization of biodiesel production - Part I: Biodiesel plant and thermoeconomic functional diagram

This work developed a methodology that uses the thermoeconomic functional diagram applied for allocating the cost of products produced by a biodiesel plant. The first part of this work discusses some definitions of exergy and thermoeconomy, with a detailed description of the biodiesel plant studied, identification of the system functions through Physical Diagram, calculation of the irreversibilities of the plant, construction of the Thermoeconomic Functional Diagram and determination of the expressions for the plant's exergetic functions. In order to calculate the exergetic increments and the physical exergy of certain flows in each step, the Chemical Engineering Simulation Software HYSYS 3.2 was used. The equipments that have the highest irreversibilities in the plant were identified after the exergy calculation. It was also found that the lowest irreversibility in the system refers to the process with a molar ratio of 6:1 and a reaction temperature of 60 °C in the transesterification process. In the second part of this work (Part II), it was calculated the thermoeconomic cost of producing biodiesel and related products, including the costs of carbon credits for the CO2 that is not released into the atmosphere, when a percentage of biodiesel is added to the petroleum diesel used by Brazil's internal diesel fleet (case study). © 2013 Elsevier Ltd. All rights reserved.

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. Structured digital abstract XfDsbC and XfDsbC bind by x ray scattering (View Interaction: 1, 2) XfDsbC and XfDsbC bind by molecular sieving (View interaction) XfDsbC and XfDsbC bind by comigration in non denaturing gel electrophoresis (View interaction) XfDsbC and XfDsbC bind by cross-linking study (View Interaction: 1, 2) XfDsbC and XfDsbC bind by dynamic light scattering (View Interaction: 1, 2)

Functional markers for gene mapping and genetic diversity studies in sugarcane

Abstract Background The database of sugarcane expressed sequence tags (EST) offers a great opportunity for developing molecular markers that are directly associated with important agronomic traits. The development of new EST-SSR markers represents an important tool for genetic analysis. In sugarcane breeding programs, functional markers can be used to accelerate the process and select important agronomic traits, especially in the mapping of quantitative traits loci (QTL) and plant resistant pathogens or qualitative resistance loci (QRL). The aim of this work was to develop new simple sequence repeat (SSR) markers in sugarcane using the sugarcane expressed sequence tag (SUCEST database). Findings A total of 365 EST-SSR molecular markers with trinucleotide motifs were developed and evaluated in a collection of 18 genotypes of sugarcane (15 varieties and 3 species). In total, 287 of the EST-SSRs markers amplified fragments of the expected size and were polymorphic in the analyzed sugarcane varieties. The number of alleles ranged from 2-18, with an average of 6 alleles per locus, while polymorphism information content values ranged from 0.21-0.92, with an average of 0.69. The discrimination power was high for the majority of the EST-SSRs, with an average value of 0.80. Among the markers characterized in this study some have particular interest, those that are related to bacterial defense responses, generation of precursor metabolites and energy and those involved in carbohydrate metabolic process. Conclusions These EST-SSR markers presented in this work can be efficiently used for genetic mapping studies of segregating sugarcane populations. The high Polymorphism Information Content (PIC) and Discriminant Power (DP) presented facilitate the QTL identification and marker-assisted selection due the association with functional regions of the genome became an important tool for the sugarcane breeding program.

Heterosis for biomass-related traits in Arabidopsis investigated by quantitative trait loci analysis of the triple testcross design with recombinant inbred lines

Arabidopsis thaliana has emerged as a leading model species in plant genetics and functional genomics including research on the genetic causes of heterosis. We applied a triple testcross (TTC) design and a novel biometrical approach to identify and characterize quantitative trait loci (QTL) for heterosis of five biomass-related traits by (i) estimating the number, genomic positions, and genetic effects of heterotic QTL, (ii) characterizing their mode of gene action, and (iii) testing for presence of epistatic effects by a genomewide scan and marker x marker interactions. In total, 234 recombinant inbred lines (RILs) of Arabidopsis hybrid C24 x Col-0 were crossed to both parental lines and their F1 and analyzed with 110 single-nucleotide polymorphism (SNP) markers. QTL analyses were conducted using linear transformations Z1, Z2, and Z3 calculated from the adjusted entry means of TTC progenies. With Z1, we detected 12 QTL displaying augmented additive effects. With Z2, we mapped six QTL for augmented dominance effects. A one-dimensional genome scan with Z3 revealed two genomic regions with significantly negative dominance x additive epistatic effects. Two-way analyses of variance between marker pairs revealed nine digenic epistatic interactions: six reflecting dominance x dominance effects with variable sign and three reflecting additive x additive effects with positive sign. We conclude that heterosis for biomass-related traits in Arabidopsis has a polygenic basis with overdominance and/or epistasis being presumably the main types of gene action.