Adriano Marocco

Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response.

Nitric oxide (NO) and reactive oxygen intermediates (ROIs) play key roles in the activation of disease resistance mechanisms both in animals and plants. In animals NO cooperates with ROIs to kill tumor cells and for macrophage killing of bacteria. Such cytotoxic events occur because unregulated NO levels drive a diffusion-limited reaction with O2− to generate peroxynitrite (ONOO−), a mediator of cellular injury in many biological systems. Here we show that in soybean cells unregulated NO production at the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate hypersensitive cell death. The HR is triggered only by balanced production of NO and ROIs. Moreover, hypersensitive cell death is activated after interaction of NO not with O2− but with H2O2 generated from O2− by superoxide dismutase. Increasing the level of O2− reduces NO-mediated toxicity, and ONOO− is not a mediator of hypersensitive cell death. During the HR, superoxide dismutase accelerates O2− dismutation to H2O2 to minimize the loss of NO by reaction with O2− and to trigger hypersensitive cell death through NO/H2O2 cooperation. However, O2− rather than H2O2 is the primary ROI signal for pathogen induction of glutathione S-transferase, and the rates of production and dismutation of O2− generated during the oxidative burst play a crucial role in the modulation and integration of NO/H2O2 signaling in the HR. Thus although plants and animals use a similar repertoire of signals in disease resistance, ROIs and NO are deployed in strikingly different ways to trigger host cell death.

Logistic regression modeling of cropping systems to predict fumonisin contamination in maize.

The aims of this research were to monitor the presence of fumonisins in maize crops in northern Italy over a 6 year period, to study the role of the cropping system on fumonisin levels, and to contribute to the development of a predictive system for fumonisin contamination. In the 6 year period from 2002 to 2007, 438 maize samples were collected in five regions, supported by agronomic data, and analyzed for fumonisin content. Fumonisin was detected in almost all of the grain samples, but 2007 was less and 2005 more contaminated compared to the other years. Preceding crop, maturity class of hybrids, nitrogen fertilization, sowing and harvest week, and grain moisture significantly affected the level of contamination. The logistic regression developed explained around 60% of variability with major roles for longitude, maturity class, and growing weeks. The function can be used to quantify the effect of these factors in a predictive system.

Impaired auxin biosynthesis in the defective endosperm18 mutant is due to mutational loss of expression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein in maize.

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.

Alternative weed control using the allelopathic effect of natural benzoxazinoids from rye mulch

In conventional agriculture, weed control by herbicides is an expensive practice and can also have a negative effect on the environment. Allelopathy permits sustainable weed management while reducing the impact of agriculture on the environment. We studied the content of 2,4-dihydroxy-1,4 (2H)-benzoxazin-3-one (DIBOA) and benzoxazolin-2(3H)-one (BOA), indicated as benzoxazinoids and considered effective for weed control, in 8 cultivars of rye and 1 of triticale grown in a greenhouse. We also tested the ability of mulches to inhibit the germination of four warm-season weeds. Our results show that all rye cultivars produced DIBOA, while BOA was found only in four of them. Benzoxazinoids were absent in triticale. Total benzoxazinoid content ranged from 177 to 545 μg g−1 and was statistically different among cultivars. Rye mulches were not able to suppress velvetleaf and common lambsquarters seedlings, while redroot pigweed and common purslane were significantly affected. Weed suppression ranged from 40% to 52% for redroot pigweed and from 40% to 74% for common purslane. The inhibitory activity of triticale mulch was observed only for common purslane, with a suppression percentage of 33%. No correlation was found between total benzoxazinoid content and the number of weed seedlings suppressed, with R2 of 0.076 for redroot pigweed and R2 of 0.003 for common purslane, indicating that benzoxazinoids are not the only source of phytotoxicity.

A resource of mapped dissociation launch pads for targeted insertional mutagenesis in the Arabidopsis genome

We describe a new resource for targeted insertional mutagenesis in Arabidopsis using a maize (Zea mays) Activator/Dissociation (Ds) two-element system. The two components of the system, T-DNA vectors carrying a Ds launch pad and a stable Activator transposase source, were designed to simplify selection of transposition events and maximize their usefulness. Because Ds elements preferentially transpose to nearby genomic sites, they can be used in targeted mutagenesis of linked genes. To efficiently target all genes throughout the genome, we generated a large population of transgenic Arabidopsis plants containing the Ds launch pad construct, identified lines containing single Ds launch pad inserts, and mapped the positions of Ds launch pads in 89 lines. The integration sites of the Ds launch pads were relatively evenly distributed on all five chromosomes, except for a region of chromosomes 2 and 4 and the centromeric regions. This resource therefore provides access to the majority of the Arabidopsis genome for targeted tagging.

A first linkage map of Cichorium intybus L. using a one-way pseudo-testcross and PCR-derived markers

We have used a one-way pseudo-testcross mapping strategy in combination with different types of PCR-based markers (RAPD, AFLP, SAMPL) to construct a first linkage map for variegated chicory (Cichorium intybus L. var. silvestre Biskoff, n=9), a self-incompatible vegetable species. The success of such a strategy depends on the presence of sufficiently high levels of heterozygosity in the individual plant which is being mapped and on the informativeness of the marker system that is used. A total of 371 markers, comprising 16 RAPDs, 72 SAMPLs and 283 AFLPs, were scored in 46 F1 individuals obtained from an interspecific cross between a C. intybus outbred individual and a C. endivia inbred line. Grouping of the markers at a LOD score of 4.0 resulted in 13 linkage groups covering 1330 cM. A framework map covering 1201.4 cM was assembled by using all markers that could be ordered with a LOD greater than 2.0. We estimate the total genome size of chicory to be ca. 1405 cM, thus considerably smaller than that estimated for lettuce (1950 cM). The usefulness of the different marker systems that were applied is analysed in terms of level of heterozygosity and marker index, i.e. number of different genetic loci that may be simultaneously analysed per experiment. Out of the 371 markers, 50 of them showed segregation distortion which is discussed in terms of the hybrid origin of the variegated chicory.

Defense responses to mycotoxin-producing fungi Fusarium proliferatum, F. subglutinans, and Aspergillus flavus in kernels of susceptible and resistant maize genotypes

Developing kernels of resistant and susceptible maize genotypes were inoculated with Fusarium proliferatum, F. subglutinans, and Aspergillus flavus. Selected defense systems were investigated using real-time reverse transcription-polymerase chain reaction to monitor the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes protective from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) at 72 h postinoculation. The study was also extended to the analysis of the ascorbate-glutathione cycle and catalase, superoxide dismutase, and cytosolic and wall peroxidases enzymes. Furthermore, the hydrogen peroxide and malondialdehyde contents were studied to evaluate the oxidation level. Higher gene expression and enzymatic activities were observed in uninoculated kernels of resistant line, conferring a major readiness to the pathogen attack. Moreover expression values of PR genes remained higher in the resistant line after inoculation, demonstrating a potentiated response to the pathogen invasions. In contrast, reactive oxygen species-scavenging genes were strongly induced in the susceptible line only after pathogen inoculation, although their enzymatic activity was higher in the resistant line. Our data provide an important basis for further investigation of defense gene functions in developing kernels in order to improve resistance to fungal pathogens. Maize genotypes with overexpressed resistance traits could be profitably utilized in breeding programs focused on resistance to pathogens and grain safety.

Use of a custom array to study differentially expressed genes during blood orange (Citrus sinensis L. Osbeck) ripening

A flesh-specific oligonucleotide custom array was designed to study gene expression during blood orange ripening. The array included 301 probes derived from a subtracted SSH library, a cDNA-AFLP collection, and a set of regulatory genes from the Harvest citrus database. The custom array was hybridized using samples of Moro, a pigmented cultivar, and Cadenera, a common cultivar, at three different ripening stages: the immature phase, the halfway point of maturation (corresponding to the start of Moro pigmentation) and the full ripening. Of the 301 probes, 27 in total, corresponding to 20 different transcripts, indicated differential expression in stage-to-stage and/or cultivar-to-cultivar comparisons. Transcripts encoding for anthocyanin biosynthesis represented most of the total over-expressed probes. The remaining differentially expressed transcripts were functionally associated with primary metabolism as flavor biosynthesis, defense and signal transduction. The expressed products associated with probes indicating differential expression were confirmed by qRT-PCR. The microarray was designed considering a small collection of sequences useful for monitoring specific pathways and regulatory genes related to fruit ripening and anthocyanin pigmentation. The main novelty of this customization is the use of expressed sequences specifically derived from blood orange flesh to study different cultivars and ripening stages, and the provision of further information about processes related to anthocyanin pigmentation in citrus fruit flesh.

Resistance to Fusarium verticillioides and fumonisin accumulation in maize inbred lines involves an earlier and enhanced expression of lipoxygenase (LOX) genes.

Fusarium verticillioides causes ear rot in maize and contaminates the kernels with the fumonisin mycotoxins. It is known that plant lipoxygenase (LOX)-derived oxylipins regulate defence against pathogens and that the host-pathogen lipid cross-talk influences the pathogenesis. The expression profiles of fifteen genes of the LOX pathway were studied in kernels of resistant and susceptible maize lines, grown in field condition, at 3, 7 and 14 days post inoculation (dpi) with F. verticillioides. Plant defence responses were correlated with the pathogen growth, the expression profiles of fungal FUM genes for fumonisin biosynthesis and fumonisin content in the kernels. The resistant genotype limited fungal growth and fumonisin accumulation between 7 and 14 dpi. Pathogen growth became exponential in the susceptible line after 7 dpi, in correspondence with massive transcription of FUM genes and fumonisins augmented exponentially at 14 dpi. LOX pathway genes resulted strongly induced after pathogen inoculation in the resistant line at 3 and 7 dpi, whilst in the susceptible line the induction was reduced or delayed at 14 dpi. In addition, all genes resulted overexpressed before infection in kernels of the resistant genotype already at 3 dpi. The results suggest that resistance in maize may depend on an earlier activation of LOX genes and genes for jasmonic acid biosynthesis.

Evaluation of broad spectrum sources of resistance to Fusarium verticillioides and advanced maize breeding lines

The fungus Fusarium verticillioides is commonly associated with maize production in temperate regions of the world, producing ear rot and grain contamination by fumonisins. Genetic resistance is the best preventive action against fumonisin contamination, although at present no commercial maize hybrids are completely resistant. Several studies of the relationship between Fusarium and other species producing ear rot suggest that these fungal species interact in similar ways with the host plant. Consequently, host plant resistance to one pathogen could be associated with resistance to another. The aim of this study was to introduce sources of resistance to Fusarium spp. into maize inbred lines and to evaluate ear rot severity and fumonisin B1 contamination in advanced breeding lines and hybrids after artificial and natural infection with F. verticillioides. Two inbred lines (CO430 and MP420) with resistance to kernel infection by Fusarium graminearum and Aspergillus flavus, respectively, were crossed and bac...