Characterization and nutritional valorization of agricultural waste corncobs from Italian maize landraces through the growth of medicinal mushrooms.

The research investigates the potential use of maize cobs (or corncobs) from five genotypes, including the B73 inbred line and four locally cultivated landraces from Northern Italy, as substrate for implementing Solid State fermentation processes with four Medicinal Mushrooms (MMs). The corncobs were characterized based on their proximate composition, lignin, phenolics content (both free and bound), and total antioxidant capacity. Among the MMs tested, Pleurotus ostreatus and Ganoderma annularis demonstrated the most robust performance. Their growth was parametrized using Image Analysis technique, and chemical composition of culture samples was characterized compared to that of corncobs alone. In all culture samples, the growth of MMs led to a significant reduction (averaging 40%) in the total phenolics contents compared to that measured in corncobs alone. However, the high content of free phenolics in the cobs negatively impacted the growth of P. ostreatus. The final MM-corncob matrix exhibited reduced levels of free sugars and starch (≤ 2.2% DW, as a sum) and increased levels of proteins (up to 5.9% DW) and soluble dietary fiber (up to 5.0% DW), with a notable trend toward higher levels of ß-glucan compared to corncobs alone. This research paves the way for the use of this matrix as an active ingredient to enhance the nutritional value of food preparations.

Phenotypic, genetic and molecular characterization of a maize low phytic acid mutant (lpa241).

Phytic acid, myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the major storage compound of phosphorous (P) in plants, predominantly accumulating in seeds (up to 4-5% of dry weight) and pollen. In cereals, phytic acid is deposited in embryo and aleurone grain tissues as a mixed "phytate" salt of potassium and magnesium, although phytates contain other mineral cations such as iron and zinc. During germination, phytates are broken down by the action of phytases, releasing their P, minerals and myo-inositol which become available to the growing seedling. Phytic acid represents an anti-nutritional factor for animals, and isolation of maize low phytic acid ( lpa) mutants provides a novel approach to study its biochemical pathway and to tackle the nutritional problems associated with it. Following chemical mutagenesis of pollen, we have isolated a viable recessive mutant named lpa 241 showing about 90% reduction of phytic acid and about a tenfold increase in seed-free phosphate content. Although germination rate was decreased by about 30% compared to wild-type, developement of mutant plants was apparentely unaffected. The results of the genetic, biochemical and molecular characterization experiments carried out by SSR mapping, MDD-HPLC and RT-PCR are consistent with a mutation affecting the MIPS1S gene, coding for the first enzyme of the phytic acid biosynthetic pathway.

The developmental expression of the maize regulatory gene Hopi determines germination-dependent anthocyanin accumulation.

The Hopi gene is a member of the maize r1 gene family. By genetic and molecular analyses we report that Hopi consists of a single gene residing on chromosome 10 approximately 4.5 cM distal to r1. Hopi conditions anthocyanin deposition in aleurone, scutellum, pericarp, root, mesocotyl, leaves, and anthers, thus representing one of the broadest specifications of pigmentation pattern reported to date of all the r1 genes. A unique feature of the Hopi gene is that seeds are completely devoid of pigment at maturity but show a photoinducible germination-dependent anthocyanin accumulation in aleurone and scutellum. Our analysis has shown that the Hopi transcript is not present in scutellum of developing seeds but is induced only upon germination and that the simultaneous presence of both C1 and Hopi mRNAs is necessary to achieve A1 activation in scutella. We conclude that the expression pattern of the Hopi gene accounts for the germination-dependent anthocyanin synthesis in scutella, whereas the developmental competence of germinating seeds to induce anthocyanin production in scutella results from the combination of the light-inducible expression of C1 and the developmentally regulated expression of the Hopi gene.

A maize anthocyanin transactivator induces pigmentation in hairy roots of dicotyledonous species.

Several dicotyledonous species were infected with an Agrobacterium rhizogenes binary vector harbouring the plasmid 121.Sn which contains the maize gene Sn under the constitutive promoter CaMV35S. In maize, Sn transactivates the anthocyanin pathway in different tissues. The aim of this work was to test the efficiency of this gene to regulate the anthocyanin pathway in heterologous systems and verify its suitability as a reporter gene. The pigmentation of the hairy roots was compared with hairy roots stained for ß-glucuronidase activity, which were used as a control. In two polymorphic genotypes of Lotus angustissimus, DNA integration and expression were assayed. The maize gene is competent to induce anthocyanin pigmentation in different species, but the complexity of the regulatory mechanisms of anthocyanin synthesis restricts the use of Sn as a reporter gene.

Ectopic anthocyanin pigmentation in maize as a tool for defining interactions between homologous regulatory factors.

The duplicated R and Sn genes are involved in the regulation of the maize anthocyanin biosynthetic pathway, encoding tissue-specific products that are homologous to the helix-loop-helix transcriptional activators. Sn determines the pigmentation of the mesocotyl, leaf basis and pericarp, while R determines pigmentation in various tissues, but not in the mesocotyl. In the progeny derived from test-crosses of R/Sn heterozygous plants, a high frequency of R plants exhibiting mesocotyl pigmentation was observed; these derivatives were defined as R*. In R* plants, the presence of this novel trait was not accompanied by the acquisition of Sn or by gross DNA rearrangements in the R profile. Accordingly, RT-PCR analysis showed that mesocotyl pigmentation in R* was attributable to the resident R gene. The occurrence of R* was observed with all R alleles tested, and was enhanced when a P component was present. The heritability of R* was shown only in the case of the standard R-r allele, which carries a functional P component. In addition, we observed that R* can influence other R alleles, transferring the ability to pigment the mesocotyl. R* is unstable, showing a tendency to return to its original state after a few generations. In R* plants there was a correlation between observed ectopic pigmentation and an increase in the level of A1 transcript but, surprisingly, not in the accumulation of R transcript. The results obtained from the analysis of test crosses of rSn/r delta plants suggest that an unlinked genetic factor accounts for the ectopic pigmentation. Concomitant occurrence of epigenetic events might explain the observed instability and reversibility noted above. Further study of this phenomenon might help to elucidate the basis of the interaction between homologous and non-homologous regulators.

Antiparallel expression of the sense and antisense transcripts of maize alpha-tubulin genes.

In all eukaryotes alpha- and beta-tubulins are encoded by small families of closely related genes and are highly conserved. In Zea mays, at least six different alpha-tubulin coding sequences are known. We describe the isolation from scutellar nodes of the maize inbred line W22 of a clone (CTM5) coding for an alpha-tubulin. On the basis of the 3' end nucleotide sequence, this clone can be assigned to the already reported tua4 gene. Northern analysis demonstrates that CTM5 encodes a 1.5 kb transcript, which is expressed in different tissues of the seed and of the seedling. In order to define the spatial and temporal expression of alpha-tubulin genes, in situ hybridization experiments were performed on these tissues. Unexpectedly, a specific signal was detected with both antisense and sense RNA strands. Temporal and spatial distribution of the two RNAs, however, shows that high levels of the two transcripts are always discordant. In tissues where sense transcripts are highly abundant (embryos at various developmental stages, root tips, pollen grains), the antisense transcripts are expressed in relatively small amounts, while in pericarp, coleoptile, leaves, and scutellar node, where antisense transcripts accumulate, the sense transcript only reaches a very low level. Northern analysis using single-stranded DNA probes confirmed the presence of an antisense transcript of 1.5 kb, prompting speculation about the role of this transcript in the regulation of the expression of alpha-tubulin genes.

Molecular homology among members of the R gene family in maize.

The R gene family determines the timing, distribution and amount of anthocyanin pigmentation in maize. This family comprises a set of regulatory genes, consisting of a cluster of several elements at the R locus, on chromosome 10, the Lc and Sn gene lying about two units R distal and B on chromosome 2. Each gene determines a tissue-specific pigmentation of different parts of the seed and plant. The proposed duplicated function of R, Sn, Lc and B loci is reflected in cDNA sequence similarity. In this paper an extensive analysis of the predicted proteins of the R, Sn, Lc and B genes together with a search for putative sites of post-translational modification is reported. A comparison with the prosite database discloses several N-glycosylation and phosphorylation sites, as well as the basic Helix-Loop-Helix (HLH) domain of transcriptional activators. Sn, Lc, and R-S show a high conservation of these sites, while B is more divergent. Analysis of the 5' leader of mRNA sequences discloses the presence of five ATG triplets with two upstream open reading frames (uORFs) of 38 and 15 amino acids and a loop structure indicating a possible mechanism of control at the translational level. It is conceivable that possible mechanisms acting at the translational and post-translational level could modulate the expression and the activation of these transcription factors. Northern analysis of various tissues of different R alleles highlights a strict correlation between pigment accumulation in different tissues and the expression of the regulatory and structural genes suggesting that the pattern of pigmentation relies on a mechanism of differential expression of the members of the R family. Analysis of the Sn promoter discloses the presence of several sequences resembling binding sites of known transcription factors (as GAGA and GT) that might be responsible for the spatial and light-induced expression of this gene. Two regions include a short sequence homologous to the consensus binding site of the B-HLH domain suggesting a self-regulatory control of the Sn gene.

Genetic and molecular analysis of Sn, a light-inducible, tissue specific regulatory gene in maize.

The Sn locus of maize is functionally similar to the R and B loci, in that Sn differentially controls the tissue-specific deposition of anthocyanin pigments in certain seedling and plant cells. We show that Sn shows molecular similarity to the R gene and have used R DNA probes to characterize several Sn alleles. Northern analysis demonstrates that all Sn alleles encode a 2.5 kb transcript, which is expressed in a tissue-specific fashion consistent with the distribution of anthocyanins. Expression of the Sn gene is light-regulated. However, the Sn: bol3 allele allows Sn mRNA transcription to occur in the dark, leading to pigmentation in dark-grown seedlings and cob integuments. We report the isolation of genomic and cDNA clones of the light-independent Sn: bol3 allele. Using Sn cDNA as a probe, the spatial and temporal expression of Sn has been examined. The cell-specific localization of Sn mRNA has been confirmed by in situ hybridization using labelled antisense RNA probes. According to its proposed regulatory role, expression of Sn precedes and, in turn, causes a coordinate and tissue-specific accumulation of mRNA of structural genes for pigment synthesis and deposition, such as A1 and C2. The functional and structural relationship between R, B, Lc and Sn is discussed in terms of an evolutionary derivation from a single ancestral gene which gave rise this diverse gene family by successive duplication events.

Sn, a light-dependent and tissue-specific gene of maize: the genetic basis of its instability.

The genetic system under investigation is defined by three major components: a gene, Sn, conferring tissue specific anthocyanin accumulation in different plant regions, light, required for color development in competent tissues, and another gene, Pl, substituting for light in its capacity to elicit pigment production. Attention is given in this paper to an Sn allele, symbolized Sn:bol3, capable of some constitutive pigmentation in seedlings and seed integuments. Sn:bol3 confers a higher pigment potential than the other alleles and is unstable. Its instability relates to its frequent changes from an original condition, indicated as Sn-s, to Sn-w, where -s and -w stand for strong and weak and refer to the two levels of seedling pigmentation. Weak derivatives arise spontaneously at a high frequency in homo- and heterozygous Sn:bol3 genotypes. In the latter, weak derivatives are also recovered on the chromosome originally devoid of Sn as if the heterozygous association had promoted "contamination" of one chromosome (recipient) with Sn coming from the other (donor). If the two chromosomes in the heterozygote are marked with contrasting alleles of R, a gene lying about two crossover units proximal to Sn, it appears that the R constitution of the recipient chromosome affects their constitution. Presence of R-r in fact leads to changes of both chromosomes in terms of Sn constitution, resulting in a majority of nonparental chromosomes, R-r Sn and r Sn-w or r sn, while replacement of R-r with R-g, a mutant derivative of R-r, leads to a drastic reduction in the yield of nonparental chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of polycyclic aromatic hydrocarbons in carbon black with reference to cancerogenic risk in tire production.

Carbon blacks used in tire production have been examined for their polycyclic aromatic hydrocarbons (P.A.H.) content. The analytical procedures commonly adopted are discussed. The results obtained show that, A benzene extraction time of 250 hours is necessary to obtain complete extraction, but for practical purposes, an extraction time of 150 hours is sufficient to extract more than 95% of the PAH. It is possible to identify two classes of blacks in regard to extractables. The one class provides extractables in the range of 200--400 micrograms/g; the second class, in the range of 1000--2000 micrograms/g. No P.A.H. were detected by direct injection of carbon black into the mass spectrograph at 200 degrees C, 10(-6) torr. This was probably due to the very strong bonding to the black particles. The P.A.H. contribute a relative constant percentage of the benzene extracts. It is reasonably possible to exclude any carcinogenic risk to personnel, in a working environment, with an airborne carbon black concentration equal to or less than 3.5 mg/m3, due to the very low P.A.H. content of carbon black and to the very strong P.A.H. bonding to the black particles.