Milvia Luisa Racchi

Expression of a metallothionein A1 gene of Pisum sativum in white poplar enhances tolerance and accumulation of zinc and copper.

Metallothioneins (MT) play an important role in heavy metal detoxification and homeostasis of intracellular metal ions in plant. In this study, two transgenic lines expressing MT type 2 gene (PsMT(A1)) from Pisum sativum, a regenerated non transformed line NT and clone AL22, selected as heavy metal tolerant, were characterized in presence of the heavy metals for the ability to accumulate zinc and copper and to activate antioxidative enzyme defences: superoxide dismutase, catalase, ascorbate peroxidase. The levels of expression of MT type 2 gene assessed by RT-qPCR confirmed the gene over-expression in transgenic lines and evidenced in NT and AL22 the up-regulation of gene transcription by zinc and copper. Transgenic poplar lines during heavy metal stress displayed increased ability to translocate and accumulate zinc and copper compared with NT and AL22. The antioxidant enzyme defence was differently activated in response to metals in the transgenic lines without a significant increase of ROS. These results suggested that PsMT(A1) could play a role in ROS scavenging leading to enhanced metal tolerance and increased zinc and copper sequestration in root and leaf.

Transcriptome changes in the cambial region of poplar (Populus alba L.) in response to water deficit

A transcriptome analysis of the Populus alba cambial region was performed with the aim of elucidating the gene network underlying the response to water deficit within the cambium and differentiating derivative cambial cells. Water stress was induced in 1-year-old P. alba plants by withholding water for 9 days. At that time, leaf predawn water potential fell to -0.8 MPa, resulting in a significant reduction in stomatal conductance, CO(2) assimilation and a consistent increment of stem shrinkage. These effects were almost fully reversed by re-hydration. The water deficit resulted in changes in gene expression that affected several functional categories, such as protein metabolism, cell wall metabolism, stress response, transporters and transcriptional regulation. The function of up- and down-regulated genes is discussed considering the physiological response of the plants to water deficit.

Differential activity of catalase and superoxide dismutase in seedlings and in vitro micropropagated oak (Quercus robur L.)

Abstract Catalase (CAT) and superoxide dismutase (SOD) activity profiles were examined by native polyacrylamide gel electrophoresis in different tissues of seedlings and microcuttings of oak (Quercus robur L.) initiated from crown material (NL100A) and from basal epicormic shoots (NL100R), which differ in rooting ability. Two CAT isoforms were differentially active in seedlings and microcuttings; in particular, CAT-2 was activated in the basal callus of rooted microshoots. SOD isoenzymes, Mn-SOD and at least four Cu/Zn-SODs were found to be present, with Mn-SODs particularly active in microcuttings. No differences were found between the electrophoretic profiles of the two lines despite their different ontogenetic origin. The strong activity of CAT-2 in rooted microshoots indicates that this isoform is a protein specifically related to rooting.

Antioxidant Defenses in Plants with Attention to Prunus and Citrus spp.

This short review briefly introduces the formation of reactive oxygen species (ROS) as by-products of oxidation/reduction (redox) reactions, and the ways in which the antioxidant defense machinery is involved directly or indirectly in ROS scavenging. Major antioxidants, both enzymatic and non enzymatic, that protect higher plant cells from oxidative stress damage are described. Biochemical and molecular features of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) are discussed because they play crucial roles in scavenging ROS in the different cell compartments and in response to stress conditions. Among the non enzymatic defenses, particular attention is paid to ascorbic acid, glutathione, flavonoids, carotenoids, and tocopherols. The operation of ROS scavenging systems during the seasonal cycle and specific developmental events, such as fruit ripening and senescence, are discussed in relation to the intense ROS formation during these processes that impact fruit quality. Particular attention is paid to Prunus and Citrus species because of the nutritional and antioxidant properties contained in these commonly consumed fruits.

A glyphosate-resistant 5-enol-pyruvyl-shikimate-3-phosphate synthase confers tolerance to a maize cell line

Abstract Among a few cell lines of maize ( Zea mays L., cv. Black Mexican Sweet (MS)) tested, one showed a natural, remarkable tolerance to glyphosate at concentrations as high as 10 mM. Two activities of 5- enol -pyruvyl-shikimate-3-phosphate (EPSP) synthase, the target enzyme of the herbicide, were separated in the tolerant culture by anion-exchange chromatography. One peak of activity was not significantly inhibited by glyphosate even at millimolar concentrations. While the glyphosate-sensitive isoform persisted throughout the growth cycle of the culture, the glyphosate-resistant EPSP synthase increased only after the onset of exponential growth and declined in the stationary phase. Tolerance to the herbicide was accompanied by a reduced affinity of the enzyme for the substrate phospho- enol -pyruvic acid.

Molecular cloning, characterisation and expression of a manganese superoxide dismutase gene from peach (Prunus persica (L.) Batsch)

Abstract. Two cDNA clones encoding mitochondrial manganese superoxide dismutases (MnSODs) from peach (Prunus persica [L.] Batsch) were identified, which show homologies to several plant MnSODs. The amino acid sequence predicted from one full-length clone (MnSOD1) showed the highest homology to an MnSOD from Nicotiana plumbaginifolia (94%) and included a 24-amino acid transit peptide typical of those used to target proteins to the mitochondria. A second, partial clone (MnSOD2) showed divergence from MnSOD1 in the 3′ untranslated region. It could therefore derive from a second gene or from an allele of MnSOD1. Southern hybridisation analysis suggests the existence of two MnSOD genes in peach. SOD isoenzyme profiles, MnSOD1 expression and protein levels were studied in aerial vegetative tissues derived from plants of different ages and in adult plants during the seasonal cycle. Zymograms revealed at least two isoforms of MnSODs in pre-shooting vegetative buds and in developing fruits. Levels of MnSODs were lower in leaves derived from apical shoots of adult plants than in leaves derived from seedlings, basal shoots or in vitro propagated juvenile plants, which are considered as juvenile-like structures. The MnSOD1 transcript and protein followed the same pattern. The results suggest that the steady-state levels of MnSOD1 mRNA in leaves vary with both the ontogenetic stage and the growth rate of the tissues examined.

Glyphosate tolerance in maize (Zea mays L.). 2. Selection and characterization of a tolerant somaclone

SummaryThe progeny of 104 regenerated maize plants were screened for tolerance to the safe broad-spectrum herbicide glyphosate during seed germination and early growth. Seven somaclones showed varying degrees of resistance to the application of the herbicide at 1.2 mM (0.1 kg a.i. in 400 1 ha-1 of water). Plants capable of a normal growth following treatment with 2.4 mM (0.2 kg ha-1) glyphosate at the three leaf stage were selfed, and their progeny analyzed. A family able to tolerate the exposure to glyphosate at 2.4 mM was isolated and shown to maintain a photosynthetic rate comparable with control after the application of the herbicide.The selfed progeny of the tolerant somaclone was characterized as to the properties of two targets of glyphosate, the shikimate pathway enzymes 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. In vitro tests ruled out the possibility that the tolerance was due to altered forms of these enzymes. Families showed significant variability with regard to EPSP and DAHP synthase levels, measured at different stages during seedling growth; however, not even these traits were correlated with in vivo response to glyphosate. The possible role of other physiological processes in determining the increased tolerance to the herbicide is discussed.

An analysis of the nutritional requirements of the Pro mutant in Zea mays

Abstract The symbol pro refers to a seedling-lethal monogenic recessive mutant identifiable on the basis of its abnormal endosperm morphology. Its requirement for proline is absolute as proved by growth of embryo, shoot and root tip cultures on media supplemented with different amino acids. By supplying mutant embryos with proline and arginine intermediates, it has been found that the pro phenotype is probably the result of a genetic block between Δ 1 -pyrroline-5-carboxilic acid (P5C) and proline.

Molecular cloning, characterisation and expression of two catalase genes from peach

Two cDNA clones encoding catalase (Cat1 and Cat2) from peach [Prunus persica (L.) Batsch] were identified, that show homologies to other plant catalases. The nucleotide sequences of the two coding regions showed 88% identity to each other. The amino acid sequences predicted from the two full-length clones showed the highest homology to a catalase from cotton and Nicotiana plumbaginifolia L. and included C-terminal tri-peptides typical of those used to target proteins to peroxisomes. Southern hybridisation analysis suggested the existence of two catalase genes in peach. The expression of Cat1 and Cat2 was determined in seeds, vegetative tissue, leaves during the seasonal cycle and in leaves in response to light / dark treatments. Cat1 had high levels of expression only in leaf tissue and was responsive to light and seasonal changes. Cat2 had high levels of expression in in vitro shoots and was also responsive to seasonal changes, but not to light. In situ hybridisations to leaf tissue indicated that the expression of Cat1 was localised mainly in palisade cells, while Cat2 mRNA was present in the vascular tissue. The results of the expression analysis and in situ hybridisation suggest a role for Cat1 in photorespiration and for Cat2 in stress responses.

Anthocyanin and proteins as biochemical markers in maize endosperm cultures.

Endosperm maize cultures derived from a strain homozygous for all genes required for anthocyanin synthesis develop an intense pigmentation. Pigmenting ability is generally maintained in successive subcultures, altough colourless areas are frequently observed in pigmented cultures. The isolated colourless cell clusters show a growth rate higher than the coloured ones. These calli nevertheless do not lose the ability to synthesize anthocyanins, and in successive subcultures turn red again.The different growth rates associated with the ability of cells to accumulate pigments suggest the existence of different physiological states of the culture. To investigate this possibility we analyzed the polypeptide patterns of coloured and colourless cultures. SDS gel electrophoresis has demonstrated differences in soluble protein fractions, among which a 26 kD peptide, characteristic of pigmented tissues, has been evidenced. Zein, the major storage protein of maize endosperm is present, although at very low levels, both in pigmented and in unpigmented cultures, confirming that its synthesis occurs continuously in vitro.