Distinct mechanisms for aerenchyma formation in leaf sheaths of rice genotypes displaying a quiescence or escape strategy for flooding tolerance.

BACKGROUND AND AIMS: Rice is one of the few crops able to withstand periods of partial or even complete submergence. One of the adaptive traits of rice is the constitutive presence and further development of aerenchyma which enables oxygen to be transported to submerged organs. The development of lysigenous aerenchyma is promoted by ethylene accumulating within the submerged plant tissues, although other signalling mechanisms may also co-exist. In this study, aerenchyma development was analysed in two rice (Oryza sativa) varieties, 'FR13A' and 'Arborio Precoce', which show opposite traits in flooding response in terms of internode elongation and survival. METHODS: The growth and survival of rice varieties under submergence was investigated in the leaf sheath of 'FR13A' and 'Arborio Precoce'. The possible involvement of ethylene and reactive oxygen species (ROS) was evaluated in relation to aerenchyma formation. Cell viability and DNA fragmentation were determined by FDA/FM4-64 staining and TUNEL assay, respectively. Ethylene production was monitored by gas chromatography and by analysing ACO gene expression. ROS production was measured by using Amplex Red assay kit and the fluorescent dye DCFH(2)-DA. The expression of APX1 was also evaluated. AVG and DPI solutions were used to test the effect of inhibiting ethylene biosynthesis and ROS production, respectively. KEY RESULTS: Both the varieties displayed constitutive lysigenous aerenchyma formation, which was further enhanced when submerged. 'Arborio Precoce', which is characterized by fast elongation when submerged, showed active ethylene biosynthetic machinery associated with increased aerenchymatous areas. 'FR13A', which harbours the Sub1A gene that limits growth during oxygen deprivation, did not show any increase in ethylene production after submersion but still displayed increased aerenchyma. Hydrogen peroxide levels increased in 'FR13A' but not in 'Arborio Precoce'. CONCLUSIONS: While ethylene controls aerenchyma formation in the fast-elongating 'Arborio Precoce' variety, in 'FR13A' ROS accumulation plays an important role.

Monoubiquitylation in the Fanconi anemia DNA damage response pathway.

The hereditary genetic disorder Fanconi anemia (FA) belongs to the heterogeneous group of diseases associated with defective DNA damage repair. Recently, several reviews have discussed the FA pathway and its molecular players in the context of genome maintenance and tumor suppression mechanisms [H. Joenje, K.J. Patel, The emerging genetic and molecular basis of Fanconi anaemia, Nat. Rev. Genet. 2 (2001) 446-457; W. Wang, Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins, Nat. Rev. Genet. 8 (2007) 735-748; L.J. Niedernhofer, A.S. Lalai, J.H. Hoeijmakers, Fanconi anemia (cross)linked to DNA repair, Cell 123 (2005) 1191-1198; K.J. Patel, Fanconi anemia and breast cancer susceptibility, Nat. Genet. 39 (2007) 142-143]. This review assesses the influence of post-translational modification by ubiquitin. We review and extract the key features of the enzymatic cascade required for the monoubiquitylation of the FANCD2/FANCI complex and attempt to include recent findings into a coherent mechanism. As this part of the FA pathway is still far from fully understood, we raise several points that must be addressed in future studies.

A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Comparative analysis of anoxic coleoptile elongation in rice varieties: relationship between coleoptile length and carbohydrate levels, fermentative metabolism and anaerobic gene expression.

Rice (Oryza sativa L.) seeds can germinate under anoxia and can show coleoptile elongation. The anoxic coleoptile is usually longer than aerobic coleoptiles. Although several hypotheses have been proposed to explain the ability of rice to elongate coleoptiles under anoxia, conclusive experimental evidence explaining this physiological trait is lacking. In order to investigate whether metabolic and molecular markers correlate with anoxic coleoptile length, we screened 141 Italian and 23 Sri Lankan rice cultivars for their ability to elongate coleoptiles under anoxia. Differences in anoxic coleoptile length were used to evaluate whether a correlation exists between coleoptile length and biochemical and molecular parameters. The expression of genes coding for glycolytic and fermentative enzymes showed a very low correlation with anoxic coleoptile length. Although differences were found in carbohydrate content between the varieties tested, this parameter also does not appear to be critical in terms of coleoptile elongation. Efficient ethanol fermentation does, however, correlate well with the elongation of coleoptiles under anoxic conditions.

[Working with families in the early stages of psychosis: a structured intervention for caregivers]. / Lavorare con le famiglie negli esordi psicotici: un intervento strutturato per i caregiver.

In the field of early psychosis psychoeducation is considered fundamental to increase coping skills with diseases and to improve the quality of life of patients and their families. The more recent and updated guidelines on schizophrenia underline the extreme importance of the families involvement in treatment of young people in the initial phases of illness. "Families are the main support for many young patients. They could be the primary carers but they have also to face individual and social consequences following the onset course. Where feasible, family members must be involved in the treatment". This work describes the components of the work with families carried on by the Centre for the early detection of psychoses and high-risk situations--Programma 2000 ("Niguarda Ca' Granda" Hospital-Milan) and is mostly focused on psychoeducation and on Expressed Emotions aspects. Even the advances suggested by the international literature drove Programma 2000 to define both the steps of caregivers assessment and intervention. During the last ten years, Programma 2000 has followed 191 caregivers. Aims of this work is to verifier the outcome of the "pilot project", started in 2007, projected specifically to increase the normally used strategies to improve the caregivers adherence and involvement in the therapeutic process. The individualized multi-componential intervention has been structured in 8 session over one years. Outcome measures used in this article are the scores of the Camberwell Family Interview and from the Psychosis Knowledge Assessement Semistructured Interview (VCP). The subjects enrolled in the structured pilot project were 25 family caregiver to young (18-30 yrs old) patients. Results shows change in the Expressed Emotion level: 13% of families moved from High Expressed Emotion to Low Expressed Emotion. Furthermore data on the knowledge of illness knowledge level illustrate a reduction in the percentage, from 47% to 18%, of carers who have just a very vague knowledge of illness, and an increase from 16% to 27% of carers who obtain a good level of specific knowledge. In conclusion we can sustain mental health expert with aim to treatment project programme individualized and multi-componential tailored for young's caregiver at the onset phase of psychosis.

C. elegans RAD-5/CLK-2 defines a new DNA damage checkpoint protein.

BACKGROUND: In response to genotoxic stress, cells activate checkpoint pathways that lead to a transient cell cycle arrest that allows for DNA repair or to apoptosis, which triggers the demise of genetically damaged cells. RESULTS: During positional cloning of the C. elegans rad-5 DNA damage checkpoint gene, we found, surprisingly, that rad-5(mn159) is allelic with clk-2(qm37), a mutant previously implicated in regulation of biological rhythms and life span. However, clk-2(qm37) is the only C. elegans clock mutant that is defective for the DNA damage checkpoint. We show that rad-5/clk-2 acts in a pathway that partially overlaps with the conserved C. elegans mrt-2/S. cerevisiae RAD17/S. pombe rad1(+) checkpoint pathway. In addition, rad-5/clk-2 also regulates the S phase replication checkpoint in C. elegans. Positional cloning reveals that the RAD-5/CLK-2 DNA damage checkpoint protein is homologous to S. cerevisiae Tel2p, an essential DNA binding protein that regulates telomere length in yeast. However, the partial loss-of-function C. elegans rad-5(mn159) and clk-2(qm37) checkpoint mutations have little effect on telomere length, and analysis of the partial loss-of-function of S. cerevisiae tel2-1 mutant failed to reveal typical DNA damage checkpoint defects. CONCLUSIONS: Using C. elegans genetics we define the novel DNA damage checkpoint protein RAD-5/CLK-2, which may play a role in oncogenesis. Given that Tel2p has been shown to bind to a variety of nucleic acid structures in vitro, we speculate that the RAD-5/CLK-2 checkpoint protein may act at sites of DNA damage, either as a sensor of DNA damage or to aid in the repair of damaged DNA.

Effect of Anoxia on Carbohydrate Metabolism in Rice Seedlings.

The metabolism of carbohydrates was investigated in rice (Oryza sativa L.) seedlings grown under anoxia. Two phases can be recognized in the utilization of carbohydrates: during the first days of germination under anoxia, the metabolism of sugars is mainly degradative, whereas after the induction of [alpha]-amylase (EC 3.2.1.1) has taken place, the increased presence of glucose and sucrose indicates that both starch degradation and sucrose synthesis operate. The analysis of the enzymes involved in carbohydrate metabolism indicates that anoxic rice seedlings possess a set of enzymes that allow the efficient metabolism of starch and sucrose to fructose-6-phosphate. We propose that cytosolic sucrose metabolism in anoxic rice seedlings takes place mainly through a sucrose synthase (EC 2.4.1.13) pathway with nucleoside diphosphate kinase (EC 2.7.4.6), allowing the cycling of urydilates needed for the operation of this pathway.

Amylolytic Activities in Cereal Seeds under Aerobic and Anaerobic Conditions.

An adequate carbohydrate supply contributes to the survival of seeds under conditions of limited oxygen availability. The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, with starch representing the main storage compound. Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes and only through the concerted action of [alpha]-amylase (EC 3.2.1.1), [beta]-amylase (EC 3.2.1.2), debranching enzyme (EC 3.2.1.41), and [alpha]-glucosidase (EC 3.2.1.20) can starch be hydrolyzed completely. We present here data concerning the complete set of starch-degrading enzymes in three cereals, rice (Oryza sativa L.), which is tolerant to anaerobiosis, and wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), which are unable to germinate under anoxia. Among the cereal seeds tested under anoxia, only rice is able to degrade nonboiled, soluble starch, reflecting the ability to degrade the starch granules in vivo. This is explained by the presence of the complete set of enzymes needed to degrade starch completely either as the result of de novo synthesis ([alpha]-amylase, [beta]-amylase) or activation of preexisting, inactive forms of the enzyme (debranching enzyme, [alpha]-glucosidase). These enzymes are either absent or inactive in wheat and barley seeds kept under anaerobic conditions.

Characterization of isoforms of hexose kinases in rice embryo.

Hexose kinases in rice embryos have been characterized. Six isoforms were detected: i.e. three glucokinases (GK1-3), two hexokinases (HK1 and HK2) and one fructokinase (FK1). Out of these, GK3, HK1 and HK2 were inhibited by mannoheptulose and glucosamine, known inhibitors of hexokinase activity. These inhibitors are also known to be modulators of sugar sensing processes. The results suggest that GK3, HK1 and HK2 may play a role in sensing the cellular sugar status in the rice embryo.

Proteolytic Activities in Alfalfa (Medicago sativa L.) Leaves.

Autodigestion in alfalfa (Medicago sativa L. cv Messe) leaves takes place at a higher rate in the acidic range of pH suggesting that acid proteases are probably involved in protein breakdown. Several lines of evidence show that alfalfa leaves contain various proteolytic activities. A carboxypeptidase active on CBZ-Phe-Met and on other CBZ-dipeptides with a pH optimum of 5.5 has been partially purified by CM-cellulose chromatography. Another acid protease, probably endopeptidase, is active on hemoglobin with an optimum at pH 4.5; perhaps it is not a single protease since it shows another peak of activity at pH 7-7.5. In the neutral-alkaline range of pH there are at least three peptidases active on naphthylamides. One of these, active on Lys-NA, is most active in very young leaves and probably is aSH dependent protease.

Arsenic uptake and translocation by plants in pot and field experiments.

A work undertaken by pot and field experiments to assess the suitability of poplars and ferns for the in-situ, phytoextraction, of a dumping site with residues from the roasting process of arseno-pyrite is reported. The main characteristic of this site is the high content of both the As metalloid and heavy metals (e.g., Al, Fe, Cu, Co, Cr, Pb). Two poplar clones (Populus deltoides 'Dvina' and Populus x canadensis 'Orion') and Pteris vittata (Chinese brake fern) were planted in the contaminated soil both ex situ in pots and in situ. Plant survival, As accumulation in plant tissues, leaf content of pigments, soluble proteins, activity of catalase and SH-groups in both roots and leaves were evaluated during a 24-month study period. Both poplar and fern plants exhibited an increase in the activity of catalase and SH group contents when grown in the presence of pyrite ashes. The results showed that the co-planting system (arsenic-hyperaccumulator fern Pteris vittata and Populus clones) was suitable for phytoextraction of multi-contaminated dumping sites. Agronomic measures such as irrigation, soil tillage and amendments also seem to be necessary for the successful establishment of poplar trees and ferns in contaminated soils in order to enhance plant growth through the improvement of soil conditions.

Regulatory interplay of the Sub1A and CIPK15 pathways in the regulation of α-amylase production in flooded rice plants.

Rice (Oryza sativa L.) can successfully germinate and grow even when flooded. Rice varieties possessing the submergence 1A (Sub1A) gene display a distinct flooding-tolerant phenotype, associated with lower carbohydrate consumption and restriction of the fast-elongation phenotype typical of flooding-intolerant rice varieties. Calcineurin B-like interacting protein kinase 15 (CIPK15) was recently indicated as a key regulator of α-amylases under oxygen deprivation, linked to both rice germination and flooding tolerance in adult plants. It is still unknown whether the Sub1A- and CIPK15-mediated pathways act as complementary processes for rice survival under O(2) deprivation. In adult plants Sub1A and CIPK15 may perhaps play an antagonistic role in terms of carbohydrate consumption, with Sub1A acting as a starch degradation repressor and CIPK15 as an activator. In this study, we analysed sugar metabolism in the stem of rice plants under water submergence by selecting cultivars with different traits associated with flooding survival. The relation between the Sub1A and the CIPK15 pathways was investigated. The results show that under O(2) deprivation, the CIPK15 pathway is repressed in the tolerant, Sub1A-containing, FR13A variety. CIPK15 is likely to play a role in the up-regulation of Ramy3D in flooding-intolerant rice varieties that display fast elongation under flooding and that do not possess Sub1A.

Differential expression of two fructokinases in Oryza sativa seedlings grown under aerobic and anaerobic conditions.

Fructokinases (EC 2.7.1.4) may play an important role in carbohydrate metabolism of Oryza sativa L. (rice) seedlings under anoxia. We present here the molecular and biochemical characterizations of two rice fructokinases, namely OsFK1 and OsFK2. The results show that, at both a transcriptional and a transductional level, OsFK1 is preferentially expressed under aerobic conditions, whereas OsFK2 is induced under anoxia. Substrate inhibition was demonstrated for OsFK1, while OsFK2 appears to be largely unaffected by fructose concentrations up to 10 mM. Sugar modulation of anoxia-induced proteins has been proposed, but our results on rice calli treated with or without glucose (10, 30 or 90 mM) for different time indicate that neither OsFK1 nor OsFK2 are sugar-regulated. We propose that OsFK2 plays a major role in fructose phosphorylation under anoxic conditions.

Proteinases and enzyme stability in crude extracts of castor bean endosperm.

The stability of catalase, fumarase, and isocitrate lyase from deliberately broken organelles in crude extracts from endosperm tissue of castor bean seedlings has been examined. These enzymes are relatively stable at 2 C in extracts from endosperm of 2-day seedlings, but rapid losses of activity occur in extracts from older seedlings. These losses are shown to be brought about by the thiol-proteinase present in the extracts. The inclusion of 35% glycerol prevented the loss of catalase, fumarase, and isocitrate lysase activity, and various inhibitors of proteinases afforded limited protection. The most striking protectant was leupeptin, an inhibitor of serine and thiol-proteinases. Leupeptin completely inhibited the loss of activity of the three enzymes in crude extracts and improved yields when included in the grinding medium.

Effects of leupeptin on proteinase and germination of castor beans.

Leupeptin, a tripeptide inhibitor of some proteinases, was shown previously to maintain the stability of several enzymes (isocitrate lyase, fumarase, and catalase) in crude extracts of castor bean endosperm. This reagent is now shown to inhibit the breakdown of water-soluble and crystalloidstorage proteins of the protein bodies isolated from castor beans by the SH-proteinase and it also inhibits the endopeptidase from mung beans. When suitably introduced into the endosperm of dry castor beans it strongly inhibits germination and seedling development. Application of leupeptin to endosperm halves removed from the seed prevents the normal development of enzymes concerned with gluconeogenesis from fat and drastically curtails sugar production. The results suggest that the SH-proteinase is intimately involved in the mobilization of storage proteins.

Gibberellins in Suspensors of Phaseolus coccineus L. Seeds.

Analysis of extracts from 6300 (1.2 grams fresh weight) Phaseolus coccineus suspensors by combined gas chromatography-mass spectrometry has demonstrated the presence of five C(19)-gibberellins, GA(1), GA(4), GA(5), GA(6), GA(8), and one C(20)-GA, GA(44). The major GAs present were GA(1) and GA(8). Data are discussed in relation to previous results obtained in P. coccineus seed as well as in the embryo-suspensor system.

Ethanol-induced injuries to carrot cells : the role of acetaldehyde.

Carrot (Daucus carota L.) cell cultures show high sensitivity to ethanol since both unorganized cell growth and somatic embryogenesis are strongly inhibited by ethanol at relatively low concentrations (10-20 millimolar). The role of acetaldehyde on ethanol-induced injuries to suspension cultured carrot cells was evaluated. When ethanol oxidation to acetaldehyde is prevented by adding an alcohol-dehydrogenase (EC 1.1.1.1) inhibitor (4-methylpyrazole) to the culture medium, no ethanol toxicity was observed, even if ethanol was present at relatively high concentrations (40-80 millimolar). Data are also presented on the effects of exogenously added acetaldehyde on both carrot cell growth and somatic embryogenesis. We conclude that the observed toxic effects of ethanol cannot be ascribed to ethanol per se but to acetaldehyde.

Effects of o(2) concentration on rice seedlings.

The ability of rice, wheat, and oat seedlings to germinate and grow as the O(2) concentration was lowered to zero was compared. The germination of rice was completely unaffected by O(2) supply, whereas that of oats and wheat was strongly retarded at levels below 5% O(2). In contrast to the coleoptiles of oats and wheat and to roots of all three species where growth was progressively diminished as the O(2) concentration was lowered, that of the rice coleoptile was progressively increased. However, the dry weight and content of protein, sugars, and cellulose were all depressed in the rice coleoptile in anoxia, and the levels of several respiratory enzymes, particularly those of mitochondria, were also much lower than those of the coleoptiles grown in air. In 1% O(2), the growth of the rice coleoptile was similar to that in air. The effect of ethanol concentration on germination and growth of rice was measured. Coleoptile growth was reduced when the ethanol concentration exceeded 40 millimolarity, and root growth was somewhat more sensitive. Coleoptiles of all three species grown in air were transferred to N(2), and ethanol accumulation was measured over 24 hours. The rate of ethanol accumulation in oats was close to that in rice, and in all three species the amounts of ethanol lost to the surrounding medium were those expected from simple diffusion from the tissue. The ability of the rice coleoptile to grow in anoxia is apparently not due to a particularly low rate of ethanol formation or to unusual ethanol tolerance. Any explanation of the success of rice in anoxia must encompass the much lower rate of ATP synthesis than that in air and account for the biochemical deficiencies of the coleoptile.

Ethanol production and toxicity in suspension-cultured carrot cells and embryos.

The process of carrot (Daucus carota L.) somatic embryogenesis is highly sensitive to exogenously added ethanol, since 5 mM ethanol inhibits this process by 50%, whereas the growth of proliferating carrot cells is inhibited to the same extent by 20 mM ethanol. This is consistent with the fact that proliferating cultures produce ethanol and release it into the medium at concentrations up to 20 mM, whereas embryogenic culture medium contains less than 1 mM ethanol. Data are presented showing the influence of cell density and 2,4-dichlorophenoxyacetic acid on ethanol production and on the presence of an alcohol-dehydrogenase (EC 1.1.1.1.) inactivator in carrot embryos.

Some data on the polygenic control of two quantitative traits in a vegetatively propagated flower plant, the carnation.

In two populations of selfed commercial varieties 4800 and 5774 ofDianthus cariophyllus as well as in the parents of 5774 two quantitative traits were examined: petal number and durability of blossoms after cutting. Both populations showed an increase in the number of petals, No. 4800 also in its durability. The estimated hereditary component was high for petals (0.47 and 0.35), lower but significant for lasting quality. The importance of an investigation on the selection for quantitative traits in cut flowers is discussed.