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SUMMARY Structural and enzymatic aspects of leaf and fruit abscission in explants treated with exogenous ethylene were studied and compared with those previously observed under field conditions. Light and electron microscopy observations reconfirm that differences in abscission occur in leaf and fruit, and in explants cell separation is accelerated, with a more dramatic degradation of the cell walls. In fruit, digestion starts from the middle lamella, and subsequently extends to the entire parietal mass. In the leaf the process involves the primary cell wall, leaving, at the end, undigested materials. In fruit the lytic activity is sustained by endocellulase, and by exo- as well as endopolygalacturonase. In the leaf only endocellulase activity has been found. Cell enlargement phenomena occur subsequent to abscission zone activation in both leaf and fruit explants, involving the cells of either the separation layer or the adjacent region.

The therapeutic muds of the Euganean Thermal District, the oldest and largest thermal site in Europe, are recognized by the Italian Health System as an excellent treatment for osteoarticular diseas...

A wheat (Triticum turgidum subsp. durum) mutant, generated with sodium azide from wild-type (WT) cv. 'Trinakria', differs in its water affinity of dry leaves, and was designated as a water-mutant. Compared with the WT, water-mutant leaves have lower rates of water uptake, while stomatal and cuticular transpiration do not differ. The nuclear magnetic resonance proton signals used for image reconstruction of leaf cross sections showed differences between these genotypes for the T1 proton spin-density and the T2 proton spin-spin relaxation time. Structural and histochemical analyses at midrib level showed that the water-mutant has thinner leaves, with more and smaller cells per unit area of mesophyll and sclerenchyma, and has altered staining patterns of lignin and pectin-like substances. Stress-strain curves to examine the rheological properties of the leaves showed a biphasic trend, which reveals that the tensile strength at break load and the elastic modulus of the second phase of the water-mutant are significantly higher than for the WT. These data support the proposal of interrelationships among local biophysical properties of the leaf, the microscopic water structure, the rheological properties and the water flux rate across the leaf. This water-mutant can be used for analysis of the genetic basis of these differences, and for identification of gene(s) that govern these traits.

Synthetic veterinary medicines are introduced routinely in the environment after animal treatment to prevent and control infectious diseases and up to 80% the administered dose can be excreted unaltered. As a consequence, the soil is the environment most contaminated by such molecules. However, information about their implications on the growth of vegetal organisms is still scarce. With the aim of better elucidating the effects of veterinary antibiotics on plants, barley was grown in a nutrient solution containing 40 μM (about 11,500 μg L−1) of two well-known sulfonamide antibiotics, sulfadimethoxine (SDM) and sulfamethazine (SZ). After 15 d of treatment, the effects on root apparatus were particularly evident, while the photosynthetic tissues remained almost unaffected. SDM and SZ stimulated root hairs and lateral root development a few mm behind the root tips. In particular, from a structural point of view, treated plants showed root shortening and an advanced differentiation in comparison to controls, later confirmed using light microscopy. At a functional level, the two active molecules were found to induce root electrolyte release, such as K+, possibly due to an impairment of membrane permeability. The research concludes that sulfonamides can have profound effects on morphology and functionality of roots of crop plants. As these alterations might have consequences on their productivity, further studies are necessary to assess effects on plants at laboratory and field conditions.

Erythronium dens-canis is an early-flowering understory lily of southern Europe with two leaves and a single flower, although a number of plants have only one leaf and do not flower. The leaves are mottled with silvery flecks and brown patches, that gradually vanish turning to a lively green color. The nature and function of this striking variegation pattern were investigated in differently colored leaf parts following the springtime color change. Tissue organization was examined by light and electron microscopy; photosynthetic pigments were analyzed by spectrophotometry and HPLC; chlorophyll fluorescence parameters were evaluated by MINI-PAM. The results showed that brown patches originated in vacuolar anthocyanins in the subepidermal cell layer while air spaces between the upper epidermis and underlying chlorenchyma resulted in silvery flecks. The two leaf areas did not differ in photosynthetic pigments, chloroplast organization and photosynthetic parameters (F(v)/F(m), NPQ, rETR). Greening of brown patches due to anthocyanin resorption was faster in non-flowering plants than in flowering ones, occurring only when young fruits were developing. Anthocyanin disappearance did not change the structural-functional features of photosynthetic tissues. As a whole the results suggest that the anthocyanin pigmentation of E. dens-canis leaves does not affect the photosynthetic light use and has no photoprotective function. It is proposed that the complex leaf color pattern may act as a camouflage to escape herbivores, while the reflective silvery spots may have a role in attracting pollinators of this early-flowering species.

During the 1960s and 1970s, a magnificent cloud forest near San Francisco de Las Pampas (near Quito, Ecuador) was converted into dairy farm pastures. One endemic composite plant locally called marco yuyo and identified as Munnozia pinnatipartita, has been considered an undesirable weed by dairy farming campesino communities because it negatively affects the introduced grass pasture and is unsuitable as cattle feed. Concurrently, M. pinnatipartita has also been utilized for its herbicidal properties as a deterrent to grasses in a project promoting cloud forest recovery in the Otonga natural park. The peculiarity of this endemic composite utilized as such a deterrent attracted our interest. Accordingly, we investigated its taxonomy, physiology, ecology, and ethnobiology, and managed to demonstrate its potential allelopathic effect on germinating grasses in greenhouse trials. Furthermore, gas-chromatography of potential volatile metabolites identified 25 compounds and, among these, terpenes seem to be primar...

Background and aims Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells. Methods Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins. Key results The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls. Conclusions The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.

The molecular properties and subcellular location of bound gamma-glutamyl transferase (GGT) were studied, and an experimental setup devised to assess its functions in barley roots. Enzyme histochemistry was used to detect GGT activity at tissue level; immunocytochemistry to localize the protein at subcellular level; and modelling studies to investigate its surface charge properties. GGT activity in vivo was measured for the first time. Functions were explored by applying chemical treatments with inhibitors and the thiol-oxidizing drug diamide, performing time-course chromatographic and spectrophotometric analyses on low-molecular-weight thiols. Gamma-glutamyl transferase activity was found to be high in the root apical region and the protein was anchored to root cell wall components, probably by basic amino acid residues. The results show that GGT is essential to the recovery of apoplastic glutathione provided exogenously or extruded by oxidative treatment. It is demonstrated that GGT activity helps to salvage extracellular glutathione and may contribute to redox control of the extracellular environment, thus providing evidence of a functional role for gamma-glutamyl cycle in roots.

This study deals with the respiratory activity of the cryophilic microalga “Chlorella” saccharophila to the growth temperatures of 4°C and 16°C. The analyses showed that this alga responded positively to the temperature rise by increasing its growth rate. Moreover, the higher temperature did not affect the ultrastructural organization of the algal cells. As regards the respiration rate, it did not show a substantial difference in algae grown at 4°C or 16°C. Respiration analyses, accomplished after treatments with inhibitors of the distinct mitochondrial electron transport pathways, as well as western analysis carried out with an antibody against the alternative oxidase (AOX), suggested that the lack of decrease in oxygen consumption at the lower temperature could depend on a great activity of the alternative cyanide-resistant pathway.

Highlight This study provides the first characterization of an R2R3 family MYB transcription factor involved in cuticle and epicuticular wax deposition, whose action is confined to maize embryogenesis and juvenile phase., The fdl1-1 mutation, caused by an Enhancer/Suppressor mutator (En/Spm) element insertion located in the third exon of the gene, identifies a novel gene encoding ZmMYB94, a transcription factor of the R2R3-MYB subfamily. The fdl1 gene was isolated through co-segregation analysis, whereas proof of gene identity was obtained using an RNAi strategy that conferred less severe, but clearly recognizable specific mutant traits on seedlings. Fdl1 is involved in the regulation of cuticle deposition in young seedlings as well as in the establishment of a regular pattern of epicuticular wax deposition on the epidermis of young leaves. Lack of Fdl1 action also correlates with developmental defects, such as delayed germination and seedling growth, abnormal coleoptile opening and presence of curly leaves showing areas of fusion between the coleoptile and the first leaf or between the first and the second leaf. The expression profile of ZmMYB94 mRNA—determined by quantitative RT-PCR—overlaps the pattern of mutant phenotypic expression and is confined to a narrow developmental window. High expression was observed in the embryo, in the seedling coleoptile and in the first two leaves, whereas RNA level, as well as phenotypic defects, decreases at the third leaf stage. Interestingly several of the Arabidopsis MYB genes most closely related to ZmMYB94 are also involved in the activation of cuticular wax biosynthesis, suggesting deep conservation of regulatory processes related to cuticular wax deposition between monocots and dicots.

Antarctic algae play a fundamental role in polar ecosystem thanks to their ability to grow in an extreme environment characterized by low temperatures and variable illumination. Here, for prolonged periods, irradiation is extremely low and algae must be able to harvest light as efficiently as possible. On the other side, at low temperatures even dim irradiances can saturate photosynthesis and drive to the formation of reactive oxygen species. Colonization of this extreme environment necessarily required the optimization of photosynthesis regulation mechanisms by algal organisms. In order to investigate these adaptations we analyzed the time course of physiological and morphological responses to different irradiances in Koliella antarctica, a green microalga isolated from Ross Sea (Antarctica). Koliella antarctica not only modulates cell morphology and composition of its photosynthetic apparatus on a long-term acclimation, but also shows the ability of a very fast response to light fluctuations. Koliella antarctica controls the activity of two xanthophyll cycles. The first, involving lutein epoxide and lutein, may be important for the growth under very low irradiances. The second, involving conversion of violaxanthin to antheraxanthin and zeaxanthin, is relevant to induce a fast and particularly strong non-photochemical quenching, when the alga is exposed to higher light intensities. Globally K. antarctica thus shows the ability to activate a palette of responses of the photosynthetic apparatus optimized for survival in its natural extreme environment.

Free porphyrins and their magnesium complexes, including chlorophylls, are potent photo-sensitizers. Plants usually accumulate these compounds bound to proteins together with protective compounds like carotenoids. Besides their protective role, carotenoids can play a structural role in these complexes. To analyze the effect of impaired carotenogenesis on plastid membranes we applied to barley seedlings the bleaching herbicide 2-(4-chlorophenylthio)triethylamine (CPTA) as a specific inhibitor for the cyclization of lycopene. To avoid interference with photo-oxidation, the essential experiments were performed on seedlings grown in darkness. While the amount of total carotenoids decreased, we found accumulation of more 6-carotene than lycopene in darkness clearly showing that CPTA inhibits the lycopene beta-cyclase more effectively than the lycopene epsilon-cyclase. The CPTA treatment resulted in accumulation of non-photoactive protochlorophyllide a; the amount of photoactive protochlorophyllide and NADPH:protochlorophyllide oxidoreductase remained constant. Further, the level of Mg protophorphyrin and its monomethyl ester increased to an extent similar to that obtained by application of 5-aminolevulinic acid (ALA). The perturbation of the ultrastructure of etioplast inner membranes, observed after CPTA-treatment, was not found after ALA-treatment; this excluded the accumulated tetrapyrroles as responsible for the perturbation. By contrast, the down-regulation of Lhcb and RbcS genes found after CPTA-treatment was compatible with the presumed role of Mg protophorphyrin as "plastid signal" for regulation of nuclear gene expression. Possible mechanisms for enhancement of tetrapyrrole accumulation by non-cyclic carotenoids are discussed.

The effects of cadmium (from 7.5 to 75 microM) on chloroplasts of rice were studied at the structural and biochemical level. Loss of pigments, reduction of thylakoids and decrease in oxygen evolution and Fv/Fm ratio occur in leaves following cadmium treatment. However, the amount of photosystem II reaction center proteins and that of its light harvesting complex is not affected, indicating that cadmium does not adversely influence the structural organization of this photosystem. In thylakoids isolated from cadmium-treated plants a loss in the capability to reduce 2,6-dichlorophenolindophenol is observed, which is partially restored if diphenylcarbazide is used as an electron donor, indicating that cadmium affects water splitting activity. In thylakoids isolated from control plants and treated with cadmium, diphenylcarbazide preserves most of the photosystem II activity lost after incubation with cadmium; most of the S(2) multiline electron paramagnetic resonance signal from the manganese cluster is lost, whereas the TyrD(+) and other signals are retained. Light-induced photosystem II damage, in vitro, is promoted by Cd-treatment as deduced from the mobility shift of the D1 protein observed by immunoblot.

Tolerance to near complete desiccation of vegetative organs is a widespread capability in bryophytes and is also shared by a small group of vascular plants known as resurrection plants. To date more than 300 species, belonging to pteridophytes and angiosperms, have been identified that possess this kind of desiccation-tolerance. The vegetative desiccation-tolerance of resurrection plants is an inductive process displayed only under environmental stress with or without the involvement of abscisic acid as molecular signal. The different problems associated with desiccation encountered by resurrection plants render the employment of many interacting mechanisms necessary. Preservation of cell order and correct structure of membranes and macromolecules is underpinned by the synthesis of large amounts of sugars, amino acids, and small polypeptides such as late embryogenesis abundant (LEA) proteins and dehydrins. Some of these compatible solutes, such as sucrose and LEA proteins, are also involved in cytoplasm v...

Although bodies of water are a major vehicle for cadmium contamination, works dealing with submerged plants directly exposed to it are still very scarce. The aim of this research was to study damaging effects of prolonged cadmium exposure on young and mature submerged leaves of Elodea canadensis treated for 1 month with 100 μM Cd added to the growth water medium. The Cd-exposed plants, compared to the controls, showed shorter and poorly pigmented shoots with thinner stems and less expanded leaves. The leaves accumulated high quantities of cadmium, most of which, about 95 and 90% in young and mature leaves, respectively, remained outside the cells, bound to the anionic cell walls. In young differentiating leaves, Cd interfered with the morphogenetic pattern, by inhibiting cell division and affecting cell enlargement. Cd hindered the division and the expansion of chloroplasts, also impairing organelle shape and thylakoid system arrangement. Cadmium, furthermore, greatly disturbed the cell wall organization, causing the demolition of the transfer cell-like frame. In differentiated leaves, Cd hastened the appearance of senescence symptoms in chloroplasts and worsened the cell wall alterations. Both young and mature leaves showed a decreased photosynthetic activity, not only ascribable to lowered chlorophyll contents.

Polygalacturonase (PG, EC 3.2.1.15) is one of the cell wall hydrolases involved in the cell separation processes which occur during ripening of some fleshy fruits (FISHER and BENNETT 1991), as well as during abscission of leaves and fruits (HUBERMANN and GOREN 1979; RASC~O et al. 1985; TAYLOR et al. 1990; BONGHI et al. 1992; TAYLOR et al. 1993). In particular, endopolygalacturonase of tomato fruit is the most widely known form of this enzyme, having been characterized at molecular and biochemical level (see literature in ZHENG et al. 1992). Endopolygalacturonase activity has also been found in peach during both abscission and development of fruits, while no such activity could be detected during leaf abscission (BONGHI et al. 1992; ZANCHIN et al. 1993). A few years ago it was observed that polyclonal antibodies raised against a tomato fruit PG (i.e., PGZA) recognized, in soft ripe fruits of peach, a polypeptide with molecular mass similar to that of PG2A. Furthermore, using as a probe a cDNA coding for tomato fruit endopolygalacturonase, the same researchers were able to clone and characterize a 3.5 kb fragment of peach genomic DNA (LEE et al. 1990). On the basis of sequence analysis it was concluded that, besides an unidentified sequence, it contained about the 3’ half of a gene which showed, in the coding regions, extensive homology with the tomato PG gene. According to the same researchers, such homology could explain the observed cross-reaction between the antibody to tomato fruit PG and a peach polypeptide, which was therefore suggested to be a peach endopolygalacturonase and the product of the partly characterized gene (LEE et al. 1990). In tomato the gene encoding the fruit endopolygalacturonase seems to be expressed during the fruit ripening, but not during the leaf abscission. In fact, despite a significative rise in PG activity, an antibody to fruit PG did not recognize any leaf abscission protein. Moreover, a cDNA encoding a tomato fruit PG gave no hybridization to mRNA obtained from activated abscission zones of tomato leaves (TAYLOR et al. 1990). In peach it has recently been shown that a cDNA coding for tomato fruit PG hybridized to mRNA obtained from fruit abscission zones but not from leaf ones where, in any case, no PG activity had been detected (BONGHI et al. 1992). In peach, cell separation events which show an involvement of endopolygalacturonase, are not restricted to fruit softening and abscission. Recently, it has been found that PG activity can also be detected throughout the fruit growth ( ZANCHIN et al. in press). On the basis of the above findings we considered it of some interest to see whether the endopolygalacturonase activity, observed in the course of different cell separation events in peach, is due to expression of the partly known PG gene (LEE et al. 1990) or, as already observed in tomato (TAYLOR et al. 1990), only some of that activity can be ascribed to expression of that gene.

Morphological, ultrastructural, and cytochemical characteristics of the red alga Plocamium cartilagineum from Antarctica have been studied and compared with those of the same species from the Mediterranean Sea. Distinct regions with possibly different functions were recognisable in the bushy thallus of Plocamium cartilagineum. Chloroplasts were concentrated in the outermost cell layer, while in the other cortex cells abundant floridean starch accumulated. No starch was found in the large medullary cells, characterised by the insertion in their walls of several large and lenticular pit connections devoid of plug caps and proteic in nature. Sulphated polysaccharides were distributed in the extracellular compartments of all the regions of the thallus, except in the surface layer. These acidic phycocolloids were particularly concentrated in the cell wall proper, which also exhibited a well organised fibrillar component probably consisting of cellulose. The distinctive feature of the Antarctic thalli,...

Acclimatization trials of the two tropical species Solanum sessiliflorum (cocona) and Solanum stramonifolium (coconilla) from Amazonas Venezuela, were carried out at the Botanical Garden of Padua. S. sessiliflorum exhibited a greater difference between the Botanical Garden and the native environment. Plant growth was reduced and pollination was difficult; despite numerous blooms few fruits were produced. Moreover, some fruit abscised early and the persistent ones were still unripe at the end of the season. On the contrary, S. stramonifolium showed good adaptation to the new environmental conditions. The plants grew well and produced a lot of ripe berries. Self-pollination as well as cross-pollination by bumblebees (Bombus terrestris) and other insects present in the open air succeeded. Thus, this latter species could be regarded as potentially exploitable in temperate climates as well as in Venezuela.

The freshwater secondarily aquatic plants, most of which are higher plants, are those returned to the water environment after spending a period of time living on land. The readaptation to living underwater has made it necessary for these plants to put in place morphological and functional strategies to cope with some major problems due to features of the aquatic environment, but also deriving from the specialized organization of their “terrestrial” bodies. The poor O2 availability underwater accounted for the evolution of wide aerenchyma tissues throughout the plant organs to improve the photosynthetic O2 flux from the shoot to the roots buried in anoxic sediments and to the neighboring rhizosphere. This favors sediment oxygenation, sustains the aerobic metabolism of roots, and improves the availability and uptake of mineral nutrients, whose delivery to the entire plants, without a transpirational flux, is ensured by an acropetal mass transport depending on root pressure, guttation from hydathodes and cha...

The effect of amitrole, known as an inhibitor of carotenoid biosynthesis, upon tetrapyrrole biosynthesis and its regulation has been studied. Etiolated barley (Hordeum vulgare L.) seedlings, grown in 125 microM amitrole, accumulated high levels of 5-aminolevulinate, Mg-protoporphyrin, Mg-protoporphyrin monomethyl ester, and protochlorophyllide. The amitrole-treated seedlings did not form paracrystalline prolamellar bodies, and the induction of Lhc and RbcS gene expression was reduced by non-photooxidative, low-intensity light. None of these events was observed upon treatment of the seedlings with 100 microM norflurazon, another inhibitor of carotenoid biosynthesis. The effect of amitrole cannot be explained solely by interaction with a presumed feedback inhibition of 5-aminolevulinate synthesis since incubation with amitrole and 5-aminolevulinate indicated that deregulation also occurs at later steps of tetrapyrrole biosynthesis. A possible relationship between this deregulation and ultrastructural changes is discussed. In connection with previously published data, we discuss Mg-protoporphyrin and its monomethyl ester as possible candidates for a "plastid signal" that operates as a negative factor, reducing the expression of Lhc and RbcS genes in this higher plant.

The chemical composition, biosynthesis and ultrastructural organization of the cell wall of cultures of Koliella antarctica (Klebsormidiales, Chlorophyta), a green microalga obtained from samples of sea water taken near the Italian Station of Terra Nova (Ross Sea) during the austral summer 1989–1990, have been studied. Purified cell walls of the microalga were sequentially treated with chemicals which, in higher plants, solubilize matrix polysaccharides (pectins and hemicelluloses) and leave an insoluble residue considered to be α-cellulose. CDTA plus Na2CO3- and KOH-solubilized polysaccharides were made up of glucosyl residues (more than 90 mol%), as well as some minor sugars (mannose and rhamnose). Linkage analysis and enzymic hydrolysis of the matrix polysaccharides indicated the presence of β-1,4-linked glucans with minor amounts of other uncharacterized polysaccharides. The insoluble residue was composed of a small amount of crystalline α-cellulose associated with mannan or glucomannan chains. The bi...

Plants of Boea hygroscopica F. Muell were dehydrated to 9% relative water content (RWC) by withholding water for 26 d, and afterward the plants were rehydrated. Leaves were taken from control plants after 7, 12, and 26 d from the beginning of dehydration, and after 6 and 48 h from rehydration. The RWC decreased by 80% during dehydration, but the leaves regained RWC with rehydration. Dehydrated plants showed lesser amounts of proteins, lipids, and chlorophyll, all of which increased following rewatering. The lipid-to-protein ratio, which decreased during dehydration, returned to control level after 48 h of rehydration. Thylakoid lipids were more unsaturated when RWC reached the value of 9%. EPR measurements of spin-labeled proteins showed the presence of three different groups of proteins with different mobility in thylakoid membranes. The rotational correlation time of groups 1 and 2 increased with dehydration and decreased upon rehydration, whereas group 3 showed little changes. Desiccation did not cause thylakoid swelling or breakage, but the membrane system assemblage showed changes in thylakoid stacking. After 48 h of rehydration the membrane system recovered completely the organization of the fully hydrated state, showing several well-defined and regularly distributed grana.

The chloroplast photo-oxidation and the expression of the Cab gene Lhcb1, encoding the Lhcb1 light-harvesting chlorophyll a/b-protein of PS II, have been studied in leaf cells of maize treated with the two bleaching herbicides norflurazon and amitrole and of the two carotenoid-free mutants vp9 and vp2 grown under high photodamaging light. Both herbicides and mutations caused severe photo-oxidation of organelles. However, the plastids of norflurazon-treated and vp2 leaves were totally devoid of thylakoids and did not contain any chlorophyll, while the organelles of amitrole-treated and vp9 leaves still had a few altered and photosynthetically unfunctional membranes and very small quantities of chlorophylls. Despite the dramatic photodamage undergone by the plastids over several days, the cells of amitrole-treated and vp9 leaves maintained a certain expression of the Lhcb1 gene which, on the contrary, was completely blocked in the cells of norflurazon-treated and vp2 leaves. The experimental results, obtained by integrating biochemical and molecular analyses with ultrastructural observations, show that the maintainance of Cab gene expression does not strictly depend on intact and functional chloroplasts. The transcription of these genes, still maintained in cells with greatly photo-oxidized organelles, seems to be inversely related to the degree of thylakoid demolition, which can affect the last steps of chlorophyll biosynthesis.

Post-translational hydroxylation of peptide- bound proline residues, catalyzed by peptidyl-prolyl-4 hydroxylase (EC 1.14.11.2) using ascorbate as co- substrate, is a key event in the maturation of a number of cell wall-associated hydroxyproline-rich glycoproteins (HRGPs), including extensins and arabinogalactan- proteins, which are involved in the processes of wall stiAening, signalling and cell proliferation. Allium cepa L. roots treated with 3,4-DL-dehydroproline (DP), a specific inhibitor of peptidyl-prolyl hydroxylase, showed a 56% decrease in the hydroxyproline content of HRGP. Administration of DP strongly aAected the organization of specialized zones of root development, with a marked reduction of the post-mitotic isodiametric growth zone, early extension of cells leaving the meristematic zone and a huge increase in cell size. Electron-microscopy analysis showed dramatic alterations both to the orga- nization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls. Moreover, DP administration inhibited cell cycle progression. Root tips grown in the presence of DP also showed an increase both in ascorbate content (+53%) and ascor- bate-specific peroxidase activity in the cytosol (+72%), and a decrease in extracellular ''secretory'' peroxidase activity ()73%). The possible interaction between HRGPs and the ascorbate system in the regulation of both cell division and extension is discussed.

AZA, 5-Acetamido-1,3,4-thiadiazole-2-sulphonamide CA, carbonic anhydrase DIC, dissolved inorganic carbon Hepes, A-(2-hydroxyethyl)-1 piperazine-ethane sulfonic acid IC, inorganic carbon PAR, photosynthetic active radiation PATAg, periodic acid-thiosemicarbazide-silver proteinate Tris, tris (hydroxymethyl)-aminomethane The structural and physiological strategies developed by the leaves of the freshwater macrophyte Ranunculus trichophyllus to adapt to submersed life were studied. Photosynthesis is carried out mainly by the epidermis cells of the numerous segments into which the leaf is finely dissected. In these cells, containing most of the chloroplasts, a peculiar organization of the wall has been identified by cytochemical tests. A thin compact outer region covers the cell surface and splits up forming large lacunae between adjacent cells. Below it, a thick and loose inner region rich in hydrophilic pectic acids occurs, which grows in along the cell sides giving rise to wide transfer areas. In this latter cell wall region, in which the cell/environment contact and exchanges are amplified, the systems for inorganic carbon supply to photosynthetic cells operate. The leaves of R. trichophyllus can rely on environmental CO2 and HCO3– as sources of inorganic carbon for photosynthesis. A mechanism for bicarbonate utilization seems to involve its conversion to CO2 by an apoplastic carbonic anhydrase, whose activity gains importance as the availability of environmental CO2 decreases. Interestingly, it has been demonstrated that in this species CO2 can also be obtained from HCO3– by a photodependent increase in plasmamembrane H+-ATPase activity in the transfer areas of the epidermis cells. This is the first time that such a mechanism has been noted in a nonpolar leaf of a submerged macrophyte.

SummaryA recessive maize mutant disrupted in seedling develop-ment was isolated following transpositional mutagenesiswith Mutator. This mutant, lilliputian (lil1), exhibits aphenotype consisting of a large primary root, extremelyreduced stature, crinkly leaves and has been mapped byTB-A translocations on the short arm of chromosome 2.Comparative histological analyses revealed an increase incell number in the mutant root and leaves, as well as anirregularity in cell shapes, suggesting that the alteredmorphology of lil1 roots and leaves is related to anuncoordinated increase in cell divisions and to a defect indivision plane alignment. On the other hand, the reducedstature of the mutant plant may be ascribed to a defectin cell elongation. In situ hybridization of primary roottissues reveals higher levels of a-tubulin transcripts in thepericycle and in cells surrounding vascular elements inmutant rather than in wild-type tissues. These featuressuggest a role of the Lil1gene in the correct functioningof microtubules. Moreover, the fact that the mutation isrecognizable even in the dormant embryo and that mutantseedlings are obtained from immature (12 DAP) lil1embryos indicates that the defect in cell division patternaccounting for the disrupted seedling morphology mani-fests itself even during embryo development.Introduction

Light, fluorescence and electron microscopical analysis of the rooted freshwater plant Ranunculus trichophyllus revealed a peculiar anatomical feature. In addition to the true endodermis encircling the root stele, endodermis-like sheaths occurred around each vascular bundle of the leaf segments and of the eustelic stem with its large central cavity, which assumed an anatomical feature resembling that of some pteridophyte stems. These impermeable sheaths, whose cells differentiate suberized walls, can play a major role in hampering the apoplastic leakage of the pressurized water solution which flows throughout the plant in xylem vessels and contains the mineral nutrients taken up by the roots from the sediment. Moreover, these sheaths can function in preventing flooding of the aerenchymatic cavities of the submerged organs. In this way the endodermis-like sheaths preserve the correct circulation of gas and nutrient solution through the entire organism and assume great significance as a structural mechanism evolved by this species to survive and grow underwater.

Phycobilisomes (PBSs) of the red alga Pterocladiella capillacea collected in the field, were characterized both in situ and in vitro by means of a transmission electron microscope (TEM) and an image analyzer. Ultrathin sections of thalli and negatively stained PBSs after isolation revealed hemi-ellipsoidal shapes. In situ PBS dimensions were 38.5 ± 0.2 nm (height) × 38.8 ± 0.2 nm (width) × 22.6 ± 0.2 nm (thickness) in good agreement with the in vitro measurements (mean diameters of 37.6 ± 0.2 nm). These dimensions, especially the width, are smaller than those so far reported for red algae. This could depend either on the ecophysiological conditions of the thalli when harvested and/or on a staggered, symmetrically rotated and compressed disposition of biliprotein rods with respect to the allophycocyanin (APC) core. Hydroxylapatite chromatography of biliproteic extracts and SDS-PAGE electrophoresis revealed that phycoerythrin type R-(λmax 565 nm>540 nm>498 nm) is formed by α (18.6 kDa), β (19.9 kDa...

Germination and post-germination events have been compared in seeds of Cercis siliquastrum whose dormancy was removed by fulfilling the natural chilling requirement or by exogenous GA3 application.

The resurrection species Sporobolus stapfianus Gandoger has been studied by LM, TEM and SEM in order to define the leaf morphology and fine structure and to analyse the cellular changes occurring during the processes of dehydration and rehydration of the plant. Some characteristics of the fully hydrated leaf and some ultrastructural and physiological events which take place during leaf wilting are discussed in relation to their possible role in plant desiccation-tolerance. The leaves of S. stapfianus show several characteristics common among xerophytic species. In the resurrection leaf they could play a role in slowing down the drying rate, thus leaving time to activate the mechanisms protecting the cell structures against drought damage. Actually, the S. stapfianus leaves do not undergo important cellular alterations during dehydration. The chloroplasts, in particular, retain part of their photosynthetic pigments and thylakoid membranes. Upon rewatering leaf recovery is rather fast and the tissue structure and cell organization of the fully hydrated state are already regained after two days.

An ultrastructural study on the reserves stored in cotyledons and endosperm and on their utilization after germination has been carried out in Cercis siliquastrum seeds. The observations have shown that all the cotyledonal reserves of proteins and lipids are used during seedling growth. However, inside the endosperm cells only the protein reserves are depleted, and large quantities of lipids remain undigested. In both cell walls and intercellular spaces of the endosperm cells huge masses of polysaccharide reserves are stored. However, only the polysaccharides inserted in the cell walls are dismantled, while those located in the intercellular spaces are not attacked by the lytic enzymes. Some hypotheses regarding the possible roles of those endospermic non-nutritional materials in the strategies of seed germination and seedling growth are suggested.

The desiccation-tolerant plant Sporobolus stapfianus was subjected to slow dehydration and to rehydration either as a silica gel-dried detached leaf or as an air-dried plant. In detached leaves dehydration resulted in a lower relative water content in comparison with leaves dried on the plant. Water loss caused a reduction in chlorophyll, carotenoid and lipid contents and an increase in conjugated dienes. In detached leaves, ultrastructure was also affected by dehydration, showing damaged cells with altered chloroplasts which retained large quantities of starch and lipid-like inclusions in the stroma. Upon rehydration a continuous degradation of the chemical composition and cell organization was observed with a further increase in peroxidation. Leaves dehydrated on the plant showed degradation of chlorophyll and lipids, whereas carotenoids increased and conjugated dienes decreased. Desiccation caused a vacuolar fragmentation and a decline in starch, whereas chloroplasts underwent slight alterations. Following rewatering a full recovery of chlorophyll and lipids occurred, while carotenoids and dienes remained constant. Starch increased in the chloroplasts and there was complete recovery of the ordered cell arrangement and chloroplast organization. Of the chloroplast polar lipids, in both sets of leaves desiccation caused a reduction only in monogalactosyldiacylglycerol, while phospholipids showed an opposite pattern, increasing in air-dried leaves and decreasing in detached leaves. Rewatering of leaves desiccated on the plant led to a complete recovery of the lipid composition, whereas detached leaves suffered a complete lipid degradation with the loss of polyunsaturated fatty acids.

Summary The effects of amitrole, a bleaching herbicide affecting carotenogenesis, were studied in barley plants grown in light at 20 °C and at 30 °C. At the lower temperature the herbicide caused dramatic damage to the leaf chloroplasts, compared with control plants. The synthesis of protective carotenoids was drastically impaired, with some accumulation of lycopene and a very low production of β-carotene and xanthophylls. In chloroplasts this effect of amitrole was related to a fall in chlorophyll content, reduction of the thylakoid system and destruction of most 70S ribosomes. Though the damaged organelles maintained a certain ability to synthesize proteins, chlorophyll-binding polypeptides encoded by plastid DNA were not found in the altered thylakoids which, however, contained chlorophyll-binding proteins and other polypeptides encoded by the nuclear DNA. Amitrole appeared to be less effective in plants grown at 30 °C. The synthesis of the protective carotenoids was greatly increased and the chloroplasts exhibited structure and composition almost similar to those of control organelles. The possible existence in the carotenoid biosynthetic pathway of alternative thermo-modulated steps, with different amitrole sensitivity, is suggested.

3-Amino-1,2,4-triazole (amitrole) provided to germinating barley at 20°C in the light led to bleached seedling leaves and photodynamic destruction of chloroplast structure, whereas normal greening and chloroplast ultrastructure was obtained when the seedlings developed in the presence of amitrole in the light at 30°C. Mass spectrometric analysis of the extractable herbicide demonstrated the same content of amitrole in leaves developed at 20 and 30°C. A very similar temperature-sensitive syndrome is characteristic for the nuclear gene mutant tigrina-o 34 in barley. Amitrole and the mutation were shown to inhibit the cyclization of lycopene, leading to severe deficiencies in β-carotene and its xanthophyll derivative lutein. Besides accumulation of lycopene, also its precursors phytoene, phytofluene and ξ-carotene accumulated. Inhibition of carotenoid biosynthesis by amitrole and the mutation at 20°C in the light led to a strong reduction of both transcript and protein levels for chloroplastic glutamine synthetase (GS 2 ) while transcript amount and protein of the cytosolic isoenzyme (GS 1 ) were unaffected. At 30°C increased levels of mRNA for the chloroplastic isoform GS 2 were observed in wild type, mutant and amitrole-treated seedlings, but protein levels remained unchanged. Turnover rates of the GS 2 protein were the same at 20 and 30°C. This extensive translational control of chloroplastic GS 2 synthesis was also observed in a heat shock experiment, which revealed transiently increased mRNA levels for chloroplastic GS 2 but unchanged protein levels. Permissive synthesis of β-carotene and chloroplastic glutamine synthetase (GS 2 ) at 30°C in the presence of amitrole or the tigrina-o 34 mutation might be due to two alternative pathways of ionone ring formation using either lycopene or neurosporene as substrates for cyclization.

The involvement of cellulase (endo-b-l,4-glucanase, EC 3.2.1.4) in a number of different cell separation events which occur in higher plants has been well established. Besides their significance for the plant growth and differentiation, these events can be economically important since they also comprise softening of fleshy fruits and abscission of fruits, flowers and leaves. In higher plants cellulase is present in a number of different biochemical isoforms which are encoded by different genes. This finding is in accordance with the wide range of physiological events which require the intervention of cellulase activity, and whose peculiarities and amplitudes can be quite various.

Cyanobacteria (phylum Cyanophyta/Cyanobacteria, class Cyanophyceae) are among the most widespread organisms and are able to adapt themselves to different extreme environments. These micro-organisms have an important ecological role, given their ability to perform oxygenic photosynthesis, and are employed in different fields based on their ability to produce several bioactive compounds. Their prokaryotic nature, the presence of many cryptic species, and the coexistence of different nomenclature systems make the taxonomic identification of cyanobacteria particularly difficult. Moreover, for several species, the original reference strains (holotypes) are lacking. Increasingly, authors are using a polyphasic approach to characterize cyanobacteria, while typification is a recent trend that is being used to solve the problem of missing holotypes in other micro-organisms. Here we focus on a filamentous cyanobacterium, isolated from the Euganean Thermal District (Padova, Italy) and temporarily named strain ETS-02, using a polyphasic approach that includes morphological, ultrastructural, biochemical (pigment and fatty acid content), physiological (nitrogen fixation), and genetic (16S rRNA, 16S-23S ITS, cpcB-IGS-cpcA, rpoC1, gyrB, rbcL, nifD loci) analyses. The description of Phormidium cf. irriguum CCALA 759 as the epitype of Phormidium irriguum was also used to complete the characterization of strain ETS-02.

The effect of different external salt concentrations, from 0 mM to 1030 mM NaCl, on photosynthetic complexes and chloroplast ultrastructure in the halophyte Arthrocnemum macrostachyum was studied. Photosystem II, but not Photosystem I or cytochrome b6/f, was affected by salt treatment. We found that the PsbQ protein was never expressed, whereas the amounts of PsbP and PsbO were influenced by salt in a complex way. Analyses of Photosystem II intrinsic proteins showed an uneven degradation of subunits with a loss of about 50% of centres in the 0 mM NaCl treated sample. Also the shape of chloroplasts, as well as the organization of thylakoid membranes were affected by NaCl concentration, with many grana containing few thylakoids at 1030 mM NaCl and thicker grana and numerous swollen thylakoids at 0 mM NaCl. The PsbQ protein was found to be depleted also in thylakoids from other halophytes.

A better understanding of the mechanisms that govern copper (Cu) uptake, distribution and tolerance in Brassica carinata plants in the presence of chelators is needed before significant progress in chelate-assisted Cu phytoextraction can be made. The aims of this study were therefore to characterise (S,S)-N,N'-ethylenediamine disuccinic acid (EDDS)-assisted Cu uptake, and to compare the spatial distribution patterns of Cu in the roots and leaves of B. carinata plants. The plants were treated with 30 μM or 150 μM CuSO(4) or CuEDDS in hydroponic solution. Quantitative Cu distribution maps and concentration profiles across root and leaf cross-sections of the desorbed plants were obtained by micro-proton induced X-ray emission. In roots, the 30 μM treatments with both CuSO(4) and CuEDDS resulted in higher Cu concentrations in epidermal/cortical regions. At 150 μM CuSO(4), Cu was mainly accumulated in root vascular bundles, whereas with 150 μM CuEDDS, Cu was detected in endodermis and the adjacent inner cortical cell layer. Under all treatments, except with a H(+)-ATP-ase inhibitor, the Cu in leaves was localised mainly in vascular tissues. The incubation of plants with 150 μM CuEDDS enhanced metal translocation to shoots, in comparison to the corresponding CuSO(4) treatment. Inhibition of H(+)-ATPase activity resulted in reduced Cu accumulation in 30 μM CuEDDS-treated roots and 150 μM CuEDDS-treated leaves, and induced changes in Cu distribution in the leaves. This indicates that active mechanisms are involved in retaining Cu in the leaf vascular tissues, which prevent its transport to photosynthetically active tissues. The physiological significance of EDDS-assisted Cu uptake is discussed.

The increase in cell size and the activities of cell wall lytic enzymes during fruit growth supported by the mesocarp cell enlargement have been studied in peach of the freestone cultivar Redhaven. Four growth stages (S1-S4) in peach fruit were observed. Cell enlargement and formation of intercellular spaces in the mesocarp were directly correlated with the increase in fruit diameter. Cellulase and exopolygalacturonase showed the highest activity during the first stage of fast fruit growth (S1), while the endopolygalacturonase activity increased during the last growth stage (S4), when the fruit reached its final size and ripening started.

The presence of pale-green flecks on leaves (speckling) is a frequent character among herbaceous species from shady places and is usually due to local loosening of palisade tissue (air space type of variegation). In the winter-green Arum italicum L. (Araceae), dark-green areas of variegated leaf blades are ca. 400 μm thick with a chlorophyll content of 1080 mg m −2 and a palisade parenchyma consisting of a double layer of oblong cells. Pale-green areas are 25% thinner, have 26% less chlorophyll and contain a single, loose layer of short palisade cells. Full-green leaves generally present only one compact layer of cylindrical palisade cells and the same pigment content as dark-green sectors, but the leaf blade is 13% thinner. A spongy parenchyma with extensive air space is present in all leaf types. Green cells of all tissues have normal chloroplasts. Assays of photosynthetic activities by chlorophyll fluorescence imaging and O 2 exchange measurements showed that variegated pale-green and dark-green sectors as well as full-green leaves have comparable photosynthetic activities on a leaf area basis at saturating illumination. However, full-green leaves require a higher saturating light with respect to variegated sectors, and pale-green sectors support relatively higher photosynthesis rates on a chlorophyll basis. We conclude that i) variegation in this species depends on number and organization of palisade cell layers and can be defined as a “variable palisade” type, and ii) the variegated habit has no limiting effects on the photosynthetic energy budget of A. italicum , consistent with the presence of variegated plants side by side to full-green ones in natural populations.

This paper deals with the characterization of a filamentous cyanobacterial strain, isolated from the microbial mats developing on the thermal muds of Ischia Thermal District (Naples, Italy). The survey was carried out by a polyphasic approach that includes morphological, ultrastructural, biochemical, and genetic analyses. For the molecular analyses the 16S rDNA gene and the rbcL gene were considered. All results showed that this filamentous cyanobacterium belongs to the order Oscillatoriales, family Pseudanabaenaceae. Moreover, the phylogenetic reconstruction suggests that strain ITD-01 might represent the first described entity belonging to a new cyanobacterial genus, whose name might be Protolyngbya.

In maize vivipary, the precocious germination of the seed while it is still attached to the ear is a reliable phenotype for the identification of mutants impaired in the biosynthesis or response to abscisic acid (ABA). Here we present the characterization of a new allele of vp10, a gene encoding for a cofactor (MoCo) required for the last step of ABA biosynthesis. The lesion in this gene leads to a reduction in the endogenous ABA level. Embryonic messenger RNAs of the ABA inducible genes glb1, lea3, and rab17 are barely detectable, although their level increases when stimulated by exogenous ABA administration. These findings confirm that the mutant can be ascribed to a defect in ABA biosynthesis. In the absence of water stress, mutant plants grow like wild-type siblings; however when mutant tissues are exposed to air they differ from non-mutant ones by showing a higher rate of water loss, of transpiration and of stomatal conductance. These events are restored to almost normal values by adding exogenous ABA. All these defects are ascribable to an impairment in the regulation of stomatal opening since, in contrast to wild-type, some of the mutant stomata exhibit partially or totally open rims. The defect in ABA biosynthesis is also associated with loss of regulation of the expression of rab17 and rab28, two genes expressed in vegetative tissues under abiotic stress. These genes are constitutively expressed in the mutant plant tissues independently of the water regime applied. Thus this mutant may provide a tool for the study of molecular mechanisms underlying drought-stress responses in crop plants.

The term “hyperaccumulator” describes a number of plants that belong to distantly related families, but share the ability to grow on metalliferous soils and to accumulate extraordinarily high amounts of heavy metals in the aerial organs, far in excess of the levels found in the majority of species, without suffering phytotoxic effects. Three basic hallmarks distinguish hyperaccumulators from related non-hyperaccumulating taxa: a strongly enhanced rate of heavy metal uptake, a faster root-to-shoot translocation and a greater ability to detoxify and sequester heavy metals in leaves. An interesting breakthrough that has emerged from comparative physiological and molecular analyses of hyperaccumulators and related non-hyperaccumulators is that most key steps of hyperaccumulation rely on different regulation and expression of genes found in both kinds of plants. In particular, a determinant role in driving the uptake, translocation to leaves and, finally, sequestration in vacuoles or cell walls of great amounts of heavy metals, is played in hyperaccumulators by constitutive overexpression of genes encoding transmembrane transporters, such as members of ZIP, HMA, MATE, YSL and MTP families. Among the hypotheses proposed to explain the function of hyperaccumulation, most evidence has supported the “elemental defence” hypothesis, which states that plants hyperaccumulate heavy metals as a defence mechanism against natural enemies, such as herbivores. According to the more recent hypothesis of “joint effects”, heavy metals can operate in concert with organic defensive compounds leading to enhanced plant defence overall. Heavy metal contaminated soils pose an increasing problem to human and animal health. Using plants that hyperaccumulate specific metals in cleanup efforts appeared over the last 20 years. Metal accumulating species can be used for phytoremediation (removal of contaminant from soils) or phytomining (growing plants to harvest the metals). In addition, as many of the metals that can be hyperaccumulated are also essential nutrients, food fortification and phytoremediation might be considered two sides of the same coin. An overview of literature discussing the phytoremediation capacity of hyperaccumulators to clean up soils contaminated with heavy metals and the possibility of using these plants in phytomining is presented. © 2010 Elsevier Ireland Ltd. All rights reserved.

In this paper we report a morphological, ultrastructural, biochemical and molecular (16S rRNA, 16S–23S ITS, rbcL and rpoC1 gene sequencing) survey on a very thin, non-heterocystous, filamentous cyanobacterium, isolated from mats covering several mud maturation tanks of the Euganean Thermal District, at temperatures ranging from 26 to 59°C. Denaturing gradient gel electrophoresis results, obtained using cyanobacterial primers targeting the 16S rRNA gene, confirmed that this cyanobacterium is one of the commonest taxa growing in the mud tanks. Comparison with Geitlerinema sp. PCC 8501 (=Phormidium laminosum Gomont ex Gomont strain OH-1-p Cl 1), a thin thermobiotic species isolated from hot springs of Oregon and morphologically similar to our isolate, led us to hypothesize that the Euganean and PCC 8501 strains are either very similar sister species or ecotypes of the same species in a yet to be defined clade, clearly distinct within the paraphyletic Leptolyngbya group.

† Background and Aims Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca 2þ and Cl 2 as essential co-factors. While most subunits form the catalytic core responsible for water oxidation, PsbO, PsbP and PsbQ form an extrinsic domain exposed to the luminal side of the membrane. In vitro studies have shown that these subunits have a role in modulating the function of Cl 2 and Ca 2þ , but their role(s) in vivo remains to be elucidated, as the relationships between ion concentrations and extrinsic polypeptides are not clear. With the aim of understanding these relationships, the photosynthetic apparatus of the extreme halophyte Salicornia veneta has been compared with that of spinach. Compared to glycophytes, halophytes have a different ionic composition, which could be expected to modulate the role of extrinsic polypeptides. † Methods Structure and function of in vivo and in vitro PSII in S. veneta were investigated and compared to spinach. Light and electron microscopy, oxygen evolution, gel electrophoresis, immunoblotting, DNA sequencing, RT– PCR and time-resolved chlorophyll fluorescence were used. † Key Results Thylakoids of S. veneta did not contain PsbQ protein and its mRNA was absent. When compared to spinach, PsbP was partly depleted (30 %), as was its mRNA. All other thylakoid subunits were present in similar amounts in both species. PSII electron transfer was not affected. Fluorescence was strongly quenched upon irradiation of plants with high light, and relaxed only after prolonged dark incubation. Quenching of fluorescence was not linked to degradation of D1 protein. † Conclusions In S. veneta the PsbQ protein is not necessary for photosynthesis in vivo. As the amount of PsbP is sub-stoichiometric with other PSII subunits, this protein too is largely dispensable from a catalytic standpoint. One possibility is that PsbP acts as an assembly factor for PSII.