The maize fused leaves1 (fdl1) gene controls organ separation in the embryo and seedling shoot and promotes coleoptile opening.

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.

Functional, histological and biomechanical characterization of wheat water-mutant leaves.

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.

Photosynthesis in extreme environments: responses to different light regimes in the Antarctic alga Koliella antarctica.

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.

Erythronium dens-canis L. (Liliaceae): an unusual case of change of leaf mottling.

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.

Use of micro-PIXE to determine spatial distributions of copper in Brassica carinata plants exposed to CuSO4 or CuEDDS.

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.

Chloroplast ultrastructure and thylakoid polypeptide composition are affected by different salt concentrations in the halophytic plant Arthrocnemum macrostachyum.

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.

Polyphasic approach and typification of selected Phormidium strains (Cyanobacteria).

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.

Variegation in Arum italicum leaves. A structural-functional study.

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 O2 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.

Heavy metal hyperaccumulating plants: how and why do they do it? And what makes them so interesting?

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.

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis.

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 extreme halophyte Salicornia veneta is depleted of the extrinsic PsbQ and PsbP proteins of the oxygen-evolving complex without loss of functional activity.

BACKGROUND AND AIMS: Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca(2+) and Cl(-) 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(-) and Ca(2+), 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.

Inhibition of lycopene cyclase results in accumulation of chlorophyll precursors.

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.

Evidence for PSII donor-side damage and photoinhibition induced by cadmium treatment on rice (Oryza sativa L.).

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.

Laticifers in Camptotheca acuminata Decne: distribution and structure.

In this paper, a system of laticifers in Camptotheca acuminata Decne (Nyssaceae) is described. Laticifers were already present in the leaf primordia of the shoot apex. In the mature leaves, laticifers were found in the midrib and in the larger veins, both in the parenchymatic region delimited by vascular bundles and in the cortex just external to the phloem. In the stem, laticifers were present in both the primary and secondary body, running parallel to the longitudinal axis. They were located in the pith and in the cortex proximal to the phloem. No laticifers were found in the roots. The histochemical analyses indicated that the main compounds accumulated in laticifers were phenols. Neutral lipids and fatty acids were also present. Ultrastructural observations showed osmiophilic globules both in the vacuoles and in the peripheral regions of the cytoplasm of the laticifer cells. Plastids were present, although altered, with some parallel membranes and lacking starch grains. The discovery in C. acuminata of a laticifer system, which had never been described for the order Cornales, could be of taxonomic value, also considering that this order has traditionally represented one of the most problematic groups of flowering plants.

Thylakoid dismantling of damaged unfunctional chloroplasts modulates the Cab and RbcS gene expression in wheat leaves.

Thylakoid membrane dismantling and Lhcb and RbcS nuclear gene expression have been analysed in leaves of wheat plants grown in high fluence rate light and deprived of photoprotective carotenoids by treatments with the two bleaching herbicides, either norflurazon or amitrole. The Lhcb transcript was not detectable in cells of norflurazon-supplied leaves, having chloroplasts totally devoid of both inner membranes and pigments. In contrast, a substantial amount of Lhcb mRNA could be found in cells of amitrole-treated leaves, whose severely damaged organelles still contained few strikingly altered and photosynthetically unfunctional thylakoids, as well as chlorophyll traces. A possible relationship between chlorophyll synthesis and Lhcb expression, with the transcript level depending on the rate of pigment production in photodamaged chloroplasts is discussed. Also the RbcS expression was linked to the chloroplast membrane photodamage. However, a detectable level of transcript was still produced in norflurazon-treated cells, despite complete thylakoid demolition. Thus, the wheat cell behaviour had to be placed between that of species, such as maize, in which the RbcS expression is broken off in these conditions, and that of species, such as pea, in which it is slightly lowered. Interestingly, the dramatically photodamaged chloroplasts still maintained the ability to synthesize proteins and this allowed SSU and LSU Rubisco subunits to be found in the organelles of both norflurazon- and amitrole-treated plants.