N. La Rocca

Amitrole treatment of etiolated barley seedlings leads to deregulation of tetrapyrrole synthesis and to reduced expression of Lhc and RbcS genes

Abstract. 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 μM 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 μM 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.

Resurrection Plants: The Puzzle of Surviving Extreme Vegetative Desiccation

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 vitrification, which occurs during the last phase of desiccation. Mechanical damage due to vacuole shrinkage in dehydrating cells is avoided by cell wall folding or by replacing the water in vacuoles with nonaqueous substances. Oxidative stress, due to enhanced production of reactive oxygen species (ROS) especially by chloroplasts, is minimized through two different strategies. The homoiochlorophyllous resurrection plants, which conserve chloroplasts with chlorophylls and thylakoids upon drying, fold leaf blades and synthesize anthocyanins, as both sunscreens and free radical scavengers, and additionally increase the activity of antioxidant systems in cells. In contrast, the chloroplasts in poikilochlorophyllous species degrade chlorophylls and thylakoid membranes yielding desiccoplasts that are devoid of any internal structures. These adaptive mechanisms preserve cells from damage by desiccation and allow them to resume vital functions once rehydrated. Even if based mainly on cell protection during drying, the vegetative desiccation-tolerance of resurrection plants also relies on systems of cell recovery and repair upon rehydration. However, most of these systems are prepared during cell dehydration.

Gamma-glutamyl transferase in the cell wall participates in extracellular glutathione salvage from the root apoplast

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.

Responses of the Antarctic microalga Koliella antarctica (Trebouxiophyceae, Chlorophyta) to cadmium contamination

Ultrastructural and physiological effects of exposure to 1 ppm and 5 ppm of cadmium (Cd) on cultured cells of Koliella antarctica, a green microalga from Antarctica, were investigated. The amount of Cd in the alga rose with the increase of the metal concentration in the growth medium and most Cd remained outside the cells, bound to the components of the cell walls. The increase of Cd in the microalga was concomitant with the decrease of other elements, mainly calcium (Ca). Exposure to 1 ppm Cd slowed culture growth by inhibiting cell division and also caused the development of some misshapen cells with chloroplast showing disordered thylakoids. However, this concentration did not substantially affect the chlorophyll (Chl) content or photosystem (PS) activity. At 5 ppm, Cd cell growth suddenly stopped and some cells lysed. After a week of Cd contamination, the cells were enlarged and severely damaged. The chloroplasts showed great ultrastructural alterations and a reduced Chl content. Cd exposure negatively affected PSII, whose activity was almost completely lost after four days.

Effects of Amitrole and Norflurazon on Carotenogenesis in Barley Plants Grown at Different Temperatures

A carotenoid deficiency can account for serious alterations of thylakoids, due to the essential roles played by these pigments in membrane protection against photo-oxidative damage (1,2). In spite of carotenoid importance in preserving the photosynthetic apparatus, information on their biosynthesis in green tissues is rather scarce, and the knowledge of carotenogenesis and its regulation mainly comes from studies on chromoplasts of ripening fruits (3,.4). Previous research on the lycopinic tigrina o 34 mutant of barley (5) and on barley plants treated with amitrole (6), a herbicide inhibiting lycopene cyclization (7), showed that plant growth temperature could affect carotenoid biosynthesis in leaf chloroplasts, suggesting the existence of thermo-modulated steps bypassing the block of mutation or herbicide. Recently, in tomato (4) and Arabidopsis (8) alternative reactions, which allow s-carotene and xanthophylls to be synthesized without involving lycopene as intermediate, have been proposed. In order to better define the relationship between temperature and carotenoid synthesis in chloroplasts, barley plants have been grown at two rather close temperatures. They have been treated with two herbicides, to analyze whether interruption of the carotenogenic pathway at distinct points would cause differentiated responses to the change in plant growth temperature. The two chemicals used were norflurazon (NF) and amitrole (AM), which inhibit phytoene desaturation and lycopene cyclization, respectively (7). In leaves of plants grown in different experimental conditions, the total and relative quantities of carotenoids synthesized and the ratios between the pigment forms were analyzed. The ultrastructural organization, chlorophyll contents and photosynthetic activity of chloroplasts were also investigated, to verify the photooxidative damage caused by the disturbed carotenoid biosynthesis.

Developmental and Photosynthetic Characteristics of a Photoautotrophic Chrysanthemum Culture

Chrysanthemum plantlets were cultivated in vitro on media with 2.0, 0.3, or 0 % sucrose, or photoautotrophically without an organic carbon source but with supplementation of the culture vessel atmosphere with 2 % CO2. The photoautotrophically cultivated plantlets showed a better growth and multiplication, higher contents of chlorophyll (Chl) and carotenoids, higher Chl a/b ratio, net photosynthetic rate and ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase activities than plantlets grown on the medium with sucrose.

Ultrastructural and cytochemical study of Plocamium cartilagineum (Plocamiales, Rhodophyta) from Ross Sea (Antarctica)

Abstract 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, compared with the Mediterranean ones, was the lack, in the former, of recognisable phycobilisomes on the thylakoid surfaces. This peculiarity had already been noticed in the other Antarctic red alga Iridaea cordata, collected, like Plocamium cartilagineum, in summer and from ice‐free waters. The loss of phycobilisomes may be a defence mechanism activated by benthic shade‐adapted red algae from Antarctica to protect the photosynthetic apparatus against damage from high light intensities.

Enzymes of ammonia assimilation, photosynthesis and respiration in alfaalfa leaves of different ages

The activities of enzymes involved in ammonia metabolism ferredoxin-dependent glutamate synthase (Fd-GOGAT), glutamine synthetase (GS) and glutamate dehydrogenase (GDH), the rates of photosynthetic oxygen evolution, dark respiration, and the activity of RuBP carboxylase (RuBPC) were determined in alfalfa (Medicago sativa L.) leaves taken from the apex (apical leaves), from the second to the fourth internode (mature leaves) and from the bottom of the canopy (basal leaves). Photosynthetic rate and the activities of RuBPC, GS and Fd-GOGAT showed their maximum in the mature leaves. The respiration rate together with amino acid and ammonium contents decreased with leaf age, whereas the opposite was true for GDH activity. Basal leaves still maintained substantial levels of chlorophylls, GS and Fd-GOGAT activities and oxygen evolution rate, thus suggesting that photosynthesis has some role in the reassimilation of the nitrogen liberated during protein degradation.

Morphological, Ultrastructural and photosynthetic features of two freshwater angiosperms.

Colonization of the aquatic environment by flowering plants has required the evolution of morphological and physiological adaptative mechanisms useful to face the numerous limiting factors which can make it difficult for a macrophyte to live in water.