Amb a 1 isoforms: Unequal siblings with distinct immunological features.

BACKGROUND: Ragweed pollen represents a major allergy risk factor. Ragweed extracts contain five different isoforms of the major allergen Amb a 1. However, the immunological characteristics of Amb a 1 isoforms are not fully investigated. Here, we compared the physicochemical and immunological properties of three most important Amb a 1 isoforms. METHODS: After purification, the isoforms were physicochemically characterized, tested for antibody binding and induction of human T-cell proliferative responses. Their immunological properties were further evaluated in vitro and in vivo in a mouse model. RESULTS: Amb a 1 isoforms exhibited distinct patterns of IgE binding and immunogenicity. Compared to Amb a 1.02 or 03 isoforms, Amb a 1.01 showed higher IgE-binding activity. Isoforms 01 and 03 were the most potent stimulators of patients' T cells. In a mouse model of immunization, Amb a 1.01 induced higher levels of IgG and IgE antibodies when compared to isoforms 02 and 03. Interestingly, ragweed-sensitized patients also displayed an IgG response to Amb a 1 isoforms. However, unlike therapy-induced antibodies, sensitization-induced IgG did not show IgE-blocking activity. CONCLUSION: The present study showed that naturally occurring isoforms of Amb a 1 possess different immunogenic and sensitizing properties. These findings should be considered when selecting sequences for molecule-based diagnosis and therapy for ragweed allergy. Due to its high IgE-binding activity, isoform Amb a 1.01 should be included in diagnostic tests. In contrast, due to their limited B- and T-cell cross-reactivity patterns, a combination of different isoforms might be a more attractive strategy for ragweed immunotherapy.

Transglutaminase as polyamine mediator in plant growth and differentiation.

Transglutaminases (TGases) are ubiquitous enzymes catalyzing many biological reactions. The best-known TGase activity, namely the transamidation of specific proteins by polyamines (PAs), has been studied in plants to verify if TGase is a mediator of PAs mechanism of action to re-interpret some of PAs effects. Usually, the TGase activity is present at basal level in plant cells, but it can be induced by internal or external events or stresses, like rehydration, wounding, light, developmental differentiation and programmed cell death (PCD). Here, two models of induced growth are presented, namely pollen apical growth and dedifferentiation followed by reacquisition of the pluripotency of already differentiated cells. Moreover, PAs and TGase involvement during the differentiation and the activity of organelles and finally during the terminal organ differentiation or self-incompatibility-induced PCD are reported. In all of these models, TGase plays a role. The enzyme was detected in several cell compartments, like cytosol, chloroplasts and possibly mitochondria, microsomal fraction, cell wall and also extracellularly. The products of TGase catalysis, modified with PAs, mainly consist of high molecular mass complexes. Among the protein substrates until now identified we mention the cytoskeletal proteins, actin and tubulin, whose PA modification also affects their interaction with motor proteins and the dynamic of cytoskeleton. The most widely studied substrates are component of chloroplast photosystems, in particular light-harvesting complexes, whose modification is light dependent and whose differentiation and size are affected by TGase, thereby conditioning photosynthetic efficiency and photoprotection. Finally, modification of cell wall substrates affects wall growth and reinforcement.

Polyamines are common players in different facets of plant programmed cell death.

Programmed cell death (PCD) is a process that occurs throughout the life span of every plant life, from initial germination of the seed to the senescence of the plant. It is a normal physiological milestone during the plant's developmental process, but it can also be induced by external factors, including a variety of environmental stresses and as a response to pathogen infections. Changes in the morphology of the nucleus is one of the most noticeable during PCD but all the components of the plant cell (cytoplasm, cytoskeleton and organelles) are involved in this fascinating process. To date, relatively little is known about PCD in plants, but several factors, among which polyamines (PAs) and plant growth regulators, have been shown to play an important role in the initiation and regulation of the process. The role of PAs in plant PCD appears to be multifaceted acting in some instances as pro-survival molecules, whereas in others seem to be implicated in accelerating PCD. The molecular mechanism is still under study. Here we present some PCD plant models, focusing on the role of the enzyme responsible for PA conjugation to proteins: transglutaminase (TGase), an enzyme linked with the process of PCD also in some animal models. The role of PAs and plant TGase in the senescence and PCD in flowers, leaf and the self-incompatibility of pollen will be discussed and examined in depth.

AtPng1 knockout mutant of Arabidopsis thaliana shows a juvenile phenotype, morpho-functional changes, altered stress response and cell wall modifications.

In order to ascertain the role of plant transglutaminases (TGase) in growth and abiotic stress response, the AtPng1 knock out (KO) line of A. thaliana has been analyzed during plant development and under heat and wound stress. Comparing wild type (WT) and KO lines a 58-kDa band was immunodetected by anti-AtPng1p antibody in the cell wall and chloroplasts only in the WT line. A residual TGase activity, not showing correlation with development nor stress response, was still present in the KO line. The KO line was less developed, with a juvenile phenotype characterized by fewer, smaller and less differentiated cells. Chloroplast TGase activity was insensitive to mutation. Data on stressed plants showed that (i) KO plants under heat stress were more juvenile compared to WT, (ii) different responses between WT and KO lines after wounding took place. TGase activity was not completely absent in the KO line, presenting high activity in the plastidial fraction. In general, the mutation affected A. thaliana growth and development, causing less differentiated cytological and anatomical features.

Chronic heat stress affects the photosynthetic apparatus of Solanum lycopersicum L. cv Micro-Tom.

Tomato (Solanum lycopersicum L.) is one of the most widely cultivated crops in the world. Tomato is a plant model and the relationship between yield and biotic/abiotic stress has attracted increasing scientific interest. Tomato cultivation under sub-optimal conditions usually negatively impacts growth and development; in particular, heat stress affects several cellular and metabolic processes, such as respiration and photosynthesis. In this work, we studied the effects of chronic heat stress on various cytological and biochemical aspects using the Micro-Tom cultivar as a model. Photosynthetic efficiency decreased during heat stress while levels of post-photosynthetic sugars (sucrose, fructose, glucose and glucose 6-phosphate) oscillated during stress. Similarly, photosynthetic pigments (lutein, chlorophyll a, chlorophyll b and ß-carotene) showed an oscillating downward trend with partial recovery during the stress-free phase. The energetic capacity of leaves (e.g. ATP and ADP) was altered, as well as the reactive oxygen species (ROS) profile; the latter increased during stress. Important effects were also found on the accumulation of Rubisco isoforms, which decreased in number. Heat stress also resulted in a decreased accumulation of lipids (oleic and linoleic acid). Photosynthetically alterations were accompanied by cytological changes in leaf structure, particularly in the number of lipid bodies and starch granules. Prolonged heat stress progressively compromised the photosynthetic efficiency of tomato leaves. The present study reports multi-approach information on metabolic and photosynthetic injuries and responses of tomato plants to chronic heat stress, highlighting the plant's ability to adapt to stress.

Determination of Transglutaminase Activity in Plants.

Transglutaminase (TGase:E.C. 2.3.2.13) catalyzes the acyl-transfer reaction between one or two primary amino groups of polyamines and protein-bound Gln residues giving rise to post-translational modifications. One increasing the positive charge on a proteins surface and the other results in the covalent crosslinking of proteins. Pioneering studies on TGase in plants started in the middle of the 1980's but the methodology designed for use with animal extracts was not directly applicable to plant extracts. Here we describe radioactive and colorimetric methods adapted to study plant TGase, as well as protocols to analyze the involvement of TGase and polyamines in the functionality of cytoskeletal proteins.