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.

Dark-induced senescence of barley leaves involves activation of plastid transglutaminases.

Transglutaminases (E.C. 2.3.2.13) catalyze the post-translational modification of proteins by establishing ε-(γ-glutamyl) lysine isopeptide bonds and by the covalent conjugation of polyamines to endo-glutamyl residues of proteins. In light of the confirmed role of transglutaminases in animal cell apoptosis and only limited information on the role of these enzymes in plant senescence, we decided to investigate the activity of chloroplast transglutaminases (ChlTGases) and the fate of chloroplast-associated polyamines in Hordeum vulgare L. 'Nagrad' leaves, where the senescence process was induced by darkness (day 0) and continued until chloroplast degradation (day 12). Using an anti-TGase antibody, we detected on a subcellular level, the ChlTGases that were associated with destacked/degraded thylakoid membranes, and beginning on day 5, were also found in the stroma. Colorimetric and radiometric assays revealed during senescence an increase in ChlTGases enzymatic activity. The MS/MS identification of plastid proteins conjugated with exogenous polyamines had shown that the ChlTGases are engaged in the post-translational modification of proteins involved in photosystem organization, stress response, and oxidation processes. We also computationally identified the cDNA of Hv-Png1-like, a barley homologue of the Arabidopsis AtPng1 gene. Its mRNA level was raised from days 3 to 10, indicating that transcriptional regulation controls the activity of barley ChlTGases. Together, the presented results deepen our knowledge of the mechanisms of the events happened in dark-induced senescence of barley leaves that might be activation of plastid transglutaminases.

Arabidopsis SHR and SCR transcription factors and AUX1 auxin influx carrier control the switch between adventitious rooting and xylogenesis in planta and in in vitro cultured thin cell layers.

BACKGROUND AND AIMS: Adventitious roots (ARs) are essential for vegetative propagation. The Arabidopsis thaliana transcription factors SHORT ROOT (SHR) and SCARECROW (SCR) affect primary/lateral root development, but their involvement in AR formation is uncertain. LAX3 and AUX1 auxin influx carriers contribute to primary/lateral root development. LAX3 expression is regulated by SHR, and LAX3 contributes to AR tip auxin maximum. In contrast, AUX1 involvement in AR development is unknown. Xylogenesis is induced by auxin plus cytokinin as is AR formation, but the genes involved are largely unknown. Stem thin cell layers (TCLs) form ARs and undergo xylogenesis under the same auxin plus cytokinin input. The aim of this research was to investigate SHR, SCR, AUX1 and LAX3 involvement in AR formation and xylogenesis in intact hypocotyls and stem TCLs in arabidopsis. METHODS: Hypocotyls of scr-1, shr-1, lax3, aux1-21 and lax3/aux1-21 Arabidopsis thaliana null mutant seedlings grown with or without auxin plus cytokinin were examined histologically, as were stem TCLs cultured with auxin plus cytokinin. SCR and AUX1 expression was monitored using pSCR::GFP and AUX1::GUS lines, and LAX3 expression and auxin localization during xylogenesis were monitored by using LAX3::GUS and DR5::GUS lines. KEY RESULTS: AR formation was inhibited in all mutants, except lax3. SCR was expressed in pericycle anticlinally derived AR-forming cells of intact hypocotyls, and in cell clumps forming AR meristemoids of TCLs. The apex was anomalous in shr and scr ARs. In all mutant hypocotyls, the pericycle divided periclinally to produce xylogenesis. Xylary element maturation was favoured by auxin plus cytokinin in shr and aux1-21. Xylogenesis was enhanced in TCLs, and in aux1-21 and shr in particular. AUX1 was expressed before LAX3, i.e. in the early derivatives leading to either ARs or xylogenesis. CONCLUSIONS: AR formation and xylogenesis are developmental programmes that are inversely related, but they involve fine-tuning by the same proteins, namely SHR, SCR and AUX1. Pericycle activity is central for the equilibrium between xylary development and AR formation in the hypocotyl, with a role for AUX1 in switching between, and balancing of, the two developmental programmes.

Increased interleukin-4-positive lymphocytes in patients with Hashimoto's thyroiditis and concurrent non-endocrine autoimmune disorders.

A prevalent T helper type 1 (Th1) subset of lymphocytes has been described in Hashimoto's thyroiditis (HT), but whether a similar polarization may characterize HT when associated with non-endocrine autoimmune disorders (NEAD) is not known. The aim of the present study was to analyse the intracellular Th1 and Th2 distinctive cytokines in patients with isolated HT or associated with non-endocrine autoimmune disorders. Intracellular cytokine expression was assessed in peripheral blood lymphocytes (PBL) of 68 out-patients (females = 55; males = 13; median age = 6 years) with HT : 33 had isolated HT and 35 had a concurrent NEAD. The percentage of interferon (IFN)-γ and interleukin (IL)-2 Th1- and IL-4 Th2-positive cells was measured by flow cytometric analysis. We found an increased percentage of IL-2-positive cells in all patients, without differences between patients with isolated HT or associated with NEAD. IFN-γ(+) cells were also increased in both groups, but the median percentage of those with isolated HT was lower than in patients with HT+NEAD (19·0 versus 29·9%; P = 0·0082). An increased number of IL-4-positive cells was observed in three of 33 (9·1%) patients with isolated HT and in 25 of 35 patients with NEAD [71%; P < 0·0001; relative risk (RR) = 3·18]. The median values of IL-4(+) cells (HT = 5·0% versus HT + NEAD = 16·8%) confirmed this large difference (P < 0·0001). A clear-cut increase of IL-4(+) lymphocytes characterizes patients with autoimmune thyroiditis who have associated non-endocrine autoimmune disorders. These findings may represent an initial tool to detect patients with autoimmune thyroiditis in which additional non-endocrine autoimmune disorders may be awaited.

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.

Compatible and self-incompatible pollination in Pyrus communis displays different polyamine levels and transglutaminase activity.

The polyamine (PA) content and the transglutaminase (TGase) activity have been investigated in Pyrus communis pollination with compatible and self-incompatible (SI) pollen in order to deepen their possible involvement in the progamic phase of plant reproduction. The PA distribution as free, perchloric acid (PCA)-soluble and PCA-insoluble fractions in ungerminated (UGP), germinating pollen (GP), styles and pollinated styles with compatible and SI pollens is discussed in the light of a possible role during pollination. Generally, the conjugated PAs both in PCA-soluble and PCA-insoluble fractions were higher than the free form. Within the conjugated PAs, the PCA-insoluble ones were the highest with the exception of the not pollinated styles. As TGase mediates some of the effects of PAs by covalently binding them to proteins, the activity of this enzyme, never checked before in styles and pollinated styles, was examined. In the SI styles, the TGase activity is higher in comparison to style-pollinated with compatible pollen, and high molecular mass cross-linked products were formed, suggesting an involvement of TGase in SI response. This is the first evidence on the presence of this enzyme activity in not pollinated and pollinated styles.

The presence of non-segmental vitiligo modifies intracellular cytokine subsets in patients with chronic lymphocytic thyroiditis.

Chronic lymphocytic thyroiditis and vitiligo often occur in association and seem to be characterized by a prevalent Th1-driven autoimmune process. The aim of this study is to analyze selected intracellular Τh1 and Th2 cytokines in patients with Hashimoto?s thyroiditis when associated with non-segmental vitiligo. We analyzed intracellular interleukin-2, interferon-gamma (Τh1) and interleukin-4 (Th2), in peripheral blood lymphocytes of 23 patients with isolated Hashimoto?s thyroiditis (group A) and of 11 patients with Hashimoto?s thyroiditis associated with non-segmental vitiligo (group B). Peripheral blood lymphocytes were stimulated and incubated with specific monoclonal antibodies. Intracellular cytokines were assayed by flow cytometric analysis. Interleukin-2 and interferon-gamma positive cells were increased in almost all patients but the median values were similar in patients with isolated Hashimoto?s thyroiditis and in those with concurrent vitiligo. In contrast, the number of patients with increased interleukin-4 positive cells was higher in patients with thyroiditis and vitiligo (9/11) than in those with isolated thyroiditis (2/23; p<0.0001). The median values of IL-4 positive cells in the two groups confirmed this difference (A: 5.8 percent, vs B: 20.6 percent; p=0.0011). Increased interleukin-4 positive lymphocytes characterize Hashimoto?s thyroiditis when associated with non-segmental vitiligo, suggesting a modified balance from highly prevalent Th1 to mixed Th1/Th2 subset.

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.

Transglutaminase activity during senescence and programmed cell death in the corolla of tobacco (Nicotiana tabacum) flowers.

Corolla life span of undetached flowers of Nicotiana tabacum was divided into stages from the closed corolla (stage 1) through anthesis (stage 5) to death (stage 9). Senescence began around stage 6 in the proximal part, concomitantly with DNA laddering. Nuclear blebbing, DNA laddering, cell wall modification, decline in protein, water, pigment content and membrane integrity were observed during senescence and PCD. Transglutaminase activity was measured as mono- and bis-derivatives of putrescine (mono-PU; bis-PU) and bis-derivatives of spermidine (bis-SD). Bis-derivatives decreased with the progression of senescence, while mono-PU increased during early senescence; derivatives were present in different amounts in the proximal and distal parts of the corolla. In excised flowers, exogenous spermine delayed senescence and PCD, and caused an increase in free and acid-soluble conjugated PA levels. Bis-PU was the most abundant PA-derivative before DNA laddering stage; thereafter, bis-PU generally decreased and mono-PU became the most abundant derivative.

Individually-tailored thyroxine requirement in the same patients before and after thyroidectomy: a longitudinal study.

OBJECTIVE: Thyroxine (T4) requirement after total thyroidectomy for differentiated thyroid carcinoma (DTC) is a debated issue. As most of the studies in the area have been retrospective and/or performed with heterogeneous therapeutic approaches, we designed our study to determine T4 requirement in the same patients and treatment settings, before and after total thyroidectomy. DESIGN, PATIENTS AND METHODS: This was a longitudinal study including 23 goitrous patients treated with T4 in an individually tailored fashion. All patients exhibited a stable TSH (median TSH = 0.28 mU/l) at a stable T4 dose for at least 1 year before surgery (median T4 dose = 1.50 µg/kg per day). The patients underwent total thyroidectomy based on cancer suspicion or compressive symptoms. Eventually diagnosed as having DTC (pT1b-pT2N0) and following surgical and radiometabolic treatment, they were treated with the same pre-surgical doses of T4. RESULTS: Three months after surgery,using the same pre-surgical dose, median TSH increased up to 5.38 mU/l (P<0.0001) and so the T4 dose had to be increased (median T4 dose = 1.95 µg/kg per day; +30%; P < 0.0001). Once divided by patients' age, we observed that, after thyroidectomy and maintaining the same pre-surgical dose, serum TSH significantly increased both in younger and in older patients (median TSH = 4.57 and 6.11 mU/l respectively). Serum TSH was restored to the pre-surgical level by increasing the dose up to 1.95 and 1.77 µg/kg per day (+25 and +21%) respectively. CONCLUSIONS: Following the same treatment regimen, a thyroidectomized patient requires one-third higher therapeutic T4 dose than before surgery. Despite this increase, the dose of T4 needed in our patients remains significantly lower than that previously described in athyreotic patients.

Identification of mitochondria in liver biopsies. A study by immunohistochemistry, immunogold and Western blot analysis.

Hepatocytes are rich in mitochondria, which play an important role in hepatic metabolism. In certain pathologic conditions (most often alcoholic liver disease) mitochondria became enlarged; nevertheless, even in these conditions they are hardly detectable on light microscopy. Recently an antimitochondrial antibody (mAM), which recognizes a 60-kDa protein, has been characterized. The purpose of the present study was to study immunoreactivity of this antibody in a series of liver biopsies. We studied 146 liver biopsies using an mAM. In 8 cases an ultrastructural study was also done, and in 2 cases Western blot analysis was performed. Cases were divided as follows: alcoholic liver disease (ALD, 31); steatosis (8); nonalcoholic steatohepatitis (NASH, 1); hepatitis C virus (HCV)-related hepatitis (83); hepatitis B virus (HBV)-related hepatitis (6); primary biliary cirrhosis (1); sclerosing cholangitis (1); haemosiderosis (1); sarcoidosis (1); alpha-1-antitrypsin deficiency (1); nonspecific findings (12). All the patients were investigated for alcohol or drug abuse, pharmacological treatment, hyperlipidaemia, hypercholesterolaemia and diabetes. Immunoreactivity was diffuse in cases of ALD, NASH and steatosis, and in patients with drug abuse. Electron microscopic immunogold and Western blot analysis confirmed that in the conditions examined the protein recognized by the mAM showed greater expression. Immunohistochemical staining was helpful in demonstrating a toxic or a metabolic insult even in cases in which the histological picture was blurred by viral infection.

Plant transglutaminases.

The identification procedures, the characteristics and the potential function of the recently detected plant transglutaminases, are discussed in the light of the knowledge of animal transglutaminases. The enzyme has been studied occasionally in lower organisms (bacteria, fungi and green algae) and more extensively in Angiosperms.

First evidence for polyamine conjugation mediated by an enzymic activity in plants.

An enzyme activity, found for the first time in plants, mainly located in the 22,000g supernatant of the crude extract of sprout apices of Helianthus tuberosus L. cv OB1 tubers, is able in vitro to covalently bind polyamines to endogenous substrates of different molecular weights. The major assay parameters, such as pH, dithiothreitol, and extract concentrations were optimized. The time course of the reaction, the dependence on putrescine concentration, its competition with histamine, the capacity to bind spermidine and spermine better than putrescine, the stability of the reaction product and analysis of the latter by sodium dodecyl sulfate polyacrylamide gel electrophoresis and thin-layer chromatography suggest that putrescine is linked to endogenous substrates by means of an enzyme reaction that shows some similarities with transglutaminase activities detected in animals. However, the activities of the crude extract and of a fraction eluted from a Sephadex G25 column were not affected by CaCl(2) concentrations lower or equal to 5 millimolar; 4 or 10 millimolar EGTA caused a very small reduction; higher concentrations of CaCl(2) and 15 millimolar or more of EDTA were inhibitory. N,N'-dimethylcasein was not recognized as a substrate. These data indicate that this activity also displays some characteristics which are different from those of animal transglutaminases.

Polyamines in chloroplasts: identification of their glutamyl and acetyl derivatives.

Incubation of chloroplasts of Helianthus tuberosus with labelled putrescine and/or spermidine and proteolytic digestion of their trichloroacetate-soluble and -insoluble proteins revealed the presence of N-(gamma-glutamyl)-putrescine, N1,N4-bis-(gamma-glutamyl)-putrescine and N1,N8-bis-(gamma-glutamyl)spermidine. This finding may be regarded as unequivocal proof of the presence of transglutaminase activity in chloroplasts. In addition, the recovery of spermidine or putrescine and acetylspermidine from chloroplasts incubated with [3H]putrescine or [3H]spermidine respectively indicates the existence of biosynthetic and oxidative pathways. These results suggest that polyamines may have an important function in chloroplasts both in their free form and by covalently binding to proteins.

Transcriptional inhibition of the operon for the spermidine uptake system by the substrate-binding protein PotD.

Inhibition of spermidine uptake in Escherichia coli, which occurs in the presence of accumulated polyamines, has been studied using the spermidine uptake operon consisting of the potA, -B, -C, and -D genes. Transcription of the potABCD operon was inhibited by PotD, a spermidine-binding protein usually found in the periplasm, and the inhibitory effect of PotD was increased by spermidine. Transcription was not affected by bovine serum albumin, PotA, or PotF, suggesting that the effects of PotD are specific to the PotD protein. In the presence of 8 mM spermidine, a 50% inhibition of transcription was observed with a molar ratio of approximately 1:500 of template DNA:PotD. It was found that PotD bound to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream of the ATG initiation codon of the potA gene. Binding of PotD to the downstream site was stimulated by spermidine. Overexpression of PotD in Escherichia coli DH5alpha inhibited the uptake of spermidine, the synthesis of PotABCD mRNA, and expression of a lacZ reporter gene fused downstream of a potA gene containing the PotD binding sites. In cells overexpressing PotD, a large amount of PotD existed as PotD precursor in spheroplasts. Our results indicate that PotD precursor can also inhibit spermidine transport. The amino acid residues in PotD that are involved in its interaction with the potABCD operon were determined using mutated PotD proteins. Thr-35 and Ser-85 of PotD were found to be important for this interaction. These results suggest that transcription of the spermidine transport (potABCD) operon is inhibited in vivo by PotD precursor rather than PotD through its binding to two regions close to the transcriptional initiation site of the operon.

Transgenic rice as a vehicle for the production of the industrial enzyme transglutaminase.

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases has increased the demand for an inexpensive, efficient and safe source of recombinant enzyme. We explored the use of plant-based systems for the production of this important industrial enzyme. Transgenic rice plants engineered with a rat prostate transglutaminase (rTGp), driven by the strong constitutive maize-1 ubiquitin promoter and its first intron, were shown to express the recombinant enzyme at the mRNA and protein levels. The Ca2+ dependence of the recombinant enzyme was confirmed by the biotin-labelled cadaverine-incorporation assay. In this communication we report the molecular and biochemical characterisation of transgenic plants expressing rTGp and this sets the stage for establishing a bioreactor system for the production of transglutaminases in plants.

A Zea mays 39-kDa thylakoid transglutaminase catalyses the modification by polyamines of light-harvesting complex II in a light-dependent way.

A transglutaminase (TGase; EC 2.3.2.13) activity, which shared many properties with the TGase activity of the Helianthus tuberosus chloroplast, was observed in the Zea mays L. chloroplast and in its fractions. This activity was found to be prevalent in thylakoids; bis-(glutamyl) spermidine and bis-(glutamyl) putrescine were the main polyamine conjugates formed. Light stimulated the endogenous thylakoid activity. Putrescine, spermidine and spermine were conjugated to the isolated light-harvesting complex of photosystem II (LHCII) with different degrees of efficiency, spermine being the polyamine most efficiently conjugated. A TGase with a light-sensitive activity was identified in the photosystem II-enriched fraction. Its partial purification on a sucrose gradient allowed the separation of a 39-kDa band, which was immunorecognised by two anti-TGase antibodies (Ab-3 and rat prostatic gland-TGase). Both a colorimetric and a radiometric assay for TGase activity, the former carried out in the presence of biotinylated cadaverine and the latter in the presence of polyamines labelled with radioactive isotopes and resulting in the isolation of glutamyl-polyamines, further confirmed that the thylakoid enzyme is indeed a calcium-dependent transglutaminase (Thyl-TGase). At variance with guinea pig liver and erythrocyte TGases, which are insensitive to light, the activity of the thylakoid transglutaminase is affected by light. Moreover, this enzyme, when tested with purified LHCII as substrate, catalysed the production of mono- and bis-glutamyl-polyamines in equal amounts, whereas the 'animal' enzymes produced mainly mono-derivatives. Herein, it is discussed whether this light sensitivity is due to the enzyme or the substrate.

Suborganellar localisation and effect of light on Helianthus tuberosus chloroplast transglutaminases and their substrates.

The light stimulation of transglutaminase (TGase EC 2.3.2.13) activity was verified by incubating isolated chloroplasts of Helianthus tuberosus L. continuously or for alternate periods of light or dark (light/dark and dark/light). The first 10 min of incubation always represented the critical period. Light-harvesting complexes of photosystem II (LHCII) were more intensely labelled by (14)C-polyamines under light and light/dark than under dark and dark/light conditions. Chloroplasts were fractionated into thylakoid- and stroma-enriched fractions in which multiple TGase forms and substrates were found. Antibodies against TGase recognised 58- and 24-kDa bands in thylakoids and a 150-kDa band in the stroma. The latter, and its 150-kDa fraction, catalysed the conjugation of 14C-polyamines to Rubisco. In both fractions (thylakoid-pre and stroma-pre) the analysis of polyamine glutamyl derivatives showed a significant light-affected conjugation of polyamines to endogenous proteins. Alternatively, entire chloroplasts were incubated and afterwards their sub-fractions were isolated (thylakoid-post and stroma-post). The PSII and LHCII complexes were more intensely immunodetected in thylakoid-post than in thylakoid-pre, especially under dark conditions. Conversely, the conjugation of polyamines to thylakoid proteins was clearly light-stimulated in thylakoid-post, and much less in thylakoid-pre. Stroma-pre proteins were poorly polyamine-conjugated and not light-affected; on the contrary, stroma-post proteins were much more polyamine-modified and strongly light-stimulated. Thus, the light-activated conjugation depends mainly on the presence of the thylakoid fraction during the assay. The protective effect on chloroplasts under photo-damage, stress or senescence conditions attributed in the literature to free polyamines is discussed with regard to the occurrence of polyamine conjugates catalysed by TGases.