Eduardo Purgatto

Chromoplast Differentiation: Current Status and Perspectives

Chromoplasts are carotenoid-accumulating plastids conferring color to many flowers and fruits as well as to some tubers and roots. Chromoplast differentiation proceeds from preexisting plastids, most often chloroplasts. One of the most prominent changes is remodeling of the internal membrane system associated with the formation of carotenoid-accumulating structures. During the differentiation process the plastid genome is essentially stable and transcriptional activity is restricted. The buildup of the chromoplast for specific metabolic characteristics is essentially dependent upon the transcriptional activity of the nucleus. Important progress has been made in terms of mediation of the chloroplast-to-chromoplast transition with the discovery of the crucial role of the Or gene. In this article we review recent developments in the structural, biochemical and molecular aspects of chromoplast differentiation and also consider the reverse differentiation of chromoplasts into chloroplast-like structures during the regreening process occurring in some fruit. Future perspectives toward a full understanding of chromoplast differentiation include in-depth knowledge of the changes occurring in the plastidial proteome during chromoplastogenesis, elucidation of the role of hormones and the search for signals that govern the dialog between the nuclear and the chromoplastic genome.

Inhibition of β-amylase activity, starch degradation and sucrose formation by indole-3-acetic acid during banana ripening

Abstract. In order to observe the effect of indole- 3-acetic acid (IAA) on carbohydrate metabolism, unripe banana (Musa acuminata AAA, cv. Nanicão) slices were infiltrated with the hormone and left to ripen under controlled conditions. The climacteric respiration burst was reduced by the action of IAA, and starch degradation and sucrose formation were delayed. Sucrose synthase (SuSy; EC 2.4.1.13) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) activities and transcript levels were not affected, indicating that prevention of sucrose accumulation was not related to sucrose-metabolizing enzymes. Impairment of sucrose synthesis could be a consequence of lack of substrate, since starch degradation was inhibited. The increase in activity and transcript level of β-amylase was delayed, indicating that this enzyme could be important in starch-to-sucrose metabolism in bananas and that it might be, at least partially, controlled at the transcriptional level. This is the first report showing that IAA can delay starch degradation, possibly affecting the activity of hydrolytic enzymes such as β-amylase (EC 3.2.1.2).

Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome.

Nitric Oxide Mediates the Hormonal Control of Crassulacean Acid Metabolism Expression in Young Pineapple Plants

Genotypic, developmental, and environmental factors converge to determine the degree of Crassulacean acid metabolism (CAM) expression. To characterize the signaling events controlling CAM expression in young pineapple (Ananas comosus) plants, this photosynthetic pathway was modulated through manipulations in water availability. Rapid, intense, and completely reversible up-regulation in CAM expression was triggered by water deficit, as indicated by the rise in nocturnal malate accumulation and in the expression and activity of important CAM enzymes. During both up- and down-regulation of CAM, the degree of CAM expression was positively and negatively correlated with the endogenous levels of abscisic acid (ABA) and cytokinins, respectively. When exogenously applied, ABA stimulated and cytokinins repressed the expression of CAM. However, inhibition of water deficit-induced ABA accumulation did not block the up-regulation of CAM, suggesting that a parallel, non-ABA-dependent signaling route was also operating. Moreover, strong evidence revealed that nitric oxide (NO) may fulfill an important role during CAM signaling. Up-regulation of CAM was clearly observed in NO-treated plants, and a conspicuous temporal and spatial correlation was also evident between NO production and CAM expression. Removal of NO from the tissues either by adding NO scavenger or by inhibiting NO production significantly impaired ABA-induced up-regulation of CAM, indicating that NO likely acts as a key downstream component in the ABA-dependent signaling pathway. Finally, tungstate or glutamine inhibition of the NO-generating enzyme nitrate reductase completely blocked NO production during ABA-induced up-regulation of CAM, characterizing this enzyme as responsible for NO synthesis during CAM signaling in pineapple plants.

The onset of starch degradation during banana ripening is concomitant to changes in the content of free and conjugated forms of indole-3-acetic acid

Summary Banana ( Musa acuminata AAA cv. Nanicao) slices were infiltrated with mannitol (control) and mannitol plus indole-3-acetic acid (IAA); then, some important ripening parameters like starch degradation, synthesis of ethylene and respiration were monitored. The contents of free-IAA and conjugated forms of IAA (ester and amide) were analyzed, by GC-MS-SIM, throughout the ripening in both banana slices and whole bananas. The starch degradation of IAA-treated slices was delayed for several days, but there was no difference between control and IAA-treated slices in the ethylene and respiration profiles. On day zero after infiltration, free-IAA levels were 500-fold higher in IAA-treated slices than in the control slices, but within 72 hours they declined to values 15-fold higher than those in the control group, with concomitant increase in IAA-ester. Similar to the banana slices, the onset of starch degradation occurred in whole bananas only when the free-IAA concentration was about 4 ng/g FW. The results herein suggest that IAA levels play a role during banana ripening in events like starch degradation with the consequence of banana sweetening.

Associations between glutathione peroxidase-1 Pro198Leu polymorphism, selenium status, and DNA damage levels in obese women after consumption of Brazil nuts

OBJECTIVE Alterations in selenium (Se) status may result in suboptimal amounts of selenoproteins, which have been associated with increased oxidative stress levels. The Pro198Leu polymorphism at the glutathione peroxidase-1 (GPx1) gene is supposed to be functional. The response of Se status, GPx activity, and levels of DNA damage to a Se supplementation trial between the genotypes related to that polymorphism was investigated. METHODS A randomized trial was conducted with 37 morbidly obese women. Participants consumed one Brazil nut, which provided approximately 290 μg of Se a day, for 8 wk. Blood Se concentrations, erythrocyte GPx activity, and DNA damage levels were measured at baseline and at 8 wk. The results were compared by genotypes. RESULTS The genotype frequencies were 0.487, 0.378, and 0.135 for Pro/Pro (the wild-type genotype), Pro/Leu, and Leu/Leu, respectively. At baseline, 100% of the subjects were Se deficient, and after the supplementation, there was an improvement in plasma Se (P < 0.001 for Pro/Pro and Pro/Leu, P < 0.05 for Leu/Leu), erythrocyte Se (P = 0.00 for Pro/Pro and Pro/Leu, P < 0.05 for Leu/Leu), and GPx activity (P = 0.00 for Pro/Pro, P < 0.00001 for Pro/Leu, P < 0.001 for Leu/Leu). In addition, the Pro/Pro group showed a decrease in DNA damage after Brazil nut consumption compared with baseline (P < 0.005), and those levels were higher in Leu/Leu subjects compared with those with the wild-type genotype (P < 0.05). CONCLUSION Consumption of one unit of Brazil nuts daily effectively increases Se status and increases GPx activity in obese women, regardless of GPx1 Pro198Leu polymorphism. However, the evaluated biomarkers showed distinct results in response to the supplementation when the polymorphism was considered.

Chemoprevention of rat hepatocarcinogenesis with histone deacetylase inhibitors: Efficacy of tributyrin, a butyric acid prodrug

Hepatocellular carcinoma (HCC) ranks in prevalence and mortality among top 10 cancers worldwide. Butyric acid (BA), a member of histone deacetylase inhibitors (HDACi) has been proposed as an anticarcinogenic agent. However, its short half‐life is a therapeutical limitation. This problem could be circumvented with tributyrin (TB), a proposed BA prodrug. To investigate TB effectiveness for chemoprevention, rats were treated with the compound during initial phases of “resistant hepatocyte” model of hepatocarcinogenesis, and cellular and molecular parameters were evaluated. TB inhibited (p < 0.05) development of hepatic preneoplastic lesions (PNL) including persistent ones considered HCC progression sites. TB increased (p < 0.05) PNL remodeling, a process whereby they tend to disappear. TB did not inhibit cell proliferation in PNL, but induced (p < 0.05) apoptosis in remodeling ones. Compared to controls, rats treated with TB presented increased (p < 0.05) hepatic levels of BA indicating its effectiveness as a prodrug. Molecular mechanisms of TB‐induced hepatocarcinogenesis chemoprevention were investigated. TB increased (p < 0.05) hepatic nuclear histone H3K9 hyperacetylation specifically in PNL and p21 protein expression, which could be associated with inhibitory HDAC effects. Moreover, it reduced (p < 0.05) the frequency of persistent PNL with aberrant cytoplasmic p53 accumulation, an alteration associated with increased malignancy. Original data observed in our study support the effectiveness of TB as a prodrug of BA and as an HDACi in hepatocarcinogenesis chemoprevention. Besides histone acetylation and p21 restored expression, molecular mechanisms involved with TB anticarcinogenic actions could also be related to modulation of p53 pathways.

Chemopreventive effects of β-ionone and geraniol during rat hepatocarcinogenesis promotion: distinct actions on cell proliferation, apoptosis, HMGCoA reductase, and RhoA

Chemopreventive activities of the dietary isoprenoids β-ionone (βI) and geraniol (GOH) were evaluated during the promotion phase of hepatocarcinogenesis. Over 5 consecutive weeks, rats received daily 16 mg/100 g body weight (b.w.) of βI (βI group), 25 mg/100 g b.w. of GOH (GOH group), or only corn oil (CO group, controls). Compared to the CO group, the following was observed: only the βI group showed a decrease in the mean number of visible hepatocyte nodules (P<.05); βI and GOH groups had reduced mean number of persistent preneoplastic lesions (pPNLs) (P<.05), but no differences regarding number of remodeling PNL (rPNLs) were observed; only the βI group exhibited smaller rPNL size and percentage of liver sections occupied by pPNLs (P<.05), whereas the GOH group displayed a smaller percentage of liver sections occupied by rPNLs (P<.05); a trend was observed in the βI group, which showed reduced cell proliferation of pPNLs (P<.10), and the GOH group had increased apoptosis in pPNLs and rPNLs (P<.05); only the βI group displayed reduced total plasma cholesterol concentrations (P<.05) and increased hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase mRNA levels (P<.05); only the GOH group had lower hepatic membrane RhoA protein levels (P<.05); both the βI- and GOH-treated groups had higher hepatic concentrations of βI and GOH, respectively (P<.05). Given these data, βI and GOH show promising chemopreventive effects during promotion of hepatocarcinogenesis by acting through distinct mechanism of actions: βI may inhibit cell proliferation and modulate HMGCoA reductase, and GOH can induce apoptosis and inhibit RhoA activation.

Carotenoid accumulation during tomato fruit ripening is modulated by the auxin-ethylene balance

BackgroundTomato fruit ripening is controlled by ethylene and is characterized by a shift in color from green to red, a strong accumulation of lycopene, and a decrease in β-xanthophylls and chlorophylls. The role of other hormones, such as auxin, has been less studied. Auxin is retarding the fruit ripening. In tomato, there is no study of the carotenoid content and related transcript after treatment with auxin.ResultsWe followed the effects of application of various hormone-like substances to “Mature-Green” fruits. Application of an ethylene precursor (ACC) or of an auxin antagonist (PCIB) to tomato fruits accelerated the color shift, the accumulation of lycopene, α-, β-, and δ-carotenes and the disappearance of β-xanthophylls and chlorophyll b. By contrast, application of auxin (IAA) delayed the color shift, the lycopene accumulation and the decrease of chlorophyll a. Combined application of IAA + ACC led to an intermediate phenotype. The levels of transcripts coding for carotenoid biosynthesis enzymes, for the ripening regulator Rin, for chlorophyllase, and the levels of ethylene and abscisic acid (ABA) were monitored in the treated fruits. Correlation network analyses suggest that ABA, may also be a key regulator of several responses to auxin and ethylene treatments.ConclusionsThe results suggest that IAA retards tomato ripening by affecting a set of (i) key regulators, such as Rin, ethylene and ABA, and (ii) key effectors, such as genes for lycopene and β-xanthophyll biosynthesis and for chlorophyll degradation.

Comprehensive Profiling of Ethylene Response Factor Expression Identifies Ripening-Associated ERF Genes and Their Link to Key Regulators of Fruit Ripening in Tomato

A small subset of ethylene response factor genes emerge as main actors in controlling fruit ripening via both ethylene-dependent and RIN/NOR-mediated mechanisms. Our knowledge of the factors mediating ethylene-dependent ripening of climacteric fruit remains limited. The transcription of ethylene-regulated genes is mediated by ethylene response factors (ERFs), but mutants providing information on the specific role of the ERFs in fruit ripening are still lacking, likely due to functional redundancy among this large multigene family of transcription factors. We present here a comprehensive expression profiling of tomato (Solanum lycopersicum) ERFs in wild-type and tomato ripening-impaired tomato mutants (Never-ripe [Nr], ripening-inhibitor [rin], and non-ripening [nor]), indicating that out of the 77 ERFs present in the tomato genome, 27 show enhanced expression at the onset of ripening while 28 display a ripening-associated decrease in expression, suggesting that different ERFs may have contrasting roles in fruit ripening. Among the 19 ERFs exhibiting the most consistent up-regulation during ripening, the expression of 11 ERFs is strongly down-regulated in rin, nor, and Nr tomato ripening mutants, while only three are consistently up-regulated. Members of subclass E, SlERF.E1, SlERF.E2, and SlERF.E4, show dramatic down-regulation in the ripening mutants, suggesting that their expression might be instrumental in fruit ripening. This study illustrates the high complexity of the regulatory network connecting RIN and ERFs and identifies subclass E members as the most active ERFs in ethylene- and RIN/NOR-dependent ripening.