Maria V. Busi

MADS-box genes expressed during tomato seed and fruit development

MADS-box genes in plants are putative transcription factors involved in regulating numerous developmental processes, such as meristem and organ identity in inflorescences and in flowers. Recent reports indicate that they are involved in other processes than flower development such as the establishment of developing embryos, seed coat and ultimately in root and fruit development. We have identified seven tomato MADS-box genes that are highly expressed during the first steps of tomato fruit development. According to comparisons of their deduced amino acid sequences, they were classified into two groups: (1) already identified tomato MADS-box genes previously defined as flower identity genes (TAG1, TDR4 and TDR6) and (2) new tomato MADS-box genes (TAGL1, TAGL2, TAGL11 and TAGL12). With the exception of TAGL12, which is expressed near uniformly in every tissue, the other genes show an induction during the tomato fruit development phase I (anthesis) and phase II, when active cell division occurs. In situ hybridization analyses show a specific expression pattern for each gene within the fruit and embryo sac tissues suggesting an important role in the establishment of tissue identity. Yeast two-hybrid analyses indicate that some of these proteins could potentially form dimers suggesting they could act together to accomplish their proposed role.

Functional and molecular characterization of the frataxin homolog from Arabidopsis thaliana

Frataxin is a highly conserved protein from bacteria to mammals that has been proposed to participate in iron–sulfur cluster assembly and mitochondrial iron homeostasis. In higher organisms, the frataxin gene is nuclear‐encoded and the protein is required for maintenance of normal mitochondrial iron levels and respiration. We describe here AtFH, a plant gene with significant homology to other members of the frataxin family. Plant frataxin has five segments of beta regions and two alpha helices, which are characteristics of human frataxin, as well as a potential N‐terminal targeting peptide for the mitochondrial localization. Transcription analysis showed that AtFH is ubiquitously expressed with high levels in flowers. Complementation of a Saccharomyces cerevisiae mutant (Δyfh) lacking the frataxin gene proved that AtFH is a functional protein, because it restored normal rates of respiration, growth and sensitivity to H2O2 of the null mutant. Our results support the involvement of AtFH in mitochondrial respiration and survival during oxidative stress in plants. This is the first report of a functional frataxin gene in plants.

Role of the N-terminal starch-binding domains in the kinetic properties of starch synthase III from Arabidopsis thaliana.

Starch synthase III (SSIII), one of the SS isoforms involved in plant starch synthesis, has been reported to play a regulatory role in the synthesis of transient starch. SSIII from Arabidopsis thaliana contains 1025 amino acid residues and has an N-terminal transit peptide for chloroplast localization which is followed by three repeated starch-binding domains (SBDs; SSIII residues 22-591) and a C-terminal catalytic domain (residues 592-1025) similar to bacterial glycogen synthase. In this work, we constructed recombinant full-length and truncated isoforms of SSIII, lacking one, two, or three SBDs, and recombinant proteins, containing three, two, or one SBD, to investigate the role of these domains in enzyme activity. Results revealed that SSIII uses preferentially ADPGlc, although UDPGlc can also be used as a sugar donor substrate. When ADPGlc was used, the presence of the SBDs confers particular properties to each isoform, increasing the apparent affinity and the V max for the oligosaccharide acceptor substrate. However, no substantial changes in the kinetic parameters for glycogen were observed when UDPGlc was the donor substrate. Under glycogen saturating conditions, the presence of SBDs increases progressively the apparent affinity and V max for ADPGlc but not for UDPGlc. Adsorption assays showed that the N-terminal region of SSIII, containing three, two, or one SBD module have increased capacity to bind starch depending on the number of SBD modules, with the D23 protein (containing the second and third SBD module) being the one that makes the greatest contribution to binding. The results presented here suggest that the N-terminal SBDs have a regulatory role, showing a starch binding capacity and modulating the catalytic properties of SSIII.

The starch‐binding capacity of the noncatalytic SBD2 region and the interaction between the N‐ and C‐terminal domains are involved in the modulation of the activity of starch synthase III from Arabidopsis thaliana

Starch synthase III from Arabidopsis thaliana contains an N‐terminal region, including three in‐tandem starch‐binding domains, followed by a C‐terminal catalytic domain. We have reported previously that starch‐binding domains may be involved in the regulation of starch synthase III function. In this work, we analyzed the existence of protein interactions between both domains using pull‐down assays, far western blotting and co‐expression of the full and truncated starch‐binding domains with the catalytic domain. Pull‐down assays and co‐purification analysis showed that the D(316–344) and D(495–535) regions in the D2 and D3 domains, respectively, but not the individual starch‐binding domains, are involved in the interaction with the catalytic domain. We also determined that the residues W366 and Y394 in the D2 domain are important in starch binding. Moreover, the co‐purified catalytic domain plus site‐directed mutants of the D123 protein lacking these aromatic residues showed that W366 was key to the apparent affinity for the polysaccharide substrate of starch synthase III, whereas either of these amino acid residues altered ADP‐glucose kinetics. In addition, the analysis of full‐length and truncated proteins showed an almost complete restoration of the apparent affinity for the substrates and Vmax of starch synthase III. The results presented here suggest that the interaction of the N‐terminal starch‐binding domains, particularly the D(316–344) and D(495–535) regions, with the catalytic domains, as well as the full integrity of the starch‐binding capacity of the D2 domain, are involved in the modulation of starch synthase III activity.

A mitochondrial dysfunction induces the expression of nuclear-encoded complex I genes in engineered male sterile Arabidopsis thaliana

To study the effect of a mitochondrial dysfunction induced by the expression of the unedited form of the subunit 9 of ATP synthase gene (u‐atp9) in Arabidopsis, we constructed transgenic plants expressing u‐atp9 under the control of three different promoters: CaMV 35S, apetala 3 and A9. The size and shape of transgenic plants bearing the apetala3::u‐atp9 and A9::u‐atp9 genes looked normal while the 35S::u‐atp9 transformed plants showed a dwarf morphology. All u‐atp9 expressing plants, independent of the promoter used, exhibited a male sterile phenotype. Molecular analysis of male sterile plants revealed the induction of the mitochondrial nuclear complex I (nCI) genes, psst, tyky and nadh binding protein (nadhbp), associated with a mitochondrial dysfunction. These results support the hypothesis that the expression of u‐atp9 can induce male sterility and reveal that the apetala3::u‐atp9 and A9::u‐atp9 plants induced the sterile phenotype without affecting the vegetative development of Arabidopsis plants. Moreover, male sterile plants produced by this procedure are an interesting model to study the global changes generated by an engineered mitochondrial dysfunction at the transcriptome and proteome levels in Arabidopsis plants.

Functional and structural characterization of the catalytic domain of the starch synthase III from Arabidopsis thaliana.

Glycogen and starch are the major energy storage compounds in most living organisms. The metabolic pathways leading to their synthesis involve the action of several enzymes, among which glycogen synthase (GS) or starch synthase (SS) catalyze the elongation of the α‐1,4‐glucan backbone. At least five SS isoforms were described in Arabidopsis thaliana; it has been reported that the isoform III (SSIII) has a regulatory function on the synthesis of transient plant starch. The catalytic C‐terminal domain of A. thaliana SSIII (SSIII‐CD) was cloned and expressed. SSIII‐CD fully complements the production of glycogen by an Agrobacterium tumefaciens glycogen synthase null mutant, suggesting that this truncated isoform restores in vivo the novo synthesis of bacterial glycogen. In vitro studies revealed that recombinant SSIII‐CD uses with more efficiency rabbit muscle glycogen than amylopectin as primer and display a high apparent affinity for ADP‐Glc. Fold class assignment methods followed by homology modeling predict a high global similarity to A. tumefaciens GS showing a fully conservation of the ADP‐binding residues. On the other hand, this comparison revealed important divergences of the polysaccharide binding domain between AtGS and SSIII‐CD. Proteins 2008.

Starch‐synthase III family encodes a tandem of three starch‐binding domains

The starch‐synthase III (SSIII), with a total of 1025 residues, is one of the enzymes involved in plants starch synthesis. SSIII from Arabidopsis thaliana contains a putative N‐terminal transit peptide followed by a 557‐amino acid SSIII‐specific domain (SSIII‐SD) with three internal repeats and a C‐terminal catalytic domain of 450 amino acids. Here, using computational characterization techniques, we show that each of the three internal repeats encodes a starch‐binding domain (SBD). Although the SSIII from A. thaliana and its close homologous proteins show no detectable sequence similarity with characterized SBD sequences, the amino acid residues known to be involved in starch binding are well conserved. Proteins 2006.

Metabolomics in Plants and Humans: Applications in the Prevention and Diagnosis of Diseases

In the recent years, there has been an increase in the number of metabolomic approaches used, in parallel with proteomic and functional genomic studies. The wide variety of chemical types of metabolites available has also accelerated the use of different techniques in the investigation of the metabolome. At present, metabolomics is applied to investigate several human diseases, to improve their diagnosis and prevention, and to design better therapeutic strategies. In addition, metabolomic studies are also being carried out in areas such as toxicology and pharmacology, crop breeding, and plant biotechnology. In this review, we emphasize the use and application of metabolomics in human diseases and plant research to improve human health.

The mitochondrial protein frataxin is essential for heme biosynthesis in plants

Frataxin, a conserved mitochondrial protein implicated in cellular iron homeostasis, has been involved as the iron chaperone that delivers iron for the Fe–S cluster and heme biosynthesis. However, its role in iron metabolism remains unclear, especially in photosynthetic organisms. In previous work, we found that frataxin deficiency in Arabidopsis results in decreased activity of the mitochondrial Fe–S proteins aconitase and succinate dehydrogenase, despite the increased expression of the respective genes, indicating an important role for Arabidopsis thaliana frataxin homolog (AtFH). In this work, we explore the hypothesis that AtFH can participate in heme formation in plants. For this purpose, we used two Arabidopsis lines, atfh‐1 and as‐AtFH, with deficiency in the expression of AtFH. Both lines present alteration in several transcripts from the heme biosynthetic route with a decrease in total heme content and a deficiency in catalase activity that was rescued with the addition of exogenous hemin. Our data substantiate the hypothesis that AtFH, apart from its role in protecting bioavailable iron within mitochondria and the biogenesis of Fe–S groups, also plays a role in the biosynthesis of heme groups in plants.

Effect of mitochondrial dysfunction on carbon metabolism and gene expression in flower tissues of Arabidopsis thaliana.

We characterized the transcriptomic response of transgenic plants carrying a mitochondrial dysfunction induced by the expression of the unedited form of the ATP synthase subunit 9. The u-ATP9 transgene driven by A9 and APETALA3 promoters induce mitochondrial dysfunction revealed by a decrease in both oxygen uptake and adenine nucleotides (ATP, ADP) levels without changes in the ATP/ADP ratio. Furthermore, we measured an increase in ROS accumulation and a decrease in glutathione and ascorbate levels with a concomitant oxidative stress response. The transcriptome analysis of young Arabidopsis flowers, validated by qRT-PCR and enzymatic or functional tests, showed dramatic changes in u-ATP9 plants. Both lines display a modification in the expression of various genes involved in carbon, lipid, and cell wall metabolism, suggesting that an important metabolic readjustment occurs in plants with a mitochondrial dysfunction. Interestingly, transcript levels involved in mitochondrial respiration, protein synthesis, and degradation are affected. Moreover, the levels of several mRNAs encoding for transcription factors and DNA binding proteins were also changed. Some of them are involved in stress and hormone responses, suggesting that several signaling pathways overlap. Indeed, the transcriptome data revealed that the mitochondrial dysfunction dramatically alters the expression of genes involved in signaling pathways, including those related to ethylene, absicic acid, and auxin signal transduction. Our data suggest that the mitochondrial dysfunction model used in this report may be useful to uncover the retrograde signaling mechanism between the nucleus and mitochondria in plant cells.