Salinity impairs photosynthetic capacity and enhances carotenoid-related gene expression and biosynthesis in tomato (Solanum lycopersicum L. cv. Micro-Tom).

Carotenoids are essential components of the photosynthetic antenna and reaction center complexes, being also responsible for antioxidant defense, coloration, and many other functions in multiple plant tissues. In tomato, salinity negatively affects the development of vegetative organs and productivity, but according to previous studies it might also increase fruit color and taste, improving its quality, which is a current agricultural challenge. The fruit quality parameters that are increased by salinity are cultivar-specific and include carotenoid, sugar, and organic acid contents. However, the relationship between vegetative and reproductive organs and response to salinity is still poorly understood. Considering this, Solanum lycopersicum cv. Micro-Tom plants were grown in the absence of salt supplementation as well as with increasing concentrations of NaCl for 14 weeks, evaluating plant performance from vegetative to reproductive stages. In response to salinity, plants showed a significant reduction in net photosynthesis, stomatal conductance, PSII quantum yield, and electron transport rate, in addition to an increase in non-photochemical quenching. In line with these responses the number of tomato clusters decreased, and smaller fruits with higher soluble solids content were obtained. Mature-green fruits also displayed a salt-dependent higher induction in the expression of PSY1, PDS, ZDS, and LYCB, key genes of the carotenoid biosynthesis pathway, in correlation with increased lycopene, lutein, ß-carotene, and violaxanthin levels. These results suggest a key relationship between photosynthetic plant response and yield, involving impaired photosynthetic capacity, increased carotenoid-related gene expression, and carotenoid biosynthesis.

Characterization of PIR1, a GATA family transcription factor involved in iron responses in the white-rot fungus Phanerochaete chrysosporium.

Iron, although toxic in excess, is an essential element for biological systems. Therefore, its homeostasis is of critical importance and tight mechanisms participate in its acquisition by microbial organisms. Lately, the relevance of this metal for biomass conversion by wood-degrading fungi has been gaining increasing attention. Iron plays a critical role as cofactor of key enzymes such as lignin and manganese peroxidases in lignin-degrading white-rot fungi, while Fe(II) also serves a pivotal role in Fenton reactions that are central in cellulose depolymerization by brown-rotters. It has been hypothesized that multicopper oxidases with ferroxidase activity might participate in controlling the bioavailability of iron in the hyphal proximity, fine-tuning Fenton chemistry and balancing lignin versus cellulose degradation. In order to further explore the dynamics of iron regulation in the well known white-rot fungus Phanerochaete chrysosporium, we analyzed the mRNA levels of the multicopper oxidases genes (mcos) in response to iron supplementation, confirming down-regulation of their expression in response to this metal. To gain a better understanding on the transcriptional mechanisms mediating this effect, we searched for a gene encoding a GATA-type transcription factor with homology to URBS1, the major transcriptional regulator of iron homeostasis in Ustilago maydis. Due to the limitation of experimental tools in P. chrysosporium, the alleged Phanerochaete iron regulator (PIR1) was studied by complementation of a Neurospora SRE/URBS1-deficient strain, where phenotypic and molecular characteristics of this transcriptional regulator could be easily assessed. In addition, using a genome-wide in silico strategy, we searched for putative cis-acting iron-responsive elements in P. chrysosporium. Some of the identified genes showed reduced transcript levels after 30 min in the presence of the metal, consistent with an SRE/URBS1-mediated mechanism, and suggesting a broad effect of iron on the regulation of several cellular processes.

The copper-dependent ACE1 transcription factor activates the transcription of the mco1 gene from the basidiomycete Phanerochaete chrysosporium.

We have previously identified and functionally characterized the transcription factor ACE1 (Pc-ACE1) from Phanerochaete chrysosporium. In Saccharomyces cerevisiae, ACE1 activates the copper-dependent transcription of target genes through a DNA sequence element named ACE. However, the possible target gene(s) of Pc-ACE1 were unknown. An in silico search led to the identification of putative ACE elements in the promoter region of mco1, one of the four clustered genes encoding multicopper oxidases (MCOs) in P. chrysosporium. Since copper exerts an effect at the transcriptional level in MCOs from several organisms, in this work we analysed the effect of copper on transcript levels of the clustered MCO genes from P. chrysosporium, with the exception of the transcriptionally inactive mco3. Copper supplementation of cultures produced an increment in transcripts from genes mco1 and mco2, but not from mco4. Electrophoretic mobility-shift assays revealed that Pc-ACE1 binds specifically to a probe containing one of the putative ACE elements found in the promoter of mco1. In addition, using a cell-free transcription system, we showed that in the presence of cuprous ion, Pc-ACE1 induces activation of the promoter of mco1, but not that of mco2.

Cloning and characterization of the genes encoding the high-affinity iron-uptake protein complex Fet3/Ftr1 in the basidiomycete Phanerochaete chrysosporium.

MCO1, a multicopper oxidase from Phanerochaete chrysosporium exhibiting strong ferroxidase activity, has recently been described. This enzyme shows biochemical and structural similarities with the yeast Fet3p, a type I membrane glycoprotein that efficiently oxidizes Fe(II) to Fe(III) for its subsequent transport to the intracellular compartment by the iron permease Ftr1p. The genome database of P. chrysosporium was searched to verify whether it includes a canonical fet3 in addition to mco1, and single copies of fet3 and ftr1 orthologues were found, separated by a divergent promoter. Pc-fet3 encodes a 628 aa protein that exhibits overall identities of about 40 % with other reported Fet3 proteins. In addition to a secretion signal, it has a C-terminal transmembrane domain, characteristic of these cell-surface-attached ferroxidases. Structural modelling of Pc-Fet3 revealed that the active site has all the residues known to be essential for ferroxidase activity. Pc-ftr1 encodes a 393 aa protein that shows about 38 % identity with several Ftr1 proteins from ascomycetes. Northern hybridization studies showed that the mRNA levels of both genes are reduced upon supplementation of the growth medium with iron, supporting the functional coupling of Fet3 and Ftr1 proteins in vivo.

Cloning and functional characterization of the gene encoding the transcription factor Ace1 in the basidiomycete Phanerochaete chrysosporium.

In this report we describe the isolation and characterization of a gene encoding the transcription factor Ace1 (Activation protein of cup 1 Expression) in the white rot fungus Phanerochaete chrysosporium. Pc-ace1 encodes a predicted protein of 633 amino acids containing the copper-fist DNA binding domain typically found in fungal transcription factors such as Ace1, Mac1 and Haa1 from Saccharomyces cerevisiae. The Pc-ace1 gene is localized in Scaffold 5, between coordinates 220841 and 222983. A S. cerevisiae ace1 null mutant strain unable to grow in high-copper medium was fully complemented by transformation with the cDNA of Pc-ace1. Moreover, Northern blot hybridization studies indicated that Pc-ace1 cDNA restores copper inducibility of the yeast cup 1 gene, which encodes the metal-binding protein metallothionein implicated in copper resistance. To our knowledge, this is first report describing an Ace1 transcription factor in basidiomycetes.

Cloning and functional characterization of the gene encoding the transcription factor Acel in the basidiomycete Phanerochaete chrysosporium

In this report we describe the isolation and characterization of a gene encoding the transcription factor Acel (Activation protein of cup 1 Expression) in the white rot fungus Phanerochaete chrysosporium. Pc-acel encodes a predicted protein of 633 amino acids containing the copper-fist DNA binding domain typically found in fungal transcription factors such as Acel, Macl and Haal from Saccharomyces cerevisiae. The Pc-acel gene is localized in Scaffold 5, between coordinates 220841 and 222983. A S. cerevisiae acel null mutant strain unable to grow in high-copper medium was fully complemented by transformation with the cDNA of Pc-acel. Moreover, Northern blot hybridization studies indicated that Pc-acel cDNA restores copper inducibility of the yeast cup 1 gene, which encodes the metal-binding protein metallothionein implicated in copper resistance. To our knowledge, this is first report describing an Acel transcription factor in basidiomycetes.