María Lorena Falcone Ferreyra

Treacher Collins syndrome (TCS) is a genetic disorder affecting facial development, primarily caused by mutations in the TCOF1 gene. TCOF1, along with NOLC1, play important roles in ribosomal RNA transcription and processing. Previously, a zebrafish model of TCS successfully recapitulated the main characteristics of the syndrome by knocking down the expression of a gene on chromosome 13 (coding for Uniprot ID B8JIY2), which was identified as the TCOF1 orthologue. However, database updates renamed this gene as nolc1 and the zebrafish database (ZFIN) identified a different gene on chromosome 14 as the TCOF1 orthologue (coding for Uniprot ID E7F9D9). NOLC1 and TCOF1 are large proteins with unstructured regions and repetitive sequences that complicate alignments and comparisons. Also, the additional whole genome duplication of teleosts sets further difficulty. In this study, we present evidence that endorses that NOLC1 and TCOF1 are paralogs, and that the zebrafish gene on chromosome 14 is a low-complexity LisH domain-containing factor that displays homology to NOLC1 but lacks essential sequence features to accomplish TCOF1 nucleolar functions. Our analysis also supports the idea that zebrafish, as has been suggested for other non-tetrapod vertebrates, lack the TCOF1 gene that is associated with tripartite nucleolus. Using BLAST searches in a group of teleost genomes, we identified fish-specific sequences similar to E7F9D9 zebrafish protein. We propose naming them “LisH-containing Low Complexity Proteins” (LLCP). Interestingly, the gene on chromosome 13 (nolc1) displays the sequence features, developmental expression patterns, and phenotypic impact of depletion that are characteristic of TCOF1 functions. These findings suggest that in teleost fish, the nucleolar functions described for both NOLC1 and TCOF1 mediated by their repeated motifs, are carried out by a single gene, nolc1. Our study, which is mainly based on computational tools available as free web-based algorithms, could help to solve similar conflicts regarding gene orthology in zebrafish.

In plants, salicylic acid (SA) hydroxylation regulates SA homoeostasis, playing an essential role during plant development and response to pathogens. This reaction is catalysed by SA hydroxylase enzymes, which hydroxylate SA producing 2,3‐dihydroxybenzoic acid (2,3‐DHBA) and/or 2,5‐dihydroxybenzoic acid (2,5‐DHBA). Several SA hydroxylases have recently been identified and characterised from different plant species, but no such activity has yet been reported in maize. In this work, we describe the identification and characterisation of a new SA hydroxylase in maize plants. This enzyme, with high sequence similarity to previously described SA hydroxylases from Arabidopsis and rice, converts SA into 2,5‐DHBA; however, it has different kinetic properties to those of previously characterised enzymes, and it also catalysers the conversion of the flavonoid dihydroquercetin into quercetin in in vitro activity assays, suggesting that the maize enzyme may have different roles in vivo to those previously reported from other species. Despite this, ZmS5H can complement the pathogen resistance and the early senescence phenotypes of Arabidopsis s3h mutant plants. Finally, we characterised a maize mutant in the S5H gene (s5h^Mu) that has altered growth, senescence and increased resistance against Colletotrichum graminicola infection, showing not only alterations in SA and 2,5‐DHBA but also in flavonol levels. Together, the results presented here provide evidence that SA hydroxylases in different plant species have evolved to show differences in catalytic properties that may be important to fine tune SA levels and other phenolic compounds such as flavonols, to regulate different aspects of plant development and pathogen defence.

In plants, salicylic acid (SA) hydroxylation regulates SA homoeostasis, playing an essential role during plant development and response to pathogens. This reaction is catalyzed by SA hydroxylase enzymes, which hydroxylate SA producing 2,3- dihydroxybenzoic acid (2,3-DHBA) and/or 2,5-dihydroxybenzoic acid (2,5-DHBA). Several SA hydroxylases have been recently identified and characterized from different plant species; however, no such activity has been previously reported in maize. In this work, we describe the identification and characterization of a new SA hydroxylase in maize plants. This enzyme, with high sequence similarity to previously described SA hydroxylases from Arabidopsis and rice, converts SA into 2,5-DHBA; however, it shows different kinetics properties to those from previously characterized enzymes, and it also catalyzes the conversion of the flavonoid dihydroquercetin into quercetin in in vitro activity assays, suggesting that the maize enzyme may have different roles in vivo as those previously reported from other species. Despite this, ZmS5H-1 can complement the resistance to pathogen and early senescence phenotypes of Arabidopsis s3h mutant plants. Finally, we characterized a maize mutant in the S5H-1 gene ( s5h-1Mu) that has altered growth, senescence and increased resistance against Colletotrichum graminicola infection, showing not only changes in SA and 2,5-DHBA but also variations in flavonol levels. Together, the results presented here provide evidence that SA hydroxylases in different plant species have evolved to show differences in catalytic properties that may be important to fine tune SA levels and other phenolic compounds such as flavonols to regulate different aspects of plant development and defense against pathogens.

Plant ribosomal proteins play universal roles in translation, although they are also involved in developmental processes and hormone signaling pathways. Among Arabidopsis RPL10 family members, RPL10A exhibits the highest expression during germination and early development, suggesting that RPL10A is the main contributor to these processes. In this work, we first analyzed RPL10A expression pattern in Arabidopsis thaliana using transgenic RPL10A_pro:GUS plants. The gene exhibits a ubiquitous expression pattern throughout the plant, but it is most strongly expressed in undifferentiated tissues. Interestingly, gene expression was also detected in stomatal cells. We then examined protein function during seedling establishment and abscisic acid (ABA) response. Heterozygous rpl10A mutant plants show decreased ABA-sensitivity during seed germination, are impaired in early seedling and root development, and exhibit reduced ABA-inhibition of stomatal aperture under light conditions. Overexpression of RPL10A does not affect the germination and seedling growth, but RPL10A-overexpressing lines are more sensitive to ABA during early plant development and exhibit higher stomatal closure under light condition both with and without ABA treatment than wild type plants. Interestingly, RPL10A expression is induced by ABA. Together, we conclude that RPL10A could act as a positive regulator for ABA-dependent responses in Arabidopsis plants.

UV‐B radiation inhibits plant growth, and this inhibition is, to a certain extent, regulated by miR396‐mediated repression of Growth Regulating transcription Factors (GRFs). Moreover, E2Fe transcription factor also modulates Arabidopsis leaf growth. Here, we provide evidence that, at UV‐B intensities that induce DNA damage, E2Fc participates in the inhibition of cell proliferation. We demonstrate that E2Fc deficient plants show a lower inhibition of leaf size under UV‐B conditions that damage DNA, decreased cell death after exposure and altered SOG1 and ATR expression. Interestingly, the previously reported participation of E2Fe in UV‐B responses, which is a transcriptional target of E2Fc, is independent and different of that described for E2Fc. On the other hand, we here demonstrate that E2Fc has an epistatic role over the miR396 pathway under UV‐B conditions. Finally, we show that inhibition of cell proliferation by UV‐B is independent of the regulation of class II TCP transcription factors. Together, our results demonstrate that E2Fc is required for miR396 activity on cell proliferation under UV‐B, and that its role is independent of E2Fe, probably modulating DNA damage responses through the regulation of SOG1 and ATR transcript levels.

Flavones, one of the largest groups of flavonoids, have beneficial effects on human health and are considered of high nutritional value. Previously, we demonstrated that maize type I flavone synthase (ZmFNSI) is one of the enzymes responsible for the synthesis of O‐glycosyl flavones in floral tissues. However, in related species such as rice and sorghum, type II FNS enzymes also contribute to flavone biosynthesis. In this work, we provide evidence that maize has both one FNSI and one FNSII flavone synthases. Arabidopsis transgenic plants expressing each FNS enzyme were generated to validate the role of flavones in protecting plants against UV‐B radiation. Here, we demostrate that ZmCYP93G7 (FNSII) has flavone synthase activity and is able to complement the Arabidopsis dmr6 mutant, restoring the susceptibility to Pseudomonas syringae. ZmFNSII expression is controlled by the C1/PL1 + R/B anthocyanin transcriptional complexes, and both ZmFNSI and ZmFNSII are regulated by UV‐B. Arabidopsis transgenic plants expressing ZmFNSI or ZmFNSII that accumulate apigenin exhibit less UV‐B‐induced damage than wild‐type plants. Together, we show that maize has two FNS‐type enzymes that participate in the synthesis of apigenin, conferring protection against UV‐B radiation.

Characterization of the steps leading to maize natural resistance against insect pests.

In this work, we investigated the role of an Arabidopsis protein, AtPDCD5, during senescence after a 24h-dark period. Previously, we demonstrated that AtPDCD5 participates in programmed cell death (PCD) after UV-B exposure and in age-induced senescence. The results presented here, together with previous data, demonstrate that AtPDCD5 not only plays an important role during DNA damage responses induced by UV-B radiation, but also takes part in PCD programs such as dark-induced senescence in Arabidopsis.

DNA damage responses have evolved to sense and react to DNA damage; the induction of DNA repair mechanisms can lead to genomic restoration or, if the damaged DNA cannot be adequately repaired, to the execution of a cell death program. In this work, we investigated the role of an Arabidopsis (Arabidopsis thaliana) protein, AtPDCD5, which is highly similar to the human PDCD5 protein; it is induced by ultraviolet (UV)-B radiation and participates in programmed cell death in the UV-B DNA damage response. Transgenic plants expressing AtPDCD5 fused to GREEN FLUORESCENT PROTEIN indicate that AtPDCD5 is localized both in the nucleus and the cytosol. By use of pdcd5 mutants, we here demonstrate that these plants have an altered antioxidant metabolism and accumulate higher levels of DNA damage after UV-B exposure, similar to levels in ham1ham2 RNA interference transgenic lines with decreased expression of acetyltransferases from the MYST family. By coimmunoprecipitation and pull-down assays, we provide evidence that AtPDCD5 interacts with HAM proteins, suggesting that both proteins participate in the same pathway of DNA damage responses. Plants overexpressing AtPDCD5 show less DNA damage but more cell death in root tips upon UV-B exposure. Finally, we here show that AtPDCD5 also participates in age-induced programmed cell death. Together, the data presented here demonstrate that AtPDCD5 plays an important role during DNA damage responses induced by UV-B radiation in Arabidopsis and also participates in programmed cell death programs.