Caracterización estructural de las proteínas FcEXPA1 y FcEXPA2 asociadas al desarrollo y maduración del fruto de Fragaria chiloensis (L). Duch

104 p.La frutilla Chilena (Fragaria chiloensis) es una especie nativa que surge como una nueva alternativa comercial, debido a sus excelentes características organolépticas y hortícolas, destacando la tolerancia frente a diferentes factores de estrés tanto biótico como abiótico. Su fruto es perecedero, de rápido ablandamiento, reflejado en la alteración de la textura del fruto, que influye negativamente en la vida de postcosecha y calidad de éste, limitando así su comercialización. Estudios previos han demostrado que el ablandamiento del fruto de F. chiloensis está relacionado con la modificación de la pared celular, mediado por un grupo de proteínas entre las que se encuentran las expansinas. Las expansinas son proteínas que inducen una disrupción reversible de los enlaces de hidrógeno existentes entre las microfibrillas de celulosa y las hemicelulosas, permitiendo la relajación de la pared celular. Hasta la fecha se desconoce a nivel molecular el modo de acción de las expansinas. Por lo tanto, con el fin de aportar nuevos antecedentes en relación a estas proteínas es que se obtuvo la estructura tridimensional de dos expansinas de F. chiloensis (FcEXPA1 y FcEXPA2), asociadas a la maduración del fruto de F. chiloensis, mediante modelado comparativo. Además, se realizó acoplamiento molecular y simulación de dinámica molecular frente a distintos sustratos del tipo hemicelulosa (XG) y celulosa, con el fin de evaluar el rol de las expansinas en la interacción con dichos sustratos. De esta forma, el estudio de la estructura de estos dos miembros de la familia de las expansinas en frutilla, nos permitió indagar comparativamente acerca de posibles diferencias estructurales y/o de interacción con sustratos entre miembros de esta familia génica./ABSTRACT:The Chilean strawberry (Fragaria chiloensis) is a native species that is emerging as a new commercial alternative, due to its excellent organoleptic and horticultural traits, highlighting tolerance to biotic and abiotic stress. Chilean strawberry fruit is perishable, showing a rapid and intense softening, reflected in the alteration of the texture of the fruit, which negatively influences the postharvest life and fruit quality, thus limiting the market. Previous studies have shown that fruit softening of F. chiloensis is related to cell wall modification mediated by expansins protein. Expansins induce a reversible disruption of the hydrogen bonds between cellulose microfibrils and hemicellulose (xyloglucan) allowing relaxation of the plant cell wall. With the aim to gain insight about the mode of action of expansins at molecular level, the three-dimensional structure of two Chilean strawberry expansins (FcEXPA1 and FcEXPA2) will be obtained by comparative modeling. Then, molecular docking and molecular dinamics with different hemicellulose and cellulose will be performed, in order to evaluate the mode of interaction of expansins with model substrates. The structural characterization of two members of the expansins family in strawberry allows us to investigate possible differences about structure and / or substrates interaction between members of the same gene famil

Priming of Defense Systems and Upregulation of MYC2 and JAZ1 Genes after Botrytis cinerea Inoculation in Methyl Jasmonate-Treated Strawberry Fruits

Several attempts have been made to study the effects of methyl jasmonate (MeJA) on plants in the past years. However, the comparative effects of the number and phenological time of MeJA applications on the activation of defense systems is currently unknown in strawberries. In the present research, we performed three field treatments during strawberry (Fragaria × ananassa ‘Camarosa’) fruit development and ripening which consisted of differential MeJA applications at flowering (M3), and the large green (M2 and M3) and red ripe (M1, M2, and M3) fruit stages. We also checked changes in gene expression related to plant defense against Botrytis cinerea inoculation post-harvest. In M3 treatment, we observed an upregulation of the anthocyanin and lignin contents and the defense-related genes, encoding for chitinases, β-1,3-glucanases and polygalacturonase-inhibiting proteins, after harvest (0 hpi), along with the jasmonate signaling-related genes FaMYC2 and FaJAZ1 at 48 h after B. cinerea inoculation (48 hpi) during postharvest storage. Although we did not find differences in gray mold incidence between the MeJA treatments and control, these results suggest that preharvest MeJA treatment from the flowering stage onwards (M3) primes defense responses mediated by the upregulation of different defense-related genes and retains the upregulation of MYC2 and JAZ1 at 48 hpi

Evolutionary Analysis of JAZ Proteins in Plants: An Approach in Search of the Ancestral Sequence

Jasmonates are phytohormones that regulate development, metabolism and immunity. Signal transduction is critical to activate jasmonate responses, but the evolution of some key regulators such as jasmonate-ZIM domain (JAZ) repressors is not clear. Here, we identified 1065 JAZ sequence proteins in 66 lower and higher plants and analyzed their evolution by bioinformatics methods. We found that the TIFY and Jas domains are highly conserved along the evolutionary scale. Furthermore, the canonical degron sequence LPIAR(R/K) of the Jas domain is conserved in lower and higher plants. It is noteworthy that degron sequences showed a large number of alternatives from gymnosperms to dicots. In addition, ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs are displayed in all plant lineages from liverworts to angiosperms. However, the cryptic MYC2-interacting domain (CMID) domain appeared in angiosperms for the first time. The phylogenetic analysis performed using the Maximum Likelihood method indicated that JAZ ortholog proteins are grouped according to their similarity and plant lineage. Moreover, ancestral JAZ sequences were constructed by PhyloBot software and showed specific changes in the TIFY and Jas domains during evolution from liverworts to dicots. Finally, we propose a model for the evolution of the ancestral sequences of the main eight JAZ protein subgroups. These findings contribute to the understanding of the JAZ family origin and expansion in land plants

Manosidasa y su participación indirecta en el desensamblaje de la pared celular de frutos de frutilla (Fragaria x ananassa)

La textura es uno de los parámetros organolépticos de la calidad de los frutos; la pérdida de esta cualidad conduce a un fruto poco atractivo para su consumo. Las enzimas que escinden los polisacáridos de la pared celular han sido foco de estudio para comprender cómo inciden el desensamblaje de esta compleja matriz y como esto se relaciona con el ablandamiento de los frutos. En Fragaria x ananassa, esta cualidad organoléptica es relevante, dado su breve periodo de vida útil donde este tipo de cambios de textura abundan, lo que conlleva a la pérdida de la calidad. La α-D-manosidasa manohidrolasa (α-man) (GH38; EC 3.2.1.24), es una enzima exoglicosidasa que escinde residuos de manosa de las N-glicosilaciones en los extremos no reducidos unidos por enlaces α-1,2, α-1,4 y α-1,6. La cuantificación de los niveles de transcritos de esta enzima en frutos como ciruela y durazno, ha reportado un incremento en los niveles de transcritos a medida que el fruto madura. Por otra parte, experimentos de RNAi de α-MAN en tomate, han evidenciado una prolongación de la vida útil del fruto hasta por 30 días. En F. x ananassa, no se cuenta con la información acerca del efecto de la enzima α-man, no se sabe cuántas isoformas es posible encontrar ni sus niveles de transcrito ni la cuantificación de la actividad enzimática durante el proceso de maduración. Es por ello, que el objetivo de esta investigación es caracterizar la familia de α-man (GH38) presentes en F. x ananassa. Para ello se realizó la búsqueda de todas las isoformas de la familia de α-MAN presentes en la pared celular de frutos de F. x ananassa, determinando su clasificación filogenética. Asimismo, se evaluó el modelo in silico de α-man en su conformación como homodímero, utilizando la estructura cristalográfica de α-man de frijol. La información anterior se complementó con la evaluación de los niveles de transcritos y la cuantificación de la actividad enzimática a través de la maduración del fruto. Como resultado, diez isoformas fueron encontradas en la búsqueda dentro del genoma de F. x ananassa, las cuales varían en sus niveles de transcritos en los diferentes estadios de maduración. Similar comportamiento se observó en los niveles de actividad enzimática total, la que se vio modificada a medida que el fruto madura. Con relación a los análisis in silico, el análisis de secuencias y de dominios conservados permitió determinar los aminoácidos clave del sitio activo, actividad catalítica y de formación de dímeros. Las diez α-man se clasificaron en diferentes grupos filogenéticos, de los cuales se discuten sus diferencias. Por otra parte, el modelamiento por homología proporcionó una construcción de la estructura terciaria con un elevado nivel de confianza de acuerdo con los softwares utilizados como apoyo. Finalmente, se sugieren proyecciones y perspectivas que ayuden a promover la vida útil del fruto.Facultad de Ciencias Agrarias y Forestale

Design and Optimization of a Self-Assembling Complex Based on Microencapsulated Calcium Alginate and Glutathione (CAG) Using Response Surface Methodology

The aim of this work was to characterize and optimize the formation of molecular complexes produced by the association of calcium alginate and reduced glutathione (GSH). The influence of varying concentrations of calcium and GSH on the production of microcapsules was analyzed using response surface methodology (RSM). The microcapsules were characterized by thermogravimetric analysis (TGA-DTG) and infrared spectroscopy (FTIR) in order to assess the hydration of the complexes, their thermal stability, and the presence of GSH within the complexes. The optimum conditions proposed by RSM to reach the maximum concentration of GSH within complexes were a 15% w/v of GSH and 1.25% w/v of CaCl2, with which a theorical concentration of 0.043 mg GSH per mg of CAG complex was reached

A new functional JAZ degron sequence in strawberry JAZ1 revealed by structural and interaction studies on the COI1–JA-Ile/COR–JAZs complexes

© The Author(s) 2020.The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates fundamental plant processes as developmental and defense responses. JA-Ile mediates the interaction between the F-box protein COI1 (part of the SCF E3 ubiquitin ligase) and a JAZ repressor leading to early jasmonate responses. The Arabidopsis JAZ1 protein contains the canonical LPIARR degron sequence, which is responsible for the stabilization of the AtCOI1-JA-Ile-AtJAZ1 complex. In strawberry (Fragaria × ananassa) JAZ family was described at the transcriptional level during fruit development but the information about the interaction mode of this complex is still scarce at the molecular level. To gain insight into the strawberry JA-Ile receptor complex, we evaluated the interaction at the structural level, and protein models were built and analyzed for FaCOI1 and FaJAZ1, FaJAZ8.1, and FaJAZ10. The interaction between FaCOI1 and FaJAZ1, FaJAZ8.1 and FaJAZ10 were explored using several ligands, through molecular docking and molecular dynamics (MD) simulations, finding the strongest interaction with (+)-7-iso-JA-Ile than other ligands. Additionally, we tested interactions between FaCOI1 and FaJAZs by yeast two-hybrid assays in the presence of coronatine (COR, a JA-Ile mimic). We detected strong COR-dependent interactions between FaCOI1 and FaJAZ1. Interestingly, FaJAZ1 contains a new non-canonical (IPMQRK) functional degron sequence, in which Arg and Lys are the key residues for maintaining the interaction of the FaCOI1–COR–FaJAZ1 complex as we observed in mutated versions of the FaJAZ1 degron. Phylogenetic analysis showed that the IPMQRK degron is only present in orthologs belonging to the Rosoideae but not in other Rosaceae subfamilies. Together, this study uncovers a new degron sequence in plants, which could be required to make an alternative and functional JA-Ile perception complex in strawberry.This research was funded by the National Research and Development Agency (ANID, Chile), grant FONDECYT/Regular 1181310 to Carlos R. Figueroa. Adrián Garrido-Bigotes acknowledges ANID (grant FONDECYT/Postdoctorado 3190894). F.V-R acknowledges Universidad de Talca for a doctoral scholarship. Work in R. Solano’s laboratory was funded by the Spanish Ministry of Economy and Competitivity grant BIO2016-77216-R (MINECO/FEDER)

Molecular Insights into FaEG1, a Strawberry Endoglucanase Enzyme Expressed during Strawberry Fruit Ripening

The endo-β-1,4-glucanases (EGs) that belong to the glycosyl hydrolase family 9 (GH9) have roles in cell wall synthesis, remodeling and degradation. Previous studies have suggested that EGs may play a key role in the ripening of different fruits including strawberries. In this study, we used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assays to determine the transcript accumulation of an endo-β-1,4-glucanase (FaEG1) during fruit development in two different strawberry ‘Camarosa’ and ‘Monterey’ with contrasting softening ratios. Phylogenetic analyses suggest that FaEG1 belongs to the α group of the GH9 family with other proteins previously described with roles in elongation, abscission and ripening. Comparative modeling was used to obtain the FaEG1 structure. The model displays a α-barrel−type structure that is typical of the GH9 enzyme family, and comprises 12 α-helices, 2 310 helices and 6 β-sheets. The catalytic residues were oriented to the solvent in the middle of an open groove. Protein−ligand interactions were explored with cellulose and two xyloglucans as ligands; the results suggest that the FaEG1-cellulose and FaEG1-XXXGXXXG (the most abundant xyloglucan in strawberries) complexes were more stable complexes than XXFGXXFG. The cell wall degradation was observed by scanning electron microscopy (SEM). The data are congruent with the probable role of the FaEG1 protein in the dissembly of the cellulose-hemicellulose fraction during the ripening of strawberry fruit

Characterization of FcXTH2, a Novel Xyloglucan Endotransglycosylase/Hydrolase Enzyme of Chilean Strawberry with Hydrolase Activity

Xyloglucan endotransglycosylase/hydrolases (XTHs) are cell wall enzymes with hydrolase (XEH) and/or endotransglycosylase (XET) activities. As they are involved in the modification of the xyloglucans, a type of hemicellulose present in the cell wall, they are believed to be very important in different processes, including growth, development, and fruit ripening. Previous studies suggest that XTHs might play a key role in development and ripening of Fragaria chiloensis fruit, and its characterization is pending. Therefore, in order to provide a biochemical characterization of the FcXTH2 enzyme to explain its possible role in strawberry development, the molecular cloning and the heterologous expression of FcXTH2 were performed. The recombinant FcXTH2 was active and displayed mainly XEH activity. The optimal pH and temperature are 5.5 and 37 °C, respectively. A KM value of 0.029 mg mL−1 was determined. Additionally, its protein structural model was built through comparative modeling methodology. The model showed a typically β-jelly-roll type folding in which the catalytic motif was oriented towards the FcXTH2 central cavity. Using molecular docking, protein-ligand interactions were explored, finding better interaction with xyloglucan than with cellulose. The data provided groundwork for understanding, at a molecular level, the enzymatic mechanism of FcXTH2, an important enzyme acting during the development of the Chilean strawberry

Involvement of the GH38 Family Exoglycosidase α-Mannosidase in Strawberry Fruit Ripening

Exoglycosidase enzymes hydrolyze the N-glycosylations of cell wall enzymes, releasing N-glycans that act as signal molecules and promote fruit ripening. Vesicular exoglycosidase α-mannosidase enzymes of the GH38 family (EC 3.2.1.24; α-man) hydrolyze N-glycans in non-reduced termini. Strawberry fruit (Fragaria × ananassa) is characterized by rapid softening as a result of cell wall modifications during the fruit ripening process. Enzymes acting on cell wall polysaccharides explain the changes in fruit firmness, but α-man has not yet been described in F. × ananassa, meaning that the indirect effects of N-glycan removal on its fruit ripening process are unknown. The present study identified 10 GH38 α-man sequences in the F. × ananassa genome with characteristic conserved domains and key residues. A phylogenetic tree built with the neighbor-joining method and three groups of α-man established, of which group I was classified into three subgroups and group III contained only Poaceae spp. sequences. The real-time qPCR results demonstrated that FaMAN genes decreased during fruit ripening, a trend mirrored by the total enzyme activity from the white to ripe stages. The analysis of the promoter regions of these FaMAN genes was enriched with ripening and phytohormone response elements, and contained cis-regulatory elements related to stress responses to low temperature, drought, defense, and salt stress. This study discusses the relevance of α-man in fruit ripening and how it can be a useful target to prolong fruit shelf life