Resumen de Caracterización funcional de mecanismos reguladores de la maduración del fruto de fresa
1. introducción o motivación de la tesis Strawberry Fragaria × ananassa ) belongs to the Rosaceae family in the genus Fragaria .
This soft fruit is cultivated in different regions of the world and is part of the diet of millions of people. Spain is the first producer of strawberries in Europe and the fifth in the world (FAO, 2018). Moreover, between the 80 90 % of the production is lo cated in Huelva, south w e st of Spain. The cultivated strawberry Fragaria × ananassa is an octoploid cultivar that results of the crossing between four different dipl oids species, F. viridis , F. nipponica , F. iinumae and F. vesca . Th e evolutionary studies re veal that F. vesca was the last genome to join and that event took place in North America (Edger et al., 2019). The genome of F.
vesca is responsible of the majorit y of transcript in the octoploid cultivar. Thus, this genome is use d in the transcriptome an alysis as its genome has been sequenced ( Edger et al ., 2017, Physiologically, strawberry is an herbaceous perennial plant which present a central stem (crow n) from roots, leaves and stolons emerge (Hancock, 1999). The inter action between temperatur e and photoperiod determines strawberry development, being able to reproduce sexually through fruits, or asexually through stolons. Strawberry fruit is a false frui t where the achenes, the true fruits, are embedded in the receptacl e. Up to 500 achenes migh t be found and those are developed through the fecundation of the carpel ( Erendorfer, 1983) and are composed in a combination of seed and ovary tissue. The receptac le presents a pith at the center, a fleshy cortex, the epidermis th at contains the achenes a nd a system of vascular bundles that connects the plant with the achenes.
The developmental process in soft fruit presents an initial phase of growth and elongation, followed by a phase of ripening. In strawberry receptacle, the first growing phase relys on the development of cortex and medulla cells and will determine the final size of the fruits (Hancock, 1999). The final size will also be determined by the numbe r and distribution of achenes in the receptacle and the percentage of fertilized carpels. Th erefore, the synthesis of auxins, mainly in the form of indole 3 acetic acid, takes place in the achenes and is the main responsible of the receptacle growth, as th e removal of the achenes at early stages of development inhibits th e fruit growing. Gibberel lins, cytokinins and abscisic acid have a limited role in the fruit growth (Nitsch,1950; Perkins Veazie, 1995).
In the elongation phase, the cortical cells presentan isodiametric growth tied to structural changes in the cell wall and subcellular structur es. On the other hand, the ripening phase takes 30 40 days to complete from anthesis and is determined by dramatic physical changes, as in the texture and color; ch emical changes, as the production of aroma and flavor compounds; as well as a variation on gwell as a variation on gene expression (Knee ene expression (Knee et al.,et al., 1977; Dennis, 1977; Dennis, 1984). This process is dependent of natural conditions, as temperature and photoperiod, 1984). This process is dependent of natural conditions, as temperature and photoperiod, and the contribution of assimilatand the contribution of assimilates and the water balance between the fruit and the plant. es and the water balance between the fruit and the plant.
All the changes produced in the changes produced in the ripening process need the synthesis of new proteins ripening process need the synthesis of new proteins (Manning, 1993). In this sense, some works have studied the evolution of transcript along (Manning, 1993). In this sense, some works have studied the evolution of transcript along developmental stages adevelopmental stages and ripening. Three evolution models have been described those nd ripening. Three evolution models have been described those that that increases along ripening,increases along ripening, those that decreases over ripening and other that peak at those that decreases over ripening and other that peak at intermediate stage and then decline (Veluthambi and Poovaiah, 1984; Reddy and intermediate stage and then decline (Veluthambi and Poovaiah, 1984; Reddy and Poovaiah, 1990; Reddy Poovaiah, 1990; Reddy et alet al.,., 1990; Manning, 1994). Recently, the use of massive 1990; Manning, 1994). Recently, the use of massive transcriptometranscriptome analysis and the sequencanalysis and the sequencing of the genome allowed us to identify the ing of the genome allowed us to identify the transcriptomic changes during development and ripening stages to select genes for further transcriptomic changes during development and ripening stages to select genes for further characterization in this thescharacterization in this thesis (Medina Puche et al., 2016, Sanchez Sevilla et al., 2017). is (Medina Puche et al., 2016, Sanchez Sevilla et al., 2017).
Fruits can be divided into cits can be divided into climacteric or nonlimacteric or non--climacteric depending if a burst of ethylene is climacteric depending if a burst of ethylene is present to trigger the ripening process. In strawberry, the ripening occurs without an present to trigger the ripening process. In strawberry, the ripening occurs without an increase onincrease on ethylene production, suggesting that this process is independent oethylene production, suggesting that this process is independent of this f this hormone (Iwata hormone (Iwata et et al.,al., 1969a, 1969b; Villareal 1969a, 1969b; Villareal et al.,et al., 2010). However, ethylene is produced 2010). However, ethylene is produced in low levels in mature fruits, suggesting that this hormone might have a secondary role iin low levels in mature fruits, suggesting that this hormone might have a secondary role in n strawberry ripening (strawberry ripening (Trainotti Trainotti et al.,et al., 2005, Villarreal 2005, Villarreal et al., et al., 22010, Merchante 010, Merchante et al., et al., 202013). 13). Thus, hormonal regulation of ripening is a very active field of study. Auxins inhibit fruit Thus, hormonal regulation of ripening is a very active field of study. Auxins inhibit fruit ripening and when their concentration decline the fruit ripening beripening and when their concentration decline the fruit ripening begins (Given gins (Given et al.,et al., 1988b). The peak concentration of gibberellins 1988b). The peak concentration of gibberellins and cytokinins is presentand cytokinins is present in achenes 7 days in achenes 7 days after anthesis. This suggests that GA might control the cell division in auxiliary buds and after anthesis. This suggests that GA might control the cell division in auxiliary buds and participates in the differentiation of the auxiliary participates in the differentiation of the auxiliary buds (Hytönen buds (Hytönen et al., et al., 2009, 2009, Teneira Teneira et al., et al., 2017). 2017). On the other hanOn the other hand, abscisic acid (ABA) pld, abscisic acid (ABA) plays many roles regulating different ays many roles regulating different physiological process in plants. It is involved in the control of seed maturation, vegetative physiological process in plants. It is involved in the control of seed maturation, vegetative growth, and in the response to difgrowth, and in the response to different abiotic stresses (Mishra ferent abiotic stresses (Mishra et al.,et al., 2006). In 2006). In strawberry, the istrawberry, the increase of ABA content, ancrease of ABA content, around 20 days post anthesis, is concomitant with round 20 days post anthesis, is concomitant with the decrease of auxins in both achenes and receptacles. The ratio ABA/auxins is the decrease of auxins in both achenes and receptacles. The ratio ABA/auxins is controlling the ripening process ancontrolling the ripening process and the genetic changes that take place in this transition d the genetic changes that take place in this transition (Perkins(Perkins--VVeazie eazie et al.,et al., 1995). The 1995). The application of exogenous ABA promotes strawberry application of exogenous ABA promotes strawberry ripening, while inhibiting its biosynthesis inhibits the process, suggesting that ABA is the ripening, while inhibiting its biosynthesis inhibits the process, suggesting that ABA is the hormone controlling thhormone controlling the process (Jia e process (Jia et al.,et al., 2011, Liao et al., 2018)2011, Liao et al., 2018) Strawberry is one of the most appreciated fof the most appreciated fruits among consumer due to its organoleptic ruits among consumer due to its organoleptic properties. The flavor is determined by the complex mixture of volatile acids and other properties. The flavor is determined by the complex mixture of volatile acids and other compounds such as sugars, organcompounds such as sugars, organic acids, phenolic acids and tannins. The receptacle is ic acids, phenolic acids and tannins. The receptacle is the depositthe deposit tissue of the plant. Comtissue of the plant. Composed by 90% and 10% soluble solids, it contains posed by 90% and 10% soluble solids, it contains many compounds much appreciated by its nutritional benefits as vitamin C, soluble sugars many compounds much appreciated by its nutritional benefits as vitamin C, soluble sugars and organic acids (Hollmanand organic acids (Hollman and Venema, 1993: Maas and Venema, 1993: Maas et alet al., 1996). Also, high ., 1996). Also, high concentrations ofconcentrations of anthocyanins and flavonoanthocyanins and flavonoids are found and compounds whose health ids are found and compounds whose health benefits are being studied nowadays benefits are being studied nowadays (Giampieri (Giampieri et alet al., 2015). Among those compounds, ., 2015). Among those compounds, carbohydrates are one of the main soluble compounds, not only providing energy for carbohydrates are one of the main soluble compounds, not only providing energy for metabolics pathways almetabolics pathways also they are important in so they are important in the flavor of the fruits. Moreover, organic the flavor of the fruits. Moreover, organic acids are responsible of the receptacle flavor and participate determining the colacids are responsible of the receptacle flavor and participate determining the color, or, inhibing some enzymatic activity as those related with cell wall dissembling. Non volatile inhibing some enzymatic activity as those related with cell wall dissembling. Non volatile acids are acids are more related with the acimore related with the acidic content of the receptacle, while volatile acids dic content of the receptacle, while volatile acids contribute to the aroma. Up to 360 volatile have been found in strawberry contribute to the aroma. Up to 360 volatile have been found in strawberry and, while being and, while being only up to 0.02% of the fresh weight, only up to 0.02% of the fresh weight, little changes in their composition may change thelittle changes in their composition may change the taste dramatically (Zabetakis and Holden, 1997).takis and Holden, 1997).
The most noticeable change along ripening is the color change from green to red The most noticeable change along ripening is the color change from green to red characteristic of richaracteristic of ripen fruits. This is due to the degradation of chlorophyll and the synthesis pen fruits. This is due to the degradation of chlorophyll and the synthesis of anthocyanins and flavonoidof anthocyanins and flavonoids (Timberlake, 1981, Perks (Timberlake, 1981, Perkinsins--Veazie, 1995). Its biosynthesis Veazie, 1995). Its biosynthesis takes place in white fruits using precursors from the shikimic acid pathway. Pelargonidin takes place in white fruits using precursors from the shikimic acid pathway. Pelargonidin 3 3 glucose is the most abundant anthocyanin in strawberry fruit, representing up to 90% of glucose is the most abundant anthocyanin in strawberry fruit, representing up to 90% of total anthocyantotal anthocyanin content. Anthocyanins in content. Anthocyanins concentration varies between different cultivars concentration varies between different cultivars but also in response to physiological conditions. but also in response to physiological conditions.
Flowering in strawberry is induced by low temperatures and shorts photoperiods as the s induced by low temperatures and shorts photoperiods as the development of the floral buds depend on thedevelopment of the floral buds depend on them. It has been described m. It has been described that that flowering locus flowering locus T T might be the responsible gene that suppress the stolon development and is associated might be the responsible gene that suppress the stolon development and is associated with independent with independent eenvironmentalnvironmental conditions flowering (conditions flowering (Perrote Perrote et alet al., 2016a). ., As sessile organism, plants are exposed to different biotic and abiotifferent biotic and abiotic agents in their ic agents in their habitat. Moreover, in the lately years the global climate change is changing the habitat. Moreover, in the lately years the global climate change is changing the environmental conditions denvironmental conditions dramatically. Those stresses might decrease strawberry ramatically. Those stresses might decrease strawberry production up to 50% (Vij y Tyagi, 2007). Thus, theproduction up to 50% (Vij y Tyagi, 2007). Thus, the studies focusing on physstudies focusing on physiological, iological, biochemical and molecular responses to those stresses are growing in an effort to reduce biochemical and molecular responses to those stresses are growing in an effort to reduce the lost produced by them.the lost produced by them. The TF The TF RdreB1BlRdreB1Bl has been identified to improve the response has been identified to improve the response to different stresses, as cold, drought and to different stresses, as cold, drought and salinity (salinity (GuGu et alet al., 2013., 2013, 2015, 2017). Moreover, , 2015, 2017). Moreover, transcriptome studies comparing plants subjected to different stresses have identified to transcriptome studies comparing plants subjected to different stresses have identified to several genseveral genes putatively involved to modulate the plant response. More studies are es putatively involved to modulate the plant response. More studies are needed to characterize the role pneeded to characterize the role played by them. layed by them. On the othOn the other hand, many diseases and er hand, many diseases and plagues also affect strawberry crops, causing a high economic impact on farmers, due to plagues also affect strawberry crops, causing a high economic impact on farmers, due to pesticide tpesticide treatment and fruit losses. Therefore, the improvement of the strawberry natural reatment and fruit losses. Therefore, the improvement of the strawberry natural resistance by genetic manresistance by genetic manipulation is an importantipulation is an important research objective. For instance, the research objective. For instance, the loci loci Rfp1Rfp1 is associated with the resistance to is associated with the resistance to Phytophthora fragariePhytophthora fragarie (Haymes (Haymes et al.,et al., 191997). 97). Moreover, both Moreover, both FaRCa1 FaRCa1 and and FaRCg1 FaRCg1 are two loci located in the chromosme 6 related with are two loci located in the chromosme 6 related with the resistancethe resistance to to Colletotrichum Colletotrichum acutatacutatumum and and C. gloeosporioidesC. gloeosporioides, respectively (Anciro , respectively (Anciro et et alal., 2018, Salinas ., 2018, Salinas et alet al., 2018). Thus, ., 2018). Thus, transgenic strawberry plants with htransgenic strawberry plants with high levels of igh levels of chitinase reduce the harm effects caused by the mildew fungus (Asao chitinase reduce the harm effects caused by the mildew fungus (Asao et al.,et al., 1997, 2003).1997, 2003).
Improving of the fruit quamproving of the fruit quality have received the most attention in breeding and lity have received the most attention in breeding and biotechnology programs. Taste, thus the combination of sweetness, aciditbiotechnology programs. Taste, thus the combination of sweetness, acidity and aroma are y and aroma are the most important quality traits to decide whether to purchase or not. Therefore, the the most important quality traits to decide whether to purchase or not. Therefore, the gegenes involved in those prones involved in those processes are being studied using transgenic plants although this cesses are being studied using transgenic plants although this technology is quite slow due to the time needed to regenerate ttechnology is quite slow due to the time needed to regenerate these plants. Moreover, the hese plants. Moreover, the european legislation is still very restrictive, blocking the using of transgeneuropean legislation is still very restrictive, blocking the using of transgenics crops. The ics crops. The developmendevelopment of the CRISPR/Cas9 technology, able to edit the DNA without introducing t of the CRISPR/Cas9 technology, able to edit the DNA without introducing exogenous material in the genome, and its applicatioexogenous material in the genome, and its application in the octoploid strawberry will allow n in the octoploid strawberry will allow to a faster genetic improvement in strawberry (to a faster genetic improvement in strawberry (Jinek Jinek et alet al., 201., 2012, Martín2, Martín--Pizarro Pizarro et alet al.,., 2018)2018)..
2.contenido de la investigación Along the development of this thesis, I have studied the the development of this thesis, I have studied the different molecular mechanisms different molecular mechanisms that regulate the ripening process in strawberry that regulate the ripening process in strawberry ((Fragaria × ananassaFragaria × ananassa)) fruit. Transcriptomic fruit. Transcriptomic analyanalysis performed in our research group allowed us to identify several transcrisis performed in our research group allowed us to identify several transcription ption factors (TFs) and regulatory proteins upfactors (TFs) and regulatory proteins up--regulated regulated alongalong the ripening process. Moreover, the ripening process. Moreover, those genes identified present a clear hormonal regulation suggesting those genes identified present a clear hormonal regulation suggesting that they are that they are ininvolved involved in the regulation of the regulation of physiological physiological pathways pathways that that contribute to modulcontribute to modulate the final ate the final organoleptic properties of the fruit. organoleptic properties of the fruit.
Among those genes, highlights FaPRE1FaPRE1, a ripening related gene that encode an atypical , a ripening related gene that encode an atypical HLH protein with high sequence homology wHLH protein with high sequence homology with ith PACLOBUTRAZOL RESISTANCE (PRE)PACLOBUTRAZOL RESISTANCE (PRE) genes. genes. bHLHbHLH TFs contain two clearly differentiated domains, a basic domain located at the TFs contain two clearly differentiated domains, a basic domain located at the amino terminus of the proteins, which contains 13amino terminus of the proteins, which contains 13--17 basic amino acids, and a HLH 17 basic amino acids, and a HLH region, located at the carboxy terminusregion, located at the carboxy terminus,, that comthat comprises two amphipathic prises two amphipathic αα--helices which helices which are ricare rich in hydrophobic amino acids and are connected by a loop of variable length.h in hydrophobic amino acids and are connected by a loop of variable length. Meanwhile, HLH proteinMeanwhile, HLH proteinss lack sequences needed lack sequences needed forfor DNADNA bindingbinding and, in consequence, and, in consequence, they did not present DNAthey did not present DNA--binding abilitybinding ability.. PRE genePRE genes are atypical bHLH proteins s are atypical bHLH proteins characterized by characterized by the the lack of lack of theirtheir DNADNA--binding domainbinding domain, thus , thus they they need to interact with need to interact with other proteins to modulate the expression of other genes.other proteins to modulate the expression of other genes.
In our work, we identified our work, we identified threethree PREPRE genes in strawberry genome that present mogenes in strawberry genome that present morere than than 90% of homology in the aminoacidic seque90% of homology in the aminoacidic sequence. Their transcriptional profile reveals thatnce. Their transcriptional profile reveals that,, while while FaPRE2 FaPRE2 and and 3 3 have their expression restricted to the vegetative tissues, have their expression restricted to the vegetative tissues, FaPRE1FaPRE1 is is induced along the ripening process, presenting null expression in vegetainduced along the ripening process, presenting null expression in vegetative tissues. tive tissues. Moreover, Moreover, FaPRE1 FaPRE1 expression is pexpression is positively regulated by ABA and negatively by auxins, ositively regulated by ABA and negatively by auxins, while gibberellins have no role in its expression. while gibberellins have no role in its expression. FaPRE1 FaPRE1 present ABA responsive present ABA responsive sequences in its promoter, but no gibberellin sequences are found. This mighsequences in its promoter, but no gibberellin sequences are found. This might explain the t explain the hormonal transcriptional response of this gene. hormonal transcriptional response of this gene. FaPRE1 FaPRE1 expression was transiently downexpression was transiently down--regulated injecting regulated injecting Agrobacterium Agrobacterium cells harboring RNAi ccells harboring RNAi construction into fruit receptacles. onstruction into fruit receptacles. Those receptacles that presentThose receptacles that presenteded higher silencing degree were shigher silencing degree were selected for further elected for further transcriptomic studies. The silenced fruits did not present any phenotypic transcriptomic studies. The silenced fruits did not present any phenotypic changes,changes, but but microarray analysis showmicroarray analysis showeded 227 genes down227 genes down--regulatregulated and 276 uped and 276 up--regulated comparing regulated comparing with control. Among the downwith control. Among the down--regulated genes, 70% were ripening rregulated genes, 70% were ripening relatedelated whilewhile,, in the upin the up--regulatedregulated genes,genes, 76% were overexpressed in immature receptacles. Those transcriptomic 76% were overexpressed in immature receptacles. Those transcriptomic analysis support an important role of FaPRE1analysis support an important role of FaPRE1whiwhichch antagonistically coordinated the antagonistically coordinated the transcription of genes transcription of genes related to both receptacle growth and rirelated to both receptacle growth and ripening.pening. Thus FaPRE1, in Thus FaPRE1, in ripened receptacle, represses the transcription of receptacle growth promoting genes while ripened receptacle, represses the transcription of receptacle growth promoting genes while activating the transcription of those geneactivating the transcription of those genes related to the receptacle ripening process.s related to the receptacle ripening process.
Transcription factors are essential for gene expression regulation in plantsion regulation in plants. Thus,. Thus, the next the next gene selected to be studied in our work was gene selected to be studied in our work was FaMYB123, FaMYB123, a a R2R3 R2R3 MYB TF. MYB TF. These TFs These TFs are are recognisable by their two imperferecognisable by their two imperfect MYB repeats that follow the R2 and R3 structure of the ct MYB repeats that follow the R2 and R3 structure of the cc--MYBMYB proteinsproteins.. To play its regulatory roTo play its regulatory role, MYB TFs interact with other TFs and proteins le, MYB TFs interact with other TFs and proteins involved in the transcription process by forming involved in the transcription process by forming anan MBW complexMBW complex that regulate many that regulate many metabolic and developmentmetabolic and developmental pathways.al pathways.
In our group,, we have previously identified and characterised other we have previously identified and characterised other MYB TF related wiMYB TF related with th the control of key pathways in the ripening process, such as FaMYB10, the control of key pathways in the ripening process, such as FaMYB10, a a master regulatormaster regulator of the phenylpropanoid/anthocyanin pathway,, and FaEOBIIand FaEOBII,, regulator of eugenol regulator of eugenol production. In this work, we report that FaMYB123 is a MYB R2R3 that production. In this work, we report that FaMYB123 is a MYB R2R3 that present the present the imperimperfect R2R3 domain in its sequence and is localized in the nucleus. The spatiofect R2R3 domain in its sequence and is localized in the nucleus. The spatio--temporal expression pattern of temporal expression pattern of FaMYB123 FaMYB123 reveal that reveal that it it is ripening related and present low is ripening related and present low expression in vegetative tissues and achenes. Moreover, itexpression in vegetative tissues and achenes. Moreover, itss expression expression is induced by ABAis induced by ABA and repressed by auxins. and repressed by auxins. These combined expression results indicate that These combined expression results indicate that FaMYB123FaMYB123 mainly regulates specific ripeningmainly regulates specific ripening--related physiological processes in the fruit receptacle.related physiological processes in the fruit receptacle.
To further investigate the physiological role played by FaMYB1233,, we generated swe generated stable table transgenics planttransgenics plantss with the expression of with the expression of FaMYB123 FaMYB123 knockknocked ed down by RNAi. Lines that down by RNAi. Lines that present a higher degree of silencing were selected for further analysispresent a higher degree of silencing were selected for further analysis (≥ 95%)(≥ 95%). Thus, . Thus, expression expression analysis revealed analysis revealed aa clear reduction of genes involved clear reduction of genes involved in anthocyanin in anthocyanin bibiosynthesisosynthesis. Those results were validated with metabolomic analysis. Those results were validated with metabolomic analysis thatthat I performed in I performed in collaboration with the Alisdair Fernie lab in two shorts stays at the Max Planck Institute of collaboration with the Alisdair Fernie lab in two shorts stays at the Max Planck Institute of Molecular Plant Physiology Molecular Plant Physiology ((Potsdam, GermanyPotsdam, Germany)). The . The metabolomic results revemetabolomic results revealaleded a a disbalance between anthocyanins and proantocyanins. While transgenics accumulatedisbalance between anthocyanins and proantocyanins. While transgenics accumulatedd lower amounts of anthocyanins, as pelargonidinlower amounts of anthocyanins, as pelargonidin--33--glucose and its malonyl derivate, a glucose and its malonyl derivate, a higher accumulation of proantocyanins were found in the trhigher accumulation of proantocyanins were found in the transgenics receptacleansgenics receptacle, wh, which ich are usually present at minor levels in ripe receptaclesare usually present at minor levels in ripe receptacles. Protein interaction studies revealed . Protein interaction studies revealed that FaMYB123 is able to bind to FabHLH3, a TF previously related to regulate that FaMYB123 is able to bind to FabHLH3, a TF previously related to regulate proanthocyanin biosynthesis through the interaction withproanthocyanin biosynthesis through the interaction with FaMYB9. This suggest thFaMYB9. This suggest that the at the complex FabHLH3complex FabHLH3--MYB9/123 MYB9/123 could could modulate the synthesis of promodulate the synthesis of pro-- and anthocyanin. and anthocyanin. Molecular and physiological studies showMolecular and physiological studies showeded that this TFthat this TF could becould be involved in the regulation involved in the regulation of structural genes of the anthocyanins or flavonols tof structural genes of the anthocyanins or flavonols that accumulate in ripen hat accumulate in ripen fruit fruit receptacles.receptacles.
Other regulatory mechanism of the gene expression is ther regulatory mechanism of the gene expression is DNA DNA methylation. Those methylation. Those epigenetics marks are usually tied to the silencing of genes, but lately there has been epigenetics marks are usually tied to the silencing of genes, but lately there has been described that DNA methylation might be requireddescribed that DNA methylation might be required for activating gene expfor activating gene expression or to bind ression or to bind TFTFss at the promoter. Epigenetics marks are related to control ripening in different fruits, but at the promoter. Epigenetics marks are related to control ripening in different fruits, but it’sit’s mainly mainly described in tomatodescribed in tomato.. DDNA methylation patterns change dramatically after the NA methylation patterns change dramatically after the application of either hyperapplication of either hyper-- or hypoor hypo--methylating drugmethylating drugs. s. 55--Azacytidine (AZA) is a chemical Azacytidine (AZA) is a chemical able to induce a general and nonspecific DNA hypomethylationable to induce a general and nonspecific DNA hypomethylation. It. It is added to DNAis added to DNA,, acting acting as a cytosine analogue, and those bases are covalently bond to DNA methyltransferases as a cytosine analogue, and those bases are covalently bond to DNA methyltransferases which would be degrwhich would be degraded thus losing methylaaded thus losing methylation marks after rounds of DNA replication. tion marks after rounds of DNA replication.
The application of AZA to halves ofhalves of fruits results in an early ripening phenotype. However, fruits results in an early ripening phenotype. However, when injected to when injected to strawberries strawberries white fruits, a complete stop of the ripening appears. white fruits, a complete stop of the ripening appears. RNARNA--seq studies determined that mseq studies determined that many TFany TFss involved in the control involved in the control of of the ripening process are the ripening process are not induced in the treated. not induced in the treated. Many transcriptsMany transcripts associated to hormone biosynthesis, aroma, associated to hormone biosynthesis, aroma, flavor and texture were either reduced or not detected in the AZAflavor and texture were either reduced or not detected in the AZA treated ptreated partsarts.. In the In the nonnon--climateric climateric strawstrawberry berry fruit, fruit, the the ripening is not controlled by ethylene but by the relation ripening is not controlled by ethylene but by the relation between ABA and auxins. After AZA treatment, kbetween ABA and auxins. After AZA treatment, key and critical genes pertaining to the ABA ey and critical genes pertaining to the ABA biosynthetic pathway were biosynthetic pathway were downregulateddownregulated whilewhile,, at the samat the same timee time,, those involved in itsthose involved in its witwithdrawal are activated.hdrawal are activated. AlsoAlso, genes related with auxins and gibberellin biosynthesis are , genes related with auxins and gibberellin biosynthesis are induced induced underunder AZAAZA treattreatmentment. . Moreover, mMoreover, metabolomic profile of AZA treated etabolomic profile of AZA treated samplessamples were were similar to the white stages of ripening rather tsimilar to the white stages of ripening rather to mature fruits. o mature fruits. Null concentratiNull concentration of on of anthocyanin was found in treated parts while the hormonal profile was similar to the early anthocyanin was found in treated parts while the hormonal profile was similar to the early stages of development. Lower concentration of bioactive ABA and higher amounts of the stages of development. Lower concentration of bioactive ABA and higher amounts of the degradation products were found. Also, hdegradation products were found. Also, higher concentration of auxins andigher concentration of auxins and gibberellins gibberellins were present in the treated part, suggesting a disbalance in hormonal content. AZA present in the treated part, suggesting a disbalance in hormonal content. AZA treatment leadtreatment leadeded to a completely stop of the ripening process due to the alterations on the to a completely stop of the ripening process due to the alterations on the hormonal content that are able to hormonal content that are able to alter the transcriptomic and metaalter the transcriptomic and metabolomic profiles.bolomic profiles. This This work provides the basiswork provides the basis revealing that methylation marks are needed for ripening in revealing that methylation marks are needed for ripening in strawberry,strawberry, but further works should be performed to fully understand this regulation. but further works should be performed to fully understand this regulation.
The last gene selected was a carboxylesterase enzyme,a carboxylesterase enzyme, FaCXEFaCXE22. C. Carboxylesterases arboxylesterases (CXE) are hydrolytic enzymes that belong to the α/β hydrolase fold superfamily of proteins(CXE) are hydrolytic enzymes that belong to the α/β hydrolase fold superfamily of proteins. . CXEs present a conserved catalytic triad, contained in the CXEs present a conserved catalytic triad, contained in the conserved sequence GXSXG, conserved sequence GXSXG, formed by formed by the the acidic amino acidacidic amino acid serineserine and a histidine, anand a histidine, and that constitutes the active site d that constitutes the active site of these enzymesof these enzymes. . In addition, theseIn addition, these enzymes are related to play several rolesenzymes are related to play several roles in in xenobiotic degradation, biotic stress responses as well as the volatile production. xenobiotic degradation, biotic stress responses as well as the volatile production.
Ripening in strawberry fruit is a complex process which carries out several changes in ch carries out several changes in flavor, color taste and softening. flavor, color taste and softening. In most fruits, as is the case with strawberry, the emission In most fruits, as is the case with strawberry, the emission of volatiles begins and increaseof volatiles begins and increase during ripening process. Only a few enzyduring ripening process. Only a few enzymes related with mes related with those process have been described inthose process have been described in strawberry. Two alcohol acetyltransferasesstrawberry. Two alcohol acetyltransferases (FaAAT1 (FaAAT1 and FaAAT2) and FaAAT2) are related with volatile production but are related with volatile production but no no CXECXE has been previously has been previously characterized in strawberry, leaving unknown the functional activities characterized in strawberry, leaving unknown the functional activities of those enzymes in of those enzymes in this this fruit.fruit. HighHigh--throughput transthroughput transcriptomic analyses allowed us to identify criptomic analyses allowed us to identify to to FaCXE2, FaCXE2, whose whose expression wasexpression was ripening related,ripening related, restricted to fruit tissues, as present high expression on restricted to fruit tissues, as present high expression on receptacle and achenesreceptacle and achenes,, and and hormonally regulated anthormonally regulated antagonistically agonistically by auxinsby auxins andand ABAABA.. The subcellular locaThe subcellular localization studies reveallization studies revealeded that this protein is located in the cytoplasm, that this protein is located in the cytoplasm, similar to other volatile related enzymes as alcohol acyltransferases and other CXE. The similar to other volatile related enzymes as alcohol acyltransferases and other CXE. The recombinant protein was obtained recombinant protein was obtained its its enzyenzymatic propertiesmatic properties were characterizedwere characterized. . The The optimal pH foptimal pH for its activity was 7.5or its activity was 7.5 and its optimal temperature between 25and its optimal temperature between 25--30ºC. 30ºC. FaCXE2 FaCXE2 showed most affinity towards the longer chainshowed most affinity towards the longer chain esters. esters.
In addition, the he FaCXE2FaCXE2 expression expression waswas transientlytransiently silenced in ripe strawberry receptaclessilenced in ripe strawberry receptacles by RNAi.by RNAi. Those receptacles with aThose receptacles with a higher silencing degree were selected for further higher silencing degree were selected for further volatile determination. Metabolomic studies revealvolatile determination. Metabolomic studies revealeded clear differences on ester content clear differences on ester content between transgenics and control fruit. Downbetween transgenics and control fruit. Downregulation of FaCXE2 resultregulation of FaCXE2 resulteded in a higher in a higher accumulation of different estersaccumulation of different esters, while alcohol compounds were in lower concentration. , while alcohol compounds were in lower concentration. Moreover, Moreover, tthe in vitro activity of he in vitro activity of FaFaCXECXE22 in hydrolyzing esters, together with the in hydrolyzing esters, together with the increaseincrease of esters in vivo caused by theof esters in vivo caused by the knoknock downck down of of FaFaCXECXE22, indicates that this CXE is a , indicates that this CXE is a regulator of volatile esteregulator of volatile ester content in r content in strawberry.strawberry.
3.conclusión 1.-- Three FaPRE genes genes withwith high homology level between themhigh homology level between them have been found in the have been found in the strawberry genomestrawberry genome. .
2.-- FaPRE1 expression pattern is expression pattern is receptaclereceptacle--specificspecific, ripening, ripening--related and related and regulated regulated by by ABA and auABA and auxinsxins whilewhile FaPRE2 FaPRE2 and and FaPRE3FaPRE3 expression is specific ofexpression is specific of vegetative tissues. vegetative tissues.
3.-- FaPRE1 antagonistically coordinateantagonistically coordinatess the transcription of genes the transcription of genes related to both related to both receptacle growth and receptacle growth and ripeningripening..
4.-- FaPRE1FaPRE1, in ripened receptacle, represses the transcription of, in ripened receptacle, represses the transcription of receptacle growth receptacle growth promoting genes while activating the transcription of those genes related to the receptacle promoting genes while activating the transcription of those genes related to the receptacle ripening process. ripening process.
5.-- FaMYB123 FaMYB123 is an R2is an R2--R3 MYB TF whR3 MYB TF whoseose gegene expression is ripening related, restricted ne expression is ripening related, restricted to the receptacle and to the receptacle and positively regulatpositively regulated by ABA and negatively by auxins.ed by ABA and negatively by auxins.
6.-- FaMYB123 control the expression of the late genes of the phenylpropanoid pathway, FaMYB123 control the expression of the late genes of the phenylpropanoid pathway, specificallyspecifically the malonyl transferases. the malonyl transferases.
7.-- In In FaMYB123FaMYB123--RNAi transgenics receptacles there is a disbalance between RNAi transgenics receptacles there is a disbalance between anthocyanin and pranthocyanin and proanthocyanin contents.oanthocyanin contents.
8..-- FaMYB123 is able to interact both FaMYB123 is able to interact both inin--vitro vitro and and inin--vivovivo with FabHLH3, a regulator of with FabHLH3, a regulator of proanthocyanin production. proanthocyanin production.
9.-- The injection of the demethylating compound 5The injection of the demethylating compound 5--azacytidine lead to a complete stop of azacytidine lead to a complete stop of the ripening process. the ripening process.
10.-- Transcriptome analysis revealed that transcriptional factor and enzymeTranscriptome analysis revealed that transcriptional factor and enzymes related with s related with ripening were repressed in the treated parts. ripening were repressed in the treated parts.
11.-- Metabolomic studies showed an alteration of the primary and secondary metabolites, Metabolomic studies showed an alteration of the primary and secondary metabolites, whose profiles were more similar to whose profiles were more similar to nonnon--ripened receptaclesripened receptacles 12.-- A disbalance between ABA and auxins was pA disbalance between ABA and auxins was produced. A higher concentration of roduced. A higher concentration of auxins and gibberellins while lower concentrations of ABA were accumulated in response auxins and gibberellins while lower concentrations of ABA were accumulated in response to AZA treatment to AZA treatment 13.-- FaCXE2 FaCXE2 isis a gene that encodesa gene that encodes an α/β hydran α/β hydrolase similar toolase similar to other carboxylesterases other carboxylesterases previously describedpreviously described 14.-- TheThe FaCXE2FaCXE2 expression is ripeningexpression is ripening--related, preferentially expressed in fruit and related, preferentially expressed in fruit and hormonally regulatedhormonally regulated positively by ABA and negatively by auxins.positively by ABA and negatively by auxins.
15.-- InIn--vitro vitro FaCXE2 activity reveals an opFaCXE2 activity reveals an optimal pH of 7.5 timal pH of 7.5 and an optical temperature of and an optical temperature of 25ºC. The recombinant protein shows most affinity towards longer chain esters. 25ºC. The recombinant protein shows most affinity towards longer chain esters.
16.-- Transient downregulation of strawberry FaCXE2 shows clear differences in the volatile Transient downregulation of strawberry FaCXE2 shows clear differences in the volatile contentcontent, presenting, presenting higher accumulationhigher accumulation of esters and aof esters and alcohol compounds were less lcohol compounds were less concentrated.concentrated.
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