Functional characterization of strawberry (fragaria x ananassa) transcription factors and transcriptional regulator during the fruit ripening, and genes with biotechnological interest
- Autores: Francisco Javier Molina Hidalgo
- Directores de la Tesis: Rosario Blanco Portales (dir. tes.), Juan Muñoz Blanco (dir. tes.)
- Lectura: En la Universidad de Córdoba (ESP) ( España ) en 2016
- Idioma: español
- Tribunal Calificador de la Tesis: Fernando Pliego Alfaro (presid.), Jesús V. Jorrin Novo (secret.), Elena Baraldi (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: Helvia
- Resumen
The strawberry (Fragaria × ananassa) belongs to the family Rosaceae 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 second one in the world after United States (FAO, 2012). The main octoploid variety Fragaria × ananassa cultivated is the result of crossing two native American species, F. virginiana and F. chiloensis (Hancock, 1999; Mabberley, 2002; Eriksson et al., 2003). The wild diploid species Fragaria vesca is also considered as an ancestor of the cultivated octoploid variety. Recently, the genome of the wild species Fragaria vesca has been sequenced (Shulaev et al., 2011). This information, together with the ESTs (expressed sequence tag) availability from cultivated species and the development of efficient transformation techniques of these varieties, will allow the development of genomics and recombinant DNA studies between different species of Rosaceae in the future (Bombarely et al., 2010).
Strawberry fruit has been classified as non-climacteric, since there is no concomitant burst of respiration and ethylene production that triggers to the ripening process. Thus, all changes related with the fruit ripening occur without a significant increase in ethylene production, which suggests that this process is independent of this hormone (Iwata et al., 1969a and 1969b; Villareal et al., 2010). The strawberry fruit has a maximum respiration at the transition between stages ripe to overripe.
Strawberries are much appreciated for their flavor, aroma and nutritional value. The mature fruit is composed of approximately 90 % water and 10% total soluble solids. Moreover, it contains many important dietary components such as vitamin C, soluble sugars such as glucose and fructose (which constitute over 80 % of total sugars), organic acids such as citric acid (88 % of total acids) and ellagic acid, which has anticancer properties (Green, 1971; Wrolstad and Shallenberger, 1981; Maas et al., 1991; Hemphill and Martin, 1992; Maas et al. 1996; Hancock, 1999).
Soft fruits have an initial phase of growth and elongation, followed by a phase of maturity. The growth of the strawberry receptacle depends of the cortex and medulla cells development while the fruit size is mainly determined for the medulla cells development and the fruit position in the inflorescence (Hancock, 1999). Moreover, the fruit development is determined by the number and distribution of achenes, the receptacle area around each achene and the percentage of fertilized carpels. In this sense, the synthesis of auxin, fundamentally indole-3-acetic acid (IAA), which takes place in the achenes, is considerate the main responsible of the receptacle growth while gibberellins, cytokinins and abscisic acid have a limited role in the fruit growth (Nitsch,1950; Perkins-Veazie, 1995).
Along the development and ripening processes, the strawberry fruit suffers important molecular changes such the removal of existing polypeptides and the synthesis of new proteins (Manning, 1994). In this sense, three evolution models of the transcripts have been described: mRNA whose concentration increases along the ripening, mRNA whose levels decrease over the ripening, and mRNA whose components exceed their maximum concentration in the intermediate stage, which then declined in stages of maturation (Veluthambi and Poovaiah, 1984; Reddy and Poovaiah, 1990; Reddy et al., 1990; Manning, 1994).
The hormonal regulation of the fruit development and maturation is one of the most studied processes in strawberry. Auxin produced by the achenes inhibits the fruit ripening in the green stage and, when the IAA concentration declines in the receptacle due to the achenes lignification, the fruit development and ripening begin (Given et al., 1988b). This suggests that the auxin stimulates the elongation of the fruit while repress its maturation. In parallel, a maximum activity of both, cytokinins and gibberellins, has been detected mainly in achenes 7 days after anthesis. It has been suggested that the GAs could control the induction of cell division in subapical tissues of axillary buds while that variations in the concentration of cytokinins are important in the process of flowering of strawberry plants (Eshghi and Tafazoli, 2007; Hytönen et al.,2009). Additionally, GAs also participates in the differentiation of axillary buds regulated by photoperiod (Hytönen et al., 2009). On the other hand, the abscisic acid (ABA) is also very important in the strawberry development. Generally, this phytohormone is involved in seed maturation, acquisition of tolerance to senescence, in vegetative growth, and in the physiological responses that confers tolerance to wáter and osmotic stress (Mishra et al., 2006). Moreover, the ABA slows down the growth in plants subjected to water stress by the restriction of ethylene production (Sharp, 2002) and acts over the biotic response (Fan et al., 2009). In strawberry fruit, this hormone is accumulated in both achenes and receptacles after 20 days post anthesis. This increase is concomitant with the decrease of IAA level in both tissues, therefore the ratio ABA /IAA might be sufficient to drive genetic changes that occurs during the transition of elongation phase to fruit ripening phase (Perkins-Veazie et al., 1995). ABA seems to play a crucial role in the regulation of fruit ripening as the application of exogenous ABA promotes the ripening of strawberry (Jia et al., 2011). The FaNCED1 gene silencing, which encodes an important protein in ABA biosynthesis, reduced the endogenous ABA levels in the strawberry fruits producing transgenic fruits without colour with the ripening inhibited (Jia et al., 2011). Moreover, the application of exogenous ABA reversed the transgenic phenotype suggesting that ABA promotes maturation of the strawberry fruit (Jia et al., 2011). The ethylene production also has a maximum level in green fruits (G1-G3) that decreases in white fruit (W) and increases again reaching a maximum in the mature stage (R) (Perkins-Veazie, 1995). This amount of ethylene produced during the strawberry ripening, although small, might be sufficient to trigger some of the physiological changes associated with this process (Trainotti et al., 2005).
Carbohydrates are one of the main soluble compounds of the soft fruits. In addition to provide energy for metabolic changes, the carbohydrates have an outstanding role in the generation of flavor. Organic acids, besides being compounds determinants of strawberry fruit flavor, also determine its color, inhibit activity of certain enzymes and change the texture of the fruit (Mussinan and Walradt, 1975). The no-volatile organic acids (citric, malic, etc.) are quantitatively the most important in determining the acidity of the fruit, while volatile organic acids contribute significantly to the aroma of fruit (Mussinan and Walradt, 1975). On the other hand, the phenolic acids provide the fruit acidity and tannins are responsible of the astringency of the fruit as result of its interaction with proteins and mucopolysaccharides of the spit (Ozawa et al., 1987; Ferrer, 1997). The flavones provide the characteristic bitter flavor of the development green stages (Hobson, 1993). These compounds are usually stored in the vacuole and its concentration varies during ripening depending on the variety and the environmental conditions of the plant.
The flavor of strawberry fruit is determined by the complex mixture of volatile compounds and other constituents (such as sugars, organic acids, phenolics and tannins), although esters are one of the most important groups of volatile compounds associated with the aroma of strawberry. Of all these compounds, about one hundred different types have been identified but only some of them contribute decisively to determine to the final fruit aroma (Zabetakis and Holden, 1997). In ripe strawberry fruit, the most abundant volatile esters are ethyl butanoate, 2-methyl-ethyl butanoate and ethyl hexanoate. One of enzymes involved in the formation of these esters is the alcohol acyl transferase (AAT) that catalyzes the transfer of the acyl group from acyl-CoA to an alcohol. The AAT expression begins in the white stage of fruit and continues increasing in the intermediate stage to its maximum expression in the red stage, coinciding with the highest levels of volatile esters in the fruit (Pérez et al., 1996; Aharoni et al., 2000). On the other hand, terpenoids are other compounds that also seem to be involved in the aroma of strawberry (Loughrin and Kasperbauer, 2002). Linalool, nerolidol, α-pinene and limonene are predominant volatile terpenes in strawberries that can be up to 20 % of total volatile fruit (Loughrin and Kasperbauer, 2002). The recombinant enzyme FaNES1 synthesized (S)-linalool and trans-(S)-nerolidol of GDP and FDP respectively. This gene is expressed strongly and specifically in fruits of cultivated varieties (octoploid) but not in wild varieties. Thus, the linalool and nerolidol are part of the final composition of the fruit aroma in strawberry cultivated varieties. Therefore, the aroma of strawberry is the result of the combination of odors "fruity" (ethyl butanoate, ethyl hexanoate and methyl 2-methylbutanoate), “green” (Z-3-hexenal), "sweet" (acid butanoic acid and 2-methylbutane), "peach" (decalactone), "candy" (4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF, furaneol) and 2.5-diethyl-4-methoxy-3 (2H)-furanone (DMMF)) (Aharoni et al., 2004). From these volatile compounds, the HDMF shows a high concentration and a low odor threshold.
Along the development process and strawberry fruit ripening, there is a color transition from the initial green color to red color characteristic of fully mature fruit. This color change is due both to degradation of chlorophyll and the synthesis of anthocyanins located in the vacuoles (Timberlake, 1981; Perkin-Veazie, 1995). Anthocyanin biosynthesis begins in the white fruit from phenylpropanoid and flavonoid by the shikimic acid pathway. The predominant anthocyanin in strawberries is pelargonidin-3- glucoside, which represents 88% of the anthocyanin in the fruit (Perkin-Veazie, 1995). The total concentration of anthocyanins varies about 16 times in the different cultivars and also in its composition. Recently, all these secondary metabolites have gained considerable importance due to its ability to prevent and protect against degenerative and cardiovascular diseases.
The strawberry plants are exposed to different abiotic agents (water deficit, high temperature, salinity, heavy metals and mechanical damage), in its natural habitat and these stress conditions can reduce crop yields by up to 50 %. Recently, physiological, biochemical and molecular studies have been performed to improve the plant tolerance to these stresses. Genes as Fcor1, 2 and 3 showed a differential expression at low temperatures (NDong et al., 1997) that was accompanied by an accumulation of the glycine betaine in different strawberry cultivars (Rajashekar et al., 1999). The synthesis of a specific group of proteins called heat shock proteins (HSP) has also been observed under high temperatures conditions (Medina-Escobar et al., 1998). On the other hand, methionine sulfoxide reductase (PMSR) is a relevant peptide in the protection of cells against oxidative damage caused by salt stress and pathogen infection (López et al., 2006).
In general, the programs of biotechnology and breeding of berries have as priority the improvement of the fruit quality. For these fruits, the taste (result of the combination of sweetness, acidity and aroma), and firmness are of great economic importance and, therefore, the genes involved in these processes are being studied by transgenesis. However and, although the evaluation of the transgenic gene function can be a valuable tool for the selection of genes, is quite slow.
Contenido de la investigación Along the development of this thesis, I have studied the transcriptomic changes that occur in the receptacle of the strawberry fruit (Fragaria × ananassa) during development and ripening using an oligo microarray platform. This analysis allowed selecting several target genes with biotechnological importance potentially involved in the process of fruit ripening, playing significant roles in various physiological processes such as cell wall degradation, water and solute transport, regulation of volatile compounds and regulation of the anthocyanins that contribute to modulate the final organoleptic properties of the fruit.
One of the selected genes, FaRGlyase1, displayed significant sequence homology with putative rhamnogalacturonan lyases from higher plants. In strawberries, the cortical parenchyma cells middle lamellae is extensively degraded throughout the ripening process, and they subsequently appear to be separated by a considerable intercellular space with little cell-to-cell contact area. Three kinds of pectins can be distinguished in plant cell walls: homogalacturonan (HGA), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II). In cell walls middle lamellae, HGA, a linear backbone of 1,4-linked galacturonic acid is predominant. Rhamnogalacturonan-degrading enzymes such as RG-hydrolases and RG-lyases (RGases) were first identified in several fungi. These enzymes digest the main RG-I chain.
In this work, we have observed that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life. Expression of this FaRGlyase1 ocurred mainly in the receptacle concurrently with ripening, and it was positively regulated by ABA and negatively by auxins.
On the other hand, FaRGLyase1 gene expression was transiently silenced by injecting live Agrobacterium cells harboring RNAi constructs into fruit receptacles. The light and electron microscopy analyses of these transiently silenced fruits revealed that this gene is involved in the degradation of pectins present in the middle lamella region between parenchymatic cells.
In addition, genetic linkage association analyses in a strawberry-segregating population have shown that FaRGLyase1 is linked to a QTL linkage group related to fruit hardness and firmness. Results show that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life.
The next gene selected to be studied was an aquaporin, FaNIP1;1, that is strongly up-regulated in ripened fruits and putatively encodes an aquaporin type NOD26-like intrinsic protein (NIP). Aquaporins belong to the family of integral membrane channel proteins known as Major Intrinsic Proteins (MIPs). MIPs are particularly abundant in plants and exhibit high multiplicity and diversity, which is likely due to the necessity of a fine-tuned water control that allows the plant to adapt to changing environmental conditions.
In the strawberry fruit, water movements are crucial since they allow for the rapid expansion of fruit. It has been reported that berry size correlates positively with the amount of irrigation water applied during flowering and fruit development. Water balances are always accompanied by water movements across biological membranes by means of diffusion or through water channels.
In this work, we report that the FaNIP1;1 protein could play an important role in the control of the strawberry fruit hydration. The analysis by qRT-PCR of FaNIP1;1 showed that this gene is mainly expressed in fruit receptacle and has a ripening-related expression pattern that was accompanied by an increase in both the abscisic acid and water content of the receptacle throughout fruit ripening. Moreover, FaNIP1;1 was induced in situations of water deficit. Additionally, we show that FaNIP1;1 expression was positively regulated by abscisic acid and negatively regulated by auxins.
The water transport capacity of FaNIP1;1 was determined by a stopped-flow spectroscopy in yeast over-expressing FaNIP1;1. Glycerol, H2O2 and boron transport were also demonstrated in yeast. On the other hand, GFP-FaNIP1;1 fusion protein was located in plasma membrane.
In conclusion, FaNIP1;1 seems to play an important role increasing the plasma membrane permeability, that allows the water accumulation in the strawberry fruit receptacle throughout the ripening process.
Transcription factors (TFs) are essential for gene expression regulation in plants. However, knowledge related to the regulatory role played by different TFs along the strawberry fruit ripening process is scarce. For that, other of the selected genes was a transcription factor belonging to the DOF family, FaDOF2, which is involved in regulating the production of phenylpropanoid volatiles. DOF transcription factors (DOF-TFs) constitute one of the multigene families of plant-specific TFs that physiologically regulate many complex and specific plant processes or metabolic pathways. I present molecular and physiological studies showing that FaDOF2 is involved in the regulation of endogenous eugenol production, a volatile phenylpropanoid in ripe fruit receptacles.
The FaDOF2 expression was ripening-related and fruit receptacle-specific, although high expression values were also found in petals. This FaDOF2 expression pattern correlated with eugenol content, a phenylpropanoid volatile, in both fruit receptacle and petals. The FaDOF2 expression was repressed by auxins, and activated by abscisic acid (ABA) during the fruit development and ripening process, respectively.
When the FaDOF2 expression was silenced in ripe strawberry receptacles, the expression of FaEOBII and FaEGS2, two key genes involved in eugenol production, were down-regulated. These fruits showed a concomitant decrease in eugenol content, which confirmed that FaDOF2 is a transcription factor involved in eugenol production in ripe fruit receptacles.
By using the yeast two-hybrid system, we have demonstrated that FaDOF2 interacts with FaEOBII, a eugenol production regulator reported in a previous study. Our results indicate that the mutual interaction between FaEOBII and FaDOF2 would determine a fine-tuning of the expression of key genes that are responsible for eugenol production. Additionally, FaEOBII expression, but not FaDOF2, was under control of FaMYB10, a master R2R3 MYB transcription factor regulating both early and late biosynthetic genes from the flavonoid/phenylpropanoid pathway.
These results provide evidences that FaDOF2 plays a subsidiary regulatory role with FaEOBII of the expression of those structural genes that control eugenol production. Taken together, our results provide new insight in regulation of the volatile phenylpropanoid pathway in ripe strawberry receptacles.
The next gene selected to be studied was a transcription regulator belonging to the BTB-ankyrins proteins of BLADE-ON-PETIOLE (BOP) class, FaBOP1. According to its aminoacidic sequence, FaBOP1 contains a BTB/POZ domain (for Broad-Complex, Tramtrack, and Bric-a-Brac/POX virus and Zinc finger) at the N-terminus, as well as four ankyrin motifs located near the C-terminus. This two conserved domain has been previously proposed as protein-protein interaction motifs.
Ripening in strawberry fruit is a complex process which carries out several changes in flavor, color taste and softening. The regulatory network that supports these molecular and metabolic changes that occur along the ripening has been scarcely studied.
High-throughput transcriptomic analyses allowed us to identify this ripening induced gene whose expression was quite fruit specific and hormonally regulated, in an antagonist way, by auxins and ABA. BTB-ankryin proteins are plant-specific transcriptional co-activators.
The FaBOP1 expression was silenced in ripe strawberry receptacles. As phenotypic effect of gene silencing, we observed a lack of colour, compared with the control receptacles. The flavonoid/phenylpropanoid pathway structural genes were down-regulated in transgenic FaBOP1 silenced fruits. This was corroborated by metabolite comparison between both control and transgenic fruits.
By using the yeast two-hybrid system, we have demonstrated that FaBOP1 interacts with FaMYB10, a master R2R3 MYB transcription factor regulating both early and late biosynthetic genes from the flavonoid/phenylpropanoid pathway.
Our study has revealed the role for the BOP-like coactivator FaBOP1, which bind to the transcription factor R2R3-MYB10 and in this way, to regulate the F/P biosynthesis. The dramatic effect in the phenotype and metabolic profile by the transient silencing clearly shows the importance of the role played, as a co-activator, by FaBOP1 in strawberry fruit receptacle. Thus, the most important molecular and metabolic changes associated with ripening, are affected by FaBOP1silencing.
Conclusiones 1. The strawberry FaRGlyase1 gene shows significant homology of sequence with a conserved rhamnogalacturonate lyase domain, which was also present in other putative RGlyase sequences deposited in the data-bases.
2. The expression of FaRGlyase1 ocurred mainly in the receptacle concurrently with ripening, and it was positively regulated by ABA and negatively by auxins.
3. FaRGlyase1 protein is involved in the degradation of cell-wall middle lamellae in fruit receptacle. The QTL analysis showed that the FaRGlyase1 gene is linked to a group of genes implied in fruit firmness.
4. FaRGLyase1 plays an important key role in the fruit ripening-related softening process that reduces the firmness and post-harvest life of the fruit.
5. FaNIP1;1 deduced protein belongs to the NIP (nodulin-26-like intrinsic proteins) subgroup of MIPs proteins.
6. FaNIP1;1 is a ripening - and drought-related gene and is positively regulated by ABA and negatively by auxins.
7. FaNIP1;1 is located in plasma membrane and transports water and some small non-ionic solute molecules.
8. FaNIP1;1 may play a key role in maintaining water cellular homeostasis increasing the membrane permeability in the ripen strawberry fruit cells.
9. FaDOF2 is a DOF-type transcription factor, located in the nucleus, and is an hormonal and ripening-related transcription factor.
10. FaDOF2 expression correlates with eugenol production in different strawberry plant tissues.
11. FaDOF2 regulates two key genes, FaEOBII and FaEGS2, related to eugenol biosynthesis in strawberry fruit receptacles.
12. In ripe receptacles, eugenol production is regulated by the physical interaction between FaDOF2 and FaEOBII, a R2R3 MYB TF, through control of the FaEGS2 expression.
13. FaBOP1 is a gene that encodes BTB-ankyrin protein of BLADE-ON-PETIOLE (BOP) class. FaBOP1 was localized in both nucleus and cytoplasm of the cells.
14. The FaBOP1 expression is ripening-related, preferentially expressed in fruit receptacle and hormonally regulated positively by ABA and negatively by auxins.
15. FaBOP1 acts as co- regulator of the expression of ripening related genes related with the secondary metabolism. FaBOP1 regulates expression of FaMYB10 TF and other genes related to F/P biosynthesis in strawberry fruit receptacles.
16. FaBOP1 interacts with FaMYB10, a R2R3 MYB transcription factor which is a major regulator of the anthocyanin production in fruit receptacle, suggesting that both would act synergistically in the regulation of phenylpropanoid pathway.
17. FaBOP1 down-regulation changes metabolite composition in strawberry fruit receptacle.
