Autophagy is required for strawberry fruit ripening

Autophagy is a catabolic and recycling pathway that maintains cellular homeostasis under normal growth and stress conditions. Two major types of autophagy, microautophagy and macroautophagy, have been described in plants. During macroautophagy, cellular content is engulfed by a double-membrane vesicle called autophagosome. This vesicle fuses its outer membrane with the tonoplast and releases the content into the vacuole for degradation. During certain developmental processes, autophagy is enhanced by induction of several autophagy-related genes (ATG genes). Autophagy in crop development has been studied in relation to leaf senescence, seed and reproductive development, and vascular formation. However, its role in fruit ripening has only been partially addressed. Strawberry is an important berry crop, representative of non-climacteric fruit. We have analyzed the occurrence of autophagy in developing and ripening fruits of the cultivated strawberry. Our data show that most ATG genes are conserved in the genome of the cultivated strawberry Fragaria x ananassa and they are differentially expressed along the ripening of the fruit receptacle. ATG8-lipidation analysis proves the presence of two autophagic waves during ripening. In addition, we have confirmed the presence of autophagy at the cellular level by the identification of autophagy-related structures at different stages of the strawberry ripening. Finally, we show that blocking autophagy either biochemically or genetically dramatically affects strawberry growth and ripening. Our data support that autophagy is an active and essential process with different implications during strawberry fruit ripening.This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie to VS-V grant agreement 844365 project FrATGaria: Identification of the role of autophagy during fruit ripening and stress resistance in Strawberry: Autophagy manipulation as a way to improve Strawberry fitness, by the Ministerio de Economía y Competitividad, co-financed by the European Regional Development Fund (grant BIO2017-82609-R) to MB and by the Plan Propio of the University of Malaga

Effect of environmental factors on wild strawberry primary metabolic profile

Climate factors such as temperature and precipitation vary significantly over continental scales, strongly structuring biomes along latitudinal gradients, and resulting in species differently adapted either genetically or plastically to cope with their local climate. However, climate change will likely alter these biomes. Thus, it is expected that Nordic regions, historically colder and rainier, will tend to have higher temperatures and less rainfall, which might lead to changes in the distribution of plants leading to novel patterns of local adaptation and maladaptation. In this study we aim to study how plant traits vary with latitude and in response to different temperature and drought conditions in order to find genetic determinants of climate adaptation. Our group is focused in determining the role of the metabolic profiling to that adaptation. For that purpose, we use the woodland strawberry (Fragaria vesca) as the model organism. In particular, we have analyzed 16 different genotypes that have been grown in five common gardens located in Belgium, Sweden, Finland, and Spain, in which drought treatments were also performed. Here, we present the chemical analysis (primary metabolism) in leaves of these genotypes in order to better understand how environmental factors can alter the primary metabolic profiles of F. vesca accessions grown in different locations.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. BiodivERsA project PlantCline -(PCI2020-120719-2) from “Programación Conjunta Internacional” program, Ministerio de Ciencia e Innovación

Generation and analysis of ESTs from strawberry (Fragaria xananassa) fruits and evaluation of their utility in genetic and molecular studies

<p>Abstract</p> <p>Background</p> <p>Cultivated strawberry is a hybrid octoploid species (<it>Fragaria xananassa </it>Duchesne ex. Rozier) whose fruit is highly appreciated due to its organoleptic properties and health benefits. Despite recent studies on the control of its growth and ripening processes, information about the role played by different hormones on these processes remains elusive. Further advancement of this knowledge is hampered by the limited sequence information on genes from this species, despite the abundant information available on genes from the wild diploid relative <it>Fragaria vesca</it>. However, the diploid species, or one ancestor, only partially contributes to the genome of the cultivated octoploid. We have produced a collection of expressed sequence tags (ESTs) from different cDNA libraries prepared from different fruit parts and developmental stages. The collection has been analysed and the sequence information used to explore the involvement of different hormones in fruit developmental processes, and for the comparison of transcripts in the receptacle of ripe fruits of diploid and octoploid species. The study is particularly important since the commercial fruit is indeed an enlarged flower receptacle with the true fruits, the achenes, on the surface and connected through a network of vascular vessels to the central pith.</p> <p>Results</p> <p>We have sequenced over 4,500 ESTs from <it>Fragaria xananassa</it>, thus doubling the number of ESTs available in the GenBank of this species. We then assembled this information together with that available from <it>F. xananassa </it>resulting a total of 7,096 unigenes. The identification of SSRs and SNPs in many of the ESTs allowed their conversion into functional molecular markers. The availability of libraries prepared from green growing fruits has allowed the cloning of cDNAs encoding for genes of auxin, ethylene and brassinosteroid signalling processes, followed by expression studies in selected fruit parts and developmental stages. In addition, the sequence information generated in the project, jointly with previous information on sequences from both <it>F. xananassa </it>and <it>F. vesca</it>, has allowed designing an oligo-based microarray that has been used to compare the transcriptome of the ripe receptacle of the diploid and octoploid species. Comparison of the transcriptomes, grouping the genes by biological processes, points to differences being quantitative rather than qualitative.</p> <p>Conclusions</p> <p>The present study generates essential knowledge and molecular tools that will be useful in improving investigations at the molecular level in cultivated strawberry (<it>F. xananassa</it>). This knowledge is likely to provide useful resources in the ongoing breeding programs. The sequence information has already allowed the development of molecular markers that have been applied to germplasm characterization and could be eventually used in QTL analysis. Massive transcription analysis can be of utility to target specific genes to be further studied, by their involvement in the different plant developmental processes.</p

Independent mutations in a single locus, the transcriptional factor MYB10, control natural variation in fruit color among Fragaria species

External and internal fruit color are important traits in strawberry (Fragaria spp.) breeding programs, where different preferences are sought depending on whether the fruits are produced for fresh consumption or processing. Therefore, there is a great interest in the development of predictive markers that effectively speed the development of new cultivars with increased consumer acceptance and/or which address processed fruit industry´s preferences. In order to identify loci controlling fruit color variation, two mapping populations were generated: one crossing diploid F. vesca parentals and another interspecific population between two octoploid species: the cultivated and the Chilean strawberry, F. x ananassa and F. chiloensis. Both populations allowed the detection of a QTL spanning a region of the F. vesca linkage group 1 (LG I) that includes the MYB10 gene, a known key regulator of anthocyanin biosynthesis. Mapping by sequencing in the F. vesca population revealed an LTR retrotransposon inserted in the third exon of FvMYB10, which produces a premature stop codon, and co-segregates with white fruits in the entire population. Genotyping by Sanger sequencing of additional white-fruited F. vesca accessions resulted in the identification of another three independent mutations in MYB10, two of them not previously described1. In octoploid strawberry, a mayor QTL on LG I-3 controls about 55% variation in internal flesh color and is associated with an insertion in the promoter region of FcMYB10. Similar insertions have been detected in other F. chiloensis accessions bearing white fruits. In all cases, transient over-expression of FvMYB10 restored anthocyanin biosynthesis and red color in fruit flesh and skin, indicating that lack of function of MYB10 was the underlying cause of white fruits in all analyzed cases

'Amiga' strawberry

‘Amiga’ is a short-day strawberry cultivar developed by the Spanish public breeding program. ‘Amiga’ has a very high fruit firmness, high production, long fruit shape, and good appearance. An agronomic and sensory characterization of this new cultivar, in comparison with the well-adapted cultivars Camarosa, Carisma, Marina and Medina, was undertaken during the 2000 and 2001 crop seasons. Most strawberries produced in Spain are shipped fresh to destinations throughout Europe. Growers therefore need cultivars producing fruit that, in addition to attractiveness and top-class sensory attributes, ensures maintenance of quality after long-distance shipment. During the crop season 2006, a total of 58.3% of the total strawberry output at Huelva (312,066 t) was shipped fresh to market (Anonymous, 2006). The main destinations were Germany, France, and the United Kingdom. As a result of the economic and social significance of strawberry production in Spain, which is currently the leading European producer of fresh-market strawberries (López-Aranda et al., 2003), a number of public institutions, including the Instituto Nacional de Investigaciones Agrarias (INIA), the Instituto de Investigacio´n y Formacio´n Agraria y Pesquera (IFAPA), and the Instituto Valenciano de Investigaciones Agrarias (IVIA), are carrying out a breeding program to develop new strawberry cultivars well adapted to growing conditions in Spanish production areas, and particularly in Huelva. ‘Amiga’ is the latest cultivar released from this program in cooperation with the private partner, Viveros California, S.L. Previous cultivars released include ‘Andana’ (Bartual et al., 1997), ‘Carisma’ (Bartual et al., 2002), ‘Marina’ (López-Aranda et al., 2004), ‘Medina’ (López-Aranda et al., 2005a), and ‘Aguedilla’ (López-Aranda et al., 2005b). The new short-day strawberry (Fragaria · ananassa Duch.) cultivar Amiga is remarkable for its very high firmness, high production in annual production systems, long fruit shape, and good appearance. Its harvest season is early, which is extremely important for economic production on the southwest coast of Spain, and its firmness is essential for long-distance shipment

Characterizing the involvement of FaMADS9 in the regulation of strawberry fruit receptacle development

FaMADS9 is the strawberry (Fragaria x ananassa) gene that exhibits the highest homology to the tomato (Solanum lycopersicum) RIN gene. Transgenic lines were obtained in which FaMADS9 was silenced. The fruits of these lines did not show differences in basic parameters, such as fruit firmness or colour, but exhibited lower Brix values in three of the four independent lines. The gene ontology MapMan category that was most enriched among the differentially expressed genes in the receptacles at the white stage corresponded to the regulation of transcription, including a high percentage of transcription factors and regulatory proteins associated with auxin action. In contrast, the most enriched categories at the red stage were transport, lipid metabolism and cell wall. Metabolomic analysis of the receptacles of the transformed fruits identified significant changes in the content of maltose, galactonic acid-1,4-lactone, proanthocyanidins and flavonols at the green/white stage, while isomaltose, anthocyanins and cuticular wax metabolism were the most affected at the red stage. Among the regulatory genes that were differentially expressed in the transgenic receptacles were several genes previously linked to flavonoid metabolism, such as MYB10, DIV, ZFN1, ZFN2, GT2, and GT5, or associated with the action of hormones, such as abscisic acid, SHP, ASR, GTE7 and SnRK2.7. The inference of a gene regulatory network, based on a dynamic Bayesian approach, among the genes differentially expressed in the transgenic receptacles at the white and red stages, identified the genes KAN1, DIV, ZFN2 and GTE7 as putative targets of FaMADS9. A MADS9-specific CArG box was identified in the promoters of these genes

Allelic Variation of MYB10 is the Major Force Controlling Natural Variation of Skin and Flesh Color in Strawberry (Fragaria spp.) fruit

Anthocyanins are the principal color-producing compounds synthesized in developing fruits of strawberry (Fragaria spp.). Substantial natural variation in color have been observed in fruits of diploid and octoploid accessions, resulting from distinct accumulation and distribution of anthocyanins in fruits. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 has been shown as the main activator in strawberry fruit. Here, we show that MYB10 mutations cause most of the anthocyanin variation observed in diploid woodland strawberry (F. vesca) and octoploid cultivated strawberry (F. ×ananassa). Using a mapping-by-sequencing approach, we identified a gypsytransposon insertion in MYB10 that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited F. vesca ecotype. Two additional lossof-function MYB10 mutations were identified among geographically diverse whitefruited F. vesca ecotypes. Genetic and transcriptomic analyses in octoploid Fragaria spp. revealed that FaMYB10-2, one of three MYB10 homoeologs identified, residing in the F. iinumae-derived subgenome, regulates the biosynthesis of anthocyanins in developing fruit. Furthermore, independent mutations in MYB10-2 are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon (FaEnSpm-2) insertion in the MYB10-2 promoter of red-fleshed accessions that was associated with enhanced expression and anthocyanin accumulation. Our findings suggest that putative cis regulatory elements provided by FaEnSpm-2 are required for high and ectopic MYB10-2 expression and induction of anthocyanin biosynthesis in fruit flesh. We developed MYB10-2 (sub-genome) specific DNA markers for marker-assisted selection that accurately predicted anthocyanin phenotypes in octoploid segregating populations

Detection of kinase domain mutations in BCR::ABL1 leukemia by ultra-deep sequencing of genomic DNA

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E−4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E−4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.Tis project was funded in part by CRIS CANCER FOUNDATION

Regulation of L-ascorbic acid content in strawberry fruits

Plants have several L-ascorbic acid (AsA) biosynthetic pathways, but the contribution of each one to the synthesis of AsA varyies between different species, organs, and developmental stages. Strawberry (Fragaria×ananassa) fruits are rich in AsA. The pathway that uses D-galacturonate as the initial substrate is functional in ripe fruits, but the contribution of other pathways to AsA biosynthesis has not been studied. The transcription of genes encoding biosynthetic enzymes such as D-galacturonate reductase (FaGalUR) and myo-inositol oxygenase (FaMIOX), and the AsA recycling enzyme monodehydroascorbate reductase (FaMDHAR) were positively correlated with the increase in AsA during fruit ripening. Fruit storage for 72 h in a cold room reduced the AsA content by 30%. Under an ozone atmosphere, this reduction was 15%. Ozone treatment increased the expression of the FaGalUR, FaMIOX, and L-galactose-1-phosphate phosphatase (FaGIPP) genes, and transcription of the L-galactono-1,4-lactone dehydrogenase (FaGLDH) and FAMDHAR genes was higher in the ozone-stored than in the air-stored fruits. Analysis of AsA content in a segregating population from two strawberry cultivars showed high variability, which did not correlate with the transcription of any of the genes studied. Study of GalUR protein in diverse cultivars of strawberry and different Fragaria species showed that a correlation between GalUR and AsA content was apparent in most cases, but it was not general. Three alleles were identified in strawberry, but any sequence effect on the AsA variability was eliminated by analysis of the allele-specific expression. Taken together, these results indicate that FaGalUR shares the control of AsA levels with other enzymes and regulatory elements in strawberry fruit