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Strawberry (Fragaria × ananassa) fruits are an excellent source of L-ascorbic acid (AsA), a powerful antioxidant for plants and humans. Identifying the genetic components underlying AsA accumulation is crucial for enhancing strawberry nutritional quality. Here, we unravel the genetic architecture of AsA accumulation using an F1 population derived from parental lines ‘Candonga’ and ‘Senga Sengana’, adapted to distinct Southern and Northern European areas. To account for environmental effects, the F1 and parental lines were grown and phenotyped in five locations across Europe (France, Germany, Italy, Poland and Spain). Fruit AsA content displayed normal distribution typical of quantitative traits and ranged five-fold, with significant differences among genotypes and environments. AsA content in each country and the average in all of them was used in combination with 6,974 markers for quantitative trait locus (QTL) analysis. Environmentally stable QTLs for AsA content were detected in linkage group (LG) 3A, LG 5A, LG 5B, LG 6B and LG 7C. Candidate genes were identified within stable QTL intervals and expression analysis in lines with contrasting AsA content suggested that GDP-L-Galactose Phosphorylase FaGGP(3A), and the chloroplast-located AsA transporter gene FaPHT4;4(7C) might be the underlying genetic factors for QTLs on LG 3A and 7C, respectively. We show that recessive alleles of FaGGP(3A) inherited from both parental lines increase fruit AsA content. Furthermore, expression of FaGGP(3A) was two-fold higher in lines with high AsA. Markers here identified represent a useful resource for efficient selection of new strawberry cultivars with increased AsA content.

The diploid woodland strawberry (

Anthocyanins are the principal color-producing compounds synthesized in developing fruits of strawberry (Fragariaspp.). 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 whichMYB10has been shown as the main activator in strawberry fruit. Here, we show thatMYB10mutations 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 agypsy-transposon insertion inMYB10that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruitedF. vescaecotype. Two additional loss-of-functionMYB10mutations were identified among geographically diverse white-fruitedF. vescaecotypes. Genetic and transcriptomic analyses in octoploidFragaria spp.revealed thatFaMYB10-2,one of threeMYB10homoeologs identified, residing in theF. iinumae-derivedsubgenome, regulates the biosynthesis of anthocyanins in developing fruit. Furthermore, independent mutations inMYB10-2are 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 theMYB10-2promoter of red-fleshed accessions that was associated with enhanced expression and anthocyanin accumulation. Our findings suggest that putative cis regulatory elements provided byFaEnSpm-2are required for high and ectopicMYB10-2expression and induction of anthocyanin biosynthesis in fruit flesh. We developedMYB10-2(sub-genome) specific DNA markers for marker-assisted selection that accurately predicted anthocyanin phenotypes in octoploid segregating populations.

The Aux/IAA proteins are auxin-sensitive repressors that mediate diverse physiological and developmental processes in plants [1, 2]. There are 29 Aux/IAA genes in Arabidopsis that exhibit unique but partially overlapping patterns of expression [3]. Although some studies have suggested that individual Aux/IAA genes have specialized function, genetic analyses of the familyhave been limited by the scarcity of loss-of-function phenotypes [4]. Furthermore, with a few exceptions, our knowledge of the factors that regulate Aux/IAA expression is limited [1, 5]. Wehypothesize that transcriptional control of Aux/IAA genes plays a central role in the establishment of the auxin-signaling pathways that regulate organogenesis, growth, and environmental response. Here, we describe a screen fortranscription factors (TFs) that regulate the Aux/IAA genes. We identify TFs from 38 families, including 26 members of the DREB/CBF family. Several DREB/CBF TFs directly promote transcription of the IAA5 and IAA19 genes in response to abiotic stress. Recessive mutations in these IAAgenes result in decreased tolerance to stress conditions, demonstrating a role for auxin in abiotic stress. Our results demonstrate that stresspathways interact with the auxin gene regulatory network (GRN) through transcription ofthe Aux/IAA genes. We propose that the Aux/IAA genes function as hubs that integrate geneticand environmental information to achieve the appropriate developmental or physiological outcome.

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors. In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that AFB4 and AFB5 are targets of the picloram family of auxinic herbicides in addition to indole-3-acetic acid. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6 (Parry et al. 2009). Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors (Dharmasiri et al. 2005; Parry et al. 2009). In this report we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that both AFB4 and AFB5 are targets of the picloram family of auxinic herbicides. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.

The plant hormone auxin is perceived by a family of F box proteins called the TIR1/auxin-signaling F box proteins (AFBs). Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling. In this report, we demonstrate a unique role for the AFB4 clade. Both AFB4 and AFB5 function as auxin receptors based on in vitro assays. However, unlike other members of the family, loss of AFB4 results in a range of growth defects that are consistent with auxin hypersensitivity, including increased hypocotyl and petiole elongation and increased numbers of lateral roots. Indeed, qRT-PCR experiments show that afb4-2 is hypersensitive to indole-3-acetic acid (IAA) in the hypocotyl, indicating that AFB4 is a negative regulator of auxin response. Furthermore, we show that AFB4 has a particularly important role in the response of seedlings to elevated temperature. Finally, we provide evidence that the AFB4 clade is the major target of the picloram family of auxinic herbicides. These results reveal a previously unknown aspect of auxin receptor function.

Seasonal changes in day length influence flowering time in many plant species. In Arabidopsis, flowering is accelerated by exposure to long day (LD). Those inductive photoperiods are perceived in leaves [1] and initiate a long-distance signaling mediated by CO and FT. CO is expressed in the phloem according to a circadian rhythm 2, 3, 4. Only under LD does CO induce FT expression as high levels of CO in the evening coincide with the external light that stabilizes CO protein 4, 5. Subsequently, FT protein travels through the phloem to the shoot apex where, together with FD, it initiates flowering 6, 7, 8, 9, 10, 11, 12. Despite the photoperiodic induction, a mechanism of floral repression is needed to avoid precocious flowering. We show that TEMPRANILLO genes (TEM1 and TEM2) act as novel direct FT repressors. Molecular and genetic analyses suggest that a quantitative balance between the activator CO and the repressor TEM determines FT levels. Moreover, developmental TEM downregulation marks the timing of flowering, as it shifts the CO/TEM balance in favor of CO activity, allowing FT transcript to reach the threshold level required to trigger flowering. We envision that this might be a general mechanism between long-day plants to ensure a tight regulation of flowering time., This work was supported by a MEC grant (BFU2006-00771) and C.C. by Junta de Andalucía and CRAG.

In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGA), are essential components for elicitation of defence responses. To investigate how modifications of pectin alter defence responses, we expressed the fruit-specific Fragaria x ananassa pectin methyl esterase FaPE1 in the wild strawberry Fragaria vesca. Pectin from transgenic ripe fruits differed from the wild-type with regard to the degree and pattern of methyl esterification, as well as the average size of pectin polymers. Purified oligogalacturonides from the transgenic fruits showed a reduced degree of esterification compared to oligogalacturonides from wild-type fruits. This reduced esterification is necessary to elicit defence responses in strawberry. The transgenic F. vesca lines had constitutively activated pathogen defence responses, resulting in higher resistance to the necrotropic fungus Botrytis cinerea. Further studies in F. vesca and Nicotiana benthamiana leaves showed that the elicitation capacity of the oligogalacturonides is more specific than previously envisaged.

The strawberry (Fragaria x ananassa) FaGAST gene encodes a small protein with 12 cysteine residues conserved in the C-terminal region similar to a group of proteins identified in other species with diverse assigned functions such as cell division, elongation, or elongation arrest. This gene is expressed in the fruit receptacle, with two peaks during ripening at the white and the red-ripe stages, both coincident with an arrest in the growth pattern. Expression is also high in the roots but confined to the cells at the end of the elongation zone. Exogenous application of gibberellin increased the transcript level of the FaGAST gene in strawberry fruits. Ectopic expression of FaGAST in transgenic Fragaria vesca under the control of the CaMV-35S promoter caused both delayed growth of the plant and fruits with reduced size. The same growth defect was observed in Arabidopsis thaliana plants overexpressing FaGAST. In addition, the transgenic plants exhibited late flowering and low sensitivity to exogenous gibberellin. Taken together, the expression pattern, the regulation by gibberellin, and the transgenic phenotypes point to a role for FaGAST in arresting cell elongation during strawberry fruit ripening.

During Arabidopsis flower development a set of homeotic genes plays a central role in specifying the distinct floral organs of the four whorls, sepals in the outermost whorl, and petals, stamens, and carpels in the sequentially inner whorls. The current model for the identity of the floral organs includes the SEPALLATA genes that act in combination with the A, B and C genes for the specification of sepals, petals, stamens and carpels. According to this new model, the floral organ identity proteins would form different complexes of proteins for the activation of the downstream genes. We show that the presence of SEPALLATA proteins is needed to activate the AG downstream gene SHATTERPROOF2, and that SEPALLATA4 alone does not provide with enough SEPALLATA activity for the complex to be functional. Our results suggest that CAULIFLOWER may be part of the protein complex responsible for petal development and that it is fully required in the absence of APETALA1 in 35S::SEP3 plants. In addition, genetic and molecular experiments using plants constitutively expressing SEPALLATA3 revealed a new role of SEPALLATA3 in activating other B and C function genes. We molecularly prove that the ectopic expression of SEPALLATA3 is sufficient to ectopically activate APETALA3 and AGAMOUS. Remarkably, plants that constitutively express both SEPALLATA3 and LEAFY developed ectopic petals, carpels and ovules outside of the floral context.

Plant hormones are small-molecule signaling compounds that are collectively involved in all aspects of plant growth and development. Unlike animals, plants actively regulate the spatial distribution of several of their hormones. For example, auxin transport results in the formation of auxin maxima that have a key role in developmental patterning. However, the spatial distribution of the other plant hormones, including gibberellic acid (GA), is largely unknown. To address this, we generated two bioactive fluorescent GA compounds and studied their distribution in Arabidopsis thaliana roots. The labeled GAs specifically accumulated in the endodermal cells of the root elongation zone. Pharmacological studies, along with examination of mutants affected in endodermal specification, indicate that GA accumulation is an active and highly regulated process. Our results strongly suggest the presence of an active GA transport mechanism that would represent an additional level of GA regulation.

In Arabidopsis, FLOWERING LOCUS T (FT) promotes flowering in response to long days in the photoperiod pathway, while signalling downstream gibberellin (GA) perception is critical for flowering under short days. Previously we have established that the TEMPRANILLO (TEM) genes have a pivotal role in the direct repression of FT. Here we show that TEM genes directly regulate the expression of the GA4 biosynthetic genes GA 3–oxidase1 and 2 (GA3OX1 and GA3OX2). Plants overexpressing TEM genes resemble GA-deficient mutants, and conversely, TEM downregulation give rise to elongated hypocotyls perhaps as a result of an increase in GA content. We consistently find that TEM1 represses GA3OX1 and GA3OX2 by directly binding a regulatory region positioned in the first exon. Our results indicate that TEM genes seem to link the photoperiod and GA-dependent flowering pathways, controlling floral transition under inductive and non-inductive day lengths through the regulation of the floral integrators., S.P.'s research group has been recognized as a Consolidated Research Group by the Catalan Government (2009 SGR 697). This work was supported by an MEC grant (BFU2009-08325) and the CRAG is supported by a Spanish Ministry grant within the CONSOLIDER Program (CSD2007-00036).

Many processes critical to plant growth and development are regulated by the hormone auxin. Auxin responses are initiated through activation of a transcriptional response mediated by the TIR1/AFB family of F-box protein auxin receptors as well as the AUX/IAA and ARF families of transcriptional regulators. However, there is little information on how auxin regulates a specific cellular response. To begin to address this question, we have focused on auxin regulation of cell expansion in the Arabidopsis hypocotyl. We show that auxin-mediated hypocotyl elongation is dependent upon the TIR1/AFB family of auxin receptors and degradation of AUX/IAA repressors. We also use microarray studies of elongating hypocotyls to show that a number of growth-associated processes are activated by auxin including gibberellin biosynthesis, cell wall reorganization and biogenesis, and others. Our studies indicate that GA biosynthesis is required for normal response to auxin in the hypocotyl but that the overall transcriptional auxin output consists of PIF-dependent and -independent genes. We propose that auxin acts independently from and interdependently with PIF and GA pathways to regulate expression of growth-associated genes in cell expansion.

The plant hormone auxin is perceived by a family of F box proteins called the TIR1/auxin-signaling F box proteins (AFBs). Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6 [1]. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling [1, 2]. In this report, we demonstrate a unique role for the AFB4 clade. Both AFB4 and AFB5 function as auxin receptors based on in vitro assays. However, unlike other members of the family, loss of AFB4 results in a range of growth defects that are consistent with auxin hypersensitivity, including increased hypocotyl and petiole elongation and increased numbers of lateral roots. Indeed, qRT-PCR experiments show that afb4-2 is hypersensitive to indole-3-acetic acid (IAA) in the hypocotyl, indicating that AFB4 is a negative regulator of auxin response. Furthermore, we show that AFB4 has a particularly important role in the response of seedlings to elevated temperature. Finally, we provide evidence that the AFB4 clade is the major target of the picloram family of auxinic herbicides. These results reveal a previously unknown aspect of auxin receptor function.

In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGA), are essential components for elicitation of defence responses. To investigate how modifications of pectin alter defence responses, we expressed the fruit-specific Fragaria x ananassa pectin methyl esterase FaPE1 in the wild strawberry Fragaria vesca. Pectin from transgenic ripe fruits differed from the wild-type with regard to the degree and pattern of methyl esterification, as well as the average size of pectin polymers. Purified oligogalacturonides from the transgenic fruits showed a reduced degree of esterification compared to oligogalacturonides from wild-type fruits. This reduced esterification is necessary to elicit defence responses in strawberry. The transgenic F. vesca lines had constitutively activated pathogen defence responses, resulting in higher resistance to the necrotropic fungus Botrytis cinerea. Further studies in F. vesca and Nicotiana benthamiana leaves showed that the elicitation capacity of the oligogalacturonides is more specific than previously envisaged.

Pectin esterases (PE, EC 3.1.1.11) catalyse the demethylation of pectin. As a result of its activity, structural interactions among cell wall components during cell wall turnover and loosening are affected. In plants, PEs are typically encoded by a gene family. This family has been studied in strawberry (Fragaria x ananassa Duch.) in order to investigate the role of distinct PE genes during fruit ripening and senescence. By a combination of a PCR-based library screening and RT-PCR four different strawberry PE cDNAs, termed FaPE1 to FaPE4, have been isolated. Differential expression of each FaPE gene in various organs and during fruit development was revealed by northern blot. FaPE1 is specifically expressed in fruit, showing an increasing expression during the ripening process up to a maximum in the turning stage. Concerning hormone regulation, auxin treatment increased FaPE1 mRNA levels in green fruit, whereas exogenous ethylene decreased FaPE1 mRNA levels in ripe and senescing fruits. It is proposed that this repression of FaPE1 expression could be involved in textural changes occurring during fruit senescence.

(Current Biology 21, 520–525; March 22, 2011)In this article, we reported that the AFB4 protein is a negative regulator of auxin response based on the auxin hypersensitivity of the afb4-2 mutant line. Recently we obtained a second afb4 allele and confirmed that it is an RNA null. This mutant is not auxin hypersensitive, indicating that our conclusion that AFB4 is a negative regulator of auxin response is incorrect. For this reason, we wish to retract the article. Other conclusions from the study, including the demonstration that AFB4 and AFB5 are auxin receptors and are the major targets of the synthetic auxin picloram, remain valid. The corresponding author wishes to apologize to the scientific community for any confusion that might have arisen from the study.