Determination of Differential Alternative Splicing Under Stress Conditions.

Alternative splicing (AS) is an important mechanism contributing to stress-induced regulation of gene expression and proteome diversity. Massive sequencing technologies allow the identification of transcripts generated via stress-responsive AS, potentially important for adaptation to stress conditions. Several bioinformatics tools have been developed to identify differentially expressed alternative splicing events/transcripts from RNA-sequencing results. This chapter describes a detailed protocol for differential alternative splicing analysis using the rMATS tool. In addition, we provide guidelines for validation of the detected splice variants by qRT-PCR based on the obtained output files.

Transcriptomic landscape of tomato traditional long shelf-life landraces under low water regimes.

'Corbarino' (COR) and 'Lucariello' (LUC) belong to the family of Mediterranean long shelf-life tomato landraces, producing high quality fruits under low water input cultivation regime in their traditional cultivation area. Understanding the morpho-physiological and molecular details of the peculiar drought stress tolerance of these two genotypes may be key to their valorization as breeding material. RNA sequencing of leaf samples of COR and LUC subjected to drought stress by water withholding in a semi-controlled greenhouse identified 3089 and 2135 differentially expressed genes respectively. These included COR- and LUC-specific annotated genes, as well as genes containing single nucleotide polymorphisms as compared to reference genome. Enriched Gene Ontology categories showed that categories such as response to water, oxidoreductase activity, nucleotide salvation and lipid biosynthesis-related processes were enriched among up-regulated DEGs. By contrast, growth and photosynthesis related genes were down-regulated after drought stress, consistent with leaf gas exchange and biomass accumulation measurements. Genes encoding cell wall degrading enzymes of the pectinase family were also down-regulated in drought stress conditions and upregulated in rewatering, indicating that cell wall composition/hardness is important for drought stress responses. Globally our results contribute to understanding the transcriptomic and physiological responses of representative tomato genotypes from Southern Italy, highlighting a promising set of genes to be investigated to improve tomato tolerance to drought.

Transcriptomic and splicing changes underlying tomato responses to combined water and nutrient stress.

Tomato is a horticultural crop of high economic and nutritional value. Suboptimal environmental conditions, such as limited water and nutrient availability, cause severe yield reductions. Thus, selection of genotypes requiring lower inputs is a goal for the tomato breeding sector. We screened 10 tomato varieties exposed to water deficit, low nitrate or a combination of both. Biometric, physiological and molecular analyses revealed different stress responses among genotypes, identifying T270 as severely affected, and T250 as tolerant to the stresses applied. Investigation of transcriptome changes caused by combined stress in roots and leaves of these two genotypes yielded a low number of differentially expressed genes (DEGs) in T250 compared to T270, suggesting that T250 tailors changes in gene expression to efficiently respond to combined stress. By contrast, the susceptible tomato activated approximately one thousand and two thousand genes in leaves and roots respectively, indicating a more generalized stress response in this genotype. In particular, developmental and stress-related genes were differentially expressed, such as hormone responsive factors and transcription factors. Analysis of differential alternative splicing (DAS) events showed that combined stress greatly affects the splicing landscape in both genotypes, highlighting the important role of AS in stress response mechanisms. In particular, several stress and growth-related genes as well as transcription and splicing factors were differentially spliced in both tissues. Taken together, these results reveal important insights into the transcriptional and post-transcriptional mechanisms regulating tomato adaptation to growth under reduced water and nitrogen inputs.

Atlas of phenotypic, genotypic and geographical diversity present in the European traditional tomato.

The Mediterranean basin countries are considered secondary centres of tomato diversification. However, information on phenotypic and allelic variation of local tomato materials is still limited. Here we report on the evaluation of the largest traditional tomato collection, which includes 1499 accessions from Southern Europe. Analyses of 70 traits revealed a broad range of phenotypic variability with different distributions among countries, with the culinary end use within each country being the main driver of tomato diversification. Furthermore, eight main tomato types (phenoclusters) were defined by integrating phenotypic data, country of origin, and end use. Genome-wide association study (GWAS) meta-analyses identified associations in 211 loci, 159 of which were novel. The multidimensional integration of phenoclusters and the GWAS meta-analysis identified the molecular signatures for each traditional tomato type and indicated that signatures originated from differential combinations of loci, which in some cases converged in the same tomato phenotype. Our results provide a roadmap for studying and exploiting this untapped tomato diversity.

Alternative splicing in plant abiotic stress responses.

Modifications of the cellular proteome pool upon stress allow plants to tolerate environmental changes. Alternative splicing is the most significant mechanism responsible for the production of multiple protein isoforms from a single gene. The spliceosome, a large ribonucleoprotein complex, together with several associated proteins, controls this pre-mRNA processing, adding an additional level of regulation to gene expression. Deep sequencing of transcriptomes revealed that this co- or post-transcriptional mechanism is highly induced by abiotic stress, and concerns vast numbers of stress-related genes. Confirming the importance of splicing in plant stress adaptation, key players of stress signaling have been shown to encode alternative transcripts, whereas mutants lacking splicing factors or associated components show a modified sensitivity and defective responses to abiotic stress. Here, we examine recent literature on alternative splicing and splicing alterations in response to environmental stresses, focusing on its role in stress adaptation and analyzing the future perspectives and directions for research.

Genetic and epigenetic dynamics affecting anthocyanin biosynthesis in potato cell culture.

Anthocyanins are antioxidant pigments widely used in drugs and food preparations. Flesh-coloured tubers of the cultivated potato Solanum tuberosum are important sources of different anthocyanins. Due to the high degree of decoration achieved by acylation, anthocyanins from potato are very stable and suitable for the food processing industry. The use of cell culture allows to extract anthocyanins on-demand, avoiding seasonality and consequences associated with land-based-tuber production. However, a well-known limit of cell culture is the metabolic instability and loss of anthocyanin production during successive subcultures. To get a general picture of mechanisms responsible for this instability, we explored both genetic and epigenetic regulation that may affect anthocyanin production in cell culture. We selected two clonally related populations of anthocyanin-producing (purple) and non-producing (white) potato cells. Through targeted molecular investigations, we identified and functionally characterized an R3-MYB, here named StMYBATV. This transcription factor can interact with bHLHs belonging to the MBW (R2R3-MYB, bHLH and WD40) anthocyanin activator complex and, potentially, may interfere with its formation. Genome methylation analysis revealed that, for several genomic loci, anthocyanin-producing cells were more methylated than clonally related white cells. In particular, we localized some methylation events in ribosomal protein-coding genes. Overall, our study explores novel molecular aspects associated with loss of anthocyanins in cell culture systems.

DRT111/SFPS Splicing Factor Controls Abscisic Acid Sensitivity during Seed Development and Germination.

RNA splicing is a fundamental mechanism contributing to the definition of the cellular protein population in any given environmental condition. DNA-DAMAGE REPAIR/TOLERATION PROTEIN111 (DRT111)/SPLICING FACTOR FOR PHYTOCHROME SIGNALING is a splicing factor previously shown to interact with phytochrome B and characterized for its role in splicing of pre-mRNAs involved in photomorphogenesis. Here, we show that DRT111 interacts with Arabidopsis (Arabidopsis thaliana) Splicing Factor1, involved in 3' splicing site recognition. Double- and triple-mutant analysis shows that DRT111 controls splicing of ABI3 and acts upstream of the splicing factor SUPPRESSOR OF ABI3-ABI5. DRT111 is highly expressed in seeds and stomata of Arabidopsis and is induced by long-term treatments of polyethylene glycol and abscisic acid (ABA). DRT111 knock-out mutants are defective in ABA-induced stomatal closure and are hypersensitive to ABA during seed germination. Conversely, DRT111 overexpressing plants show ABA-hyposensitive seed germination. RNA-sequencing experiments show that in dry seeds, DRT111 controls expression and splicing of genes involved in osmotic-stress and ABA responses, light signaling, and mRNA splicing, including targets of ABSCISIC ACID INSENSITIVE3 (ABI3) and PHYTOCHROME INTERACTING FACTORs (PIFs). Consistently, expression of the germination inhibitor SOMNUS, induced by ABI3 and PIF1, is upregulated in imbibed seeds of drt111-2 mutants. Together, these results indicate that DRT111 controls sensitivity to ABA during seed development, germination, and stomatal movements, and integrates ABA- and light-regulated pathways to control seed germination.

Salinity and ABA Seed Responses in Pepper: Expression and Interaction of ABA Core Signaling Components.

Abscisic acid (ABA) plays an important role in various aspects of plant growth and development, including adaptation to stresses, fruit development and ripening. In seeds, ABA participates through its core signaling components in dormancy instauration, longevity determination, and inhibition of germination in unfavorable environmental conditions such as high soil salinity. Here, we show that seed germination in pepper was delayed but only marginally reduced by ABA or NaCl with respect to control treatments. Through a similarity search, pepper orthologs of ABA core signaling components PYL (PYRABACTIN RESISTANCE1-LIKE), PP2C (PROTEIN PHOSPHATASE2C), and SnRK2 (SUCROSE NONFERMENTING1 (SNF1)-RELATED PROTEIN KINASE2) genes were identified. Gene expression analyses of selected members showed a low abundance of PYL and SnRK2 transcripts in dry seeds compared to other tissues, and an up-regulation at high concentrations of ABA and/or NaCl for both positive and negative regulators of ABA signaling. As expected, in hydroponically-grown seedlings exposed to NaCl, only PP2C encoding genes were up-regulated. Yeast two hybrid assays performed among putative pepper core components and with Arabidopsis thaliana orthologs confirmed the ability of the identified proteins to function in ABA signaling cascade, with the exception of a CaABI isoform cloned from seeds. BiFC assay in planta confirmed some of the interactions obtained in yeast. Altogether, our results indicate that a low expression of perception and signaling components in pepper seeds might contribute to explain the observed high percentages of seed germination in the presence of ABA. These results might have direct implications on the improvement of seed longevity and vigor, a bottleneck in pepper breeding.

TIP41 network analysis and mutant phenotypes predict interactions between the TOR and ABA pathways.

Environmental conditions inform the rate of plant growth and development. The target of rapamycin (TOR) signalling pathway is a central regulator of plant growth in response to nutrients and energy, while abscisic acid (ABA) is a main mediator of abiotic stress responses. We recently characterized Arabidopsis TIP41, a predicted TOR pathway component involved in the ABA-mediated response to abiotic stress. Here, we report the ABA sensitivity of tip41 mutants, supporting the relation between TIP41 and the hormone pathway. The analysis of predicted TIP41 functional network identified several protein phosphatases. In particular, candidate protein interactors included catalytic subunits of type 2A protein phosphatases and protein phosphatases 6, which regulate different developmental processes and responses to environmental stimuli. These results provide important information on the role of TIP41 in the cross talk between TOR and ABA pathways.

The PP2A-interactor TIP41 modulates ABA responses in Arabidopsis thaliana.

Modulation of growth in response to environmental cues is a fundamental aspect of plant adaptation to abiotic stresses. TIP41 (TAP42 INTERACTING PROTEIN OF 41 kDa) is the Arabidopsis thaliana orthologue of proteins isolated in mammals and yeast that participate in the Target-of-Rapamycin (TOR) pathway, which modifies cell growth in response to nutrient status and environmental conditions. Here, we characterized the function of TIP41 in Arabidopsis. Expression analyses showed that TIP41 is constitutively expressed in vascular tissues, and is induced following long-term exposure to NaCl, polyethylene glycol and abscisic acid (ABA), suggesting a role of TIP41 in adaptation to abiotic stress. Visualization of a fusion protein with yellow fluorescent protein indicated that TIP41 is localized in the cytoplasm and the nucleus. Abolished expression of TIP41 results in smaller plants with a lower number of rosette leaves and lateral roots, and an increased sensitivity to treatments with chemical TOR inhibitors, indicating that TOR signalling is affected in these mutants. In addition, tip41 mutants are hypersensitive to ABA at germination and seedling stage, whereas over-expressing plants show higher tolerance. Several TOR- and ABA-responsive genes are differentially expressed in tip41, including iron homeostasis, senescence and ethylene-associated genes. In yeast and mammals, TIP41 provides a link between the TOR pathway and the protein phosphatase 2A (PP2A), which in plants participates in several ABA-mediated mechanisms. Here, we showed an interaction of TIP41 with the catalytic subunit of PP2A. Taken together, these results offer important insights into the function of Arabidopsis TIP41 in the modulation of plant growth and ABA responses.

Whole-genome re-sequencing of two Italian tomato landraces reveals sequence variations in genes associated with stress tolerance, fruit quality and long shelf-life traits.

Tomato is a high value crop and the primary model for fleshy fruit development and ripening. Breeding priorities include increased fruit quality, shelf life and tolerance to stresses. To contribute towards this goal, we re-sequenced the genomes of Corbarino (COR) and Lucariello (LUC) landraces, which both possess the traits of plant adaptation to water deficit, prolonged fruit shelf-life and good fruit quality. Through the newly developed pipeline Reconstructor, we generated the genome sequences of COR and LUC using datasets of 65.8 M and 56.4 M of 30-150 bp paired-end reads, respectively. New contigs including reads that could not be mapped to the tomato reference genome were assembled, and a total of 43, 054 and 44, 579 gene loci were annotated in COR and LUC. Both genomes showed novel regions with similarity to Solanum pimpinellifolium and Solanum pennellii. In addition to small deletions and insertions, 2, 000 and 1, 700 single nucleotide polymorphisms (SNPs) could exert potentially disruptive effects on 1, 371 and 1, 201 genes in COR and LUC, respectively. A detailed survey of the SNPs occurring in fruit quality, shelf life and stress tolerance related-genes identified several candidates of potential relevance. Variations in ethylene response components may concur in determining peculiar phenotypes of COR and LUC.

Subfunctionalization of duplicate MYB genes in Solanum commersonii generated the cold-induced ScAN2 and the anthocyanin regulator ScAN1.

Wild potato species are useful sources of allelic diversity and loci lacking in the cultivated potato. In these species, the presence of anthocyanins in leaves has been associated with a greater tolerance to cold stress. However, the molecular mechanisms that allow potatoes to withstand cold exposure remain unclear. Here, we show that the expression of AN2, a MYB transcription factor, is induced by low temperatures in wild, cold-tolerant Solanum commersonii, and not in susceptible Solanum tuberosum varieties. We found that AN2 is a paralog of the potato anthocyanin regulator AN1, showing similar interaction ability with basic helix-loop-helix (bHLH) co-partners. Their sequence diversity resulted in a different capacity to promote accumulation of phenolics when tested in tobacco. Indeed, functional studies demonstrated that AN2 is less able to induce anthocyanins than AN1, but nevertheless it has a strong ability to induce accumulation of hydroxycinnamic acid derivatives. We propose that the duplication of R2R3 MYB genes resulted in subsequent subfunctionalization, where AN1 specialized in anthocyanin production and AN2 conserved the ability to respond to cold stress, inducing mainly the synthesis of hydroxycinnamic acid derivatives. These results contribute to understanding the evolutionary significance of gene duplication on phenolic compound regulation.

Chloroplast proteome response to drought stress and recovery in tomato (Solanum lycopersicum L.).

BACKGROUND: Drought is a major constraint for plant growth and crop productivity that is receiving an increased attention due to global climate changes. Chloroplasts act as environmental sensors, however, only partial information is available on stress-induced mechanisms within plastids. Here, we investigated the chloroplast response to a severe drought treatment and a subsequent recovery cycle in tomato through physiological, metabolite and proteomic analyses. RESULTS: Under stress conditions, tomato plants showed stunted growth, and elevated levels of proline, abscisic acid (ABA) and late embryogenesis abundant gene transcript. Proteomics revealed that water deficit deeply affects chloroplast protein repertoire (31 differentially represented components), mainly involving energy-related functional species. Following the rewatering cycle, physiological parameters and metabolite levels indicated a recovery of tomato plant functions, while proteomics revealed a still ongoing adjustment of the chloroplast protein repertoire, which was even wider than during the drought phase (54 components differentially represented). Changes in gene expression of candidate genes and accumulation of ABA suggested the activation under stress of a specific chloroplast-to-nucleus (retrograde) signaling pathway and interconnection with the ABA-dependent network. CONCLUSIONS: Our results give an original overview on the role of chloroplast as enviromental sensor by both coordinating the expression of nuclear-encoded plastid-localised proteins and mediating plant stress response. Although our data suggest the activation of a specific retrograde signaling pathway and interconnection with ABA signaling network in tomato, the involvement and fine regulation of such pathway need to be further investigated through the development and characterization of ad hoc designed plant mutants.

Opportunities for genome editing in vegetable crops.

Vegetables include high-value crops with health-promoting effects and reduced environmental impact. The availability of genomic and biotechnological tools in certain species, coupled with the recent development of new breeding techniques based on precise editing of DNA, provides unique opportunities to finally take advantage of the past decades of detailed genetic analyses, thus making improvement of traits related to quality and stress tolerance achievable in a reasonable time frame. Recent reports of such approaches in vegetables illustrate the feasibility of obtaining multiple homozygous mutations in a single generation, heritable by the progeny, using stable or transient transformation approaches, which may not rely on the integration of unwanted foreign DNA. Application of these approaches to currently non-sequenced/tissue culture recalcitrant crops will contribute to meet the challenges posed by the increase in population and climate change.

Distinct gene networks drive differential response to abrupt or gradual water deficit in potato.

Water-limiting conditions affect dramatically plant growth and development and, ultimately, yield of potato plants (Solanum tuberosum L.). Therefore, understanding the mechanisms underlying the response to water deficit is of paramount interest to obtain drought tolerant potato varieties. Herein, potato 10K cDNA array slides were used to profile transcriptomic changes of two potato cell populations under abrupt (shocked cells) or gradual exposure (adapted cells) to polyethylene glycol (PEG)-mediated water stress. Data analysis identified >1000 differentially expressed genes (DEGs) in our experimental conditions. Noteworthy, our microarray study also suggests that distinct gene networks underlie the cellular response to shock or gradual water stress. On the basis of our experimental findings, it is possible to speculate that DEGs identified in shocked cells participate in early protective and sensing mechanisms to environmental insults, while the genes whose expression was modulated in adapted cells are directly involved in the acquisition of a new cellular homeostasis to cope with water stress conditions. To validate microarray data obtained for potato cells, the expression analysis of 21 selected genes of interest was performed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Intriguingly, the expression levels of these transcripts in 4-week old potato plants exposed to long-term water-deficit. qRT-PCR analysis showed that several genes were regulated similarly in potato cells cultures and tissues exposed to drought, thus confirming the efficacy of our simple experimental system to capture important genes involved in osmotic stress response. Highlighting the differences in gene expression between shock-like and adaptive response, our findings could contribute to the discussion on the biological function of distinct gene networks involved in the response to abrupt and gradual adaptation to water deficit.

Transcriptomic Changes Drive Physiological Responses to Progressive Drought Stress and Rehydration in Tomato.

Tomato is a major crop in the Mediterranean basin, where the cultivation in the open field is often vulnerable to drought. In order to adapt and survive to naturally occurring cycles of drought stress and recovery, plants employ a coordinated array of physiological, biochemical, and molecular responses. Transcriptomic studies on tomato responses to drought and subsequent recovery are few in number. As the search for novel traits to improve the genetic tolerance to drought increases, a better understanding of these responses is required. To address this need we designed a study in which we induced two cycles of prolonged drought stress and a single recovery by rewatering in tomato. In order to dissect the complexity of plant responses to drought, we analyzed the physiological responses (stomatal conductance, CO2 assimilation, and chlorophyll fluorescence), abscisic acid (ABA), and proline contents. In addition to the physiological and metabolite assays, we generated transcriptomes for multiple points during the stress and recovery cycles. Cluster analysis of differentially expressed genes (DEGs) between the conditions has revealed potential novel components in stress response. The observed reduction in leaf gas exchanges and efficiency of the photosystem PSII was concomitant with a general down-regulation of genes belonging to the photosynthesis, light harvesting, and photosystem I and II category induced by drought stress. Gene ontology (GO) categories such as cell proliferation and cell cycle were also significantly enriched in the down-regulated fraction of genes upon drought stress, which may contribute to explain the observed growth reduction. Several histone variants were also repressed during drought stress, indicating that chromatin associated processes are also affected by drought. As expected, ABA accumulated after prolonged water deficit, driving the observed enrichment of stress related GOs in the up-regulated gene fractions, which included transcripts putatively involved in stomatal movements. This transcriptomic study has yielded promising candidate genes that merit further functional studies to confirm their involvement in drought tolerance and recovery. Together, our results contribute to a better understanding of the coordinated responses taking place under drought stress and recovery in adult plants of tomato.

Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling.

The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling.

The Arabidopsis RNA-binding protein AtRGGA regulates tolerance to salt and drought stress.

Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

Phenylpropanoids Accumulation in Eggplant Fruit: Characterization of Biosynthetic Genes and Regulation by a MYB Transcription Factor.

Phenylpropanoids are major secondary metabolites in eggplant (Solanum melongena) fruits. Chlorogenic acid (CGA) accounts for 70-90% of total phenolics in flesh tissues, while anthocyanins are mainly present in the fruit skin. As a contribution to the understanding of the peculiar accumulation of these health-promoting metabolites in eggplant, we report on metabolite abundance, regulation of CGA and anthocyanin biosynthesis, and characterization of candidate CGA biosynthetic genes in S. melongena. Higher contents of CGA, Delphinidin 3-rutinoside, and rutin were found in eggplant fruits compared to other tissues, associated to an elevated transcript abundance of structural genes such as PAL, HQT, DFR, and ANS, suggesting that active in situ biosynthesis contributes to anthocyanin and CGA accumulation in fruit tissues. Putative orthologs of the two CGA biosynthetic genes PAL and HQT, as well as a variant of a MYB1 transcription factor showing identity with group six MYBs, were isolated from an Occidental S. melongena traditional variety and demonstrated to differ from published sequences from Asiatic varieties. In silico analysis of the isolated SmPAL1, SmHQT1, SmANS, and SmMyb1 promoters revealed the presence of several Myb regulatory elements for the biosynthetic genes and unique elements for the TF, suggesting its involvement in other physiological roles beside phenylpropanoid biosynthesis regulation. Transient overexpression in Nicotiana benthamiana leaves of SmMyb1 and of a C-terminal SmMyb1 truncated form (SmMyb1Δ9) resulted in anthocyanin accumulation only of SmMyb1 agro-infiltrated leaves. A yeast two-hybrid assay confirmed the interaction of both SmMyb1 and SmMyb1Δ9 with an anthocyanin-related potato bHLH1 TF. Interestingly, a doubled amount of CGA was detected in both SmMyb1 and SmMyb1Δ9 agro-infiltrated leaves, thus suggesting that the N-terminal region of SmMyb1 is sufficient to activate its synthesis. These data suggest that a deletion of the C-terminal region of SmMyb1 does not limit its capability to regulate CGA accumulation, but impairs anthocyanin biosynthesis. To our knowledge, this is the first study reporting a functional elucidation of the role of the C-term conserved domain in MYB activator proteins.

High AN1 variability and interaction with basic helix-loop-helix co-factors related to anthocyanin biosynthesis in potato leaves.

AN1 is a regulatory gene that promotes anthocyanin biosynthesis in potato tubers and encodes a R2R3 MYB transcription factor. However, no clear evidence implicates AN1 in anthocyanin production in leaves, where these pigments might enhance environmental stress tolerance. In our study we found that AN1 displays intraspecific sequence variability in both coding/non-coding regions and in the promoter, and that its expression is associated with high anthocyanin content in leaves of commercial potatoes. Expression analysis provided evidence that leaf pigmentation is associated to AN1 expression and that StJAF13 acts as putative AN1 co-regulator for anthocyanin gene expression in leaves of the red leaf variety 'Magenta Love,' while a concomitant expression of StbHLH1 may contribute to anthocyanin accumulation in leaves of 'Double Fun.' Yeast two-hybrid experiments confirmed that AN1 interacts with StbHLH1 and StJAF13 and the latter interaction was verified and localized in the cell nucleus by bimolecular fluorescence complementation assays. In addition, transgenic tobacco (Nicotiana tabacum) overexpressing a combination of either AN1 with StJAF13 or AN1 with StbHLH1 showed deeper purple pigmentation with respect to AN1 alone. This further confirmed AN1/StJAF13 and AN1/StbHLH1 interactions. Our findings demonstrate that the classical loci identified for potato leaf anthocyanin accumulation correspond to AN1 and may represent an important step to expand our knowledge on the molecular mechanisms underlying anthocyanin biosynthesis in different plant tissues.