Investigating molecular mechanisms controlling phenylpropanoid production in potato and tomato

Plant phenylpropanoids draw an outstanding interest due to their effect in both human nutrition and plant defence. In fact, compounds produced through the phenylpropanoid biochemical pathway represent indispensable elements for plants to face environmental stresses. Moreover, thanks to their strong antioxidant activity, these compounds are important components of functional food. In this work we focused on phenylpropanoid pathway, with a particular interest for the braches of flavonoids. In particular, the major aim of our research was to investigate the genetic mechanism controlling the production of flavonoids in order to clarify either their role in plant stress tolerance or to enhance the production of useful metabolites in plant derived foods. As regard the role of flavonoids in plant environmental tolerance, we focused on the ability of anthocyanin pigments to induce cold stress tolerance in potato. Since no molecular information was available on anthocyanin regulation in potato leaves, the first part of our research focused onto the determination of MYB/bHLH complex that influences anthocyanin production in vegetative tissues of the cultivated potato Solanum tuberosum. In particular, we found that StAN1, a gene codifying a MYB factor, displays intraspecific sequence variability in both coding/non-coding regions and in the promoter. In addition, expression analysis suggested that leaf pigmentation is associated with StAN1 expression and that a bHLH, named StJAF13, acts as putative StAN1 co-regulator for anthocyanin gene expression in leaves of red-leaf varieties. Functional analysis through protein/protein interaction and ectopic transgenic expression, further confirmed AN1/StJAF13 interaction complex to induce anthocyanin accumulation. Once characterized this anthocyanin complex, we compared the anthocyanin genes of the wild cold tolerant species Solanum commersonii with those of cultivated varieties under cold stress condition. As suggested by functional and metabolic analysis in Nicotiana benthamiana, ScAN2, a paralog gene of ScAN1, evolved differently between cultivated and wild species. In S. commersonii, ScAN2 seemed to keep a pleiotropic and ancestral function with respect to ScAN1, inducing a multiple activation of several phenylpropanoid branches to respond to cold injury. Consistently, we found that ScAN2 was up regulated after cold treatment only in the cold tolerant S. commersonii. In addition, metabolic and microscopy analyses suggested that ScAN2 is connected to the production of phenolic compounds located on plasma membrane and cell wall of transformed tobacco cells. The third part of our work was mainly concentrated on the group of flavonols which are flavonoids with a strong anti-inflammatory activity as well as protective role against cardiovascular diseases. In this study, we tried to understand which were the genes that normally contribute to flavonol accumulation in tomato flesh. We choose an eQTLs approach to move in the intricate gene regulation architecture that may influence flavonol accumulation in tomato flesh. The material used was the introgression population developed from crosses between S. pennellii and the cultivated tomato S. lycopersicum, cultivar “M82”. The most significant outcome from this research was the identification of two potential negative regulators we named SlMYB4 and SlELVIRA. The function of these two genes was studied using VIGS (virus induced gene silencing) approach. The transient silencing of the SlMYB4 and SlELVIRA resulted in an increase of flavonols as well as of chlorogenic acid in tomato flesh. This suggested a negative action of these two genes in flavonoid regulation. Ultimately, we believe that these studies may provide a new framework to explore how phenylpropanoid genes regulate the different branches of phenylpropanoid pathway to either increase plant tolerance to external stresses or to enhance the accumulation of human beneficial metabolites in important crops

Coexpression gene network analysis of cold-tolerant Solanum commersonii reveals new insights in response to low temperatures

AbstractAmong abiotic stressors, cold is one of the most harmful for the cultivated potato (Solanum tuberosum L.), a frost‐sensitive crop. RNA sequencing (RNA‐seq) profiling of two different clones of wild potato (S. commersonii Dun.) contrasting in their capacity to withstand low temperatures revealed a higher number of differentially expressed genes (DEGs) under nonacclimated conditions (NAC) in tolerant clone cmm1T vs. the susceptible cmm6‐6 (1,002 and 8,055 DEGs, respectively). By contrast, the number of DEGs was much more comparable when both genotypes were under acclimated conditions (AC). Indeed, a total of 5,650 and 8,936 DEGs were detected in the tolerant genotype vs. the susceptible. Gene ontology (GO) classification under NAC showed a significant role for transcription regulation, lignin catabolic genes, and regulation of plant type secondary cell wall in the cold‐tolerant genotypes, suggesting an important role in conferring tolerance response. By contrast, response to stress and response to stimuli were enriched GO categories in both clones under AC. Unsigned weighted correlation networks analysis (WGCNA) allowed identification of coexpressed hub genes with possible main regulatory functions and major impacts on the phenotype. Among those identified, we clarified the role of CBF4. This gene showed contrasting expression profiles in the two clones under NAC, being induced in cold‐tolerant cmm1T but suppressed in susceptible cmm6‐6. By contrast, under AC, CBF4 was upregulated in both clones. Our study provides a global understanding of mechanisms involved following exposure to NAC and AC in S. commersonii. The mechanisms described here will inform future investigations for detailed validation in studies regarding cold tolerance in plants

A basic Helix-Loop-Helix (SlARANCIO), identified from a Solanum pennellii introgression line, affects carotenoid accumulation in tomato fruits

Abstract Carotenoid accumulation in tomato (Solanum lycopersicum) fruits is influenced by environmental stimuli and hormonal signals. However, information on the relative regulatory mechanisms are scanty since many molecular players of the carotenoid biosynthetic pathway are still unknown. Here, we reported a basic Helix-Loop-Helix transcription factor, named SlARANCIO (SlAR), whose silencing influences carotenoid accumulation in tomato fruits. The SlAR gene was found in the S. pennellii introgression line (IL) 12-4SL that holds the carotenoid QTL lyc12.1. We observed that the presence of the wild region in a cultivated genetic background led to a decrease in total carotenoid content of IL12-4SL fruits. To get insights into the function of SlAR, a quick reverse genetic approach was carried out. Virus-induced gene silencing of SlAR in S. lycopersicum M82 and MicroTom fruits reproduced the same phenotype observed in IL12-4SL, i.e. decreased content of lycopene and total carotenoids. Vice versa, the overexpression of SlAR in Nicotiana benthamiana leaves increased the content of total carotenoids and chlorophylls. Our results, combined with public transcriptomic data, highly suggest that SlAR acts indirectly on the carotenoid pathway and advances current knowledge on the molecular regulators controlling lyc12.1 and, potentially, precursors of carotenoid biosynthesis

Anthocyanins are Key Regulators of Drought Stress Tolerance in Tobacco

Abiotic stresses will be one of the major challenges for worldwide food supply in the near future. Therefore, it is important to understand the physiological mechanisms that mediate plant responses to abiotic stresses. When subjected to UV, salinity or drought stress, plants accumulate specialized metabolites that are often correlated with their ability to cope with the stress. Among them, anthocyanins are the most studied intermediates of the phenylpropanoid pathway. However, their role in plant response to abiotic stresses is still under discussion. To better understand the effects of anthocyanins on plant physiology and morphogenesis, and their implications on drought stress tolerance, we used transgenic tobacco plants (AN1), which over-accumulated anthocyanins in all tissues. AN1 plants showed an altered phenotype in terms of leaf gas exchanges, leaf morphology, anatomy and metabolic profile, which conferred them with a higher drought tolerance compared to the wild-type plants. These results provide important insights for understanding the functional reason for anthocyanin accumulation in plants under stress

DNA-based technologies for grapevine biodiversity exploitation: state of the art and future perspectives

The cultivated grapevine, Vitis vinifera subsp. vinifera L., is represented by an enormous population of varieties and clones. They arise from the accumulation of gametic and somatic mutations during centuries of sexual and asexual propagation. These varieties represent a vast reservoir of traits/alleles that could be useful in improving the berry quality as well as against environmental stresses. However, most of them are still unexploited. For this reason, an efficient characterization system is essential to define the varietal identity, avoid cases of synonymy (identical genotypes but different names) and homonymy (same names but different genotypes) and deepen our understanding of the existing diversity within the grape germplasm. The plethora of DNA-based high-throughput technologies currently available provides promising tools for the analysis of diversity, overcoming many of the limitations of phenotypic-based diversity analyses. However, the analysis of intra-varietal diversity remains challenging. In this scenario, after summarizing the causes and consequences of grapevine genetic inter- and intra-varietal diversity, we review the DNA-based technologies used for varietal genotyping, emphasizing those able to distinguish clones within a variety. This review provides an update on the technologies used to explore grapevine diversity, the knowledge of which is necessary for an efficient exploitation and conservation of the grapevine germplasm

Genotype-specific changes associated to early synthesis of autotetraploids in wild potato species

Polyploidy is an important factor in plant evolution that may trigger drastic genome reorganization and phenotypic differentiation. In the last decade, extensive studies have been carried to understand the consequences of allopolyploidization, where the effects of ploidy change may be confounded by interspecific hybridization. By contrast, less is known on autopolyploidization, which only involves doubling of homologous chromosomes. This study was undertaken to assess leaf anatomical modifications and gene expression changes occurring after doubling the somatic chromosome complement of diploid (2n = 2x = 24) potato species Solanum commersonii Dunal and S. bulbocastanum Dunal. Polyploidization did not induce qualitative changes in leaf structure and, for several leaf traits, anatomic modifications were stochastic. In addition, in both species a diploid superiority was generally observed, suggesting the occurrence of a high-ploidy syndrome. Expression change study was carried out on eight important cell cycle-regulatory genes in plant. It revealed a strong alteration of the expression patterns in the 4x genotypes with respect to the 2x parents. Changes often exceed the twofold, with no consistent trend towards up- or down regulation when comparing 2x vis-à-vis 4x. We discuss the possible relevance of epigenetic changes in controlling the expression of duplicated genes

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. This article is protected by copyright. All rights reserved

Genes involved in stress signals: The cbls-cipks network in cold tolerant solanum commersonii

Several studies revealed the important contribution of calcineurin B-like (CBLs) and CBL-interacting kinase (CIPKs) genes in transmitting stress signals in plants. Taking advantage from the genome sequences of the cultivated potato Solanum tuberosum and its wild relatives S. commersonii and S. chacoense, we identified for the first time 10 CBLs and 26 CIPKs genes in each species. The CBLs and CIPKs derived from tandem duplications indicate that these gene families in potato mainly arise through amplification mechanisms. Once annotated, we compared the par excellence model of Arabidopsis thaliana with S. commersonii, the potato model species for studying cold tolerance. We found that four ScCBL proteins (ScCBL1, ScCBL4a, ScCBL4b, and ScCBL9) started with a conserved N-myristoylation motif (MGXXXS/T), which might function in membrane targeting of the CBLs-CIPKs complex. Additionally, expression analyses of S. commersonii CBL and CIPK genes based on RNAseq revealed diverse expression patterns following various abiotic and biotic stresses and in the four tissues analyzed (flowers, leaf, roots, and tubers). Data also suggest that the ScCBLs-ScCIPKs complex may be more responsive to abiotic rather than biotic stimuli. Overall, the results described in the present work will be useful for future investigations and for functional characterization of individual CBLs and CIPKs in Solanum

Relationships between composition, microstructure and cooking performances of six potato varieties

Potatoes tubers are the raw materials of many processed food, such as cooked potatoes in hot water, baked potatoes and the most popular fried potatoes. The objective of this work was to study the impact of boiling, baking and frying on microstructure and properties of six potato varieties (Agata, Agria, Innovator, Lady Rosetta, Musica and Spunta) with different origin. Scanning Electron Microscopy revealed significant differences between varieties and tuber microstructure changes following all cooking processes. Differential Scanning Calorimeter analysis showed that the transition temperatures (ranging between 60 °C and 85 °C) and enthalpies of gelatinization (2.1 J/g – 3.9 J/g) of tubers were also variety dependent. In addition, the elasticity modulus of cooked samples depended on process type and followed the order: baked potatoes > boiled >fried potatoes. In particular, baked Lady Rosetta (224.3 kPa) showed the least decrease in rigidity between thermal processes. Fried Agria and Spunta, (56.3 and 61 kPa, respectively) had the smallest value of Young’s modulus. Molecular marker analyses provided a genetic fingerprinting of our varieties, allowing the identification of diagnostic markers. Innovator revealed an important genetic distance from the other varieties. Such distance corresponded to its exclusive phenotypic traits, that are known to affect thermochemical properties. The information obtained in this work may be useful to further study and associate genetic sequences with appreciable food technological traits