Complex petal spot formation in the Beetle Daisy (Gorteria diffusa) relies on spot-specific accumulation of malonylated anthocyanin regulated by paralogous GdMYBSG6 transcription factors.

Gorteria diffusa has elaborate petal spots that attract pollinators through sexual deception, but how G. diffusa controls spot development is largely unknown. Here, we investigate how pigmentation is regulated during spot formation. We determined the anthocyanin composition of G. diffusa petals and combined gene expression analysis with protein interaction assays to characterise R2R3-MYBs that likely regulate pigment production in G. diffusa petal spots. We found that cyanidin 3-glucoside pigments G. diffusa ray floret petals. Unlike other petal regions, spots contain a high proportion of malonylated anthocyanin. We identified three subgroup 6 R2R3-MYB transcription factors (GdMYBSG6-1,2,3) that likely activate the production of spot pigmentation. These genes are upregulated in developing spots and induce ectopic anthocyanin production upon heterologous expression in tobacco. Interaction assays suggest that these transcription factors regulate genes encoding three anthocyanin synthesis enzymes. We demonstrate that the elaboration of complex spots in G. diffusa begins with the accumulation of malonylated pigments at the base of ray floret petals, positively regulated by three paralogous R2R3-MYB transcription factors. Our results indicate that the functional diversification of these GdMYBSG6s involved changes in the spatial control of their transcription, and modification of the duration of GdMYBSG6 gene expression contributes towards floral variation within the species.

Complete biosynthesis of the potent vaccine adjuvant QS-21.

QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure-activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

AtIAR1 is a Zn transporter that regulates auxin metabolism in Arabidopsis thaliana.

Root growth in Arabidopsis is inhibited by exogenous auxin-amino acid conjugates, and mutants resistant to one such conjugate [indole-3-acetic acid (IAA)-Ala] map to a gene (AtIAR1) that is a member of a metal transporter family. Here, we test the hypothesis that AtIAR1 controls the hydrolysis of stored conjugated auxin to free auxin through zinc transport. AtIAR1 complements a yeast mutant sensitive to zinc, but not manganese- or iron-sensitive mutants, and the transporter is predicted to be localized to the endoplasmic reticulum/Golgi in plants. A previously identified Atiar1 mutant and a non-expressed T-DNA mutant both exhibit altered auxin metabolism, including decreased IAA-glucose conjugate levels in zinc-deficient conditions and insensitivity to the growth effect of exogenous IAA-Ala conjugates. At a high concentration of zinc, wild-type plants show a novel enhanced response to root growth inhibition by exogenous IAA-Ala which is disrupted in both Atiar1 mutants. Furthermore, both Atiar1 mutants show changes in auxin-related phenotypes, including lateral root density and hypocotyl length. The findings therefore suggest a role for AtIAR1 in controlling zinc release from the secretory system, where zinc homeostasis plays a key role in regulation of auxin metabolism and plant growth regulation.

Exploring the metabolic and physiological roles of HQT in S. lycopersicum by gene editing.

The most abundant phenolic compound in Solanaceous plants is chlorogenic acid (CGA), which possesses protective properties such as antimicrobial and antioxidant activities. These properties are particularly relevant when plants are under adverse conditions, such as pathogen attack, excess light, or extreme temperatures that cause oxidative stress. Additionally, CGA has been shown to absorb UV-B light. In tomato and potato, CGA is mainly produced through the HQT pathway mediated by the enzyme hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase. However, the absence of natural or induced mutants of this gene has made it unclear whether other pathways contribute to CGA production and accumulation. To address this question, we used CRISPR technology to generate multiple knock-out mutant lines in the tomato HQT gene. The resulting slhqt plants did not accumulate CGA or other caffeoylquinic acids (CQAs) in various parts of the plant, indicating that CQA biosynthesis depends almost entirely on the HQT pathway in tomato and, likely, other Solanaceous crops. We also found that the lack of CGA in slhqt plants led to higher levels of hydroxycinnamoyl-glucose and flavonoids compared to wild-type plants. Gene expression analysis revealed that this metabolic reorganization was partly due to flux redirection, but also involved modulation of important transcription factor genes that regulate secondary metabolism and sense environmental conditions. Finally, we investigated the physiological role of CGA in tomato and found that it accumulates in the upper epidermis where it acts as a protector against UV-B irradiation.

Pangenomic analysis reveals plant NAD+ manipulation as an important virulence activity of bacterial pathogen effectors.

Nicotinamide adenine dinucleotide (NAD+) has emerged as a key component in prokaryotic and eukaryotic immune systems. The recent discovery that Toll/interleukin-1 receptor (TIR) proteins function as NAD+ hydrolases (NADase) links NAD+-derived small molecules with immune signaling. We investigated pathogen manipulation of host NAD+ metabolism as a virulence strategy. Using the pangenome of the model bacterial pathogen Pseudomonas syringae, we conducted a structure-based similarity search from 35,000 orthogroups for type III effectors (T3Es) with potential NADase activity. Thirteen T3Es, including five newly identified candidates, were identified that possess domain(s) characteristic of seven NAD+-hydrolyzing enzyme families. Most Pseudomonas syringae strains that depend on the type III secretion system to cause disease, encode at least one NAD+-manipulating T3E, and many have several. We experimentally confirmed the type III-dependent secretion of a novel T3E, named HopBY, which shows structural similarity to both TIR and adenosine diphosphate ribose (ADPR) cyclase. Homologs of HopBY were predicted to be type VI effectors in diverse bacterial species, indicating potential recruitment of this activity by microbial proteins secreted during various interspecies interactions. HopBY efficiently hydrolyzes NAD+ and specifically produces 2'cADPR, which can also be produced by TIR immune receptors of plants and by other bacteria. Intriguingly, this effector promoted bacterial virulence, indicating that 2'cADPR may not be the signaling molecule that directly initiates immunity. This study highlights a host-pathogen battleground centered around NAD+ metabolism and provides insight into the NAD+-derived molecules involved in plant immunity.

Loss of PROTEIN TARGETING TO STARCH 2 has variable effects on starch synthesis across organs and species.

Recent work has identified several proteins involved in starch granule initiation, the first step of starch synthesis. However, the degree of conservation in the granule initiation process remains poorly understood, especially among grass species differing in patterns of carbohydrate turnover in leaves, and granule morphology in the endosperm. We therefore compared mutant phenotypes of Hordeum vulgare (barley), Triticum turgidum (durum wheat), and Brachypodium distachyon defective in PROTEIN TARGETING TO STARCH 2 (PTST2), a key granule initiation protein. We report striking differences across species and organs. Loss of PTST2 from leaves resulted in fewer, larger starch granules per chloroplast and normal starch content in wheat, fewer granules per chloroplast and lower starch content in barley, and almost complete loss of starch in Brachypodium. The loss of starch in Brachypodium leaves was accompanied by high levels of ADP-glucose and detrimental effects on growth and physiology. Additionally, we found that loss of PTST2 increased granule initiation in Brachypodium amyloplasts, resulting in abnormal compound granule formation throughout the seed. These findings suggest that the importance of PTST2 varies greatly with the genetic and developmental background and inform the extent to which the gene can be targeted to improve starch in crops.

Biofortified tomatoes provide a new route to vitamin D sufficiency.

Poor vitamin D status is a global health problem; insufficiency underpins higher risk of cancer, neurocognitive decline and all-cause mortality. Most foods contain little vitamin D and plants are very poor sources. We have engineered the accumulation of provitamin D3 in tomato by genome editing, modifying a duplicated section of phytosterol biosynthesis in Solanaceous plants, to provide a biofortified food with the added possibility of supplement production from waste material.

Subtelomeric assembly of a multi-gene pathway for antimicrobial defense compounds in cereals.

Non-random gene organization in eukaryotes plays a significant role in genome evolution. Here, we investigate the origin of a biosynthetic gene cluster for production of defence compounds in oat-the avenacin cluster. We elucidate the structure and organisation of this 12-gene cluster, characterise the last two missing pathway steps, and reconstitute the entire pathway in tobacco by transient expression. We show that the cluster has formed de novo since the divergence of oats in a subtelomeric region of the genome that lacks homology with other grasses, and that gene order is approximately colinear with the biosynthetic pathway. We speculate that the positioning of the late pathway genes furthest away from the telomere may mitigate against a 'self-poisoning' scenario in which toxic intermediates accumulate as a result of telomeric gene deletions. Our investigations reveal a striking example of adaptive evolution underpinned by remarkable genome plasticity.

The branched-chain amino acid aminotransferase TaBCAT1 modulates amino acid metabolism and positively regulates wheat rust susceptibility.

Plant pathogens suppress defense responses to evade recognition and promote successful colonization. Although identifying the genes essential for pathogen ingress has traditionally relied on screening mutant populations, the post-genomic era provides an opportunity to develop novel approaches that accelerate identification. Here, RNA-seq analysis of 68 pathogen-infected bread wheat (Triticum aestivum) varieties, including three (Oakley, Solstice and Santiago) with variable levels of susceptibility, uncovered a branched-chain amino acid aminotransferase (termed TaBCAT1) as a positive regulator of wheat rust susceptibility. We show that TaBCAT1 is required for yellow and stem rust infection and likely functions in branched-chain amino acid (BCAA) metabolism, as TaBCAT1 disruption mutants had elevated BCAA levels. TaBCAT1 mutants also exhibited increased levels of salicylic acid (SA) and enhanced expression of associated defense genes, indicating that BCAA regulation, via TaBCAT1, has a key role in SA-dependent defense activation. We also identified an association between the levels of BCAAs and resistance to yellow rust infection in wheat. These findings provide insight into SA-mediated defense responses in wheat and highlight the role of BCAA metabolism in the defense response. Furthermore, TaBCAT1 could be manipulated to potentially provide resistance to two of the most economically damaging diseases of wheat worldwide.

Formation and diversification of a paradigm biosynthetic gene cluster in plants.

Numerous examples of biosynthetic gene clusters (BGCs), including for compounds of agricultural and medicinal importance, have now been discovered in plant genomes. However, little is known about how these complex traits are assembled and diversified. Here, we examine a large number of variants within and between species for a paradigm BGC (the thalianol cluster), which has evolved recently in a common ancestor of the Arabidopsis genus. Comparisons at the species level reveal differences in BGC organization and involvement of auxiliary genes, resulting in production of species-specific triterpenes. Within species, the thalianol cluster is primarily fixed, showing a low frequency of deleterious haplotypes. We further identify chromosomal inversion as a molecular mechanism that may shuffle more distant genes into the cluster, so enabling cluster compaction. Antagonistic natural selection pressures are likely involved in shaping the occurrence and maintenance of this BGC. Our work sheds light on the birth, life and death of complex genetic and metabolic traits in plants.

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants.

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.

Fulvic acid increases forage legume growth inducing preferential up-regulation of nodulation and signalling-related genes.

The use of potential biostimulants is of broad interest in plant science for improving yields. The application of a humic derivative called fulvic acid (FA) may improve forage crop production. FA is an uncharacterized mixture of chemicals and, although it has been reported to increase growth parameters in many species including legumes, its mode of action remains unclear. Previous studies of the action of FA have lacked appropriate controls, and few have included field trials. Here we report yield increases due to FA application in three European Medicago sativa cultivars, in studies which include the appropriate nutritional controls which hitherto have not been used. No significant growth stimulation was seen after FA treatment in grass species in this study at the treatment rate tested. Direct application to bacteria increased Rhizobium growth and, in M. sativa trials, root nodulation was stimulated. RNA transcriptional analysis of FA-treated plants revealed up-regulation of many important early nodulation signalling genes after only 3 d. Experiments in plate, glasshouse, and field environments showed yield increases, providing substantial evidence for the use of FA to benefit M. sativa forage production.

Hairy root transformation of Brassica rapa with bacterial halogenase genes and regeneration to adult plants to modify production of indolic compounds.

During the last years halogenated compounds have drawn a lot of attention. Metabolites with one or more halogen atoms are often more active than their non-halogenated derivatives like indole-3-acetic acid (IAA) and 4-Cl-IAA. Within this work, bacterial flavin-dependent tryptophan halogenase genes were inserted into Brassica rapa ssp. pekinensis (Chinese cabbage) with the aim to produce novel halogenated indole compounds. It was investigated which tryptophan-derived indole metabolites, such as indole glucosinolates or potential degradation products can be synthesized by the transgenic root cultures. In vivo and in vitro activity of halogenases heterologously produced was shown and the production of chlorinated tryptophan in transgenic root lines was confirmed. Furthermore, chlorinated indole-3-acetonitrile (Cl-IAN) was detected. Other tryptophan-derived indole metabolites, such as IAA or indole glucosinolates were not found in the transgenic roots in a chlorinated form. The influence of altered growth conditions on the amount of produced chlorinated compounds was evaluated. We found an increase in Cl-IAN production at low temperatures (8 °C), but otherwise no significant changes were observed. Furthermore, we were able to regenerate the wild type and transgenic root cultures to adult plants, of which the latter still produced chlorinated metabolites. Therefore, we conclude that the genetic information had been stably integrated. The transgenic plants showed a slightly altered phenotype compared to plants grown from seeds since they also still expressed the rol genes. By this approach we were able to generate various stably transformed plant materials from which it was possible to isolate chlorinated tryptophan and Cl-IAN.

RNA-seq, de novo transcriptome assembly and flavonoid gene analysis in 13 wild and cultivated berry fruit species with high content of phenolics.

BACKGROUND: Flavonoids are produced in all flowering plants in a wide range of tissues including in berry fruits. These compounds are of considerable interest for their biological activities, health benefits and potential pharmacological applications. However, transcriptomic and genomic resources for wild and cultivated berry fruit species are often limited, despite their value in underpinning the in-depth study of metabolic pathways, fruit ripening as well as in the identification of genotypes rich in bioactive compounds. RESULTS: To access the genetic diversity of wild and cultivated berry fruit species that accumulate high levels of phenolic compounds in their fleshy berry(-like) fruits, we selected 13 species from Europe, South America and Asia representing eight genera, seven families and seven orders within three clades of the kingdom Plantae. RNA from either ripe fruits (ten species) or three ripening stages (two species) as well as leaf RNA (one species) were used to construct, assemble and analyse de novo transcriptomes. The transcriptome sequences are deposited in the BacHBerryGEN database (http://jicbio.nbi.ac.uk/berries) and were used, as a proof of concept, via its BLAST portal (http://jicbio.nbi.ac.uk/berries/blast.html) to identify candidate genes involved in the biosynthesis of phenylpropanoid compounds. Genes encoding regulatory proteins of the anthocyanin biosynthetic pathway (MYB and basic helix-loop-helix (bHLH) transcription factors and WD40 repeat proteins) were isolated using the transcriptomic resources of wild blackberry (Rubus genevieri) and cultivated red raspberry (Rubus idaeus cv. Prestige) and were shown to activate anthocyanin synthesis in Nicotiana benthamiana. Expression patterns of candidate flavonoid gene transcripts were also studied across three fruit developmental stages via the BacHBerryEXP gene expression browser (http://www.bachberryexp.com) in R. genevieri and R. idaeus cv. Prestige. CONCLUSIONS: We report a transcriptome resource that includes data for a wide range of berry(-like) fruit species that has been developed for gene identification and functional analysis to assist in berry fruit improvement. These resources will enable investigations of metabolic processes in berries beyond the phenylpropanoid biosynthetic pathway analysed in this study. The RNA-seq data will be useful for studies of berry fruit development and to select wild plant species useful for plant breeding purposes.

Linking a rapid throughput plate-assay with high-sensitivity stable-isotope label LCMS quantification permits the identification and characterisation of low ß-L-ODAP grass pea lines.

BACKGROUND: Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties. RESULTS: A plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for ß-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled ß-L-ODAP) allowing accurate quantification of ß-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any ß-L-ODAP in these species. The LCMS method was also used to quantify ß-L-ODAP accurately in different tissues of grass pea. CONCLUSIONS: The plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and ß-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of ß-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new 'gold standard' for ß-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-ß-L-ODAP genotypes.

Discovery of an RmlC/D fusion protein in the microalga Prymnesium parvum and its implications for NDP-ß-l-rhamnose biosynthesis in microalgae.

The 6-deoxy sugar l-rhamnose (l-Rha) is found widely in plant and microbial polysaccharides and natural products. The importance of this and related compounds in host-pathogen interactions often means that l-Rha plays an essential role in many organisms. l-Rha is most commonly biosynthesized as the activated sugar nucleotide uridine 5'-diphospho-ß-l-rhamnose (UDP-ß-l-Rha) or thymidine 5'-diphospho-ß-l-rhamnose (TDP-ß-l-Rha). Enzymes involved in the biosynthesis of these sugar nucleotides have been studied in some detail in bacteria and plants, but the activated form of l-Rha and the corresponding biosynthetic enzymes have yet to be explored in algae. Here, using sugar-nucleotide profiling in two representative algae, Euglena gracilis and the toxin-producing microalga Prymnesium parvum, we show that levels of UDP- and TDP-activated l-Rha differ significantly between these two algal species. Using bioinformatics and biochemical methods, we identified and characterized a fusion of the RmlC and RmlD proteins, two bacteria-like enzymes involved in TDP-ß-l-Rha biosynthesis, from P. parvum Using this new sequence and also others, we explored l-Rha biosynthesis among algae, finding that although most algae contain sequences orthologous to plant-like l-Rha biosynthesis machineries, instances of the RmlC-RmlD fusion protein identified here exist across the Haptophyta and Gymnodiniaceae families of microalgae. On the basis of these findings, we propose potential routes for the evolution of nucleoside diphosphate ß-l-Rha (NDP-ß-l-Rha) pathways among algae.

Two CYP82D Enzymes Function as Flavone Hydroxylases in the Biosynthesis of Root-Specific 4'-Deoxyflavones in Scutellaria baicalensis.

Baicalein, wogonin, and their glycosides are major bioactive compounds found in the medicinal plant Scutellaria baicalensis Georgi. These flavones can induce apoptosis in a variety of cancer cell lines but have no effect on normal cells. Furthermore, they have many additional benefits for human health, such as anti-oxidant, antiviral, and liver-protective properties. Here, we report the isolation and characterization of two CYP450 enzymes, SbCYP82D1.1 and SbCYP82D2, which function as the flavone 6-hydroxylase (F6H) and flavone 8-hydroxylase (F8H), respectively, in S. baicalensis. SbCYP82D1.1 has broad substrate specificity for flavones such as chrysin and apigenin and is responsible for biosynthesis of baicalein and scutellarein in roots and aerial parts of S. baicalensis, respectively. When the expression of SbCYP82D1.1 is knocked down, baicalin and baicalein levels are reduced significantly while chrysin glycosides accumulate in hairy roots. SbCYP82D2 is an F8H with high substrate specificity, accepting only chrysin as its substrate to produce norwogonin, although minor 6-hydroxylation activity can also be detected. Phylogenetic analysis suggested that SbCYP82D2 might have evolved from SbCYP82D1.1 via gene duplication followed by neofunctionalization, whereby the ancestral F6H activity is partially retained in the derived SbCYP82D2.

Genetic dissection of cyclic pyranopterin monophosphate biosynthesis in plant mitochondria.

Mitochondria play a key role in the biosynthesis of two metal cofactors, iron-sulfur (FeS) clusters and molybdenum cofactor (Moco). The two pathways intersect at several points, but a scarcity of mutants has hindered studies to better understand these links. We screened a collection of sirtinol-resistant Arabidopsis thaliana mutants for lines with decreased activities of cytosolic FeS enzymes and Moco enzymes. We identified a new mutant allele of ATM3 (ABC transporter of the mitochondria 3), encoding the ATP-binding cassette transporter of the mitochondria 3 (systematic name ABCB25), confirming the previously reported role of ATM3 in both FeS cluster and Moco biosynthesis. We also identified a mutant allele in CNX2, cofactor of nitrate reductase and xanthine dehydrogenase 2, encoding GTP 3',8-cyclase, the first step in Moco biosynthesis which is localized in the mitochondria. A single-nucleotide polymorphism in cnx2-2 leads to substitution of Arg88 with Gln in the N-terminal FeS cluster-binding motif. cnx2-2 plants are small and chlorotic, with severely decreased Moco enzyme activities, but they performed better than a cnx2-1 knockout mutant, which could only survive with ammonia as a nitrogen source. Measurement of cyclic pyranopterin monophosphate (cPMP) levels by LC-MS/MS showed that this Moco intermediate was below the limit of detection in both cnx2-1 and cnx2-2, and accumulated more than 10-fold in seedlings mutated in the downstream gene CNX5 Interestingly, atm3-1 mutants had less cPMP than wild type, correlating with previous reports of a similar decrease in nitrate reductase activity. Taken together, our data functionally characterize CNX2 and suggest that ATM3 is indirectly required for cPMP synthesis.

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.

Profiling of Sugar Nucleotides.

Sugar nucleotides are essential building blocks for the glycobiology of all living organisms. Detailed information on the types of sugar nucleotides present in a particular cell and how they change as a function of metabolic, developmental, or disease status is vital. The extraction, identification, and quantification of sugar nucleotides in a given sample present formidable challenges. In this chapter, currently used techniques for sugar nucleotide extraction from cells, separation from complex biological matrices, and detection by optical and mass spectrometry methods are discussed.