Nonsense-mediated mRNA decay pathway in plants under stress: general gene regulatory mechanism and advances.

MAIN CONCLUSION: Nonsense-mediated mRNA decay in eukaryotes is vital to cellular homeostasis. Further knowledge of its putative role in plant RNA metabolism under stress is pivotal to developing fitness-optimizing strategies. Nonsense-mediated mRNA decay (NMD), part of the mRNA surveillance pathway, is an evolutionarily conserved form of gene regulation in all living organisms. Degradation of mRNA-bearing premature termination codons and regulation of physiological RNA levels highlight NMD's role in shaping the cellular transcriptome. Initially regarded as purely a tool for cellular RNA quality control, NMD is now considered to mediate various aspects of plant developmental processes and responses to environmental changes. Here we offer a basic understanding of NMD in eukaryotes by explaining the concept of premature termination codon recognition and NMD complex formation. We also provide a detailed overview of the NMD mechanism and its role in gene regulation. The potential role of effectors, including ABCE1, in ribosome recycling during the translation process is also explained. Recent reports of alternatively spliced variants of corresponding genes targeted by NMD in Arabidopsis thaliana are provided in tabular format. Detailed figures are also provided to clarify the NMD concept in plants. In particular, accumulating evidence shows that NMD can serve as a novel alternative strategy for genetic manipulation and can help design RNA-based therapies to combat stress in plants. A key point of emphasis is its function as a gene regulatory mechanism as well as its dynamic regulation by environmental and developmental factors. Overall, a detailed molecular understanding of the NMD mechanism can lead to further diverse applications, such as improving cellular homeostasis in living organisms.

The R2R3-MYB transcription factor EVER controls the emission of petunia floral volatiles by regulating epicuticular wax biosynthesis in the petal epidermis.

The epidermal cells of petunia (Petunia × hybrida) flowers are the main site of volatile emission. However, the mechanisms underlying the release of volatiles into the environment are still being explored. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by virus-induced gene silencing and clustered regularly interspaced short palindromic repeat (CRISPR), and metabolomics, we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)-a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. To generate ever knockout lines, we developed a viral-based CRISPR/Cas9 system for efficient gene editing in plants. These knockout lines, together with transient-suppression assays, revealed EVER's involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in volatile production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. Liquid chromatography/gas chromatography-MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. We reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a regulatory layer in the scent-emission machinery in petunia flowers.

Polygalacturonase gene family analysis identifies FcPG12 as a key player in fig (Ficus carica L.) fruit softening.

BACKGROUND: The fig (Ficus carica L.) tree has high economic value. However, its fruit have a short shelf life due to rapid softening. Polygalacturonases (PGs) are essential hydrolases, responsible for the pectin degradation that plays a key role in fruit softening. However, fig PG genes and their regulators have not yet been characterized. RESULTS: In this study, 43 FcPGs were identified in the fig genome. They were non-uniformly distributed on 13 chromosomes, and tandem repeat PG gene clusters were found on chromosomes 4 and 5. Ka/Ks calculation and collinear analysis indicated negative selection as the main driver of FcPG family expansion. Fourteen FcPGs were found expressed in fig fruit with FPKM values > 10, of which seven were positively correlated, and three, negatively correlated with fruit softening. Eleven FcPGs were upregulated and two downregulated in response to ethephon treatment. FcPG12, a member of the tandem repeat cluster on chromosome 4, was selected for further analyses due to its sharp increment in transcript abundance during fruit softening and its response to ethephon treatment. Transient overexpression of FcPG12 led to decreased fig fruit firmness and increased PG enzyme activity in the tissue. Two ethylene response factor (ERF)-binding GCC-box sites were found on the FcPG12 promoter. Yeast one-hybrid and dual luciferase assays showed that FcERF5 binds directly to the FcPG12 promoter and upregulates its expression. Transient overexpression of FcERF5 upregulated FcPG12 expression, thereby increasing PG activity and fruit softening. CONCLUSIONS: Our study identified FcPG12 as a key PG gene in fig fruit softening, and its direct positive regulation by FcERF5. The results provide new information on the molecular regulation of fig fruit softening.

Developmental and temporal changes in petunia petal transcriptome reveal scent-repressing plant-specific RING-kinase-WD40 protein.

In moth-pollinated petunias, production of floral volatiles initiates when the flower opens and occurs rhythmically during the day, for optimal flower-pollinator interaction. To characterize the developmental transcriptomic response to time of day, we generated RNA-Seq databases for corollas of floral buds and mature flowers in the morning and in the evening. Around 70% of transcripts accumulating in petals demonstrated significant changes in expression levels in response to the flowers' transition from a 4.5-cm bud to a flower 1 day postanthesis (1DPA). Overall, 44% of the petal transcripts were differentially expressed in the morning vs. evening. Morning/evening changes were affected by flower developmental stage, with a 2.5-fold larger transcriptomic response to daytime in 1DPA flowers compared to buds. Analyzed genes known to encode enzymes in volatile organic compound biosynthesis were upregulated in 1DPA flowers vs. buds-in parallel with the activation of scent production. Based on analysis of global changes in the petal transcriptome, PhWD2 was identified as a putative scent-related factor. PhWD2 is a protein that is uniquely present in plants and has a three-domain structure: RING-kinase-WD40. Suppression of PhWD2 (termed UPPER - Unique Plant PhEnylpropanoid Regulator) resulted in a significant increase in the levels of volatiles emitted from and accumulated in internal pools, suggesting that it is a negative regulator of petunia floral scent production.

SCARECROW-like GRAS protein PES positively regulates petunia floral scent production.

Emission of scent volatiles by flowers is important for successful pollination and consequently, reproduction. Petunia (Petunia hybrida) floral scent is formed mainly by volatile products of the phenylpropanoid pathway. We identified and characterized a regulator of petunia scent production: the GRAS protein PHENYLPROPANOID EMISSION-REGULATING SCARECROW-LIKE (PES). Its expression increased in petals during bud development and was highest in open flowers. Overexpression of PES increased the production of floral volatiles, while its suppression resulted in scent reduction. We showed that PES upregulates the expression of genes encoding enzymes of the phenylpropanoid and shikimate pathways in petals, and of the core regulator of volatile biosynthesis ODORANT1 by activating its promoter. PES is an ortholog of Arabidopsis (Arabidopsis thaliana) PHYTOCHROME A SIGNAL TRANSDUCTION 1, involved in physiological responses to far-red (FR) light. Analyses of the effect of nonphotosynthetic irradiation (low-intensity FR light) on petunia floral volatiles revealed FR light as a scent-activating factor. While PHYTOCHROME A regulated scent-related gene expression and floral scent production under FR light, the influence of PES on volatile production was not limited by FR light conditions.

A whiff of the future: functions of phenylalanine-derived aroma compounds and advances in their industrial production.

Plants produce myriad aroma compounds-odorous molecules that are key factors in countless aspects of the plant's life cycle, including pollinator attraction and communication within and between plants. For humans, aroma compounds convey accurate information on food type, and are vital for assessing the environment. The phenylpropanoid pathway is the origin of notable aroma compounds, such as raspberry ketone and vanillin. In the last decade, great strides have been made in elucidating this pathway with the identification of numerous aroma-related biosynthetic enzymes and factors regulating metabolic shunts. These scientific achievements, together with public acknowledgment of aroma compounds' medicinal benefits and growing consumer demand for natural products, are driving the development of novel biological sources for wide-scale, eco-friendly, and inexpensive production. Microbes and plants that are readily amenable to metabolic engineering are garnering attention as suitable platforms for achieving this goal. In this review, we discuss the importance of aroma compounds from the perspectives of humans, pollinators and plant-plant interactions. Focusing on vanillin and raspberry ketone, which are of high interest to the industry, we present key knowledge on the biosynthesis and regulation of phenylalanine-derived aroma compounds, describe advances in the adoption of microbes and plants as platforms for their production, and propose routes for improvement.

Genome recoding strategies to improve cellular properties: mechanisms and advances.

The genetic code, once believed to be universal and immutable, is now known to contain many variations and is not quite universal. The basis for genome recoding strategy is genetic code variation that can be harnessed to improve cellular properties. Thus, genome recoding is a promising strategy for the enhancement of genome flexibility, allowing for novel functions that are not commonly documented in the organism in its natural environment. Here, the basic concept of genetic code and associated mechanisms for the generation of genetic codon variants, including biased codon usage, codon reassignment, and ambiguous decoding, are extensively discussed. Knowledge of the concept of natural genetic code expansion is also detailed. The generation of recoded organisms and associated mechanisms with basic targeting components, including aminoacyl-tRNA synthetase-tRNA pairs, elongation factor EF-Tu and ribosomes, are highlighted for a comprehensive understanding of this concept. The research associated with the generation of diverse recoded organisms is also discussed. The success of genome recoding in diverse multicellular organisms offers a platform for expanding protein chemistry at the biochemical level with non-canonical amino acids, genetically isolating the synthetic organisms from the natural ones, and fighting viruses, including SARS-CoV2, through the creation of attenuated viruses. In conclusion, genome recoding can offer diverse applications for improving cellular properties in the genome-recoded organisms.

Papain-Like Cysteine Protease Gene Family in Fig (Ficus carica L.): Genome-Wide Analysis and Expression Patterns.

The papain-like cysteine proteases (PLCPs) are the most abundant family of cysteine proteases in plants, with essential roles in biotic/abiotic stress responses, growth and senescence. Papain, bromelain and ficin are widely used in food, medicine and other industries. In this study, 31 PLCP genes (FcPCLPs) were identified in the fig (Ficus carica L.) genome by HMM search and manual screening, and assigned to one of nine subfamilies based on gene structure and conserved motifs. SAG12 and RD21 were the largest subfamilies with 10 and 7 members, respectively. The FcPCLPs ranged from 1,128 to 5,075 bp in length, containing 1-10 introns, and the coding sequence ranged from 624 to 1,518 bp, encoding 207-505 amino acids. Subcellular localization analysis indicated that 24, 2, and 5 PLCP proteins were targeted to the lysosome/vacuole, cytoplasm and extracellular matrix, respectively. Promoter (2,000 bp upstream) analysis of FcPLCPs revealed a high number of plant hormone and low temperature response elements. RNA-seq revealed differential expression of 17 FcPLCPs in the inflorescence and receptacle, and RD21 subfamily members were the major PLCPs expressed in the fruit; 16 and 5 FcPLCPs responded significantly to ethylene and light, respectively. Proteome analyses revealed 18 and 5 PLCPs in the fruit cell soluble proteome and fruit latex, respectively. Ficins were the major PLCP in fig fruit, with decreased abundance in inflorescences, but increased abundance in receptacles of commercial-ripe fruit. FcRD21B/C and FcALP1 were aligned as the genes encoding the main ficin isoforms. Our study provides valuable multi-omics information on the FcPLCP family and lays the foundation for further functional studies.

Spatial patterning of scent in petunia corolla is discriminated by bees and involves the ABCG1 transporter.

Floral guides are patterned cues that direct the pollinator to the plant reproductive organs. The spatial distribution of showy visual and olfactory traits allows efficient plant-pollinator interactions. Data on the mechanisms underlying floral volatile patterns or their interactions with pollinators are lacking. Here we characterize the spatial emission patterns of volatiles from the corolla of the model plant Petunia × hybrida and reveal the ability of honeybees to distinguish these patterns. Along the adaxial epidermis, in correlation with cell density, the petal base adjacent to reproductive organs emitted significantly higher levels of volatiles than the distal petal rim. Volatile emission could also be differentiated between the two epidermal surfaces: emission from the adaxial side was significantly higher than that from the abaxial side. Similar emission patterns were also observed in other petunias, Dianthus caryophyllus (carnation) and Argyranthemum frutescens (Marguerite daisy). Analyses of transcripts involved in volatile production/emission revealed lower levels of the plasma-membrane transporter ABCG1 in the abaxial versus adaxial epidermis. Transient overexpression of ABCG1 enhanced emission from the abaxial epidermis to the level of the adaxial epidermis, suggesting its involvement in spatial emission patterns in the epidermal layers. Proboscis extension response experiments showed that differences in emission levels along the adaxial epidermis, that is, petal base versus rim, detected by GC-MS are also discernible by honeybees.

Tomatoes expressing thaumatin II retain their sweet taste after salting and pickling processing.

BACKGROUND: Thaumatin II, a supersweet protein from the African plant katemfe (Thaumatococcus daniellii Benth.), shows promise as a zero-calorie sweetener for use in the food and pharmaceutical industries and for improving the taste of fruit. RESULTS: We report on the stability of thaumatin in salted and pickled tomatoes, as well as on the effect of thaumatin on the taste quality of processed tomatoes. Fruit of tomato cv. Yalf, transformed with the thaumatin II gene were salted and pickled and then stored for 6 months. Western blot analysis showed relative thaumatin II stability at salting; its content in processed fruits was 62-83% of the initial level depending in the studied line. In pickled tomatoes, thaumatin II content was decreased by up to 25% of the initial amount. Both salted and pickled tomatoes had a sweet taste with a typical thaumatin aftertaste. Salted tomatoes were characterized as being sweeter than pickled tomatoes. The overall taste of pickled tomatoes was rated by panellists as significantly better compared to that of salted or non-processed ones. CONCLUSION: In the present study, we have shown that tomatoes expressing supersweet protein thaumatin II can be used for processing under mild conditions, including salting and pickling. © 2021 Society of Chemical Industry.

ACE2 Co-evolutionary Pattern Suggests Targets for Pharmaceutical Intervention in the COVID-19 Pandemic.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spillover infection in December 2019 has caused an unprecedented pandemic. SARS-CoV-2, as other coronaviruses, binds its target cells through the angiotensin-converting enzyme 2 (ACE2) receptor. Accordingly, this makes ACE2 research essential for understanding the zoonotic nature of coronaviruses and identifying novel drugs. Here we present a systematic analysis of the ACE2 conservation and co-evolution protein network across 1,671 eukaryotes, revealing an unexpected conservation pattern in specific metazoans, plants, fungi, and protists. We identified the co-evolved protein network and pinpointed a list of drugs that target this network by using data integration from different sources. Our computational analysis found widely used drugs such as nonsteroidal anti-inflammatory drugs and vasodilators. These drugs are expected to perturb the ACE2 network affecting infectivity as well as the pathophysiology of the disease.

Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes.

Artemisinin-based drugs are the most effective medicine for the malaria treatment. To date, the main method of artemisinin production is its extraction from wormwood plants Artemisia annua L. Due to the limitation of this source, considerable efforts are now directed to the development of methods for artemisinin production using heterologous expression systems. Artemisinin is a sesquiterpene lactone, synthesized through the cyclization of farnesyl diphosphate involved in other sesquiterpene biosynthetic systems. Chrysanthemum species as well as A. annua, belong to Asteraceae family, and had been characterized by containing highly content of sesquiterpenes and their precursors. This makes chrysanthemum a promising target for the production of artemisinin in heterologous host plants. Chrysanthemum (C. morifolium Ramat.) was transformed by Agrobacterium tumefaciens carrying with the binary vectors p1240 and p1250, bearing artemisinin biosynthesis genes coding: amorpha-4,11-diene synthase, artemisinic aldehyde Δ11(13) reductase, amorpha-4,11-diene monooxygenase (p1240 was targeted to the mitochondria and p1250 was targeted to the cytosol), cytochrome P450 reductase from A. annua, as well as yeast truncated 3-hydroxy-3-methylglutarylcoenzyme A reductase. This study obtained 8 kanamycin-resistant lines after transformation with the p1240 and 2 lines from p1250. All target genes were detected in 2 and 1 transgenic lines of the 2 vectors. The transformation frequency of all target genes were 0.33% and 0.17% for p1240 and p1250, relative to the total transformed explant numbers. RT-PCR analysis revealed the transcription of all transferred genes in two lines obtained after transformation with the p1240 vector, confirming the possibility of transferring genetic modules encoding entire biochemical pathways into the chrysanthemum genome. This holds promise for the development of a chrysanthemum-based expression system to produce non-protein substances, such as artemisinin.

Ectopic Expression of PAP1 Leads to Anthocyanin Accumulation and Novel Floral Color in Genetically Engineered Goldenrod (Solidago canadensis L.).

Floral pigmentation is of major importance to the ornamental industry, which is constantly searching for cultivars with novel colors. Goldenrod (Solidago canadensis) has monochromatic yellow carotenoid-containing flowers that cannot be modified using classical breeding approaches due to a limited gene pool. To generate Solidago with novel colors through metabolic engineering, we first developed a procedure for its regeneration and transformation. Applicability of different cytokinins for adventitious regeneration was examined in the commercial cv. Tara, with zeatin yielding higher efficiency than 6-benzylaminopurine or thidiazuron. A comparison of regeneration of commercial cvs. Tara, Golden Glory and Ivory Glory revealed Tara to be the most potent, with an efficiency of 86% (number of shoots per 100 leaf explants). Agrobacterium-based transformation efficiency was highest for cv. Golden Glory (5 independent transgenic shoots per 100 explants) based on kanamycin selection and the GUS reporter gene. In an attempt to promote anthocyanin biosynthesis, we generated transgenic Solidago expressing snapdragon (Antirrhinum majus) Rosea1 and Delila, as well as Arabidopsis thaliana PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) transcription factors. Transgenic cv. Golden Glory expressing cauliflower mosaic virus 35S-driven PAP1 generated red flowers that accumulated delphinidin and its methylated derivatives, as compared to control yellow flowers in the GUS-expressing plants. The protocol described here allows efficient engineering of Solidago for novel coloration and improved agricultural traits.

Retracing the molecular basis and evolutionary history of the loss of benzaldehyde emission in the genus Capsella.

The transition from pollinator-mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate-CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer Capsella orientalis. We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found within and outside of Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent.

Proteome and transcriptome analyses reveal key molecular differences between quality parameters of commercial-ripe and tree-ripe fig (Ficus carica L.).

BACKGROUND: Fig fruit are highly perishable at the tree-ripe (TR) stage. Commercial-ripe (CR) fruit, which are harvested before the TR stage for their postharvest transportability and shelf-life advantage, are inferior to TR fruit in size, color and sugar content. The succulent urn-shaped receptacle, serving as the protective structure and edible part of the fruit, determines fruit quality. Quantitative iTRAQ and RNA-Seq were performed to reveal the differential proteomic and transcriptomic traits of the receptacle at the two harvest stages. RESULTS: We identified 1226 proteins, of which 84 differentially abundant proteins (DAPs) were recruited by criteria of abundance fold-change (FC) ≥1.3 and p < 0.05 in the TR/CR receptacle proteomic analysis. In addition, 2087 differentially expressed genes (DEGs) were screened by ≥2-fold expression change: 1274 were upregulated and 813 were downregulated in the TR vs. CR transcriptomic analysis. Ficin was the most abundant soluble protein in the fig receptacle. Sucrose synthase, sucrose-phosphate synthase and hexokinase were all actively upregulated at both the protein and transcriptional levels. Endoglucanase, expansin, beta-galactosidase, pectin esterase and aquaporins were upregulated from the CR to TR stage at the protein level. In hormonal synthesis and signaling pathways, high protein and transcriptional levels of aminocyclopropane-1-carboxylate oxidase were identified, together with a few diversely expressed ethylene-response factors, indicating the potential leading role of ethylene in the ripening process of fig receptacle, which has been recently reported as a non-climacteric tissue. CONCLUSIONS: We present the first delineation of intra- and inter-omic changes in the expression of specific proteins and genes of TR vs. CR fig receptacle, providing valuable candidates for further study of fruit-quality formation control and fig cultivar innovation to accommodate market demand.

Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants.

The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3' end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB-M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB-M130 accumulation ranging from 0.25-2.5 µg/g fresh weight (0.0006-0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB-M130 in the aggregate form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 µg duckweed-derived RTB-M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of nti-M2e IgG was 1,024. It was confirmed that oral immunization with RTB-M130 induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.

Isolation of Intact Vacuoles from Petunia Petals and Extraction of Sequestered Glycosylated Phenylpropanoid Compounds.

Plant vacuoles are the largest compartment in plant cells, occupying more than 80% of the cell volume. A variety of proteins, sugars, pigments and other metabolites are stored in these organelles ( Paris et al., 1996 ; Olbrich et al., 2007 ). Flowers produce a variety of specialized metabolites, some of which are unique to this organ, such as components of pollination syndromes, i.e., scent volatiles and flavonoids ( Hoballah et al., 2007; Cna'ani et al., 2015). To study the compounds stored in floral vacuoles, this compartment must be separated from the rest of the cell. To enable isolation of vacuoles, protoplasts were first generated by incubating pierced corollas with cellulase and macrozyme enzymes. After filtering and several centrifugation steps, protoplasts were separated from the debris and damaged/burst protoplasts, as revealed by microscopic observation. Concentrated protoplasts were lysed, and vacuoles were extracted by Ficoll-gradient centrifugation. Vacuoles were used for quantitative GC-MS analyses of sequestered metabolites. This method allowed us to identify vacuoles as the subcellular accumulation site of glycosylated volatile phenylpropanoids and to hypothesize that conjugated scent compounds are sequestered in the vacuoles en route to the headspace (Cna'ani et al., 2017).

Regulation of Fig (Ficus carica L.) Fruit Color: Metabolomic and Transcriptomic Analyses of the Flavonoid Biosynthetic Pathway.

Combined metabolomic and transcriptomic analyses were carried out with fig cultivar Green Peel and its color mutant "Purple Peel." Five and twenty-two metabolites were identified as having significantly different contents between fruit peels of the two cultivars at young and mature stages, respectively. Cyanidin O-malonylhexoside demonstrated a 3,992-fold increase in the mature purple peel, the first identification of a major cyanidin in fig fruit; cyanidin 3-O-glucoside, cyanidin O-malonylhexoside O-hexoside and cyanidin-3,5-O-diglucoside were upregulated 100-fold, revealing the anthocyanins underlying the purple mutation. Beyond the visible differences, there was very significant accumulation of the colorless flavonoids procyanidin B1, luteolin-3',7-di-O-glucoside, epicatechin and quercetin-3-O-rhamnoside in the mature "Purple Peel" compared to "Green Peel." At the young stage, only cyanidin O-malonylhexoside, cyanidin O-malonylhexoside O-hexoside and esculetin were upregulated a few fold in the mutant. Transcriptome analysis revealed a downregulated expression trend of genes encoding phenylpropanoid and flavonoid biosynthetic pathway enzyme in the young "Purple Peel" compared to the young "Green Peel," whereas significant and simultaneous upregulation was revealed in almost all of the flavonoid and anthocyanin pathway components and relevant transcription factors in the mature-stage mutant. The role of R2R3-MYB transcription factors in the color morph mutation and its possible relation to the activity of retrotransposons are discussed. Moreover, large-scale upregulation of small heat-shock protein genes was found in the mature mutant. This is the first work to reveal comprehensive metabolome and transcriptome network changes underlying a fig mutation in a single horticultural attribute, and its profound effects on fruit nutrition and quality.

Phenylpropanoid Scent Compounds in Petunia x hybrida Are Glycosylated and Accumulate in Vacuoles.

Floral scent has been studied extensively in the model plant Petunia. However, little is known about the intracellular fate of scent compounds. Here, we characterize the glycosylation of phenylpropanoid scent compounds in Petunia x hybrida. This modification reduces scent compounds' volatility, reactivity, and autotoxicity while increasing their water-solubility. Gas chromatography-mass spectrometry (GC-MS) analyses revealed that flowers of petunia cultivars accumulate substantial amounts of glycosylated scent compounds and that their increasing level parallels flower development. In contrast to the pool of accumulated aglycones, which drops considerably at the beginning of the light period, the collective pool of glycosides starts to increase at that time and does not decrease thereafter. The glycoside pool is dynamic and is generated or catabolized during peak scent emission, as inferred from phenylalanine isotope-feeding experiments. Using several approaches, we show that phenylpropanoid scent compounds are stored as glycosides in the vacuoles of petal cells: ectopic expression of Aspergillus niger ß-glucosidase-1 targeted to the vacuole resulted in decreased glycoside accumulation; GC-MS analysis of intact vacuoles isolated from petal protoplasts revealed the presence of glycosylated scent compounds. Accumulation of glycosides in the vacuoles seems to be a common mechanism for phenylpropanoid metabolites.

GA as a regulatory link between the showy floral traits color and scent.

Emission of volatiles at advanced stages of flower development is a strategy used by plants to lure pollinators to the flower. We reveal that GA negatively regulates floral scent production in petunia. We used Agrobacterium-mediated transient expression of GA-20ox in petunia flowers and a virus-induced gene silencing approach to knock down DELLA expression, measured volatile emission, internal pool sizes and GA levels by GC-MS or LC-MS/MS, and analyzed transcript levels of scent-related phenylpropanoid-pathway genes. We show that GA has a negative effect on the concentrations of accumulated and emitted phenylpropanoid volatiles in petunia flowers; this effect is exerted through transcriptional/post-transcriptional downregulation of regulatory and biosynthetic scent-related genes. Both overexpression of GA20-ox, a GA-biosynthesis gene, and suppression of DELLA, a repressor of GA-signal transduction, corroborated GA's negative regulation of floral scent. We present a model in which GA-dependent timing of the sequential activation of different branches of the phenylpropanoid pathway during flower development may represent a link between the showy traits controlling pollinator attraction, namely color and scent.