Ketocarotenoid production in tomato triggers metabolic reprogramming and cellular adaptation: The quest for homeostasis.

Plants are sessile and therefore have developed an extraordinary capacity to adapt to external signals. Here, the focus is on the plasticity of the plant cell to respond to new intracellular cues. Ketocarotenoids are high-value natural red pigments with potent antioxidant activity. In the present study, system-level analyses have revealed that the heterologous biosynthesis of ketocarotenoids in tomato initiated a series of cellular and metabolic mechanisms to cope with the formation of metabolites that are non-endogenous to the plant. The broad multilevel changes were linked to, among others, (i) the remodelling of the plastidial membrane, where the synthesis and storage of ketocarotenoids occurs; (ii) the recruiting of core metabolic pathways for the generation of metabolite precursors and energy; and (iii) redox control. The involvement of the metabolites as regulators of cellular processes shown here reinforces their pivotal role suggested in the remodelled 'central dogma' concept. Furthermore, the role of metabolic reprogramming to ensure cellular homeostasis is proposed.

The chemotype core collection of genus Nicotiana.

Sustainable production of chemicals and improving these biosources by engineering metabolic pathways to create efficient plant-based biofactories relies on the knowledge of available chemical/biosynthetic diversity present in the plant. Nicotiana species are well known for their amenability towards transformation and other new plant breeding techniques. The genus Nicotiana is primarily known through Nicotiana tabacum L., the source of tobacco leaves and all respective tobacco products. Due to the prevalence of the latter, N. tabacum and related Nicotiana species are one of the most extensively studied plants. The majority of studies focused solely on N. tabacum or other individual species for chemotyping. The present study analysed a diversity panel including 17 Nicotiana species and six accessions of Nicotiana benthamiana and created a data set that effectively represents the chemotype core collection of the genus Nicotiana. The utilisation of several analytical platforms and previously published libraries/databases enabled the identification and measurement of over 360 metabolites of a wide range of chemical classes as well as thousands of unknowns with dedicated spectral and chromatographic properties.

The potential of metabolomics in assessing global compositional changes resulting from the application of CRISPR/Cas9 technologies.

Exhaustive analysis of genetically modified crops over multiple decades has increased societal confidence in the technology. New Plant Breeding Techniques are now emerging with improved precision and the ability to generate products containing no foreign DNA and mimic/replicate conventionally bred varieties. In the present study, metabolomic analysis was used to compare (i) tobacco genotypes with and without the CRISPR associated protein 9 (Cas9), (ii) tobacco lines with the edited and non-edited DE-ETIOLATED-1 gene without phenotype and (iii) leaf and fruit tissue from stable non-edited tomato progeny with and without the Cas9. In all cases, multivariate analysis based on the difference test using LC-HRMS/MS and GC-MS data indicated no significant difference in their metabolomes. The variations in metabolome composition that were evident could be associated with the processes of tissue culture regeneration and/or transformation (e.g. interaction with Agrobacterium). Metabolites responsible for the variance included quantitative changes of abundant, well characterised metabolites such as phenolics (e.g. chlorogenic acid) and several common sugars such as fructose. This study provides fundamental data on the characterisation of gene edited crops, that are important for the evaluation of the technology and its assessment. The approach also suggests that metabolomics could contribute to routine product-based analysis of crops/foods generated from New Plant Breeding approaches.

Metabolic effects of agro-infiltration on N. benthamiana accessions.

Over the recent years, Nicotiana benthamiana has gained great importance as a chassis for the production of high value, low volume pharmaceuticals and/or active pharmaceutical ingredients (APIs). The process involving infiltration of the N. benthamiana leaves with Agrobacterium spp, harbouring vectors with the gene of interest, facilitates transient expression. To date, little information is available on the effect of the agro-infiltration process on the metabolome of N. benthamiana, which is necessary to improve the process for large-scale, renewable manufacturing of high value compounds and medical products. Hence, the objective of the present study was to assess metabolic adaptation of N. benthamiana as a response to the presence of Agrobacterium. The present study elucidated changes of the steady-state metabolism in the agroinfiltrated leaf area, the area around the infection and the rest of the plant. Furthermore, the study discusses the phenotypic advantages of the N. benthamiana lab strain, optimised for agro-infiltration, compared to three other wild accessions. Results showed that the lab strain has a different metabolic composition and showed less alterations of the phenylpropanoid pathway and cell wall remodelling in the agroinfiltrated leaf areas, for example chlorogenic acid, cadaverine and C18:0-2-glycerol ester. In conclusion, both of these alterations present potential candidates to improve the phenotype of the N. benthamiana lab strain for a more efficient transient expression process.

Engineering of tomato for the sustainable production of ketocarotenoids and its evaluation in aquaculture feed.

Ketocarotenoids are high-value pigments used commercially across multiple industrial sectors as colorants and supplements. Chemical synthesis using petrochemical-derived precursors remains the production method of choice. Aquaculture is an example where ketocarotenoid supplementation of feed is necessary to achieve product viability. The biosynthesis of ketocarotenoids, such as canthaxanthin, phoenicoxanthin, or astaxanthin in plants is rare. In the present study, complex engineering of the carotenoid pathway has been performed to produce high-value ketocarotenoids in tomato fruit (3.0 mg/g dry weight). The strategy adopted involved pathway extension beyond ß-carotene through the expression of the ß-carotene hydroxylase (CrtZ) and oxyxgenase (CrtW) from Brevundimonas sp. in tomato fruit, followed by ß-carotene enhancement through the introgression of a lycopene ß-cyclase (ß-Cyc) allele from a Solanum galapagense background. Detailed biochemical analysis, carried out using chromatographic, UV/VIS, and MS approaches, identified the predominant carotenoid as fatty acid (C14:0 and C16:0) esters of phoenicoxanthin, present in the S stereoisomer configuration. Under a field-like environment with low resource input, scalability was shown with the potential to deliver 23 kg of ketocarotenoid/hectare. To illustrate the potential of this "generally recognized as safe" material with minimal, low-energy bioprocessing, two independent aquaculture trials were performed. The plant-based feeds developed were more efficient than the synthetic feed to color trout flesh (up to twofold increase in the retention of the main ketocarotenoids in the fish fillets). This achievement has the potential to create a new paradigm in the renewable production of economically competitive feed additives for the aquaculture industry and beyond.

The road to astaxanthin production in tomato fruit reveals plastid and metabolic adaptation resulting in an unintended high lycopene genotype with delayed over-ripening properties.

Tomato fruit are an important nutritional component of the human diet and offer potential to act as a cell factory for speciality chemicals, which are often produced by chemical synthesis. In the present study our goal was to produce competitive levels of the high value ketocarotenoid, astaxanthin, in tomato fruit. The initial stage in this process was achieved by expressing the 4, 4' carotenoid oxygenase (crtW) and 3, 3' hydroxylase (crtZ) from marine bacteria in tomato under constitutive control. Characterization of this genotype showed a surprising low level production of ketocarotenoids in ripe fruit but over production of lycopene (~3.5 mg/g DW), accompanied by delayed ripening. In order to accumulate these non-endogenous carotenoids, metabolite induced plastid differentiation was evident as well as esterification. Metabolomic and pathway based transcription studies corroborated the delayed onset of ripening. The data also revealed the importance of determining pheno/chemotype inheritance, with ketocarotenoid producing progeny displaying loss of vigour in the homozygous state but stability and robustness in the hemizygous state. To iteratively build on these data and optimize ketocarotenoid production in this genotype, a lycopene ß-cyclase was incorporated to avoid precursor limitations and a more efficient hydroxylase was introduced. These combinations resulted in the production of astaxanthin (and ketocarotenoid esters) in ripe fruit at ~3 mg/g DW. Based on previous studies, this level of product formation represents an economic competitive value in a Generally Regarded As Safe (GRAS) matrix that requires minimal downstream processing.

Construction of a fusion enzyme for astaxanthin formation and its characterisation in microbial and plant hosts: A new tool for engineering ketocarotenoids.

The high-value ketocarotenoid astaxanthin, a natural red colorant with powerful antioxidant activity, is synthesised from ß-carotene by a hydroxylase and an oxygenase enzyme, which perform the addition of two hydroxyl and keto moieties, respectively. Several routes of intermediates, depending on the sequence of action of these enzymes, lead to the formation of astaxanthin. In the present study, the enzyme activities of 3, 3' ß-carotene hydroxylase (CRTZ) and 4, 4' ß-carotene oxygenase (CRTW) have been combined through the creation of "new to nature" enzyme fusions in order to overcome leakage of non-endogenous intermediates and pleotropic effects associated with their high levels in plants. The utility of flexible linker sequences of varying size has been assessed in the construction of pZ-W enzyme fusions. Frist, in vivo color complementation assays in Escherichia coli have been used to evaluate the potential of the fusion enzymes. Analysis of the carotenoid pigments present in strains generated indicated that the enzyme fusions only possess both catalytic activities when CRTZ is attached as the N-terminal module. Astaxanthin levels in E. coli cells were increased by 1.4-fold when the CRTZ and CRTW enzymes were fused compared to the individual enzymes. Transient expression in Nicotiana benthamiana was then performed in order to assess the potential of the fusions in a plant system. The production of valuable ketocarotenoids was achieved using this plant-based transient expression system. This revealed that CRTZ and CRTW, transiently expressed as a fusion, accumulated similar levels of astaxanthin compared to the expression of the individual enzymes whilst being associated with reduced ketocarotenoid intermediate levels (e.g. phoenicoxanthin, canthaxanthin and 3-OH-echinenone) and a reduced rate of leaf senescence after transformation. Therefore, the quality of the plant material producing the ketocarotenoids was enhanced due to a reduction in the stress induced by the accumulation of high levels of heterologous ketocarotenoid intermediates. The size of the linkers appeared to have no effect upon activity. The potential of the approach to production of valuable plant derived products is discussed.

Carotenoid biosynthesis and sequestration in red chilli pepper fruit and its impact on colour intensity traits.

The exploitation of diverse natural variation has been a key progenitor of crop breeding over the last decade. However, commercial practice is now turning to the use of accessions with less extreme phenotypes as genetic donors. In the present study, the carotenoid formation in a red-fruited discovery panel of Capsicum annuum (chilli pepper) has been characterized. The data indicated that colour intensity correlated with the amount of capsanthin and its esters, along with transcript levels of the 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and phytoene synthase-1 (PSY-1) genes. Quantification of carotenoids through development and ripening suggested the presence of separate biosynthesis and accumulation phases. Subplastid fractionation demonstrated the differential sequestration of pigments in high- and low-intensity lines and revealed the PSY protein to be most active in the membrane fractions when abundance was highest in the fibril fractions. Carotenoid accumulation was associated with the esterification of xanthophylls, expression of a putative carotenoid acyl transferase, and increased fibril content within the plastid. Interrogation of TEM images and carotenoid analysis of subplastid fractions suggest that the plastoglobuli are likely to be the progenitor of the characteristic fibrils found in pepper fruit. Collectively, these data provide an insight into the underpinning molecular, biochemical, and cellular mechanisms associated with the synthesis and sequestration of carotenoids in chromoplast-containing fruits, in addition to providing potential tools and resources for the breeding of high red colour intensity pepper varieties.

The regulation of carotenoid formation in tomato fruit.

Carotenoid biosynthesis in plants includes a complex series of desaturation/isomerisation reactions, catalyzed by four independent enzymes. In bacteria and fungi one desaturase/isomerase enzyme completes the same series of reactions. In the present study, a bacterial desaturase (crtI) from Pantoea ananatis has been overexpressed in the tangerine mutant of tomato (Solanum lycopersicon) which accumulates cis-carotene isomers in the fruit due to a defective isomerase (CRTISO) and the old gold crimson (ogc ) tomato mutant, which is defective in the fruit-enhanced lycopene ß-cyclase (CYCB). Comprehensive molecular and biochemical characterization of the resulting lines expressing crtI has revealed negative feedback mechanisms, acting predominantly at the level of phytoene synthase-1 (PSY1), and feed-forward mechanisms inducing cyclisation. In both cases, altered transcription appears to be the progenitor, with subsequent post-transcriptional modulation highlighting the complexity of the processes involved in modulating carotenoid homeostasis in plant tissues.

Subchromoplast sequestration of carotenoids affects regulatory mechanisms in tomato lines expressing different carotenoid gene combinations.

Metabolic engineering of the carotenoid pathway in recent years has successfully enhanced the carotenoid contents of crop plants. It is now clear that only increasing biosynthesis is restrictive, as mechanisms to sequestrate these increased levels in the cell or organelle should be exploited. In this study, biosynthetic pathway genes were overexpressed in tomato (Solanum lycopersicum) lines and the effects on carotenoid formation and sequestration revealed. The bacterial Crt carotenogenic genes, independently or in combination, and their zygosity affect the production of carotenoids. Transcription of the pathway genes was perturbed, whereby the tissue specificity of transcripts was altered. Changes in the steady state levels of metabolites in unrelated sectors of metabolism were found. Of particular interest was a concurrent increase of the plastid-localized lipid monogalactodiacylglycerol with carotenoids along with membranous subcellular structures. The carotenoids, proteins, and lipids in the subchromoplast fractions of the transgenic tomato fruit with increased carotenoid content suggest that cellular structures can adapt to facilitate the sequestration of the newly formed products. Moreover, phytoene, the precursor of the pathway, was identified in the plastoglobule, whereas the biosynthetic enzymes were in the membranes. The implications of these findings with respect to novel pathway regulation mechanisms are discussed.

Integrative transcript and metabolite analysis of nutritionally enhanced DE-ETIOLATED1 downregulated tomato fruit.

Fruit-specific downregulation of the DE-ETIOLATED1 (DET1) gene product results in tomato fruits (Solanum lycopersicum) containing enhanced nutritional antioxidants, with no detrimental effects on yield. In an attempt to further our understanding of how modulation of this gene leads to improved quality traits, detailed targeted and multilevel omic characterization has been performed. Metabolite profiling revealed quantitative increases in carotenoid, tocopherol, phenylpropanoids, flavonoids, and anthocyanidins. Qualitative differences could also be identified within the phenolics, including unique formation in fruit pericarp tissues. These changes resulted in increased total antioxidant content both in the polar and nonpolar fractions. Increased transcription of key biosynthetic genes is a likely mechanism producing elevated phenolic-based metabolites. By contrast, high levels of isoprenoids do not appear to result from transcriptional regulation but are more likely related to plastid-based parameters, such as increased plastid volume per cell. Parallel metabolomic and transcriptomic analyses reveal the widespread effects of DET1 downregulation on diverse sectors of metabolism and sites of synthesis. Correlation analysis of transcripts and metabolites independently indicated strong coresponses within and between related pathways/processes. Interestingly, despite the fact that secondary metabolites were the most severely affected in ripe tomato fruit, our integrative analyses suggest that the coordinated activation of core metabolic processes in cell types amenable to plastid biogenesis is the main effect of DET1 loss of function.

A xanthophyll-derived apocarotenoid regulates carotenogenesis in tomato chromoplasts.

Carotenoids possess important biological functions that make them essential components of the human diet. ß-Carotene and some other carotenoids have vitamin A activity while lutein and zeaxanthin, typically referred to as the macular pigments, are involved in good vision and in delaying the onset of age-related eye diseases. In order to create a zeaxanthin-producing tomato fruit, two transgenic lines, one with a high ß-carotene cyclase activity and the other with a high ß-carotene hydroxylase activity, have been genetically crossed. Ripe fruits from the resulting progeny contained significant levels of violaxanthin, antheraxanthin, and xanthophyll esters. However, their zeaxanthin content was not as high as expected, and the total level of carotenoids was only 25% of the carotenoids found in ripe fruits of the comparator line. Targeted transcript analysis and apocarotenoids determinations indicated that transcriptional regulation of the pathway or degradation of synthesized carotenoids were not responsible for the low carotenoid content of hybrid fruits which instead appeared to result from a substantial reduction of carotenoid biosynthesis. Notably, the content of an unidentified hydroxylated cyclic (C13) apocarotenoid was 13 times higher in the hybrid fruits than in the control fruits. Furthermore, a GC-MS-based metabolite profiling demonstrated a perturbation of carotenogenesis in ripening hybrid fruits compatible with a block of the pathway. Moreover, carotenoid profiling on leaf, fruit, and petal samples from a set of experimental lines carrying the hp3 mutation, in combination with the two transgenes, indicated that the carotenoid biosynthesis in petal and fruit chromoplasts could be regulated. Altogether the data were consistent with the hypothesis of the regulation of the carotenoid pathway in tomato chromoplasts through a mechanism of feedback inhibition mediated by a xanthophyll-derived apocarotenoid. This chromoplast-specific post-transcriptional mechanism was disclosed in transgenic fruits of HU hybrid owing to the abnormal production of zeaxanthin and antheraxanthin, the more probable precursors of the apocarotenoid signal. A model describing the regulation of carotenoid pathway in tomato chromoplasts is presented.

Isolation and characterization of sub-plastidial fractions from carotenoid rich fruits.

Carotenoid biosynthesis and sequestration in higher plants occurs in the plastid organelle. Among diverse germplasm collections displaying natural variation for carotenoids and outputs from metabolic engineering experiments it has become clear that plastid type and numbers can have important implications on the quantitative composition of carotenoids accumulating. Therefore, it is important to characterize these organelles to fully evaluate the potential of the germplasm to enhance carotenoids and create nutrient dense fruits and vegetables. In this article the procedures used to isolate sub-plastidial structures from carotenoid-rich Solanaceae fruits (tomato and Capsicum) are described.

Metabolomic approaches for the characterization of carotenoid metabolic engineering in planta.

Carotenoid biosynthesis has now been subjected to metabolic engineering for over two decades. The outputs clearly show that carotenoid formation is an integral component of metabolism. Perturbations can affect intermediary metabolism and other isoprenoids. The advances in omic technologies have enabled the quantitative assessment of changes in the transcriptome, proteome and metabolome in response to altered carotenoid biosynthesis. In the present article, the approaches and procedures relating to the capture of the metabolome in response to modulation of the carotenoid biosynthetic pathway are described. These data will contribute to the fundamental understanding of metabolic biology, underpinning future rationale design of New Plant Breeding Techniques (NPBTs) and associated regulatory affairs.

Metabolic changes in leaves of N. tabacum and N. benthamiana during plant development.

Dwindling fossil fuel reserves and poor environmental credentials of chemical synthesis means, new renewable sources for the production and manufacture of valuable chemicals and pharmaceuticals are required. Presently, tobacco is an underutilised non-food crop with the potential to act as a biofactory. In this study, metabolite profiling across vegetative development has been carried out to provide a quantitative baseline of metabolites, their formation and interaction. Two tobacco platforms have been used, Nicotiana benthamiana and Nicotiana tabacum. Our data generated has provided the quantitative and qualitative baseline levels for exploitable pathways and metabolites, across two complementary Nicotiana species. N. benthamiana is the chassis of choice for transient expression. The metabolite data obtained for N. benthamiana highlighted that before flower emergence, the increased central carbon metabolism and high amino acid levels are available for the biosynthesis of endogenous or heterologous metabolites. In the future, engineering pathways or biocatalysts into N. benthamiana could add value to the process presently used to produce low volume, high cost pharmaceuticals. Similar outputs were obtained for N. tabacum, which has the advantage of providing a large biomass and hence, high product yield. These data provide an insight into the metabolite pools available in tobacco for future exploitation by emerging New Plant Breeding Techniques.

The esterification of xanthophylls in Solanum lycopersicum (tomato) chromoplasts; the role of a non-specific acyltransferase.

The esterification of carotenoids has been associated with high-level accumulation, greater stability and potentially improved dietary bioavailability. Engineering the formation of ketocarotenoids into tomato fruit has resulted in the esterification of these non-endogenous metabolites. A genotype of tomato was created that contains; (i) the mutant pale yellow petal (pyp)1-1 allele, which is responsible for the absence of carotenoid esters in tomato flowers and (ii) the heterologous enzymes for ketocarotenoid formation. Analysis of the resulting progeny showed altered quantitative and qualitative differences in esterified carotenoids. For example, in ripe fruit tissues, in the presence of the pyp mutant allele, non-endogenous ketocarotenoid esters were absent while their free forms accumulated. These data demonstrate the involvement of the PYP gene product in the esterification of diverse xanthophylls.

Understanding colour retention in red chilli pepper fruit using a metabolite profiling approach.

Carotenoids are the pigments responsible for conferring the characteristic deep red colour to chilli pepper. The post-harvest retention of this colour is a key trait that governs the price of the produce. Determining colour retention and the associated underlying biochemical mechanisms are important issues that require investigation. In this present study, the ability of image analysis to determine colour change in ground chilli fruit was evaluated. This method enabled differentiation of extreme retention phenotypes whilst also reducing the duration of storage required to make accurate determinations. The analysis of volatiles indicated different levels of lipid and carotenoid derived volatiles in lines with different retention properties. Metabolite profiling of intermediary metabolism supported these findings, with increased levels of unsaturated fatty acids present in lines with low retention properties. Collectively, these data have led us to propose that in chilli fruit lipid peroxidation is one of the progenitors of carotenoid degradation.

New plant breeding techniques and their regulatory implications: An opportunity to advance metabolomics approaches.

Over the previous decades, biotechnological innovations have led to improved agricultural productivity, more nutritious foods and lower chemical usage. Both in western societies and Low Medium Income Countries (LMICs). However, the projected increases in the global population, means the production of nutritious food stuffs must increase dramatically. Building on existing genetic modification technologies a series of New Plant Breeding Technologies (NPBT) has recently emerged. These approaches include, Agro-infiltration, grafting, cis and intragenesis and gene editing technologies. How these new techniques should be regulated has fostered considerable debate. Concerns have also been raised, to ensure over-regulation does not arise, creating administrative and economic burden. In this article the existing landscape of genetically modified crops is reviewed and the potential of several New Plant Breeding Techniques (NPBT) described. Metabolomics is an omic technology that has developed in a concurrent manner with biotechnological advances in plant breeding. There is potentially further opportunities to advance our metabolomic technologies to characterise the outputs of New Plant Breeding Technologies, in a manner that is beneficial both from an academic, biosafety and industrial perspective.

Analysis of Tomato Post-Harvest Properties: Fruit Color, Shelf Life, and Fungal Susceptibility.

A wide variety of fresh market and processing tomatoes (Solanum lycopersicum) is grown and consumed worldwide. Post-harvest losses are a major contributing factor to losses in crop productivity and can account for up to 50% of the harvest. To select and breed elite tomato varieties, it is important to characterize fruit quality and evaluate the post-harvest properties of tomato fruits. This includes the analysis of shelf life (the period during which a fruit remains suitable for consumption without qualitative deterioration), color, and pathogen susceptibility. Tomato shelf life depends upon the rate of fruit softening which accompanies fruit ripening and exacerbates damage during transport and handling. Furthermore, the susceptibility of tomatoes to fruit pathogens is also often linked to fruit ripening, especially for necrotrophic fungi such as Botrytis cinerea, also known as gray mold. The methods described here are critical for determining fruit quality and fungal susceptibility during storage. © 2020 The Authors. Basic Protocol 1: Fruit color as a determinant of fruit quality Basic Protocol 2: Shelf life test of tomato fruits Basic Protocol 3: Botrytis cinerea pathogen test of tomato fruits Support Protocol: Preparation of Botrytis spore inoculum.

Rocket Science: The Effect of Spaceflight on Germination Physiology, Ageing, and Transcriptome of Eruca sativa Seeds.

In the 'Rocket Science' project, storage of Eruca sativa (salad rocket) seeds for six months on board the International Space Station resulted in delayed seedling establishment. Here we investigated the physiological and molecular mechanisms underpinning the spaceflight effects on dry seeds. We found that 'Space' seed germination vigor was reduced, and ageing sensitivity increased, but the spaceflight did not compromise seed viability and the development of normal seedlings. Comparative analysis of the transcriptomes (using RNAseq) in dry seeds and upon controlled artificial ageing treatment (CAAT) revealed differentially expressed genes (DEGs) associated with spaceflight and ageing. DEG categories enriched by spaceflight and CAAT included transcription and translation with reduced transcript abundances for 40S and 60S ribosomal subunit genes. Among the 'spaceflight-up' DEGs were heat shock proteins (HSPs), DNAJ-related chaperones, a heat shock factor (HSFA7a-like), and components of several DNA repair pathways (e.g., ATM, DNA ligase 1). The 'response to radiation' category was especially enriched in 'spaceflight-up' DEGs including HSPs, catalases, and the transcription factor HY5. The major finding from the physiological and transcriptome analysis is that spaceflight causes vigor loss and partial ageing during air-dry seed storage, for which space environmental factors and consequences for seed storage during spaceflights are discussed.