Biology subjects

Fraser, P. D.

Publications and source records attributed to Fraser, P. D..

10 recordsLinked to original sources

bioRxiv2025

Metabolomics characterisation of cassava pre-breeding populations with enhanced whitefly tolerance

Cassava (Manihot esculenta Crantz) provides food security for over 500 million people in Sub-Saharan Africa (SSA). Whitefly (Bemisia tabaci) is a pest in this region that result in ca. 50% crop yield losses. Thus, it is important to develop approaches that will generate new varieties tolerant to this pest to advance food security in the region. Two parental cassava varieties, ECU72 tolerant to whiteflies and COL2246 a susceptible line have been used to generate bi-parental populations. The F1 generation has been screened for whitefly resistance and progeny identified displaying enhanced tolerance. From designated F1 tolerant progeny, F2 families have been generated and phenotyped. The tolerance to whiteflies in the F2 population was further enhanced. Untargeted metabolomics was used to characterise whitefly susceptible and tolerant sub-groups. PCA of the molecular features generated clustering of accessions into whitefly resistant and susceptible groups and differentiating metabolite biomarkers were identified. The most significant metabolite marker for resistance being the chemical feature 316.0924. Although not consistent among all whitefly resistance sub-groups targeted LC-MS analysis revealed several pathways displaying perturbed levels. These include cyanogenic glycosides, apocarotenoids and phenylpropanoid super-pathway comprising of hydroxycinnamic acids, flavonoids and proanthocyanidins. Thus, the generation of a bi-parental population for whitefly tolerance/susceptibility enabled the identification of quantitative metabolite markers, the pathways contributing to tolerance, the underlying modes of action associated with resistance and the potential for the development of future high-throughput low-cost proxy markers. The approach also provides generic insights into future breeding strategies utilising bi-parental progeny for the enhancement of traits. SUMMARY. SIGNIFICANCE STATEMENT.

plant biology↗

bioRxiv2023

Carotenoid composition and sequestration in cassava (Manihot esculentum Crantz) root

Cassava (Manihot esculentum Crantz) is a staple food source for many developing countries. Its edible roots are high in starch but lack micronutrients such as {beta}-carotene. In the present study, analysis of pedigree breeding populations has led to the identification of cassava accessions with enhanced {beta}-carotene contents up to 40 g/g DW. This represents 0.2% of the Recommended Daily Allowance (RDA) for vitamin A. The {beta}-branch of the carotenoid pathway predominates in cassava roots, with dominant levels of {beta}-carotene followed by other minor epoxides of {beta}-ring derived carotenoids. Metabolomic analysis revealed that steady state levels of intermediary metabolism were not altered by the formation of carotenoids, similar to starch and carbohydrate levels. Apocarotenoids appeared to be independent of their carotenoid precursors. Lipidomic analysis provided evidence of a significant positive correlation between carotenoid and lipid content, in particular plastid specific galactolipids. Proteomic analysis of isolated amyloplasts revealed an abundance of carbohydrate/starch biosynthetic associated proteins (e.g. glucose-1-phosphate adenylyltransferase). No carotenoid related proteins were detected even in the highest carotenoid containing lines. Carotenoids were associated with fractions typically annotated as plastoglobuli and plastid membranes (particularly the envelope). Proteomic analysis confirmed these structures apart from plastoglobuli, thus potentially amyloplast structures may not contain classical plastoglobuli structures. HighlightCassava genotypes with enhanced provitamin A content ({beta}-carotene) reveals interconnectivity between the carotenoid pathway, starch and lipid biosynthesis.

plant biology↗

bioRxiv2023

Carotenoid retention during post-harvest storage of Capsicum annuum: the role of the fruit surface structure.

Degradation of carotenoids in food crops during post-harvest storage results in major economic and nutritional losses. In this study, a pepper (Capsicum annuum) panel for post-harvest carotenoid retention was studied to elucidate underlying mechanisms associated with this commercial trait of interest. Quantitative determination of carotenoid pigments and concurrent cellular analysis indicated that those pepper fruit with thicker lipid exocarp layers and smooth surfaces, following post-harvest drying and storage, possessed increased carotenoid retention. Total cutin monomer content increased in high carotenoid retention fruits and sub-epidermal cutin deposits were responsible for the difference in exocarp thickness. Cutin biosynthesis and cuticle precursor transport genes were differentially expressed between high and low carotenoid retention genotypes, and this supports the finding that fruit cuticle biosynthesis is associated with carotenoid retention. Carotenoids were located within cells embedded within the sub-epidermal cutin layer, and these carotenoids were protected from degradation due to the lack of permeability of the fruit surface to reactive oxygen species, and their precursors. The identification of a novel role for the pepper fruit surface in protecting against carotenoid degradation serves as an important discovery for the function of the fruit cuticle and provides an exploitable resource to enhance fruit quality. Highlight statementCarotenoid pigments in Chilli pepper confer post-harvest colour and nutritional quality. Analysis of diverse commercial genotypes indicates the involvement of the fruit surface in carotenoid retention

plant biology↗

bioRxiv2023

The characterisation of phytoene synthase-1 and 2, and 1-D-deoxy-xylulose 5-phosphate synthase genes from red chilli pepper (Capsicum annuum)

The red colouration of ripe chilli pepper fruit is due to the presence of the carotenoid capsanthin. 1-D-deoxy-xylulose 5-phosphate synthase (DXS) and phytoene synthase(s) (PSY) are influential biosynthetic steps in the carotenoid pathway. A panel of chilli accessions for varying fruit colour intensity revealed the correlation of carotenoid content with PSY and DXS transcript levels. The PSY and DXS genes were sequenced from high and low carotenoid genotypes to deduce potential allelic variation. PSY-1 and -2 showed tissue specific expression, with PSY-1and 2 expressed in chromoplast and chloroplast containing tissues, respectively. Protein modelling, phylogenetic analysis and transcription factor analysis was conducted to understand how the allelic variation identified could affect carotenoid biosynthesis and regulation, and subsequently colour intensity phenotype. A candidate mutation located in the transit peptide of the PSY-1 protein sequence was identified in the low colour intensity genotype. Within the promoter region of the DXS gene present in the high colour intensity genotype sequence variation was determined in the glyceraldehyde 3-phosphate binding motif (GAPF) of the promoter region. These data can be exploited to develop tools and resources for the breeding of high colour intensity chilli pepper fruit. Key policy highlightsUsing the molecular findings disclosed in this article the potential exists to develop molecular markers for the breeding varieties of chilli peppers with improved aesthetic consumer preference and nutritional attributes.

molecular biology↗

bioRxiv2023

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 role of the metabolites as regulators of cellular processes shown here, reinforces their pivotal role suggested in the remodelled "central dogma" concept.

systems biology↗

bioRxiv2023

The FIBRILLIN multigene family in tomato, their roles in plastoglobuli structure and metabolism

Plastoglobuli (PG) are plant lipoprotein compartments, present in plastid organelles. They are involved in the formation and/or storage of lipophilic metabolites. FIBRILLINs (FBN) are one of the main PG-associated proteins and are particularly abundant in carotenoid-enriched chromoplasts found in ripe fruits and flowers. To address the contribution of different FBNs to isoprenoid sequestration and PG function, a multiplex gene editing approach was undertaken. Analysis of single and high-order fbn mutants for the major PG-related FBNs in tomato, namely SlFBN1, SlFBN2a, SlFBN4, SlFBN7a, revealed functional redundancy. High order fbn mutants displayed phenotypes associated with abnormal isoprenoid accumulation, and aberrant PG formation and morphology. Lipidomic analysis highlighted broader changes in lipid metabolism. Paralog-specific roles were also observed and included the regulation of specific isoprenoids (e.g., plastochromanol) and control of plastidial esterification capability by SlFBN7a. Collectively, the results support both structural and regulatory roles of SlFBNs in PGs. Our findings expose fundamental aspects of metabolic compartmentalisation in plant cells and the importance of lipoprotein particles for their plastid metabolism/physiology. Significance statementIn the chromoplast of ripe tomato fruit and flower, plastoglobuli (PGs) are associated with several important biotechnological traits, due to their functional involvement in metabolism, developmental transitions, and environmental adaption. FIBRILLINS (FBN) are a multigene family of proteins that are collectively major components of the PG. Using a multiplex CRISPR-Cas9 approach single and high-order fbn mutants have been developed. Functional redundancy amongst the members of the FBN multigene family was evident, but also paralog specific functions/influence. Aberrant plastoglobuli formation and altered lipid metabolism are evident among fbn mutants. Characterisation of this resource has shed light on the functional role of FBN and their role in PG formation. This strategy offers new potential for the development of nutritional enhanced and climate resilient crops.

plant biology↗

bioRxiv2022

Integrated genetic and metabolic characterisation of diverse Latin American cassava (Manihot esculenta Crantz) germplasm; implications for future breeding strategies

Cassava is an important staple crop for food security in Africa and South America. The present study describes an integrated genomic and metabolomic approach to the characterisation of Latin American cassava germplasm. Classification based on genotyping and the leaf metabolome correlates, the key finding being the adaption to specific eco-geographical environments. In contrast the root metabolome does not relate to the genotypic clustering, suggesting different spatial regulation of this tissues metabolome. The data has been used to generate pan-metabolomes for specific tissues and the inclusion of phenotypic data has enabled the identification of metabolic sectors underlying traits of interest. For example, tolerance to whiteflies was not linked to cyanide content but to cell wall related phenylpropanoids or apocarotenoids. Collectively, these data advance the communities resources and provides a valuable insight into new parental breeding materials with traits of interest directly related to combating food security. Significance statementCassava is a staple crop in developing countries of sub-tropical regions. Traditionally, cassava has been considered as a subsistence crop. However recently it has become a sustainable solution to fulfil both hunger and malnutrition needs, and drive economic development. Varietal improvement via classic breeding has successfully delivered products into the Asian market by including/exchanging germplasm from original Latin American collections. Conversely, modest progress has been achieved in Sub-Saharan countries since genetic resources are biased towards exploitation of local landraces and uncharacterised parental material. The present work explores the genetic and metabolic diversity of Latin American cassavas genebank, one of the largest and most complete worldwide. These data provide a robust characterisation of valuable germplasm that can be exploited in breeding programmes.

plant biology↗

bioRxiv2022

Dually biofortified cisgenic tomatoes with increased flavonoids and branched-chain amino acids content

Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched-chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant-derived foods contain only small amounts of BCAAs and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically-engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide-resistant version of an acetolactate synthase (mSlALS) gene expressed broadly, and a MYB12-like transcription factor (SlMYB12) expressed in a fruit-specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21-fold compared to wild type levels), Valine (9-fold), Isoleucine (3-fold), and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64-fold) and quercetin (45-fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption.

bioengineering↗

bioRxiv2022

Potato virus X -delivered CRISPR activation programs lead to strong endogenous gene induction and transient metabolic reprogramming in Nicotiana benthamiana.

Programmable transcriptional regulators based on CRISPR architecture are promising tools for the control of plant gene expression. In plants, CRISPR gene activation (CRISPRa) has been shown effective in modulating development processes, such as the flowering time, or customising biochemical composition. The most widely used method for delivering the CRISPR components into the plant is Agrobacterium tumefaciens-mediated genetic transformation, either transient or stable. However, due to their versatility and their ability to move, virus-derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular the gRNA, which represents the variable component in CRISPR strategies. In this work we describe a Potato virus X (PVX)-derived vector that, upon agroinfection in N. benthamiana, serves as a vehicle for gRNAs delivery, producing a highly specific Virus-Induced Gene Activation (VIGA). The system works in combination with a Nicotiana benthamiana transgenic line carrying the remaining complementary CRISPRa components, specifically the dCasEV2.1 cassette, which has previously been shown to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non-invasive spraying method, we show here that gRNAs-mediated activation programs move locally and systemically generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this PVX-based virus-induced gene reprogramming (VIGR) strategy results in program-specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid-enriched metabolite profiles.

synthetic biology↗

bioRxiv2018

The genome and metabolome of the tobacco tree, Nicotiana glauca: a potential renewable feedstock for the bioeconomy

BackgroundGiven its tolerance to stress and its richness in particular secondary metabolites, the tobacco tree, Nicotiana glauca, has been considered a promising biorefinery feedstock that would not be competitive with food and fodder crops.\n\nResultsHere we present a 3.5 Gbp draft sequence and annotation of the genome of N. glauca spanning 731,465 scaffold sequences, with an N50 size of approximately 92 kbases. Furthermore, we supply a comprehensive transcriptome and metabolome analysis of leaf development comprising multiple techniques and platforms.\n\nThe genome sequence is predicted to cover nearly 80% of the estimated total genome size of N. glauca. With 73,799 genes predicted and a BUSCO score of 94.9%, we have assembled the majority of gene-rich regions successfully. RNA-Seq data revealed stage-and/or tissue-specific expression of genes, and we determined a general trend of a decrease of tricarboxylic acid cycle metabolites and an increase of terpenoids as well as some of their corresponding transcripts during leaf development.\n\nConclusionThe N. glauca draft genome and its detailed transcriptome, together with paired metabolite data, constitute a resource for future studies of valuable compound analysis in tobacco species and present the first steps towards a further resolution of phylogenetic, whole genome studies in tobacco.

plant biology↗