Biology subjects

Drapal, M.

Publications and source records attributed to Drapal, M..

5 recordsLinked to original sources

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

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↗