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Analyzing Mass Spectrometry Imaging Data of ¹³C-Labeled Phospholipids in Camelina sativa and Thlaspi arvense (Pennycress) Embryos.

ABSTRACT: The combination of ¹³C-isotopic labeling and mass spectrometry imaging (MSI) offers an approach to analyze metabolic flux in situ. However, combining isotopic labeling and MSI presents technical challenges ranging from sample preparation, label incorporation, data collection, and analysis. Isotopic labeling and MSI individually create large, complex data sets, and this is compounded when both methods are combined. Therefore, analyzing isotopically labeled MSI data requires streamlined procedures to support biologically meaningful interpretations. Using currently available software and techniques, here we describe a workflow to analyze ¹³C-labeled isotopologues of the membrane lipid and storage oil lipid intermediate-phosphatidylcholine (PC). Our results with embryos of the oilseed crops, Camelina sativa and Thlaspi arvense (pennycress), demonstrated greater ¹³C-isotopic labeling in the cotyledons of developing embryos compared with the embryonic axis. Greater isotopic enrichment in PC molecular species with more saturated and longer chain fatty acids suggest different flux patterns related to fatty acid desaturation and elongation pathways. The ability to evaluate MSI data of isotopically labeled plant embryos will facilitate the potential to investigate spatial aspects of metabolic flux in situ.

SUBMITTER: Romsdahl TB

PROVIDER: S-EPMC7999836 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pennycress [Thlaspi arvense L (Brassicaceae)] is being domesticated as a renewable biodiesel feedstock that also provides crucial ecosystems services, including as a nutritional resource for pollinators. However, its flowers produce significantly less nectar than other crop relatives in the Brassicaceae. This study was undertaken to understand the basic biology of the pennycress nectary as an initial step toward the possibility of enhancing nectar output from its flowers.Pennycress flowers contain four equivalent nectaries located extrastaminally at the base of the insertion sites of short and long stamens. Like other Brassicaceae, the nectaries have open stomates on their surface, which likely serve as the sites of nectar secretion. The nectaries produce four distinct nectar droplets that accumulate in concave structures at the base of each of the four petals. To understand the molecular biology of the pennycress nectary, RNA was isolated from 'immature' (pre-secretory) and 'mature' (secretory) nectaries and subjected to RNA-seq. Approximately 184 M paired-end reads (368 M total reads) were de novo assembled into a total of 16,074 independent contigs, which mapped to 12,335 unique genes in the pennycress genome. Nearly 3700 genes were found to be differentially expressed between immature and mature nectaries and subjected to gene ontology and metabolic pathway analyses. Lastly, in silico analyses identified 158 pennycress orthologs to Arabidopsis genes with known enriched expression in nectaries. These nectary-enriched expression patterns were verified for select pennycress loci by semi-quantitative RT-PCR.Pennycress nectaries are unique relative to those of other agriculturally important Brassicaceae, as they contain four equivalent nectaries that present their nectar in specialized cup-shaped structures at the base of the petals. In spite of these morphological differences, the genes underlying the regulation and production of nectar appear to be largely conserved between pennycress and Arabidopsis thaliana. These results provide a starting point for using forward and reverse genetics approaches to enhance nectar synthesis and secretion in pennycress.

Project description:BackgroundUnderstanding how organisms evolve and adapt to extreme habitats is of crucial importance in evolutionary ecology. Altitude gradients are an important determinant of the distribution pattern and range of organisms due to distinct climate conditions at different altitudes. High-altitude regions often provide extreme environments including low temperature and oxygen concentration, poor soil, and strong levels of ultraviolet radiation, leading to very few plant species being able to populate elevation ranges greater than 4000 m. Field pennycress (Thlaspi arvense) is a valuable oilseed crop and emerging model plant distributed across an elevation range of nearly 4500 m. Here, we generate an improved genome assembly to understand how this species adapts to such different environments.ResultsWe sequenced and assembled de novo the chromosome-level pennycress genome of 527.3 Mb encoding 31,596 genes. Phylogenomic analyses based on 2495 single-copy genes revealed that pennycress is closely related to Eutrema salsugineum (estimated divergence 14.32-18.58 Mya), and both species form a sister clade to Schrenkiella parvula and genus Brassica. Field pennycress contains the highest percentage (70.19%) of transposable elements in all reported genomes of Brassicaceae, with the retrotransposon proliferation in the Middle Pleistocene being likely responsible for the expansion of genome size. Moreover, our analysis of 40 field pennycress samples in two high- and two low-elevation populations detected 1,256,971 high-quality single nucleotide polymorphisms. Using three complementary selection tests, we detected 130 candidate naturally selected genes in the Qinghai-Tibet Plateau (QTP) populations, some of which are involved in DNA repair and the ubiquitin system and potential candidates involved in high-altitude adaptation. Notably, we detected a single base mutation causing loss-of-function of the FLOWERING LOCUS C protein, responsible for the transition to early flowering in high-elevation populations.ConclusionsOur results provide a genome-wide perspective of how plants adapt to distinct environmental conditions across extreme elevation differences and the potential for further follow-up research with extensive data from additional populations and species.

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Analyzing Mass Spectrometry Imaging Data of ¹³C-Labeled Phospholipids in Camelina sativa and Thlaspi arvense (Pennycress) Embryos.

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Analyzing Mass Spectrometry Imaging Data of 13C-Labeled Phospholipids in Camelina sativa and Thlaspi arvense (Pennycress) Embryos.

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Analyzing Mass Spectrometry Imaging Data of ¹³C-Labeled Phospholipids in Camelina sativa and Thlaspi arvense (Pennycress) Embryos.