Elevated Temperature Effects on Carotenoid Biosynthesis in the Diploid Strawberry, Fragaria vesca

Carotenoids, a subfamily of the isoprenoids, are one of the most diverse classes of secondary metabolites distributed throughout nature. They are lipophilic in nature, and include over 600 tetraterpenoid compounds synthesized by plants, bacteria, and fungi. Carotenoids, as the major pigment responsible for the red, yellow, and orange colors of fruits and vegetable, help promote human health and wellness by serving as antioxidants and precursors to vitamin A. Climate changes that threaten plant reproduction, negatively impact crop production worldwide. Little is understood about the chemistry of carotenoids in plant reproductive structures. Insight into the metabolic roles and functions of carotenoids in plant reproduction and, the effects of abiotic stresses on carotenoid biosynthesis in these structures would globally impact agriculture production by reducing yield loss. The potential for these metabolites to protect the reproductive structures under elevated temperature stress was assessed using biochemical analysis, genomics, and genetic studies. Fourteen candidate genes involved in carotenoid biosynthesis were identified, revealing three small gene families. Quantitative real-time polymerase chain reaction (qPCR) expression analysis of these genes and targeted metabolic profiling using liquid chromatography-high resolution mass spectrometry (LC-HRMS) throughout plant development under control and moderately elevated temperature stress showed that gene expression and metabolite accumulation are tissue specific and differentially responsive to elevated temperature stress. Three phytoene synthase genes were identified and characterized. Genomic analyses revealed that the PSY gene family exhibits functional diversity in plant tissues, both with respect to location and stage of development, as well as in response to abiotic stress

Growth of wildtype and mutant E. coli strains in minimal media for optimal production of nucleic acids for preparing labeled nucleotides

Since RNAs lie at the center of most cellular processes, there is a need for synthesizing large amounts of RNAs made from stable isotope-labeled nucleotides to advance the study of their structure and dynamics by nuclear magnetic resonance (NMR) spectroscopy. A particularly effective means of obtaining labeled nucleotides is to harvest these nucleotides from bacteria grown in defined minimal media supplemented with 15NH4Cl and various carbon sources. Given the high cost of carbon precursors required for labeling nucleic acids for NMR studies, it becomes important to evaluate the optimal growth for commonly used strains under standard minimal media conditions. Such information is lacking. In this study, we characterize the growth for Escherichia coli strains K12, K10zwf, and DL323 in three minimal media with isotopic-labeled carbon sources of acetate, glycerol, and glycerol combined with formate. Of the three media, the LeMaster-Richards and the Studier media outperform the commonly used M9 media and both support optimal growth of E. coli for the production of nucleotides. However, the growth of all three E. coli strains in acetate is reduced almost twofold compared to growth in glycerol. Analysis of the metabolic pathway and previous gene array studies help to explain this differential growth in glycerol and acetate. These studies should benefit efforts to make selective 13C-15N isotopic-labeled nucleotides for synthesizing biologically important RNAs

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems