A General Protocol for Accurate Gene Expression Analysis in Plants

Keyser, Ellen De; Desmet, Laurence; Losschaert, Magali; Riek, Jan De

doi:10.1007/978-1-4939-9833-3_9

Cited by 2 publications

(2 citation statements)

References 15 publications

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“…In addition, the expression of several known defense related genes in lettuce and DEGs selected from RNA-Seq analysis were studied by RT-qPCR (described in detail in Supplementary Text S1 ), using the method described previously ( De Keyser et al., 2020 ).…”

Section: Methodsmentioning

confidence: 99%

Assessment of transcriptional reprogramming of lettuce roots in response to chitin soil amendment

Kaufmann

Makechemu

et al. 2023

Front. Plant Sci.

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Chitin soil amendment is known to improve soil quality, plant growth and stress resilience, but the underlying mechanisms are not well understood. In this study, we monitored chitin’s effect on lettuce physiology every two weeks through an eight-week growth period, analyzed the early transcriptional reprogramming and related metabolomic changes of lettuce, in response to crab chitin treatment in peat-based potting soil. In commercial growth conditions, chitin amendment still promoted lettuce growth, increased chlorophyll content, the number of leaves and crop head weight from week six. The flavonoid content in lettuce leaves was altered as well, showing an increase at week two but a decrease from week six. Transcriptomic analysis showed that over 300 genes in lettuce root were significantly differentially expressed after chitin soil treatment. Gene Ontology-term (GO) enrichment analysis revealed statistical overrepresentation of GO terms linked to photosynthesis, pigment metabolic process and phenylpropanoid metabolic process. Further analysis of the differentially expressed genes (DEGs) showed that the flavonoid pathway was mostly upregulated whereas the bifurcation of upstream phenylpropanoid pathway towards lignin biosynthesis was mostly downregulated. Metabolomic analysis revealed the upregulation of salicylic acid, chlorogenic acid, ferulic acid, and p-coumaric acid in chitin-treated lettuce seedlings. These phenolic compounds (PCs) mainly influence the phenylpropanoid biosynthesis pathway and may play important roles in plant defense reactions. Our results suggest that chitin soil amendments might activate induced resistance by priming lettuce plants and promote lettuce growth via transcriptional changes.

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Section: Methodsmentioning

confidence: 99%

Assessment of transcriptional reprogramming of lettuce roots in response to chitin soil amendment

Kaufmann

Makechemu

et al. 2023

Front. Plant Sci.

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“…RNA was extracted from 150 to 200 mg ground plant material, using the 3% CTAB RNA extraction protocol ( Luypaert et al., 2017 ). RNA extraction, quantification and RT-qPCR were performed as previously described ( De Keyser et al. (2020) .…”

Section: Methodsmentioning

confidence: 99%

Identification and characterization of CYP71 subclade cytochrome P450 enzymes involved in the biosynthesis of bitterness compounds in Cichorium intybus

et al. 2023

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Industrial chicory (Cichorium intybus var. sativum) and witloof (C. intybus var. foliosum) are crops with an important economic value, mainly cultivated for inulin production and as a leafy vegetable, respectively. Both crops are rich in nutritionally relevant specialized metabolites with beneficial effects for human health. However, their bitter taste, caused by the sesquiterpene lactones (SLs) produced in leaves and taproot, limits wider applications in the food industry. Changing the bitterness would thus create new opportunities with a great economic impact. Known genes encoding enzymes involved in the SL biosynthetic pathway are GERMACRENE A SYNTHASE (GAS), GERMACRENE A OXIDASE (GAO), COSTUNOLIDE SYNTHASE (COS) and KAUNIOLIDE SYNTHASE (KLS). In this study, we integrated genome and transcriptome mining to further unravel SL biosynthesis. We found that C. intybus SL biosynthesis is controlled by the phytohormone methyl jasmonate (MeJA). Gene family annotation and MeJA inducibility enabled the pinpointing of candidate genes related with the SL biosynthetic pathway. We specifically focused on members of subclade CYP71 of the cytochrome P450 family. We verified the biochemical activity of 14 C. intybus CYP71 enzymes transiently produced in Nicotiana benthamiana and identified several functional paralogs for each of the GAO, COS and KLS genes, pointing to redundancy in and robustness of the SL biosynthetic pathway. Gene functionality was further analyzed using CRISPR/Cas9 genome editing in C. intybus. Metabolite profiling of mutant C. intybus lines demonstrated a successful reduction in SL metabolite production. Together, this study increases our insights into the C. intybus SL biosynthetic pathway and paves the way for the engineering of C. intybus bitterness.

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A General Protocol for Accurate Gene Expression Analysis in Plants

Cited by 2 publications

References 15 publications

Assessment of transcriptional reprogramming of lettuce roots in response to chitin soil amendment

Assessment of transcriptional reprogramming of lettuce roots in response to chitin soil amendment

Identification and characterization of CYP71 subclade cytochrome P450 enzymes involved in the biosynthesis of bitterness compounds in Cichorium intybus

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