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Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data.

ABSTRACT: Tiger nut (Cyperus esculentus), a perennial C₄ plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

SUBMITTER: Bai X

PROVIDER: S-EPMC7961719 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCyperus esculentus is unique in that it can accumulate significant amounts of oil, starch and sugar as major storage reserves in tubers with high tuber yield and therefore considered as a novel model to study carbon allocation into different storage reserves in underground sink tissues such as tubers and roots. Sucrose (Suc) plays a central role in control of carbon flux toward biosynthesis of different storage reserves; however, it remains unclear for the molecular mechanism underlying Suc metabolism in underground oil-rich storage tissues. In the present study, a comprehensive transcriptome analysis of C. esculentus oil tuber compared to other plant oil- or carbohydrate-rich storage tissues was made for the expression patterns of genes related to the Suc metabolism.ResultsThe results revealed some species-specific features of gene transcripts in oil tuber of C. esculentus, indicating that: (i) the expressions of genes responsible for Suc metabolism are developmentally regulated and displayed a pattern dissimilar to other plant storage tissues; (ii) both of Suc breakdown and biosynthesis processes might be the major pathways associated with Suc metabolism; (iii) it was probably that Suc degradation could be primarily through the action of Suc synthase (SUS) other than invertase (INV) during tuber development. The orthologs of SUS1, SUS3 and SUS4 are the main SUS isoforms catalyzing Suc breakdown while the vacuolar INV (VIN) is the leading determinant controlling sugar composition; (iv) cytosolic hexose phosphorylation possibly relies more on fructose as substrate and uridine diphosphate glucose pyrophosphorylase (UGP) plays an important role in this pathway; (v) it is Suc-phosphate synthase (SPS) B- and C-family members rather than SPS A that are the principal contributors to SPS enzymes and play crucial roles in Suc biosynthesis pathway.ConclusionsWe have successfully identified the Suc metabolic pathways in C. esculentus tubers, highlighting several conserved and distinct expressions that might contribute to sugar accumulation in this unique underground storage tissue. The specific and differential expression genes revealed in this study might indicate the special molecular mechanism and transcriptional regulation of Suc metabolism occurred in oil tubers of C. esculentus.

Project description:BackgroundEngineering triacylglycerol (TAG) accumulation in vegetative tissues of non-food crops has become a promising way to meet our increasing demand for plant oils, especially the renewable production of biofuels. The most important target modified in this regard is diacylglycerol acyltransferase (DGAT) enzyme responsible for the final rate-limiting step in TAG biosynthesis. Cyperus esculentus is a unique plant largely accumulating oleic acid-enriched oil in its underground tubers. We speculated that DGAT derived from such oil-rich tubers could function more efficiently than that from oleaginous seeds in enhancing oil storage in vegetative tissues of tobacco, a high-yielding biomass crops.ResultsThree CeDGAT genes namely CeDGAT1, CeDGAT2-1 and CeDGAT2-2 were identified in C. esculentus by mining transcriptome of developing tubers. These CeDGATs were expressed in tissues tested, with CeDGAT1 highly in roots, CeDGAT2-1 abundantly in leaves, and CeDGAT2-2 predominantly in tubers. Notably, CeDGAT2-2 expression pattern was in accordance with oil dynamic accumulation during tuber development. Overexpression of CeDGAT2-2 functionally restored TAG biosynthesis in TAG-deficient yeast mutant H1246. Oleic acid level was significantly increased in CeDGAT2-2 transgenic yeast compared to the wild-type yeast and ScDGA1-expressed control under culture with and without feeding of exogenous fatty acids. Overexpressing CeDGAT2-2 in tobacco led to dramatic enhancements of leafy oil by 7.15- and 1.7-fold more compared to the wild-type control and plants expressing Arabidopsis seed-derived AtDGAT1. A substantial change in fatty acid composition was detected in leaves, with increase of oleic acid from 5.1% in the wild type to 31.33% in CeDGAT2-2-expressed tobacco and accompanied reduction of saturated fatty acids. Moreover, the elevated accumulation of oleic acid-enriched TAG in transgenic tobacco exhibited no significantly negative impact on other agronomic traits such as photosynthesis, growth rates and seed germination except for small decline of starch content.ConclusionsThe present data indicate that CeDGAT2-2 has a high enzyme activity to catalyze formation of TAG and a strong specificity for oleic acid-containing substrates, providing new insights into understanding oil biosynthesis mechanism in plant vegetative tissues. Overexpression of CeDGAT2-2 alone can significantly increase oleic acid-enriched oil accumulation in tobacco leaves without negative impact on other agronomy traits, showing CeDGAT2-2 as the desirable target gene in metabolic engineering to enrich oil and value-added lipids in high-biomass plants for commercial production of biofuel oils.

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Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data.

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