Abscisic acid metabolic genes of wheat (Triticum aestivum L.): identification and insights into their functionality in seed dormancy and dehydration tolerance

Abstract

The three homeologues of wheat NCED2 were identified; the wheat NCED2A and CYP707A1B affect seed ABA level and dormancy but not leaf ABA level and transpirational water loss in Arabidopsis. Biosynthesis and catabolism of abscisic acid (ABA) in plants are primarily regulated by 9-cis-epoxycarotenoid dioxygenases (NCEDs) and ABA 8'-hydroxylase (ABA8'OH), respectively. The present study identified the complete coding sequences of a second NCED gene, designated as TaNCED2, and its homeologues (TaNCED2A, TaNCED2B and TaNCED2D) in hexaploid wheat, and characterized its functionality in seed dormancy and leaf dehydration tolerance using the TaNCED2A homeologue. The study also investigated the role of the B genome copy of the cytochrome P450 monooxygenase 707A1 (CYP707A1) gene of hexaploid wheat (TaCYP707A1B), which encodes ABA8'OH, in regulating the two traits as this has not been studied before. Ectopic expression of TaNCED2A and TaCYP707A1B in Arabidopsis resulted in altered seed ABA level and dormancy with no effect on leaf ABA content and transpirational water loss. To gain insights into the physiological roles of TaNCED2 and TaCYP707A1 in wheat, the study examined their spatiotemporal expression patterns and determined the genomic contributions of transcripts to their total expression.

A Simple and Efficient Approach to Elucidate Genomic Contribution of Transcripts to a Target Gene in Polyploids: The Case of Hexaploid Wheat (Triticum aestivum L.)

“…The full length cDNA sequences of the three homeologs for the target genes of hexaploid wheat, the ABA biosynthetic gene, 9-cis-epoxycarotenoid dioxygenases ( NCED ) ( TaNCED2 ; TaNCED2A, TaNCED2B , and TaNCED2D ), and the ABA catabolic gene, cytochrome P450 monooxygenase 707A1 ( CYP707A1 ) ( TaCYP707A1 ; TaCYP707A1A, TaCYP707A1B , and TaCYP707A1D ), were identified as described previously (Chono et al, 2013; Son et al, 2016). The total expression of the TaNCED2 and TaCYP707A1 in the flag leaf, peduncle, and internode tissues of two hexaploid wheat genotypes used as experimental plant materials in this study, AC Domain and RL4452, was determined using real time quantitative polymerase chain reaction (qPCR) (Figures 1A, 2A), and gene specific primers designed from the coding regions conserved across the three homeologs of each target gene (Table 1).…”

“…This study involved the use of the three homeologs of ABA biosynthetic [ TaNCED2 ( TaNCED2A , GenBank ID: KX711890; TaNCED2B , GenBank ID: KX711891; TaNCED2B , GenBank ID: KX711892; Son et al, 2016)] and ABA catabolic [ TaCYP707A1 ( TaCYP707A1A , GenBank ID: AB714577; TaCYP707A1B , GenBank ID: AB714578; TaCYP707A1D , GenBank ID: AB714579; Chono et al, 2013)] genes of hexaploid wheat as target genes. The forward and reverse primer sequences that can amplify fragments conserved across all three homeologs of each target gene and thereby detect their collective expression are described previously (Son et al, 2016; Table 1).…”

“…The forward and reverse primer sequences that can amplify fragments conserved across all three homeologs of each target gene and thereby detect their collective expression are described previously (Son et al, 2016; Table 1). The wheat β -actin ( Ta β -actin ) gene was used as reference gene for expression analysis of the target genes and it was amplified using primers described before (Son et al, 2016; Table 1). The real time qPCR assays for the target and reference genes were performed as described before (Nguyen et al, 2016).…”

“…The forward and reverse primers spanning regions that are polymorphic across the three homeologs of each target gene ( TaNCED2 and TaCYP707A1 ) and thereby amplify fragments unique to each homeolog of the target genes are described previously (Son et al, 2016), and these primers were used to perform the PCR assays. The PCR reactions for TaNCED2 consists of 5 μl of cDNA (50 ng), 5 μl of 5x high GC enhancer, 5 μl of 5x Q5 buffer, 0.5 μl of 10 mM dNTPs, 1 μl of forward primer (5 mM), 0.5 μl reverse primer (5 mM), 0.25 μl of Q5 high fidelity DNA polymerase (2000 U; New England BioLabs, Ipswich, MA, USA), and nuclease-free water to make a total reaction volume of 25 μl.…”

“…Elucidating the contribution of each homeolog or genome to the total expression of a given target gene has a paramount significance for detailed dissection of the molecular mechanisms underlying the regulation of the associated trait of economic importance. Here, we report a strategy that is able to determine genomic contributions to the total expression of individual target genes in hexaploid wheat (Deol et al, 2013; Mukherjee et al, 2015; Son et al, 2016) using the homeologs of two recently reported genes encoding enzymes that catalyze specific steps in the metabolic pathway of abscisic acid (ABA) (Chono et al, 2013; Son et al, 2016), a plant hormone implicated in regulating several traits of agronomic importance including seed dormancy (Gao and Ayele, 2014). …”