Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant.

Ling-Xia Guo,Xiao Liu,Yong-Zhong Liu,Huan Yang,Long-Fei Jing,Cai-Yun Shi,Dong-Yuan Ning

doi:10.1038/srep29343

Abstract

‘Hong Anliu’ (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of ‘Anliu’ (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between ‘HAL’ and ‘AL’. Compared with ‘AL’, the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in ‘HAL’. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H+-ATPase, vacuolar H+-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in ‘HAL’. These results implied that ‘HAL’ has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with ‘AL’. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in ‘HAL’.

Highlights

The TCA cycle begins with the condensation of acetyl-CoA and OAA to form citrate catalyzed by citrate synthase (CS, EC2.3.3.1)
The products of degrading citrate relate to the biosynthesis of γ-aminobutyric acid (GABA)[12], isoprenoids, flavonoids and fatty acid extension[13,14]
Transcript analysis confirmed that the H+/citrate symporter CsCit[125], the cytosolic Aco, cyt-IDH or NADP-IDH, glutamate decarboxylase (GAD, EC 4.1.1.15), and ACL participate in citrate catabolism as the fruit ripens[12,16,26,27,28,29,30,31]

Summary

Introduction

The TCA cycle begins with the condensation of acetyl-CoA and OAA to form citrate catalyzed by citrate synthase (CS, EC2.3.3.1). In the purine or pyrimidine nucleosides and analogues groups, all identified metabolites, including guanosine, deoxyinosine, uridine, uracil, and hypoxanthine, had significantly higher levels in ‘HAL’ compared with ‘AL’. The enzyme activity analysis showed that the activities of myt-IDH (Fig. 3b1) and cyt-IDH (Fig. 3c1) were both significantly higher in ‘HAL’ than ‘AL’.

Results

Conclusion