Objective This study aims to select the most suitable reference genes, which are stably expressed during the process from the opening to senescence and different tissues in blooming period of peony cut flowers, and to deeply analyze the function of ACS and ACO genes in ethylene-mediated process of peony cut flower opening to senescence.

Method (1) With cultivar ‘Taohuafeixue’ as materials, the variations of expression levels of 13 candidate internal reference genes at different stages from blooming to senescence and in different tissues of the full bloom stage in herbaceous peony cut flowers were analyzed by qRT-PCR. The expression stability of 13 candidate internal reference genes was assessed by ΔCt and 3 softwares including geNorm, NormFinder, and BestKeeper. The comprehensive rankings of expression stability of candidate internal reference genes were obtained by online RefFinder analysis. (2) Based on the selected double internal reference genes, the expression profile of ACS and ACO, key genes of ethylene biosynthesis were quantitatively analyzed at different stages from blooming to senescence and in different tissues of full bloom stage in herbaceous peony cut flowers.

Result (1) geNorm analysis showed that the expression stability of GAPDH and RAN3 at different stages was best. NormFinder analysis indicated that the expression stability of RAN3 was optimal. BestKeeper analysis illustrated that 18S rRNA expression was the most stable. RefFinder analysis indicated that RAN3 and GAPDH were the best suitable internal reference genes at different stages. (2) geNorm analysis indicated that the expression of ARF and PP2A in different tissues had the best expression stability. NormFinder analysis showed that the expression of UBQ was the most stable. BestKeeper analysis illustrated that GAPDH was best. RefFinder analysis indicated that UBQ and ARF were the best internal reference genes in different tissues in herbaceous peony cut flowers. (3) From opening to senescence process of peony cut flowers, the expression of ACS gene was significantly lower than that of ACO gene, and ACS was the ‘switch’ for the regulation of ethylene synthesis. ACO mainly played a role in pre-opening stage of cut flowers (0−12 h). ACS expression played an important role both in the bloom stage and in the senescence stage (6 h and 144 h), and played a limiting role in ethylene synthesis during the bloom stage. (4) ACS expression was significantly lower than ACO in different tissues of peony cut flowers during blooming period, there was tissue variability in the expression of ACS and ACO, and both of them were higher in stems and calyxes.

Conclusion This study screens double internal reference gene combinations for qPCR quantitative expression analysis in different tissues of herbaceous peony cut flowers at varied periods from opening to senescence (RAN3, GAPDH) and at full bloom (UBQ, ARF). ACO gene, a key gene of ethylene biosynthesis mainly plays a role in the blooming process, and ACS plays an important role in both blooming and senescence process in herbaceous peony cut flowers. The finding will provide a reference for the improvement of cut flower longevity.