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. 2019 May 1:6:51.
doi: 10.1038/s41438-019-0133-7. eCollection 2019.

Transcriptomic analysis of interstock-induced dwarfism in Sweet Persimmon (Diospyros kaki Thunb.)

Yanying Shen  1   2 , Weibing Zhuang  3 , Xutong Tu  1 , Zhihong Gao  1 , Aisheng Xiong  1 , Xinyi Yu  1 , Xuehan Li  1 , Feihong Li  1 , Shenchun Qu  1   2
Affiliations

Transcriptomic analysis of interstock-induced dwarfism in Sweet Persimmon (Diospyros kaki Thunb.)

Yanying Shen et al. Hortic Res. .

Abstract

Growth monitoring indicated that the height of 'Kanshu' plants with 'Nantong-xiaofangshi' as an interstock was significantly shorter than that of 'Kanshu' plants with no interstock. A transcriptome analysis of the two graft combinations ('Kanshu'/Diospyros lotus and 'Kanshu'/'Nantong-xiaofangshi'/Diospyros lotus) was conducted to explore the dwarfing genes related to the use of the 'Nantong-xiaofangshi' interstock. Hormone levels and water conductance were also measured in these two graft combinations. The results indicated that the levels of both IAA and GA were lower in 'Kanshu' that had been grafted onto the 'Nantong-xiaofangshi' interstock than in 'Kanshu' with no interstock; additionally, the water conductance was lower in grafts with interstocks than in grafts without interstocks. The expression of AUX/IAA and auxin-responsive GH3 genes was enhanced in scions grafted on the interstock and was negatively correlated with the IAA content and growth of scions. The expression of GA2ox, DELLA, and SPINDLY genes were also upregulated and associated with a decrease in the level of GA in scions grafted on the interstock. Since one of the GA2ox unigenes was annotated as DkGA2ox1 in Diospyros kaki, but was not functionally validated, a functional analysis was conducted in transgenic tobacco. Overexpression of DkGA2ox1 in transgenic plants resulted in a dwarf phenotype that could be recovered by the exogenous application of GA3. We conclude that the 'Nantong-xiaofangshi' interstock affects the water conductance and expression of genes related to the metabolism and transduction of IAA and GA in the grafted scion and thus regulates phytohormone levels, producing dwarfing.

Keywords: Plant development; Plant hormones; RNA sequencing.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Stem tissues of the two graft combinations prepared for transcriptomic sequencing and analyses.
KS1, Nx, and DL1: scion, interstock and rootstock in the combination of ‘Kanshu’/N.x/Diospyros lotus; KS2 and DL2: scion and rootstock in ‘Kanshu’/Diospyros lotus
Fig. 2
Fig. 2. Changes in the ‘Kanshu’ scion growth rate for different graft combinations.
Error bars indicate the standard error of five biological replicates. Asterisks indicate significant differences; one asterisk represents P < 0.05, and two asterisks represents P < 0.01
Fig. 3
Fig. 3. Phytohormone levels in different tissues of ‘Kanshu’ persimmon with different graft combinations.
a Indole-3-acetic acid (IAA), (b) gibberellin (GA4), and (c) abscisic acid (ABA). Ks1, Nx, and Dl1 represent ‘Kanshu’, ‘Nantong-xiaofangshi’, and Diospyros lotus, respectively, in ‘Kanshu’/‘Nantong-xiaofangshi’/Diospyros lotus grafting plants. Ks2 and Dl2 represent ‘Kanshu’ and Diospyros lotus, respectively, in ‘Kanshu’/Diospyros lotus grafting plants. Error bars: the standard error of three biological replicates. Different letters: significantly different at P < 0.01 by Duncan’s multiple range tests
Fig. 4
Fig. 4. RT-qPCR confirmation of 31 differentially expressed unigenes.
a Unigenes related to gibberellin (GA) metabolism and signal transduction. b Unigenes related to auxin (IAA) signaling. c Unigenes related to brassinosteroid (BR), NAC transcription factor, phytochrome-interacting factor (PIF), phenol-containing compound (PHC) and peroxidase (POD). Error bars indicate the standard error of three biological replicates. Note: double asterisk (**) values are significantly different (P < 0.01)
Fig. 5
Fig. 5. Phylogenetic analysis of DkGA2ox1.
A neighbor joining phylogenetic tree of TR54490|c1_g1_i1 (DkGA2ox1) produced with 1000 bootstrap replications. DkGA2ox1 is highlighted by a pale yellow box
Fig. 6
Fig. 6. Genetic transformation and functional analysis of DkGA2ox1 in tobacco.
a Plant height of tobacco. b Tobacco plants 90 days after planting. c Transgenic plants before GA3 treatment. d Reversal of normal phenotype of transgenic plants by GA3 treatment 30 days after transplanting. e GA content in transgenic and wild-type tobacco. f Expression of associated genes in transgenic and wild-type tobacco. WT: nontransgenic tobacco plant; 1–1,1–3: two independent transgenic lines of DkGA2ox1. Note: means with double asterisk (**) are significantly different (P < 0.01) by Duncan’s multiple range tests. Each expression analysis utilized three biological and technical replicates. Error bars indicate the standard error of three biological replicates
Fig. 7
Fig. 7. Cleaved amplified polymorphic sequence (CAPS) analysis of RT-PCR products of the persimmons.
a Fragment sizes predicted by ApaI digestion in ‘Nantong-xiaofangshi’, Diospyros lotus and ‘Kanshu’. b Polyacrylamide gel electrophoresis of restrictive digestion of different RT-PCR products. c Samples from an 82 cm tall grafted tree with ‘Kanshu’ as scion, ‘Nantong-xiaofangshi’ as interstock and Diospyros lotus as rootstock. d CAPS analysis on purified RT-PCR products of different tissues in ‘Kanshu’/‘Nantong-xiaofangshi’/Diospyros lotus. e Comparison of total DkGA2ox1/DLGA2ox1/KSGA2ox1 transcripts in ‘Kanshu’/‘Nantong-xiaofangshi’/Diospyros lotus. DkGA2ox1, DLGA2ox1 and KSGA2ox1 are the sequences in ‘Nantong-xiaofangshi’, Diospyros lotus and ‘Kanshu’, respectively; N.x: ‘Nantong-xiaofangshi’/Diospyros lotus, DL: Diospyros lotus, KS: ‘Kanshu’, M’: mixture of cDNA from N.x, DL and KS2, KS1: ‘Kanshu’/‘Nantong-xiaofangshi’/Diospyros lotus, KS2: ‘Kanshu’/Diospyros lotus, GU1: graft union of Diospyros lotus and ‘Nantong-xiaofangshi’, IN: ‘Nantong-xiaofangshi’ interstock, GU2: graft union of ‘Nantong-xiaofangshi’ and ‘Kanshu’, 1-3: ‘Kanshu’ scion every 10 cm from the GU2, 4: young leaves at the top of the scion. M: DNA ladder marker 500. Scale bars = 5 cm. Note: double asterisk (**) indicates a significant difference (P < 0.01). The purified PCR products were diluted to the same concentration for enzyme cutting
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