Changes of Small GTPases Activity During Cucumber Tendril Winding

doi:10.11983/CBB22058

Abstract

Abstract: Tendrils of climbing plants are highly sensitive to mechanical stimuli. Small GTPases signals can be activated by mechanical stimulation which regulate development in Arabidopsis, but whether small GTPases signals play a role in the entanglement of tendrils response to mechanical stimuli is unknown. In this study, the activity of small GTPases in cucumber tendril after mechanical stimulation was investigated using Cucumis sativus, and ROP activity detection results showed that the activity of small GTPases CsROP6 was significantly enhanced during tendril winding. Meanwhile, mass spectrometry revealed that auxin content increased significantly during tendril winding. Further experiments showed that exogenous auxin treatment increased the activity of CsROP6, suggesting mechanical stimulation might activate ROP6 activity through auxin during tendril winding. This study suggests that mechanical stimulation of small GTPases signals may be conserved in different plants.

Key words: auxin, cucumber tendrils, mechanical signal, small GTPases

Binqi Li, Jiahui Yan, Hao Li, Wei Xin, Yunhe Tian, Zhenbiao Yang, Wenxin Tang. Changes of Small GTPases Activity During Cucumber Tendril Winding[J]. Chinese Bulletin of Botany, 2022, 57(3): 299-307.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22058

https://www.chinbullbotany.com/EN/Y2022/V57/I3/299

Figures/Tables 7

Figure 1 Mechanical contact response of cucumber tendril (A) Natural winding: the white arrows show the winding process of the tendril after touching the stick; 20:00, 20:30, 21:00, and 22:00 indicate the time points of the observation day (bars=3 cm); (B) Winding after manual touching: after 30 seconds’ (30 touches) touch with a wooden stick, tendrils were photographed and recorded every 30 seconds, and tendrils were curled at about 180° at 3 minutes; white arrow indicates the stick touch point (bars=2 cm).

Figure 2 Evolutionary analysis and sequence alignment of ROP family members in cucumber (A) The phylogenetic tree of ROPs in cucumber and Arabidopsis thaliana; (B) Comparison of amino acid sequences of ROP6 between cucumber and Arabidopsis thaliana by DNAMAN.

Table 1 Primer sequences for RT-PCR in cucumber

Primer name	Forward primer (5′→3′)	Reverse primer (5′→3′)
Cucsa.322750	CAAATGTAGTTGTGAATGGGAGC	TTCCAACCAGAACAATAGGCACT
Cucsa.393880	TTTCCCACGGACTACGTGCCTAC	AACTGCTTGTCATCCCGAAGATC
Cucsa.234270	AGGCTTGTGGGATACTGCTGGTC	CTTCTTTCGCTTCTTCCGCTTTG
Cucsa.309440	TTCAGTGCTAATGTGGTGGTGGA	GATGGCTAGTCAGAAATTGCTTGTCGT
Cucsa.046650	TCAATCTTGGCTTATGGGACACT	GGAGCTGCATTCTATGTATGTAATT
Cucsa.197080	TTCGTTAGTTAGCCGAGCGAGTT	TGATTTCACATTCTGCTGGGTTT
Cucsa.256740	TGGTTAATGGGCAGTCTGTGAAT	TGGTCCCTACAAGAATAATGGGT
Cucsa.332390	TGTGGCTTTCTCGTTAATCAGCA	GTTCCTCCTCTTCCTTGGCATCT
Cucsa.116450	TACTGTTGGAGATGGAGCGGTTGG	TTAGCATATCGACGGTCCTCACG
Cucsa.322750	CAAATGTAGTTGTGAATGGGAGC	TTCCAACCAGAACAATAGGCACT

Figure 3 Tissue expression pattern of ROPs gene in cucumber (A) Detect the expression abundance of ROPs in cucumber tendrils, stems, leaves, fruits and flowers by RT-PCR; (B) Detect the expression of ROPs in cucumber tendrils by qRT-PCR (n=4).

Figure 4 Changes of ROP activity during cucumber tendril winding 0C, 1C and 2C represent 0, 1 and 2 cycle of winding, respectively. n=5; significance analysis was performed by Student’s t test. **P<0.01

Figure 5 Auxin treatment significantly enhanced mechanical stimulation sensitivity of cucumber tendrils (A) Determination of auxin content before and after tendril winding by liquid mass spectrometry (0C, 0.5C, 1C, 2C represent 0, 0.5, 1 and 2 cycles of winding, respectively); (B), (C) The effects of auxin on mechanical sensitivity of cucumber tendril (bars=3 cm); n=10; significance analysis was performed using One-way ANOVA; *P<0.05; **P<0.01; NS: No significant difference

Figure 6 Auxin induces the increase of ROP6 activity in cucumber tendrils n=5; significance analysis was performed by Student’s t test. **P<0.01

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