AtGH3.17-mediated Regulation of Auxin and Brassinosteroid Response in Arabidopsis thaliana

doi:10.11983/CBB22063

Abstract

Abstract: Brassinosteroid and auxin play important roles in plant growth and development. There is a complex crosstalk between the two plant hormones. In order to study whether GH3.17 is involved in the crosstalk between auxin and brassinosteroid (BR), we created overexpression and CRISPR gene loss mutants of Arabidopsis GH3.17. The root length of GH3.17 CRISPR mutants was slightly longer than that of wild-type plants. However, the GH3.17 overexpression seedlings had typical auxin deficiency phenotypes with decreased height, curly leaves and shorten roots and petioles. The root length of GH3.17 overexpression seedlings was substantially increased by the application of low concentrations of indole acetic acid (IAA) or brassinolide (BL), and the overexpression plants were more sensitive to IAA and BR than the wild type plants. We also found that the expression of auxin signaling, and BR synthesis pathway-related genes IAA12 and IAA16 was inhibited in the overexpression plants, while the expression abundance of brassinosteroid biosynthetic genes DWF4 and CPD was increased, suggesting that the inhibition of auxin signal transduction caused by the deficiency of active auxin may be compensated by BR compounds accumulation.

Key words: auxin, brassinosteroid, early auxin-response gene, GH3.17

Shuyao Zhou, Jianming Li, Juan Mao. AtGH3.17-mediated Regulation of Auxin and Brassinosteroid Response in Arabidopsis thaliana[J]. Chinese Bulletin of Botany, 2023, 58(3): 373-384.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22063

https://www.chinbullbotany.com/EN/Y2023/V58/I3/373

Figures/Tables 7

Table 1 Primers used in this study

Primer name	Primer sequence (5′-3′)
pBRI1:gGH3.17-F	CAAACTCTTGAGAAGGTACCATGATA- CCAAGTTACGACCC
pBRI1:gGH3.17-R	GTGTCGACTCTAGAGGATCCAGAAT- CTAAACCAAGTGGTT
GH3.17-DT1-BsF	ATATATGGTCTCGATTGGATCTCATGTCCTAAGAGTGTTTTAGAGCTAGAAATAGC
GH3.17-DT1-BsR	ATTATTGGTCTCGAAACGCAGAGACTCGGTCTTGTCCAATCTCTTAGTCGACTCTAC
U6-26p-F	TGTCCCAGGATTAGAATGATTAGGC
U6-26p-R	AGCCCTCTTCTTTCGATCCATCAAC
CAS 9-F	ATCCAATCTTCGGCAACAT
CAS 9-R	TATCCAGGTCATCGTCGTA
GH3.17-ba-F	AGATGAGGGAAAAGGGATGT
GH3.17-ba-R	TCAAGATTCCTTCCCACGAC
GH3.17-qPCR-F	CGATGTATGCTTCCTCTGAGTG
GH3.17-qPCR-R	ATCTCTTCGTGGGATTTGTCG
IAA12-qPCR-F	TGGGTTACACAGGATGAACAG
IAA12-qPCR-R	AACCCTAAGCCCTGAACTTTC
IAA16-qPCR-F	TGAAGATAAAGATGGCGACTGG
IAA16-qPCR-R	AAGTCCGATTGCTTCTGATCC
IAA20-qPCR-F	GTACTCGAAACCTAAGCACGG
IAA20-qPCR-R	CACATATTCCGCATCCTCTACC
DWF4-qPCR-F	CATTGCTCTCGCTATCTTCTTC
DWF4-qPCR-R	GACTCTCCTAGTTCCTTCTTGG
CPD-qPCR-F	TCCTTGTGGGTCTAGTGTTTG
CPD-qPCR-R	TTGAACCATTGAAGCAGAAGAG
BES1-qPCR-F	CAGCCATTCTCTGCCTCTATG
BES1-qPCR-R	ACTCGGAGCTTTGACCAATC
Actin2-qPCR-F	GGTAACATTGTGCTCAGTGGTGG
Actin2-qPCR-R	AACGACCTTAATCTTCATGCTGC

Figure 1 Tissue-specific expression of GH3.17 in Arabidopsis thaliana Error bars denote SEM. *** indicate significant differences at P<0.001 (Student’s t-test).

Figure 2 Phenotype of Arabidopsis thaliana wild type (WT), GH3.17 CRISPR mutant and overexpression seedlings (A) Generation of knockout alleles of GH3.17 (gh3.17-10-13 and gh3.17-4-9) by CRISPR/Cas9 (exons are represented by dark green boxes, introns by black lines and 5' and 3' untranslated region (UTR) by light green boxes; the mutation site and sequence are presented in the figure); (B) The aerial part phenotype of 20-day-old soil-grown WT, GH3.17 CRISPR mutant and overexpression lines under normal conditions (bar=1 cm); (C) Petiole length statistics of the first pair of true leaves of WT and GH3.17 CRISPR mutant (n=20); (D) Statistics of petiole length of the second pair of true leaves of WT and GH3.17 CRISPR mutant (n=20); (E), (G) The primary root length phenotype and statistics of 7-day-old WT, GH3.17 CRISPR mutant and overexpression seedlings vertically grown on 1/2MS medium under normal conditions (bar=1 cm) (the data was obtained through three replicates of 30 seedlings each); (F), (H) The hypocotyls phenotype and statistics of 5-day-old dark-grown WT, GH3.17 CRISPR mutant and overexpression seedlings grown on 1/2MS medium under normal conditions (bar=1 cm) (the data was obtained through three replicates of 30 seedlings each); (I) The above-ground phenotypes of 50-day-old soil-grown WT, GH3.17 CRISPR mutant and overexpression lines under normal conditions (bar=5 cm); (J) qRT-PCR analysis of GH3.17 transcriptional level in WT and GH3.17 overexpression plants; (K) Immunoblot analysis of GH3.17-Flag fusion protein in GH3.17 overexpression transgenic lines. Error bars in (C), (D), (G) and (H) denote SD; error bars in (J) denote SEM. ** indicate significant differences at P<0.01; *** indicate significant differences at P<0.001 (Student’s t-test).

Figure 3 The IAA-Glu and oxIAA content of Arabidopsis tha- liana wild type (WT), GH3.17 overexpression and CRISPR mutant (A) The IAA-Glu content of WT, GH3.17 overexpression and CRISPR mutant; (B) The oxIAA content of WT, GH3.17 overexpression and CRISPR mutant. IAA level in 12-day-old Arabidopsis thaliana seedlings grown on 1/2MS solid medium is obtained from extraction of three independent tissue samples. Error bars indicate SD. * indicates significant difference at P<0.05; *** indicate significant differences at P<0.001 (Student’s t-test).

Figure 4 Response of Arabidopsis thaliana wild type (WT), GH3.17 CRISPR mutant and overexpression seedlings to auxin (A) The relative expression level of IAA12, IAA16 and IAA20 in WT and OEGH3.17-1-2 overexpression plants (error bars represent SEM of three technical duplicates); (B) The root phenotype of WT, GH3.17 CRISPR mutant and overexpression seedling on 1/2MS medium with different concentrations of IAA for 7 days (bars=1 cm); (C) The statistical analysis of WT, GH3.17 CRISPR mutant and overexpression seedling root length grown on 1/2MS medium with different concentrations of IAA for 7 days (the data was obtained through three replicates of 30 seedlings each, error bars represent SD); (D) Root length curves of WT, GH3.17 CRISPR mutant and overexpression seedling showing their response to different concentrations of IAA (the data was obtained through three replicates of 30 seedlings each, error bars represent SD). * indicate significant differences at P<0.05; *** indicate significant differences at P<0.001 (Student’s t-test).

Figure 5 The transcription level of Arabidopsis thaliana GH3.17 was inhibited by brassinolide (BL) treatment Error bars represent SEM of three technical duplicates. * indicates significant difference at P<0.05; ** indicates significant difference at P<0.01 (Student’s t-test). WT: Wild type

Figure 6 Response of Arabidopsis thaliana wild type (WT), GH3.17 overexpression and CRISPR mutant seedlings to brassinosteroid (BR) (A) The relative expression level of DWF4, CPD and BES1 in WT and OEGH3.17-1-2 overexpression plants by qRT-PCR (error bars represent SEM of three technical duplicates); (B) The root phenotype of WT, OEGH3.17-1-2 and gh3.17-10-13 grown on 1/2MS solid medium with different concentrations of brassinolide (BL) for 9 days (bars=1 cm); (C) The statistical analysis of WT, OEGH3.17-1-2 and gh3.17-10-13 root length grown on 1/2MS solid medium with different concentrations of BL for 9 days (the data was obtained through three replicates of 30 seedlings each, error bars represent SD); (D) Root length curves of WT, OEGH3.17-1-2 and gh3.17-10-13 showing their response to different concentrations of BL (the data was obtained through three replicates of 30 seedlings each, error bars represent SD). * indicate significant differences at P<0.05; ** indicate significant differences at P<0.01; *** indicate significant differences at P<0.001 (Student’s t-test).

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