The SAUR41 subfamily of SMALL AUXIN UP RNA genes is abscisic acid inducible to modulate cell expansion and salt tolerance in Arabidopsis thaliana seedlings

Abstract

Background and aims: Most primary auxin response genes are classified into three families: AUX/IAA, GH3 and SAUR genes. Few studies have been conducted on Arabidopsis thaliana SAUR genes, possibly due to genetic redundancy among different subfamily members. Data mining on arabidopsis transcriptional profiles indicates that the SAUR41 subfamily members of SMALL AUXIN UP RNA genes are, strikingly, induced by an inhibitory phytohormone, abscisic acid (ABA). We aimed to reveal the physiological roles of arabidopsis SAUR41 subfamily genes containing SAUR40, SAUR41, SAUR71 and SAUR72.

Methods: Transcriptional responses of arabidopsis SAUR41 genes to phytohormones were determined by quantitative real-time PCR. Knock out of SAUR41 genes was carried out with the CRISPR/Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing technique. The saur41/40/71/72 quadruple mutants, SAUR41 overexpression lines and the wild type were subjected to ultrastructural observation, transcriptome analysis and physiological characterization.

Key results: Transcription of arabidopsis SAUR41 subfamily genes is activated by ABA but not by gibberellic acids and brassinosteroids. Quadruple mutations in saur41/40/71/72 led to reduced cell expansion/elongation in cotyledons and hypocotyls, opposite to the overexpression of SAUR41; however, an irregular arrangement of cell size and shape was observed in both cases. The quadruple mutants had increased transcription of calcium homeostasis/signalling genes in seedling shoots, and the SAUR41 overexpression lines had decreased transcription of iron homeostasis genes in roots and increased ABA biosynthesis in shoots. Notably, both the quadruple mutants and the SAUR41 overexpression lines were hypersensitive to salt stress during seedling establishment, whereas specific expression of SAUR41 under the ABA-responsive RD29A (Responsive to Desiccation 29A) promoter in the quadruple mutants rescued the inhibitory effect of salt stress.

Conclusions: The SAUR41 subfamily genes of arabidopsis are ABA inducible to modulate cell expansion, ion homeostasis and salt tolerance. Our work may provide new candidate genes for improvement of plant abiotic stress tolerance.

Keywords: Arabidopsis thaliana; SMALL AUXIN UP RNA genes; CRISPR/Cas9; abscisic acid; cell expansion; ion homeostasis; salt tolerance; transcription profiling.

Fig. 1.

Relative expression levels of SAUR41 genes in 5-day-old arabidopsis seedlings. (A) Relative expression levels of SAUR41 genes in seedling shoots. The transcript level of SAUR40 is set to 1.0. SAUR71 displays the highest expression level. (B) Relative expression levels of SAUR41 genes in seedling roots. The transcript level of SAUR40 is set to 1.0. SAUR41 is expressed in both shoots and roots, while SAUR72 is highly active in roots. Error bars represent the s.d. **P < 0.01, *P < 0.05, Student’s t-test.

Fig. 2.

The SAUR41 subfamily genes are induced by ABA treatment. (A) Transcriptional responses of SAUR41 genes and SAUR19 to NAA treatment in seedling shoots. Five-day-old seedlings were incubated in liquid medium containing 10 μm NAA for 0.5, 1.0, 2.0, 3.0 and 4.0 h. SAUR19 seems dramatically auxin responsive. (B) Transcriptional responses of SAUR41 genes and SAUR19 to NAA treatment in roots. (C) Transcriptional responses of SAUR41 genes and SAUR19 to ABA treatment in seedling shoots. Five-day-old seedlings were incubated in liquid medium containing 10 μm ABA for 0.5, 1.0, 2.0, 3.0 and 4.0 h. SAUR19 is repressed by ABA, unlike SAUR41 genes. (D) Transcriptional responses of SAUR41 genes and SAUR19 to ABA treatment in roots. Again, SAUR41 genes are induced by ABA but SAUR19 is repressed by ABA. (E) Transcriptional responses of SAUR41 genes and SAUR19 to GA treatment in seedling shoots. Five-day-old seedlings were incubated in liquid medium containing 10 μm GA for 0.5, 1.0, 2.0, 3.0 and 4.0 h. SAUR19 is slightly induced by GA while SAUR41 genes are not induced by GA. (F) Transcriptional responses of SAUR41 genes and SAUR19 to GA treatment in seedling roots. Each treatment contained three biological replicates. In all cases, the transcript levels of each gene without hormone treatment were set to 1.0. Error bars represent the s.d. **P < 0.01, *P < 0.05, Student’s t-test.

Fig. 3.

A set of single mutants for each SAUR41 family member was generated by CRISPR/Cas9 genomic editing. Protospacers (guide RNA targets) are marked in green and protospacer-adjacent motifs (PAMs) are marked in red. Heritable mutations were identified by Sanger sequencing.

Fig. 4.

Inactivation of SAUR41 subfamily genes leads to reduced cell expansion. (A) Seven-day-old light-grown seedlings of the wild type, the saur41 quadruple mutants saur41/40/71/72 and the SAUR41 overexpression lines OE-SAUR41. (B) Statistical comparison of cotyledon areas in 7-day-old seedlings of three genotypes. (C) Statistical comparison of hypocotyl length in 7-day-old seedlings. (D) Statistical comparison of root length in 7-day-old seedlings. (E) Statistical comparison of epidermal cell and cortical cell length in 7-day-old hypocotyls. The quadruple mutants saur41-1sauir40-1saur71-1saur72-1 and saur41-5sauir40-1saur71-2saur72-1 are abbreviated as saur41/40/71/72#1 and saur41/40/71/72#4, while the 35S::SAUR41-MYC overexpression lines are abbreviated as OE-SAUR41#1 and #2. Each treatment contained approx. 30 seedlings or cells and was replicated three times. Error bars represent the s.d. **P < 0.01, *P < 0.05, Student’s t-test.

Fig. 5.

Both inactivation of SAUR41 genes and overexpression of SAUR41 led to irregular arrangement of cell size and shape in endodermis and pericycles of arabidopsis hypocotyls. (A) Optical section of hypocotyls of 6-day-old wild-type (Col-0) seedlings. (B) Optical section of the quadruple mutants saur41/40/71/72. (C) Optical section of the SAUR41 overexpression OE-SAUR41 lines. (D) Enlargement of an optical section of the wild type. (E) Enlargement of an optical section of the quadruple mutant. (F) Enlargement of an optical section of the SAUR41 overexpression line. (G) TEM of the wild type. (H) TEM of the quadruple mutant. (I) TEM of the SAUR41 overexpression line. en, endodermis; pe, pericycle cells; ph, phloem.

Fig. 6.

Transcriptome profiling analysis of saur41 quadruple mutants and SAUR41 overexpression lines. (A–D) Venn diagrams illustrating differentially expressed genes in wild-type shoot vs. mutant shoot (A), wild-type root vs. mutant root (B), wild-type shoot vs. overexpression shoot (C) and wild-type root vs. overexpression root (D). Shoot samples had far more differentially expressed genes than root samples. (E–H) In 5-day-old seedlings, qRT-PCR validation of differentially expressed genes identified by transcriptome analysis. For each gene, the expression level in Col-0 was set to l.0. (E) In the shoots of saur41/40/71/72, a set of calcium homeostasis/signalling genes are upregulated. (F) A set of ABA signalling-related genes are differentially expressed in the shoos of the quadruple mutants. (G) Overexpression of SAUR41 decreases the transcription of iron homeostasis genes in roots. (H) Overexpression of SAUR41 increases the transcription of ABA biosynthesis/signalling genes in shoots. Each treatment contained three biological replicates. Error bars represent the s.d. **P < 0.01, *P < 0.05, Student’s t-test.

Fig. 7.

Measurement of metal ion and ABA contents in 7-day-old seedling shoots. (A) There are no difference in calcium content among Col-0, saur41/40/71/72 and OE-SAUR41. (B) The SAUR41 overexpression lines have the highest levels of iron. (C) The SAUR41 overexpression lines accumulate much more potassium. (D) The saur41/40/71/72 mutants contain the lowest levels of sodium. (E) The SAUR41 overexpression lines have 2-fold increased ABA contents as compared with the wild type. Each treatment contained three biological replicates. Error bars represent the s.d. **P < 0.01, *P < 0.05, Student’s t-test.

Fig. 8.

Both the quadruple mutants and the SAUR41 overexpression lines were hypersensitive to salt stress. (A) Seedling establishment and growth of the wild-type Col-0, quadruple mutants saur41/40/71/72, the SAUR41 overexpression OE-SAUR41 lines and the complementary lines Comp, grown on B5 media containing 100 or 150 mm NaCl for 7 d. The Comp lines were generated by specific expression of SAUR41-EGFP under the RD29A promoter (proRD29A::SAUR41-EGFP) in the quadruple mutant saur41/40/71/72#1. (B) Fresh weight of 7-day-old seedlings of four genotypes under salt stress. The labelled percentages are the rate of reduction of the fresh weight under salt stress. (C) Statistical comparison of percentage seedling establishment of four genotypes under salt stress. All the control treatments for the various lines had a 100 % seedling establishment rate. (D) qRT-PCR assay of the accumulation of SOS1 encoding an Na⁺/H⁺ antiporter and a salt tolerance determinant. Each treatment contained approx. 30 seedlings or approx. 90 seeds and was replicated three times. Error bars represent the s.d. **P < 0.01, *P < 0.05, Student’s t-test.

Fig. 9.

Overexpression of SAUR41 led to extensive rhizosphere acidification. Seedlings were grown on B5 medium with normal (A) or one-fifth (B) iron concentrations for 7 d and then transferred to B5 plates containing the pH sensor bromocresol purple (0.003 %) for 2 d. Extensive medium acidification was observed for the roots of OE-SAUR41, especially when the seedlings were from the one-fifth iron concentration medium. A pH colorimetric panel is illustrated.

Fig. 10.

A working model for the function of SAUR41 genes. ABA induces the expression of SAUR41 genes to fine-tune seedling establishment and salt tolerance by modulating cell expansion and ion homeostasis.