Inter-regulation of the unfolded protein response and auxin signaling

Abstract

The unfolded protein response (UPR) is a signaling network triggered by overload of protein-folding demand in the endoplasmic reticulum (ER), a condition termed ER stress. The UPR is critical for growth and development; nonetheless, connections between the UPR and other cellular regulatory processes remain largely unknown. Here, we identify a link between the UPR and the phytohormone auxin, a master regulator of plant physiology. We show that ER stress triggers down-regulation of auxin receptors and transporters in Arabidopsis thaliana. We also demonstrate that an Arabidopsis mutant of a conserved ER stress sensor IRE1 exhibits defects in the auxin response and levels. These data not only support that the plant IRE1 is required for auxin homeostasis, they also reveal a species-specific feature of IRE1 in multicellular eukaryotes. Furthermore, by establishing that UPR activation is reduced in mutants of ER-localized auxin transporters, including PIN5, we define a long-neglected biological significance of ER-based auxin regulation. We further examine the functional relationship of IRE1 and PIN5 by showing that an ire1 pin5 triple mutant enhances defects of UPR activation and auxin homeostasis in ire1 or pin5. Our results imply that the plant UPR has evolved a hormone-dependent strategy for coordinating ER function with physiological processes.

Keywords: Arabidopsis thaliana; IRE1; PIN5; auxin response; endoplasmic reticulum stress; unfolded protein response.

Figure 1. ER stress alters the expression of auxin regulators

(a) RT-qPCR analyses of TIR1, AFB1, AFB2, and AFB3 expression in ten-day-old Col-0 Arabidopsis seedlings after treatment with 5 μg/ml Tm for 0.5, 1, or 4 h. Error bars represent standard error of the mean (SEM) from three independent biological replicates. P-values were calculated by Student’s two-tailed t test against expression levels at 4 h relative to 0 h: TIR1 (P = 0.00036), AFB1 (P = 0.00041), AFB2 (P = 0.00048), AFB3 (P = 0.00032). (b) The levels of DII-VENUS fusion proteins increase upon exposure to ER stress. Ten-day-old DII-VENUS transgenic plants were treated with 5 μg/ml Tm or DMSO for 6 h. Proteins were extracted from root tissues and the fusion proteins were detected by immunoblot analysis using anti-GFP serum (upper panel). Coomassie blue staining gel used as loading control (lower panel). (c) Ten-day-old transgenic plants expressing DII-VENUS were treated with 5 μg/ml Tm or DMSO for 6 h. Primary root tips were subjected to confocal microscopy analyses. Scale bars = 50 μm. (d) PIN mRNA levels decrease upon exposure to ER stress. RT-qPCR analyses of PIN family transcripts in ten-day-old wild-type Col-0 seedlings during treatment with 5 μg/ml Tm for 0.5, 1, or 4 h. Error bars represent SEM from three independent biological replicates. P-values were calculated against expression levels at 4 h relative to 0 h: PIN1 (P = 0.00221), PIN2 (P = 0.00316), PIN3 (P = 5.4E-05), PIN4 (P = 4.9E-05), PIN5 (P = 6.4E-05), PIN6 (P = 0.00012), PIN7 (P = 0.00353). The transcriptional level of ETR1, an ER-associated ethylene receptor, was unchanged after treatment with Tm for 0.5, 1, or 4 h.

Figure 2. IRE1 and TIR1/AFBs play fine-tuning roles in ER stress-induced down-regulation of auxin regulators

(a) RT-qPCR analyses of TIR1, AFB1, AFB2, and AFB3 expression in ten-day-old atire1a atire1b (ire1) Arabidopsis seedlings after treatment with 5 μg/ml Tm for 0.5, 1, or 4 h. (b) PIN mRNA levels decrease upon exposure to ER stress in ten-day-old ire1 or tir1 afb1 afb2 afb3 (tir1 afb). RT-qPCR analyses of PIN family transcripts in ten-day-old ire1 or tir1 afb Arabidopsis seedlings after treatment with 5 μg/ml Tm for 0.5, 1, or 4 h. (c) Transcriptional repression of PINs after treatment with 5 μg/ml Tm for 4 h in ten-day-old Col-0, ire1, or tir1 afb Arabidopsis seedlings. Error bars represent SEM from three independent biological replicates.

Figure 3. atire1a atire1b (ire1) exhibits compromised auxin responses

(a–c) In ire1, root growth is largely resistant to treatments with auxin (NAA and IAA) or an auxin transport inhibitor (NPA). Relative primary root length of ten-day-old Col-0 and ire1 Arabidopsis seedlings grown in the presence 50, 100, 200 nM NAA (a), or 1, 2, 3, 4, 5 μM IAA (b), or 50, 100 nM NPA (c) compared to those grown in the absence of the chemicals. Error bars represent standard error of the mean (SEM), n > 30. Scale bars = 1 cm. P-values are relative to Col-0: 100, 200 nM NAA (P < 0.00078), 1, 2, 3, 4, 5 μM IAA (P < 0.00050), 50, 100 nM NPA (P < 0.00344). (d) RT-qPCR analyses of IAA5 and GH3.6 expression in ten-day-old Col-0 and ire1 Arabidopsis seedlings after a 2- or 4-h treatment with 10 μM NAA. Error bars represent SEM from three independent biological replicates. P-values are relative to Col-0: IAA5 (P < 0.00016), GH3.6 (P < 0.00391). (e) Free IAA concentration in ten-day-old Col-0 and ire1 roots. Error bars represent SEM from three independent biological replicates. P-value is relative to Col-0: P = 4.9E-05.

Figure 4. Mutants impaired in intracellular auxin transport display a defective UPR phenotype

(a) RT-qPCR analyses of BiP1/2 and PDI6 in ten-day-old pin5-5 (pin5), pin6-4 (pin6), pils2-2 (pils2), pils5-2 (pils5), pils2-2 pils5-2 (pils2 pils5), abp1-5 (abp1), YUC, and tir1 afb1 afb2 afb3 (tir1 afb) relative to wild-type Col-0 Arabidopsis seedlings after a 1-h treatment with 5 μg/ml Tm. Error bars represent standard error of the mean (SEM) from three independent biological replicates. P-values are relative to Col-0: pin5 (P = 0.00029), pin6 (P = 0.00095), pils2 (P = 0.00093), pils5 (P = 0.00067), pils2 pils5 (P = 0.00089), abp1 (P = 0.00215), YUC (P = 0.00014), tir1 afb (P = 0.00026). (b) RT-qPCR analyses of BiP1/2 and PDI6 in ten-day-old pin1-1 (pin1), eir1-1 (pin2), pin3-4 (pin3), pin4-3 (pin4), pin7-2 (pin7), pin3-4 pin4-3 pin7-2 (pin3 pin4 pin7), OxPIN1, and aux1-22 (aux1) relative to wild-type Col-0 Arabidopsis seedlings after a 1-h treatment with 5 μg/ml Tm. Error bars represent SEM from three independent biological replicates.

Figure 5. pin5 enhances the auxin and ER stress response phenotype in atire1a atire1b (ire1)

(a) pin5-5 (pin5) enhances the short root phenotype of ire1. Relative primary root length of pin5, ire1, and ire1 pin5 compared to Col-0 under unstressed conditions. Error bars represent standard error of the mean (SEM), n > 30. P-value is ire1 pin5 relative to ire1: *P = 0.00226. (b) Free IAA measurement in the roots of ten-day-old Col-0, pin5, ire1, and ire1 pin5 seedlings. Error bars represent SEM from three independent biological replicates. P-value is ire1 pin5 relative to ire1: *P = 0.00182. (c–e) Relative primary root length of ten-day-old Col-0, pin5, ire1, and ire1 pin5 Arabidopsis seedlings grown in the presence 50, 100, 200 nM NAA (c), or 1, 2, 3, 4, 5 μM IAA (d), or 50, 100 nM NPA (e) compared to those grown in the absence of the chemicals. Error bars represent SEM, n > 30. P-values are ire1 pin5 relative to ire1: 50, 100 or 200 nM NAA (P < 0.00032), 1, 2, 3, 4, or 5 μM IAA (P < 0.00075), 50 or 100 nM NPA (P < 0.00149). Scale bars = 1 cm. (f) pin5 enhances the UPR defects in ire1 under ER stress. RT-qPCR analyses of BiP1/2 and PDI6 in ten-day-old Col-0, pin5, ire1, and ire1 pin5 relative to DMSO mock control after a 1-h treatment with 5 μg/ml Tm. Error bars represent SEM from three independent biological replicates. P-value is ire1 pin5 relative to ire1: *P = 0.01856.

Figure 6. Working model

(a) IRE1 is required for the auxin responses upon external auxin application. IRE1, ER- and PM-localized PINs are involved in the maintenance of auxin homeostasis without chemical induction of ER stress. (b) ER stress triggers down-regulation of auxin receptors TIR1/AFBs, ER- and PM-localized PINs. IRE1 and ER-localized PINs are required for the optimal induction of UPR target genes.