Systemic signaling contributes to the unfolded protein response of the plant endoplasmic reticulum

Abstract

The unfolded protein response (UPR) of the endoplasmic reticulum constitutes a conserved and essential cytoprotective pathway designed to survive biotic and abiotic stresses that alter the proteostasis of the endoplasmic reticulum. The UPR is typically considered cell-autonomous and it is yet unclear whether it can also act systemically through non-cell autonomous signaling. We have addressed this question using a genetic approach coupled with micro-grafting and a suite of molecular reporters in the model plant species Arabidopsis thaliana. We show that the UPR has a non-cell autonomous component, and we demonstrate that this is partially mediated by the intercellular movement of the UPR transcription factor bZIP60 facilitating systemic UPR signaling. Therefore, in multicellular eukaryotes such as plants, non-cell autonomous UPR signaling relies on the systemic movement of at least a UPR transcriptional modulator.

Fig. 1

Intercellular translocation of sbZIP60 induces BiP3 expression in systemic tissues. a Confocal laser scanning microscopy of Col-0; pSHR-GFP, shr; pSHR-SHR-GFP, and bzip28/60; pSHR-sbZIP60-GFP at the primary root tips of 5-day-old transgenics reveals stele (St) accumulation of GFP, and stele and endodermis (En) distribution of SHR-GFP; noticeably, sbZIP60-GFP is localized in the stele, endodermis, cortex (Co) and epidermis (Ep). Similarly to SHR-GFP, sbZIP60-GFP is localized in nuclei (arrows). As also reported earlier, we did not find SHR-GFP localization in the nuclei of the cortex and epidermis. Propidium iodide (PI) was used for counterstaining. Scale bar: 50 μm. b Expression of pBIP3:GUS in bzip28/60, Col-0 and bzip28/60;pSHR-sbZIP60-GFP seedlings grown vertically on half LS agar medium for 11 days. X-Gluc was used for histochemical staining to monitor GUS activity. Scale bar: 100 μm. c Longitudinal confocal optical sections of the regions along the primary root shown in b. Epidermis: Ep; Co: cortex; En: endodermis; St: stele. The indications upper, middle and lower refer to the a, b, and c zones indicated in panel b. Scale bar: 20 μm

Fig. 2

Root-expressed sbZIP60 transcripts are translocated to the shoot. a, b Quantitative RT-PCR analyses of sbZIP60 (a) and BiP3 (b) in 14-day-old wild type (Col-0), bzip28/60, and bzip28/60; pRoot::sbZIP60 seedlings treated with DMSO or 0.5 µM Tm (Tunicamycin) at the root in the shoot–root split system for 24 h. Transcription of UBQ10 was used as internal control. Error bars represent s.e.m among three biological replicates. Data significantly different from the corresponding control are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Unpaired t-test). Black asterisks designate differences between bzip28/60 and bzip28/60; pRoot::sbZIP60 lines under Tm non-treatment condition. Red asterisks designate differences between bzip28/60 and bzip28/60; pRoot::sbZIP60 under Tm treatment condition

Fig. 3

Local ER stress ignites the UPR systemically, mostly in a shoot-ward direction. a Diagrams illustrating the Arabidopsis shoot–root split culture system in which the shoot and root of an intact seedling are exposed to separate growth media with different chemical conditions: mock DMSO (D) or 0.5 µM Tm (T, Tunicamycin). T/D denotes shoot on Tm-containing medium and root on DMSO-containing medium; conversely, D/T denotes shoot on DMSO-containing medium and root on Tm-containing medium. b Quantitative RT-PCR analyses of UPR markers in 14-day-old wild-type seedlings treated with DMSO or 0.5 μM Tm for 24 h as described in a. Values are presented relative to non-treated control (0 h), which was set to 1. Transcription of UBQ10 was used as internal control. Error bars represent s.e.m among three biological replicates. Data significantly different from the corresponding control are indicated by asterisks (*P < 0.05, ****P < 0.0001, NS, nonsignificant; Unpaired t-test). c Quantitative HPLC/MS analyses of Tm content in shoot and root of seedlings after treatments as described in a in a shoot–root split culture system. The numbers over the histograms express ng g⁻¹ fresh weight (F.W.). Data significantly different from the corresponding control are indicated by asterisks (**P < 0.01, NS, nonsignificant; Unpaired t-test)

Fig. 4

Micro-grafting analyses support the existence of endogenous systemic UPR signals. a–c Plots of the mean values (dots) of the quantitative RT-PCR analyses of the transcripts of sbZIP60 (a) BiP3 (b) and bZIP28 (c) in scion and rootstock of 4-week-old grafted seedlings of wild-type and bzip28/60 backgrounds treated with DMSO or 0.5 μM Tm (Tunicamycin) for 48 h as described in Fig. 3a. The average of all the values is indicated by a black line. Error bars represent mean with 95% confidence interval among all grafts. Error bars above and below indicate the 95th and 5th percentiles. Average values significantly different from the values of wild-type (Col-0) and bzip28/60 self-grafts are indicated by red asterisks (*P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001, NS, nonsignificant; Mann-Whitney test). n, total number of grafted unions

Fig. 5

Long-distance of UPR signaling relies on the PD availability. a, b Quantitative RT-PCR analyses of sbZIP60 and BiP3 in 14-day-old wild type (Col-0) (a) and a conditional PD block mutant (pMDC7-icals3m) (b) seedlings treated with DMSO (Mock), 10 μM 17-β-estradiol (estrogen), 0.5 µM Tm (Tunicamycin), and 10 μM 17-β-estradiol (estrogen) in combination with 0.5 µM Tm (Tunicamycin) at the root in the shoot–root split system for 24 h. Transcription of UBQ10 was used as internal control. Error bars represent s.e.m among three biological replicates. Data significantly different from the corresponding control are indicated by asterisks (*P < 0.05, NS, nonsignificant; Unpaired t-test)