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Editor-in-Chief Li Jiayang, Beijing Institute of Genetics and Developmental Biology, China, Approved on October 8, 2021 (Received on February 22, 2021)

For plants that grow in crowded environments, phytochrome interaction factor 7 (PIF7) plays a key role by initiating a series of adaptive growth responses. Here, we demonstrate that in addition to transcriptional activity and subcellular localization, the strictly regulated PIF7 protein level is also important for the shade avoidance response. We have identified two ubiquitin-specific proteases, UBP12 and UBP13, which positively regulate the rapid growth of plants in response to shading light. These two ubiquitin proteases directly interact with PIF7 and protect the latter from the 26S proteasome. The dynamic changes in the abundance of PIF7 regulated by UBP12 and UBP13 provide insights into the role of PIF7 post-translational modifications in integrating environmental changes and endogenous responses.

The change in light quality caused by the presence of neighbors regulates many growth and development processes of plants. The subcellular localization, DNA binding properties, and protein abundance of Photochromic Interaction Factor 7 (PIF7) are regulated in a photoreversible manner, and it plays a central role in connecting shadow light perception and growth response. How the activity of PIF7 is regulated during the shade avoidance reaction has been well studied, and many factors involved in this process have been identified. However, the detailed molecular mechanism of shaded light regulating PIF7 protein levels is still largely unknown. Here, we show that PIF7 protein level regulation is important for shade-induced growth. By increasing the level of PIF7 protein, two ubiquitin-specific proteases UBP12 and UBP13 were identified as positive regulators of the shade avoidance response. The sensitivity of the ubp12-2w/13-3 double mutant to shadow-induced cell elongation and acceleration of reproduction was significantly impaired. Our genetic and biochemical analysis showed that UBP12 and UBP13 act downstream of phyB and directly interact with PIF7 through deubiquitination to maintain the stability and abundance of PIF7.

Light is the key environmental factor for all life on earth. In addition to providing energy for photosynthesis, sunlight also acts as a signal that regulates plant growth and development (1). When growing in a crowded environment, the presence of adjacent vegetation will reduce the red/far-red light (R/FR) ratio (2), thereby changing the light quality experienced by plants. In order to reduce the degree of existing or future shadows caused by neighbors, many adaptive responses in plant structure and function are triggered, including rapid elongation of stems and petioles, upward bending of leaves, reduced leaf expansion, and early flowering. Collectively, these morphological adaptations are called the shade avoidance response (SAR) (2, 3).

Plants use photoreceptors to sense changes in the surrounding light environment (4⇓ –6). The decrease in the R/FR ratio of light is mainly perceived by the photoreceptor phytochrome B (phyB) (4, 5). After being sensed by phyB, shadow light (SL) converts phyB from the active form (Pfr) that absorbs far red light to the inactive form (Pr) that absorbs red light (7⇓ –9), allowing dephosphorylation and a The accumulation of a subset of basic helix-loop-helix transcription factors, phytochrome interaction factor (PIF) (10, 11). The activated PIF binds to the promoter of the shade response gene to promote their expression. Among the reported PIFs, PIF4, PIF5, and PIF7 redundantly promote SAR, while PIF7 played a major role (11⇓ ⇓ –14). The abundance and phosphorylation of PIF7 are regulated by light quality; however, the detailed molecular mechanism of how to regulate the post-translational modification of PIF7 is still largely unknown (11, 15).

Protein ubiquitination is a key post-translational modification that mediates a large number of eukaryotic cell processes and signaling pathways. The protein polyubiquitinated by the specific ubiquitin E3 ligase is then sent to the 26S proteasome for degradation, which is essential for protein turnover and signal pathway reset (16, 17). Like phosphorylation, protein ubiquitination is a dynamic and reversible process. Ubiquitinated proteins can be deubiquitinated by deubiquitinating enzyme (DUB) to rescue the target protein from destruction (18). The Arabidopsis genome encodes more than 1,000 E3 ligases, but not more than 100 DUBs (19, 20). This indicates that DUB may have multiple goals. UBP12 and UBP13 are two homologous proteins in the ubiquitin-specific protease (UBPs/USPs) subfamily (19, 21, 22). They regulate many aspects of plant growth and development, including pathogen immunity, leaf senescence, and photoperiod Flowering, biological clock, cell size, root meristem maintenance and JA signaling pathway (22⇓ ⇓ ⇓ ⇓ ⇓ ⇓ –29). In order to better understand the ubiquitination kinetics of plant growth and development, other functions and substrates of UBP12 and UBP13 need to be identified and characterized. Using genetic and molecular methods, we hereby demonstrate that UBP12 and UBP13 play a key role in accelerating SAR. These UBPs promote shadow-mediated changes in plant structure by directly binding to PIF7 and preventing its degradation.

UBP12 and UBP13 loss-of-function plants exhibit short petiole and dwarfism phenotypes (22, 23), indicating that these two UBPs are involved in regulating cell elongation during plant development. Light is the most important environmental factor that regulates cell elongation. The change in light quality greatly changes the stem growth and lowness of plants. In order to examine whether UBP12 and UBP13 are involved in regulating the growth and development mediated by changes in light quality, we analyzed the effects of various UBP12/UBP13 mutants and overexpressors on SL with reduced R/FR ratios (low R/FR). reaction. Under normal white light (WL) and SL conditions, the hypocotyl elongation of the ubp13-3 single mutant is not different from that of the wild type (WT). However, ubp12-2w is a weak double mutant of UBP12 and UBP13 (22), which is less sensitive to SL-induced hypocotyl growth. In addition, ubp12-2w/13-3 is a stronger double mutant, showing a more severe phenotype than ubp12-2w, and it is almost completely insensitive to SL (Figure 1A and B). Scanning electron microscopy results showed that the short hypocotyl phenotype of the ubp12-2w/13-3 double mutant under shading conditions was mainly due to the inhibition of cell elongation rather than the decrease in cell number (SI appendix, Figure S1). Compared with ubp12-2w/13-3, transgenic plants overexpressing UBP12 and UBP13 showed slightly longer hypocotyls than WT when grown under SL (SI Appendix, Figure S2). We also studied the petiole length and flowering time of WT and ubp12-2w/13-3 under WL and SL conditions. Consistent with our observations on seedlings, compared with WT, the petiole elongation and flowering acceleration induced by SL in ubp12-2w/13-3 were greatly impaired (Figure 1 CF). In summary, our physiological results indicate that UBP12 and UBP13 act as positive regulators of SAR.

UBP12 and UBP13 are positive regulators of SAR. (A and B) Hypocotyl phenotype (A) and hypocotyl length (B) of 8-day-old seedlings grown under WL or SL conditions. (Scale bar: 1 cm.) The displayed value is the mean ± SD (n ≥ 20). *P <0.05 and **P <0.01; based on student's t-test. (C and D) The phenotype of 3-week-old WT (Col-0) and ubp12-2w/13-3 grown under WL or SL conditions. Plants grown for 10 days under WL were transferred to SL or kept under WL for another 10 days. (Scale bar: 1 cm.) (E and F) The petiole length and leaf number (F) of the third and fourth leaves (E) of the plants analyzed in C and D. The displayed value is the mean ± SD (n ≥ 10). *P <0.05 and **P <0.01; based on Student's t-test.

In order to further determine whether the cool reaction requires the deubiquitination catalytic activity of UBP12, we used UBP12 (WT) and UBP12 (C208S) to supplement the defects of ubp12-2w/13-3, UBP12 (22, 24). Transgenic plants with equivalent UBP12 expression levels were used to examine their response to SL (SI appendix, Figure S3E). UBP12 (WT), but not UBP12 (C208S), can partially but significantly save the hypocotyl and petiole response of ubp12-2w/13-3 to SL (SI appendix, Figure S3 AD). These results indicate that the DUB activity of UBP12 is critical to its function in SAR.

We also studied the transcription and protein levels of UBP12 and UBP13 under WL and SL. 8 hours after the transfer from WL to SL, the transcription levels of UBP12 and UBP13 decreased slightly (SI Appendix, Figure S4 A and B); however, the protein abundance of UBP12 and UBP13 did not change significantly due to SL treatment (SI Appendix, Figure S4 A and B). S4 C and D). These results indicate that SL may regulate the functions of UBP12 and UBP13 by increasing their catalytic activity or their binding to substrates rather than their expression levels.

Among the five phytochrome photoreceptors, phyB plays a major role in light quality detection (2, 4, 5). Even under high R/FR light conditions, phyB loss-of-function mutants showed extended hypocotyls and petioles and early flowering. In order to determine the genetic relationship between phyB and UBP12/UBP13, we generated a triple mutant phyB/ubp12-2w/13-3 and compared its sensitivity to SL with phyB. We found that ubp12-2w/13-3 greatly saved the constitutive shadow response of phyB (Figure 2A and B and SI appendix, Figure S5), indicating that UBP12 and UBP13 act downstream of phyB in SAR.

UBP12 and UBP13's promotion of shade response depends on the function of PIF7. (A) The phenotypes of WT (Col-0), phyB, ubp12-2w/13-3 and phyB/ubp12-2w/13-3 under WL and SL conditions. (Scale bar: 1 cm.) (B) The hypocotyl length of the plant shown in A. The displayed value is the mean ± SD (n ≥ 20). *P <0.05 and **P <0.01; based on student's t-test. (C) WT (Col-0) and ubp12-2w/13-3 grown under WL and SL respond to hypocotyls treated with PIC. The seedlings were grown on media with different concentrations of PIC (0, 0.01, 0.1, 1.0, and 2.0 µM) and kept under WL for 4 days, then transferred to a cool place or kept under WL for another 4 days. (Scale bar: 1 cm.) (D and E) The hypocotyl length of the seedling indicated in C under WL (D) and SL (E). The displayed value is the mean ± SD (n ≥ 20). *P <0.05 and **P <0.01; based on student's t-test. (FI) Under WL and SL conditions, the expression of shadow-induced genes in WT (Col-0) and ubp12-2w/13-3. The displayed value is the mean ± SD. *P <0.05 and **P <0.01; based on student's t-test. (J and K) The genetic interaction between PIF7 and UBP12/UBP13 in SAR. The hypocotyl phenotype and length are shown in J and K, respectively. (Scale bar: 1 cm.) The displayed value is the mean ± SD (n ≥ 20). *P <0.05 and **P <0.01; ns, not significant (P≥0.05); based on student's t-test. (L and M) The expression of shade-responsive genes in the various genotypes shown in J and K. The plants were grown under WL for 7 days, and then half of them were treated under SL for 4 hours. The displayed value is the mean ± SD. *P <0.05 and **P <0.01; ns, not significant (P≥0.05); based on student's t-test.

Previous studies have shown that after being sensed by phyB, SL greatly and rapidly increases the accumulation of auxin by activating auxin biosynthesis genes (11, 30, 31). In order to study whether UBP12/UBP13-mediated SAR depends on the biosynthesis of auxin, we analyzed the response of WT and ubp12-2w/13-3 to different concentrations of pyrroline (PIC) (an auxin analog) . Figure 2 CE shows that under WL and SL conditions, the hypocotyl of ubp12-2w/13-3 is more sensitive to PIC than the hypocotyl of WT. The application of exogenous PIC significantly saved the short hypocotyl phenotype of ubp12-2w/13-3 under SL (Figure 2C and E). Consistent with physiological results, the transcription levels of auxin biosynthesis genes YUC8 and YUC9 and auxin-responsive genes IAA19 and IAA29 in WT were induced 4 hours after transfer to SL. However, the induction of ubp12-2w/13-3 was largely impaired (Figure 2 FI). Our results demonstrate the key role of UBP12 and UBP13 in linking SL perception and auxin biosynthesis.

The accumulation of newly synthesized auxin is necessary to respond to the rapid growth of SL, and it is regulated by a set of PIF, of which PIF7 plays an important role (11). SL releases the inhibition of phyB and increases the activity and abundance of PIF7. PIF7 directly binds to the promoter regions of many auxin biosynthesis genes to activate their expression (11, 15). In order to determine the upper relationship between PIF7 and UBP12/UBP13, we crossed pif7 with ubp12-2w/13-3, UBP12-OE (UBQ10::UBP12-HA) and UBP13-OE (UBQ10::UBP13-HA), And homozygous plants are used for shade response analysis. Consistent with previous reports, the pif7 single mutant is significantly less sensitive to shadow-induced growth, although it can still respond to SL (Figure 2J and K), which may be caused by the functional redundancy of PIF. The response of ubp12-2w/13-3 hypocotyl to SL was even more severe than that of pif7, and almost lost all shadow reactivity (Figure 2J and K), indicating that in addition to PIF7, UBP12 and UBP13 may also regulate other PIF functions. The hypocotyl phenotype of ubp12-2w/13-3 under WL and SL was not significantly changed by pif7 (Figure 2J and K), indicating that PIF7 and UBP12/UBP13 act in the same way in regulating shade-induced growth . In addition, UBP12-OE and UBP13-OE cannot save the insensitive phenotype of pif7 to SL (Figure 2 J and K). Consistent with the genetic results, compared with UBP12-OE and UBP13-OE, the shade-induced expression of YUC8 and IAA19 in pif7/UBP12-OE and pif7/UBP13-OE was significantly impaired, but it was similar to that in pif7 (Figure 2L and Meter). In conclusion, all these data support the view that SAR regulation mediated by UBP12/UBP13 depends on PIF7 function.

The genetic relationship between PIF7 and UBP12/UBP13 indicates that the proteins they encode may have the possibility of a direct biochemical relationship. To explore this hypothesis, we examined possible interactions between PIF7 and UBP12 or UBP13. The yeast two-hybrid test showed that PIF7 interacted with UBP12 and UBP13 in yeast cells (Figure 3A). This interaction was confirmed by in vitro assays using GST-UBP12 or GST-UBP13 to pull down the purified MBP-PIF protein. PIF7 and PIF3 are closely related to UBP12 and UBP13, while PIF4 and PIF5 interact weakly with UBP13, but have nothing to do with UBP12 (Figure 3B). Therefore, in addition to PIF7, UBP12/UBP13 can also adjust the functions of several other PIFs. In summary, these results support the view that PIF7 can directly interact with UBP12/UBP13. In addition to in vitro interactions, in the Nicotiana benthamiana bimolecular fluorescence complementation (BiFC) assay, a strong fluorescence signal was observed in the nuclei co-transformed with PIF7 combined with UBP12 or UBP13 (Figure 3C). In addition, co-immunoprecipitation (co-IP) analysis in Arabidopsis further confirmed the in vivo association between UBP12 and PIF7 under WL and SL conditions (Figure 3D).

PIF7 interacts with UBP12 and UBP13 in vitro and in vivo. (A) The interaction between PIF7 and UBP12 or UBP13 in the yeast two-hybrid test. (B) In vitro pull-down assay of UBP12 and UBP13 and PIF transcription factors. * Indicates the main band of each MBP-PIF protein. (C) BIFC assay showed that PIF7 interacted with UBP12 and UBP13 in tobacco. (D) Co-IP analysis of extracts from WT (Col-0) and 35S::PIF7-FLASH plants that express PI with FLASH tags (3×FLAG, 6×MYC and 6×HIS)​ ​F7. Anti-FLAG M2 affinity agarose gel is used for immunoprecipitation. The input and immunoprecipitation products are detected by anti-MYC and anti-UBP12 antibodies, respectively. P-PIF7, the phosphorylated form of PIF7.

Previous studies have shown that post-translational modifications are essential for PIF7 to regulate shade avoidance (11, 15). SL treatment leads to rapid dephosphorylation of PIF7 (15), which promotes the transfer of PIF7 from the cytoplasm to the nucleus. In addition to phosphorylation, ubiquitination is another important modification of PIF transcription factors, which regulates the stability of PIFs (10, 32⇓ –34). A previous study showed that SL increases the number of PIF7, which can be reversed by WL (11). To clarify whether PIF7 abundance is important for its function, we overexpressed PIF7 with MYC tag in WT (35S::PIF7-MYC), and analyzed the phenotype and PIF7 protein level of four independent transgenic lines. Transgenic lines #12 and #13 with lower PIF7 protein levels showed similar phenotypes to WT, while lines #14 and #16 with much higher PIF7 levels than #12 and #13, even under WL The hypocotyls exhibited significantly elongated (SI appendix, Figure S6), indicating that there is a close relationship between the abundance of PIF7 and its function of promoting cell elongation. To determine whether PIF7 protein levels respond to SL dynamic regulation, we used 35S::PIF7-FLASH (35S::PIF7-9×Myc-6×His-3×FLAG) transgenic plants to study WL and SL. In the presence of cycloheximide (CHX), which blocks the synthesis of new proteins, PIF7 protein levels are reduced under both WL and SL, but a faster degradation rate is observed under WL (SI appendix, Figure S7). PIF7 degradation was prevented by MG132, indicating the involvement of 26S proteasome (SI appendix, Figure S7). Our results indicate that PIF7 is an unstable protein, but SL can protect it from 26S proteasome degradation. Previous studies have shown that phosphorylation is necessary for the subsequent ubiquitination and degradation of many transcription factors (32⇓ –34). PIF7 can also be phosphorylated, which is essential for its subcellular localization and function (15). To examine the possible relationship between PIF7 phosphorylation and ubiquitination in SAR, we generated 35S::PIF7(2A)-MYC transgenic plants in which S139 and S141 were mutated to alanine to mimic the unphosphorylation of these residues Status (15). We found that compared with 35S::PIF7-MYC#12, which has a similar phenotype to WT (Col-0), 35::PIF7(2A)-MYC showed significantly longer hypocotyls under both WL and SL (SI appendix, Figure 2). S8 A and B). In addition, 35::PIF7(2A)-MYC plants express much higher levels of PIF7-MYC protein than 35S::PIF7-MYC#12 (SI appendix, Figure S8C). Please note that the transcription level of PIF7 in 35::PIF7(2A)-MYC is lower than 35S::PIF7-MYC#12 (SI Appendix, Figure S8D). Our evidence suggests that phosphorylation of PIF7 is essential for its stability regulation. Shading promotes the dephosphorylation of PIF7, thereby enhancing its stability, resulting in higher PIF7 abundance to promote rapid growth induced by shading.

Considering that UBP12 and UBP13 are DUBs that directly interact with PIF7, these two UBPs are likely to modulate SAR by removing ubiquitin from polyubiquitinated PIF7 to prevent damage. To examine this possibility, we produced UBP12-OE/35S::PIF7-FLASH plants by crossing 35S::PIF7-FLASH with pUBQ10::UBP12-HA. The transcription level of PIF7 did not change significantly under different genetic backgrounds (SI Appendix, Figure S9A). Then we compared the abundance of FLASH-labeled PIF7 protein in WT and UBP12-OE under WL and SL. Consistent with previous studies (11, 15), SL promoted the conversion of PIF7 from phosphorylated form to dephosphorylated form. SL treatment significantly increased PIF7 protein levels in WT, and MG132 could prevent PIF7 degradation in WL (Figure 4A). In contrast, in the UBP12-OE background, PIF7 protein levels were greatly increased compared with WT, and MG132 treatment had a smaller effect on PIF7 protein levels (Figure 4A). These results indicate that PIF7 in UBP12-OE is more stable than that in WT. In order to compare the PIF7 protein levels in WT and ubp12-2w/13-3, we also generated ubp12-2w/13-3/35S::PIF7 by crossing ubp12-2w/13-3 with 35S::PIF7-FLASH -FLASH, but the level of PIF7 with the FLASH tag in the double mutant background is too low to be detected. In order to circumvent this problem, we crossed ubp12-2w/13-3 with another transgenic plant 35S::PIF7-MYC#14 with higher expression levels of PIF7 (SI appendix, Figure S6). We found that compared with WT, the abundance of PIF7-MYC in the background of ubp12-2w/13-3 was significantly reduced; however, the abundance of PIF7-MYC in the double mutant could be increased by MG132, indicating its instability ( Figure 4B). Note that MG132 treatment did not significantly change the PIF7 transcription level (SI Appendix, Figure S9B). In order to confirm the important role of UBP12 in protecting PIF7 from destruction, we added CHX to inhibit new protein biosynthesis, and studied the time course of PIF7 protein degradation under WL and SL under the background of WT and UBP12-OE. The results of western blotting showed that the degradation of PIF7 in UBP12-OE was significantly hindered compared with WT under WL and SL conditions (SI appendix, Figure S10). In order to determine whether the role of UBP12 in regulating the stability of PIF7 requires the deubiquitination catalytic activity of UBP12, we compared PIF7- in WT, UBP12-OE and UBP12(C208S)-OE plants carrying 35S::PIF7-FLASH. FLASH protein level transgenic. The transcription level of PIF7 is comparable in plants of these three genotypes (SI appendix, Figure S11A). Compared with WT, the protein level of PIF7 with FLASH tag in UBP12-OE is greatly increased (Figure 4C). However, in UBP12(C208S)-OE, which expressed the same UBP12-HA protein level as UBP12-OE, compared with WT, the PIF7-FLASH abundance did not change significantly (Figure 4C). We further examined the ubiquitination status of PIF7 in the background of WT, UBP12-OE and UBP12(C208S)-OE. In order to retain polyubiquitinated PIF7, we added MG132 to block 26S proteasome activity. We found that the PIF7-FLASH protein in the WT background is highly polyubiquitinated under WL compared to SL. When UBP12 was expressed instead of the UBP12 (C208S) mutant, the polyubiquitination of PIF7 was significantly reduced (Figure 4D). In addition, overexpression of UBP12 instead of UBP12 (C208S) mutants increased the hypocotyl elongation of 35S::PIF7-FLASH under WL and SL (SI Appendix, Figure S11 B and C), which is in line with the increased PIF7 protein in UBP12 The same level -OE/35S::PIF7-FLASH plant (Figure 4C). In summary, our results confirm the view that UBP12 and UBP13 require UBP12 and UBP13 to deubiquitinate PIF7 and maintain its abundance in SAR.

UBP12 and UBP13 increase the stability of PIF7. (A) PIF7 protein levels in the background of WT (Col-0) and UBP12-OE under WL and SL conditions. At 7 days of age, the 35S::PIF7-FLASH and UBP12-OE/35S::PIF7-FLASH seedlings grown under WL were moved to a cool place or under WL with or without 50 µM MG132 for 4 hours, and then the samples were harvested Protein extraction. P-PIF7, the phosphorylated form of PIF7. (B) PIF7 protein levels in the background of WT (Col-0) and ubp12-2w/13-3 under WL and SL conditions. 35S::PIF7-MYC and ubp12-2w/13-3/35S::PIF7-MYC plants are treated as shown in A. (C) PIF7 protein levels in WT (Col-0), 35S::PIF7-FLASH, UBP12-OE/35S::PIF7-FLASH and UBP12(C208S)-OE/35S::PIF7- under WL and SL FLASH. At 7 days of age, the WL-grown seedlings are transferred to SL or placed under WL for 4 hours, and then collected for protein extraction. (D) The polyubiquitination status of PIF7 in various genotypes. The seedlings were grown under WL for 10 days and then treated with 50 µM MG132 for 4 hours under SL or WL. The protein extract was immunoprecipitated (IP) using Ni-NTA nickel beads (Qiagen). The IP products were analyzed by Western blot using anti-ubiquitin and anti-MYC antibodies. (Bottom) The exposure time in the middle is shorter. The molecular weight standards are indicated. (E and F) Hypocotyl phenotype (E) and hypocotyl length ( The measured value of F) 3/35S::PIF7-MYC#14 grows under WL and SL. (Scale bar: 1 cm.) The displayed value is the mean ± SD (n ≥ 20). *P <0.05 and **P <0.01; based on student's t-test. (G and H) Shade-induced gene expression under WL and SL in plants indicated in E and F. The displayed value is the mean ± SD. *P <0.05 and **P <0.01; based on student's t-test.

Because UBP12 and UBP13 directly interact with PIF7 to promote its stability, we hypothesized that the insensitivity of ubp12-2w/13-3 to SL treatment is caused by the decrease of PIF7 protein level. To confirm this hypothesis, we crossed ubp12-2w/13-3 with 35S::PIF7-MYC#14, which expressed PIF7 protein at high levels (SI appendix, Figure S6) and showed a constitutive shadow response. We found that 35S::PIF7-MYC#14 can significantly save the phenotype of ubp12-2w/13-3 (Figure 4 E and F). Consistent with the genetic results, compared with ubp12-2w/13-3, the expression of shade-inducing genes YUC8 and IAA19 in ubp12-2w/13-3/PIF7-OE#14 under SL also increased (Figure 4) G And H). In summary, our data indicate that the regulation of PIF7 protein stability mediated by UBP12 and UBP13 is essential for shadow-induced adaptive growth.

Protein instability is a hallmark of transcription factors, because fine-tuning their levels can quickly respond and adapt to changing cell conditions. After transferring plants from WL to SL, PIF7 transcription levels did not change significantly, indicating that PIF7 regulation in response to shading is mainly a post-translational event (11). Here, we demonstrated that the dynamic regulation of PIF7 protein abundance is essential for plants to respond quickly in a cool environment. PIF7, which is unstable in WL, becomes stable when transferred to SL. MG132 can inhibit the decline of PIF7 levels (SI Appendix, Figure S7 and Figure 4 A and B). Previous studies have shown that changes in light quality can induce rapid and reversible phosphorylation of PIF7, which is critical for its subcellular localization (15). In this study, we showed that phosphorylation of PIF7 also plays an important role in regulating the stability of PIF7 (SI Appendix, Figure S8). SL induces the conversion of PIF7 from the phosphorylated form to the dephosphorylated stable form, resulting in an increase in PIF7 levels.

The dynamics of protein abundance depend on the balance between ubiquitination and deubiquitination, which is usually mediated by E3 ligase and UBP, respectively. In Arabidopsis, members of the UBP subfamily are involved in different cellular processes and signaling pathways. A large number of studies have shown that UBPs are involved in embryonic development (35), pollen development and dissemination (36), chromatin modification (37), pathogen defense (27), leaf development and senescence (21, 23), and peptide and hormone signal transduction ( 26, 29). Although UBPs have multiple functions in the plant life cycle, the role of UBPs in integrating environmental changes into plant endogenous responses is still largely unknown. Here, we have determined UBP12 and UBP13 as the DUB of PIF7 in SAR. The ubp12-2w/13-3 mutant was blocked in shade-induced cell elongation and flowering acceleration (Figure 1). Our in-depth genetic analysis showed that UBP12 and UBP13 act as positive regulators of the phyB-PIF7-auxin cascade in SAR, and their function depends on PIF7 (Figure 2 and SI appendix, Figure S5). These two UBPs form a complex with PIF7 in the nucleus (Figure 3) and deubiquitinate PIF7 to prevent degradation (Figure 4).

Our research reveals the important role of PIF7 stability regulation in SAR, and proves that UBP12 and UBP13 play a key role in regulating the abundance of PIF7 in response to changes in the light environment. Under normal light conditions, phyB is converted by red light from inactive Pr light to active Pfr, which interacts with PIF7, resulting in rapid phosphorylation of PIF7. This post-translational modification of PIF7 promotes its polyubiquitination, leading to the degradation of most PIF7 proteins by the 26S proteasome. In this process, UBP12 and UBP13 are required to maintain the basic PIF7 level and the expression of auxin biosynthesis genes. Under shading, as the R/FR ratio decreases, phyB is inactivated by far-red light, and PIF7 protein is converted from phosphorylated form to dephosphorylated form. UBP12/UBP13-mediated enhancement of PIF7 deubiquitination leads to increased accumulation of PIF7 protein in the nucleus, which activates the expression of auxin biosynthesis genes (SI Appendix, Figure S12).

When plants grow in a crowded environment, the photoreceptors will perceive a decrease in the R/FR ratio of ambient light, and these plants will quickly grow adaptively to avoid the stress caused by shadows. However, if plants are kept under shading for a long time, they are vulnerable to pathogens. In addition, the flowering time is accelerated, resulting in reduced fertilization, which affects crop yields. Our results and previously reported results indicate that UBP12 and UBP13 are positive regulators of SAR, but negative regulators of plant defense (27), indicating that these two UBPs maintain the balance between plant defense and SAR-induced rapid growth Plays an important role. We hope that they can also stimulate the development of new strategies to design crops with shade tolerance and enhanced immunity.

All plants used in this study are Col-0 germplasm. pif7-1 (CS68809) and phyB-9 (CS6217) were obtained from ABRC (Arabidopsis Biological Resource Center). The ubp12-2w (GABI_244E11), ubp13-3 (SALK_132368) and ubp12-2w/13-3 double mutants were obtained from the Cao Xiaofeng laboratory, and their characteristics have been previously described (22). 35S::PIF7-FLASH is kindly provided by Lin Li, Fudan University, China (11). UBQ10::UBP12-HA and UBQ10::UBP13-HA have been described previously (23, 26). For constitutive overexpression, DNA fragments including full-length open reading frame (ORF) in the form of PIF7, PIF7(2A) mutants and UBP12(C208S) mutants with HA tags are generated by PCR using designated primers ( SI appendix, Table S1) and cloned into pBA-35S::GWR-MYC or UBQ10::GWR vector by using the gateway cloning method (Invitrogen). These constructs were introduced into the GV3101 strain of Agrobacterium and transformed into WT (Col-0) or ubp12-2w/13-3 plants using the flower dip method (38). The Agrobacterium carrying UBQ10::UBP12-HA and UBQ10::UBP12(C208S)-HA was transformed into ubp12-2w/13-3. pif7/ubp12-2w/13-3, pif7/UBP12-OE and pif7/UBP13-OE were obtained by hybridizing pif7-1 with ubp12-2w/13-3, UBQ10::UBP12-HA and UBQ10::UBP13 -HA, respectively. The phyB/ubp2-2w/13-3 triple mutant was produced by crossing phyB-9 and ubp12-2w/13-3. UBP12-OE/35S::PIF7-FLASH and UBP12(C208S)-OE/35S::PIF7-FLASH plants were obtained by crossing 35S::PIF7-FLASH with UBQ10::UBP12-HA and UBQ10::UBP12(C208S) -HA, respectively. ubp12-2w/13-3/PIF7-MYC#14 is produced by crossing ubp12-2w/13-3 with 35S::PIF7-MYC#14 transgenic plants. All plants are grown under 22°C long-day conditions (16 hours of light/8 hours of darkness) for general growth and seed harvesting. For phenotypic and molecular analysis, we use constant WL (50 μE ⋅ m−2 ⋅ s−1) or simulated shading as described above (11, 30).

The surface-sterilized seeds were planted on 1/2 Murashige and Skoog (MS) medium containing 1% sucrose and 0.8% agar. After 2 days of vernalization, the plate was placed under WL for 4 days, then transferred to SL or kept under WL for another 4 days before measurement. PIC (Sigma) processing has been described previously (31). In short, the seeds inoculated on 1/2 MS medium containing different concentrations of PIC were cultured under WL for 4 days, and then transferred to SL or WL conditions. ImageJ software is used to quantify the hypocotyl length. The experiment was carried out in three biological replicates, and each independent experiment measured at least 20 seedlings.

The seedlings grown under WL for 7 days are transferred to SL or kept under WL for 4 hours. Use the plant total RNA extraction kit (Qiagen) to extract total RNA. A total of 1 μg of total RNA was used for complementary DNA (cDNA) synthesis using the Bio-Rad Reverse Transcriptase Kit. According to the manufacturer's instructions, use SYBR Premix Ex Taq II (Bio-Rad) on the Bio-Rad real-time PCR system for real-time PCR. Three biological replicates were performed for each experimental group. Similar results were obtained, and a representative set of results were shown after normalization for ACTIN2.

Seedlings grown for 7 days under WL conditions were transferred to SL or kept under WL for another 4 hours. About 20 seedlings were harvested for protein extraction. Plant tissue is ground into a fine powder in liquid nitrogen. Use protein extraction buffer (100 mM Tris HCl pH 7.8, 4 M urea, 5% sodium dodecyl sulfate [SDS], 15% glycerol, 1 mM DTT, 1 mM phenylmethanesulfonyl fluoride [PMSF] and 1 mM Protease inhibitors) extract the total protein mixture). For CHX treatment, 7-day-old seedlings grown under WL were transferred to SL for 4 hours to accumulate PIF7 protein to a high level. The seedlings were then transferred to WL or kept under SL conditions in the presence of 200 μM C​​HX with or without 50 μM MG132 for the specified time. Use the designated antibody to detect protein levels. Tubulin probed with anti-α-tubulin antibody (Sigma) was used as an internal loading control. The experiment was repeated 3 times with independent biological samples, and similar results were obtained. Shows a representative set of results.

The full-length UBP12 and UBP13 cDNA was cloned into the pGBKT7 vector, and the full-length PIF7 cDNA was cloned into the pGADT7 vector using the fusion method (Clontech). Perform the yeast two-hybrid test as described in (39).

The Gateway method was used to clone the full-length PIF7 cDNA into pEarley Gate201-nYFP, and the full-length cDNA of UBP12 and UBP13 into pEarley Gate202-cYFP. Perform BiFC measurement as described in (39).

By cloning into the pMAL-GWR-MYC vector, the full-length coding sequences of PIF3, PIF4, PIF5 and PIF7 are fused in frame with the sequence encoding the MBP tag. To prepare GST-UBP12 and GST-UBP13 fusion proteins, UBP12 and UBP13 were cloned into the pGEX-GWR-HA vector. MBP-labeled PIF3, PIF4, PIF5 and PIF7 were expressed in E. coli BL21 and purified using amylose resin (NEB). The purified GST-UBP12 and GST-UBP13 proteins retained on Glutathione Sepharose beads were incubated with the same amount of MBP, MBP-PIF3, MBP-PIF4, MBP-PIF5 or MBP-PIF7 at 4°C for 4 hours. After washing 4 times with washing buffer, add 50 µL of 2×SDS loading buffer to each sample, and boil at 95°C for 10 minutes. After centrifugation, the pull-down product was detected by immunoblotting with anti-MBP or anti-GST antibody.

The co-IP was performed as previously described (39). WT (Col-0) and 35S::PIF7-FLASH seedlings are used to detect the possible interaction between PIF7 and UBP12. Before analysis, seedlings that were 10 days old and grown under WL conditions were transferred to SL or kept under WL for 4 hours. The tissue was ground into a fine powder in liquid nitrogen, and in IP buffer (50 mM Tris HCl pH 7.5, 1 mM ethylenediaminetetraacetic acid [EDTA], 75 mM NaCl, 0.5% Triton X-100, 5% glycerol, 2 mM DTT and 1 mM protease inhibitor mixture). After centrifugation at 13,000 rpm at 4°C for 15 minutes, the supernatant was mixed with 40 μL of anti-FLAG M2 affinity agarose gel (Sigma) and incubated at 4°C for 4 hours. Wash the beads five times with washing buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl and 0.1% Triton X-100). The bound protein is eluted from the affinity beads using 2×SDS loading buffer boiled at 95°C for 10 minutes. Use anti-MYC or anti-UBP12 antibody to analyze immunoprecipitated products by western blot.

All research data is included in the article and/or SI appendix.

Thanks to Dr. Xiaofeng Cao from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences for providing ubp12-2w (GABI_244E11), ubp13-3 (SALK_132368) and ubp12-2w/13-3 double mutants and 35S::PIF7- from Dr. Lin Li of Fudan University, China FLASH genetically modified seeds. This research was supported by the Research Excellence and Technology Enterprise Campus (CREATE) program of the National Research Foundation (NRF) of the Prime Minister’s Office of Singapore. The disruptive and sustainable technology of agricultural precision is an interdisciplinary research team of the Singapore-MIT Research and Technology Center Alliance, supported by the Singapore Prime Minister’s Office NRF under its CREATE program.

Author contributions: YZ and N.-HC design research; YZ, S.-HP and MYS conducted research; YZ, S.-HP and N.-HC analysis data; YZ and N.-HC wrote this paper .

The author declares no competing interests.

This article is directly contributed by PNAS.

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