A selective autophagy cargo receptor NBR1 modulates abscisic acid signalling in Arabidopsis thaliana.

The plant selective autophagy cargo receptor neighbour of breast cancer 1 gene (NBR1) has been scarcely studied in the context of abiotic stress. We wanted to expand this knowledge by using Arabidopsis thaliana lines with constitutive ectopic overexpression of the AtNBR1 gene (OX lines) and the AtNBR1 Knock-Out (KO lines). Transcriptomic analysis of the shoots and roots of one representative OX line indicated differences in gene expression relative to the parental (WT) line. In shoots, many differentially expressed genes, either up- or down-regulated, were involved in responses to stimuli and stress. In roots the most significant difference was observed in a set of downregulated genes that is mainly related to translation and formation of ribonucleoprotein complexes. The link between AtNBR1 overexpression and abscisic acid (ABA) signalling was suggested by an interaction network analysis of these differentially expressed genes. Most hubs of this network were associated with ABA signalling. Although transcriptomic analysis suggested enhancement of ABA responses, ABA levels were unchanged in the OX shoots. Moreover, some of the phenotypes of the OX (delayed germination, increased number of closed stomata) and the KO lines (increased number of lateral root initiation sites) indicate that AtNBR1 is essential for fine-tuning of the ABA signalling pathway. The interaction of AtNBR1 with three regulatory proteins of ABA pathway (ABI3, ABI4 and ABI5) was observed in planta. It suggests that AtNBR1 might play role in maintaining the balance of ABA signalling by controlling their level and/or activity.


Results
AtNBR1 overexpression changes the gene expression profiles of both shoots and roots. Several NBR1-OX and nbr1-KO lines were obtained and verified by testing for the presence of the corresponding transcripts and proteins ( Supplementary Fig. S1). A large set of differentially expressed genes (DEGs) were identified when the transcriptomes of 4-week-old OX7.5 and WT plants were compared, despite the lack of obvious morphological differences between the lines under the applied growth conditions (not shown). DEGs were defined as those with at least a 1.5-fold difference (p ≤ 0.05) relative to WT. In shoots, 294 of the 510 DEGs were upregulated, while 216 were downregulated (Supplementary Table S1), whereas in roots 416 of 919 DEGs were upregulated and 503 were downregulated (Supplementary Table S2). Remarkably, there were very few DEGs common to the two plant parts, prompting us to analyse the two data sets separately (Fig. 1). Among the GO categories associated with DEGs in shoots, 'response to stress' and 'response to stimulus' were over-represented among both up-and down-regulated genes, whereas 'regulation of gene expression' , 'transcription factor activity' , 'DNA binding' and 'regulation of metabolic and cellular processes' were only associated with genes upregulated in shoots ( Supplementary Fig. S2). In roots, upregulated genes were associated with the categories 'mitochondrion' and 'macromolecular complex' , whereas the categories 'translation' , 'gene expression' and 'nitrogen compound metabolic processes' were strongly associated with downregulated genes (Supplementary Fig. S2). Thus, AtNBR1 overexpression affected shoots and roots differently and seems to influence multiple cellular processes and functions.
In order to gain insight into the regulatory networks responsible for the observed changes in the transcriptome of the OX7.5 line overexpressing AtNBR1, we analysed known interactions of DEGs from the shoots and roots. Looking for putative regulatory hubs, we identified 64 nodes that interacted with at least 5 DEGs (Supplementary  Table S3). However these hubs also had the highest total numbers of interactions, so we looked for hubs that were enriched in interactions with DEGs in order to identify hubs specifically involved in transcriptional responses to AtNBR1 overexpression. The selection was made on the basis of the ratio of the number of interacting DEGs to the total number of interacting nodes (Supplementary Table S4), and the calculated enrichment values were plotted against the number of interacting DEGs ( Supplementary Fig. S3). This analysis identified the four hubs that were most enriched in interactions with DEGs: ABI5 (AT2G36270; bZIP transcription factor), RGL3 (AT5G17490; DELLA subfamily member), MYB49 (AT5G54230; MYB domain transcription factor) and AO (AT5G14760; aspartate oxidase catalysing the first key step of NAD+ biosynthesis). When the network of these four hubs was visualised along with interacting DEGs, two more putative regulatory hubs emerged: SR1 (AT5G01820; serine/ threonine protein kinase 1, SnRK3.15; CIPK14) and AIB1 (AT1G10585; bHLH transcription factor) (Fig. 2). Both SR1 and AIB1 have been shown to be a part of the SnRK3-related abscisic acid (ABA) signalling network 55 . Transcript analysis revealed that many ABA upregulated genes 56 were also upregulated by NBR1 overexpression, particularly in shoots (Fig. 3), whereas ABA downregulated genes were much less affected by NBR1 overexpression ( Supplementary Fig. S4). It should be emphasised that although all DEGs were used for network construction, shoot DEGs were mainly responsible for the proposed association between AtNBR1 and ABA response, further indicating that AtNBR1 overexpression affects the shoot and root transcriptomes differently.
Changes in expression of ABA-related genes in shoots and roots of OX7.5, KO1 and KO3 lines in comparison to the WT are shown in Supplementary Table S5. The expression of genes encoding enzymes involved in ABA biosynthesis or ABA catabolism was not changed significantly, except 4-fold downregulation of UGT71B7 in shoots of KO1. This result suggested that ABA metabolism was rather unaffected in OX and KO lines. To verify this conclusion, we assayed the level of endogenous ABA and its degradation products, PA and DPA in shoots of 4-week-old plants. The levels of PA and DPA were significantly lower in both OX lines than in the WT and KO lines, however, the levels of ABA were similar in all lines (Fig. 4, Supplementary Fig. S5). Therefore, we conclude that the gene expression changes observed in the shoots of OX7.5 did not results from increased ABA production or accumulation but from enhanced ABA sensing and/or signalling.
Differences in seed germination suggest that OX lines have increased ABA sensitivity. Since ABA is known to regulate seed germination, we decided to check if the presumed enhanced ABA signalling in OX lines would have an impact on this process. Indeed, germination was delayed in OX seeds relative to WT seeds, but only in freshly collected batches of seeds (Fig. 5A). Such differences in germination disappeared when seeds were stored for as little as 10 days and when fresh seeds were kept at 4 o C for 3-5 days before sowing. Besides, the OX lines were slightly more sensitive to ABA during germination. These results suggest an increase in seed dormancy due to AtNBR1 overexpression. Subsequently we monitored expression of several ABA related transcripts in fresh and cold-stratified seeds. We found that many of them were misregulated in both fresh and cold-stratified seeds of OX7.5 and OX2.3.5 lines (Fig. 5B, Supplementary Fig. S6). In some cases (PGIP1, PGIP2, EM1, ARR15 and ARR4) the changes in gene expression were similar in the two OX lines, but in others the differences between the lines were quite substantial. For example, ABI4 was similarly expressed in the WT and OX2.3.5 lines, but strongly enhanced in cold-stratified OX7.5 seeds. Since it was difficult to correlate the observed transcriptional changes with germination delays observed in fresh seeds from the OX lines, so the exact role of AtNBR1 during seed germination requires further investigation. Surprisingly, the stratified seeds from OX lines had lower level of ABA than those from WT (Fig. 5C, Supplementary Fig. 7). These results suggest that AtNBR1 overexpression enhances ABA signalling.
Reduction of stomatal opening in OX lines. Endogenous ABA is considered a main regulator of stomatal movement. Examination of stomatal aperture in seedlings in the absence of ABA indicated that the lines overexpressing AtNBR1 had significantly reduced stomatal aperture than the WT and KO lines (Fig. 6A, Supplementary  Fig. S8). This result supports our assumption that NBR1 overexpression enhances ABA-related responses. Interestingly, the application of ABA stimulated stomatal closure in all investigated lines, however, the difference in the KO lines between the ABA-treated and non-treated material was statistically insignificant. These results suggest that NBR1 might be also involved in modulation of ABA-dependent stomata movement. In addition, a high concentration of yellow fluorescent protein (YFP) in guard cells of the transgenic line overexpressing NBR1-YFP points to an involvement of AtNBR1 in stomatal movement (Fig. 6B).
Enhanced lateral roots initiation in KO lines. The OX and KO lines did not show significant differences from the WT in primary roots growth in the absence or presence of 10 and 20 µM ABA (results not shown). However, some interesting differences were found in lateral root formation in 10-day-old seedlings grown in a hydroponic medium. Several sites of lateral root initiation (1-4 per root) were found in the KO lines, but no such sites were observed in the WT and OX lines. The location of initiation sites varied, with no apparent rule or preferred location (Fig. 7A). No differences between the lines were observed in this experiment with respect to primary root length (Fig. 7B), thus the increased formation of lateral roots in the nbr1-KO lines cannot be attributed to differences in the size or developmental stage of the primary roots.
NBR1 interacts with regulators of ABA signalling. The response to ABA is regulated by a complex network including transcriptional factors, of which the best known are ABI3, ABI4 and ABI5 (ABA-insensitive 3, 4 and 5, respectively), whose direct targets have already been identified 57,58 . Transcript analysis of genes directly regulated by ABI3, ABI4 and ABI5 indicated that only some of them were differently expressed in OX7.5; based on these results it is impossible to identify the (single) factor responsible for the gene expression changes in OX7.5. Given that AtNBR1 acts as a selective autophagy cargo receptor it seemed probable that it would interact physically with some transcriptional regulators of ABA signalling. We therefore tested whether ABI3, ABI4 and ABI5, interact with AtNBR1 in a bimolecular fluorescence complementation (BiFC) in planta experiment. Indeed, AtNBR1 can interact with all of them. Interestingly, binding to ABI3 and ABI4, in contrast to ABI5 binding, did  www.nature.com/scientificreports www.nature.com/scientificreports/ not require the ubiquitin-associated (UBA) domain of AtNBR1 (Fig. 8A) that is involved in binding to Ub-tagged proteins 28 . This implies that binding of NBR1 to ABI5 probably requires prior ubiquitination of ABI5, whereas binding of NBR1 to ABI3 and ABI4 appears to be independent of ubiquitination. The binding of ABI3 and ABI4 to both forms of NBR1 (full-length NBR1 and NBR1ΔUBA) was confirmed by the pull-down assay using the recombinant proteins purified from E.coli cells (Fig. 8B, Fig. S9). The pull-down assay did not confirm the binding of ABI5 to NBR1 highlighting the importance of potential ubiquitination for such interaction. Further clarification of the significance and molecular details of the NBR1-ABI3, NBR1-ABI4 and NBR1-ABI5 interactions and the physical binding of NBR1 to other proteins of the ABA signal transduction pathway is required.

Discussion
The aim of this study was to characterise the NBR1-OX lines and compare their leaf and root transcriptomes with those of the WT. We report for the first time the existence of crosstalk between AtNBR1 and ABA signalling (Fig. 2). Two hubs of the interaction network of the differentially expressed genes (DEGs) in OX7.5 are involved in ABA signalling pathway: (i) ABI5 (AT2G36270, bZIP TF), which is the main regulator of ABA signalling and is also involved in controlling seed dormancy and germination 38,59 and (ii) AIB1 (AT1G10585, bHLH TF), which has been identified as an element of the SnRK3 (SnRK3.15/SnRK3.22) network and as a central hub of the ABA-regulated transcriptional network 55 . In fact, other elements of the SNRK3 network were also identified as www.nature.com/scientificreports www.nature.com/scientificreports/ hubs in our interaction network: SR1 (AT5G01820, SnRK3.15), RGL3 (AT5G17490, DELLA family), and MYB49 (AT5G54230). RGL3 integrates ABA, auxin and gibberellin (GA) signalling [60][61][62][63] , whilst MYB49 has recently been linked with cadmium resistance 64 .
We then examined the potential effects of NBR1 overexpression on ABA-regulated processes, such as seed germination and regulation of stomata aperture and root growth. Delayed germination of OX lines' fresh seeds (Fig. 5) could be attributed to their enhanced ABA sensitivity, despite displaying lower amount of ABA in the seeds (Fig. 5C). Stomatal closure was also significantly enhanced in three independent AtNBR1 overexpressors (Fig. 6A). In line with the positive role of AtNBR1in ABA signalling, the reduced expression of several ABA-up regulated genes were observed in shoots of two KO lines (Fig. 3). Moreover, the careful microscopic investigation of the roots of seedlings grown in a hydroponic medium indicated the presence of several lateral root initiation sites (from 1 to 4 per plantlet) in all the seedlings from all three tested deletions lines (KO1-3) (Fig. 7). In contrast, www.nature.com/scientificreports www.nature.com/scientificreports/ such sites were almost absent from WT and OX seedlings grown under the same conditions (liquid medium). These results, along with the lack of evidence for an increase in ABA level in the shoots of OX lines, strongly suggest that AtNBR1 is involved in modulating ABA signalling. The OX lines showed enhanced ABA sensitivity, whereas ABA signalling seems to be reduced in KO lines, however it is yet unclear which stages of ABA regulation pathway are modulated by NBR1.
Published data suggest that complex interplay among regulatory factors is responsible for the tested phenotypes. The main regulators, ABI3, ABI4 and ABI5, have been linked to seed dormancy 54,65,66 , stomata closure 67 and initiation and growth of lateral roots 68,69 . Although the details of how AtNBR1 is involved in ABA signalling remain to be determined, the finding that AtNBR1 is capable of binding to ABI3, ABI4 and ABI5 in planta is the first report of such interaction (Fig. 8). It is unclear whether AtNBR1 affects the stability or function of these proteins and if it targets them at autophagosomes. ABI5 binds only a full-length AtNBR1, whereas ABI3 and ABI4 also binds truncated AtNBR1 (without UBA domains; NBR1ΔUBA), which suggests that ABI3 and ABI4 may not need to be ubiquitinated in order to interact with AtNBR1; it is possible that a different type of interaction from the classical 'ubiquitinated target -autophagy receptor' is involved. Despite of the interaction of AtNBR1 with ABI3, ABI4 and ABI5, we failed to detect significant changes of expression of direct targets of ABI3, ABI4 or ABI5 that had been identified before 57,58 . Instead, interaction network analysis indicated that the AIB1 (AT1G10585) and SnRK3 networks act as important for regulation of the OX DEGs. It is also possible that AtNBR1 acts as an autophagy receptor for other elements of the ABA pathway, for example it could stimulate autophagic degradation of some of the PP2C phosphatases that act as negative regulators of ABA signalling 40 .
In summary, our results indicate that OX lines are oversensitive to ABA. Based on the observed interaction between AtNBR1 and such ABA-related transcription factors as ABI3, ABI4 and ABI5, it is tempting to speculate that AtNBR1 is involved in maintaining the balance of the regulatory elements involved in ABA signalling, however, we cannot exclude that it could also fine tune other key elements of ABA sensing/signalling pathway. In a hypothetical model we propose that the observed enhancement of ABA signalling in the OX lines is due to direct modulation of the ABA pathway by AtNBR1 (Fig. 9). However, our recent results suggest that AtNBR1 overexpression enhances autophagy and modulates ribosome function 70 . Therefore, we cannot exclude that AtNBR1 www.nature.com/scientificreports www.nature.com/scientificreports/ overexpression can also modulate other aspects of TOR signalling, acting upstream or downstream of TOR, or at both levels.

Materials and methods
Plant material. The plant lines used in this study are described in Table 1 and Supplementary Fig. S1.
Plant growth conditions. The composition of the plant media, 0.5 x Hoagland (used in most experiments) and 0.5 x AB (used only for the microarray experiment with OX line) is provided in Supplementary material (Table S6). Seeds were dry sterilised as described previously 71 and placed on the solid medium with 0.8% Agargel (Sigma-Aldrich Sp. z o.o., Poland). All seeds were usually stratified (4 days, 4 o C) unless specified differently, and maintained in a growth chamber. Germinated seeds were counted in the indicated time (24,48,72   or NBR1 with a C-terminal deletion of 87 amino acids (NBR1ΔUBA) and ABI3, ABI4 or ABI5. Interaction was considered to have taken place if a YFP signal is detected. (B) Pull-down assays using GST and c-myc-tagged proteins expressed in E. coli. Precipitated proteins were detected by immunoblotting. Input shows the presence of the GST-tagged proteins in crude extracts. After affinity purification through the anti-c-myc gel with attached ABI3, ABI4, or ABI5, the GST-tagged proteins NBR1 and NBR1ΔUBA (lanes 1, 4 and 2, 5, respectively) are detected due to interaction. There is no interaction detected between ABI5 and NBR1 nor NBRΔUBA (lanes 7 and 8, respectively). The negative controls, SLIM1Nt with the GST-tag are shown in lanes 3, 6 and 9. The molecular marker (M) with the size (kDa) of the proteins is indicated. Expected sizes of the fusion proteins: c-myc-ABI3, 109 kDa; c-myc-ABI4, 66 kDa; c-myc-ABI5, 77 kDa; GST-NBR1, 103 kDa; GST-NBR1ΔUBA, 93 kDa; GST-SLIM1Nt, 62 kDa. The primary antibodies recognizing the GST and myc tags are indicated as α-GST and α-myc, respectively. (2020) 10:7778 | https://doi.org/10.1038/s41598-020-64765-z www.nature.com/scientificreports www.nature.com/scientificreports/ and Biophysics of the Polish Academy of Sciences. Raw data were normalised and subjected to a three-way ANOVA to create lists of genes expressed differently in the biological variants (fold changes of at least ±1.5, p < 0.05). Real-time quantitative polymerase chain reactions. The quantitative PCR (qPCR) was performed in a PikoReal Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) with Luminaris Color HiGreen qPCR Master Mix (Thermo Fisher Scientific). Actin 2 (At3g18780), a constitutively expressed gene, was used to normalise the quantity of total RNA present in each sample. Relative gene expression levels were calculated using the delta-delta Ct method. Operator variance was assessed using qPCR in 3 biological replicates, each in triplicate.  www.nature.com/scientificreports www.nature.com/scientificreports/ ABA assay. ABA level in seeds was assayed according to a previously published procedure 72 , using the Phytodetek ABA Test Kit (Agdia, Elkhart, IN, USA). The assay was done in 3 biological replicates, each performed as 3 technical replicates. ABA, PA and DPA level in shoots was assayed using the liquid chromatography coupled to mass spectrometry (LC-MS) method. The samples were flash frozen in liquid nitrogen. Plant hormones were extracted, purified and quantified according to reported procedures 73,74 . Briefly, samples were extracted with extraction buffer methanol/H 2 O/formic acid (15:4:1, v-v:v) supplemented with stable isotope-labelled internal standards (10 pmol per sample). The extracts were purified by solid phase extraction (SPE) using Oasis MCX cartridges (Waters, Milford, MA, USA). The SPE eluates were evaporated to dryness and pellets dissolved in 30 μl of 5% methanol in water (v/v). Quantification was done on an Ultimate 3000 high-performance liquid chromatograph (HPLC; Dionex, Bannockburn, IL, USA) coupled to a 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA, USA).) using isotope dilution method. Data processing was carried out with Analyst 1.5 software (Applied Biosystems).

Confocal microscopy.
All observations were made using a Eclipse TE2000-E inverted confocal microscope (Nicon Instruments Inc., New Yourk, NY, USA). Acidic speckles (such as autophagosmomes) were stained for 15 min with 1 μg/ml acridine orange (Invitrogen), and observed using a 543 nm laser (helium-neon laser; Melles Griot, NY, USA) and 605/75 filter. Stomata were observed in visual light and counted using the saved photographs. Figures for stomatal aperture are based on results from at least 3 independent seedlings (at least 5 slides per plant). The leaves used to assess stomata were floated on the buffer (50 mM KCl, 0.1 mM CaCl 2 , 10 mM MES pH6.5) for 90 min. To assess the influence of ABA on stomata aperture the leaves were then transferred to buffer supplemented with 10 µM ABA for an additional 90 min. Fluorescence from NBR1-YFP was observed in fresh leaves using a 488 nm laser (Sapphire 488-20 CDRH, Coherent, Santa Clara, CA, USA) and 515/30 filter. Bimolecular fluorescence complementation (BiFC), used to test protein-protein interactions in plants, was monitored 2 days after agroinfiltration of Nicotiana benthamiana leaves with Agrobacterium tumefaciens cells (strain GV3101) transformed with the indicated combination of plasmids. The plasmids encoded the N-terminal (pSITE-nEYFP-C1) or C-terminal (pSITE-cEYFP-C1) part of YFP 75 fused to the proteins or protein fragments being investigated. Bacteria were grown for 24 h at 28 °C in YEB medium supplemented with 10 μg/ml rifampicin and 50 μg/ml spectinomycin prior to agroinfiltration. Interactions were tested using a 488 nm laser (Sapphire 488-20 CDRH, Coherent, Santa Clara, CA, USA) and 515/30 filter. Seedlings for observation of lateral roots were stained with 0.05% Ruthenium red for 8 min and washed twice in H 2 O.
Pull-down experiments. The plasmids encoding the N-terminal GST or c-myc tag linked to the proteins being investigated are listed in Supplementary Table S8. All GST and c-myc-tagged proteins were expressed in Escherichia coli Rosetta (DE3). Total proteins containing c-myc fusion proteins (ABI3, ABI4, ABI5) were extracted from bacteria by sonication in RIPA buffer (ThermoFisher Scientific, Waltham, MA, USA) supplemented with Proteinase Inhibitor Cocktail (Sigma-Aldrich Sp. z o.o., Poland) and purified on EZview Red Anti-c-myc Affinity Gel (Sigma-Aldrich Sp. z o.o., Poland) according to manufacturer's protocol. Next, the crude extracts containing GST fusion proteins (NBR1, NBR1ΔUBA and control protein SLIM1Nt) were incubated with above prepared raisins in 4 °C overnight with gentle rocking. The raisins were washed 6 times with RIPA buffer, boiled with 2x SDS-PAGE gel loading buffer, subjected to SDS-PAGE and next immunoblotted using mouse monoclonal anti-GST IgG and anti-c-myc IgG (Sigma-Aldrich Sp. Bioinformatic tools. The statistical significance of group differences was assessed with the Fisher's Least Significant Difference (LSD) test of ANOVA or Tukey's Honest Significant Difference (HSD) test, using Statistica 12 software (StatSoft Polska Sp. z o.o., Poland). Differences were considered significant at p < 0.05. Function was ascribed to the obtained lists of genes using the AgriGO v2.0 analysis tool [http://systemsbiology.cau.edu. cn/agriGOv2/] with TAIR10_2017 background and Plant GO Slim list of GO terms. Interaction networks were analysed with Cytoscape (ver. 3.7.1) software 76 and visualised using the enhanced Graphics (ver. 1.2.0) plugin 77 .

Data availability
The transcriptomic data generated in this study are accessible through GEO Series accession numbers GSE122705 and GSE147124.