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. 2022 Sep 28:13:998961.
doi: 10.3389/fpls.2022.998961. eCollection 2022.

Interaction of BES1 and LBD37 transcription factors modulates brassinosteroid-regulated root forging response under low nitrogen in arabidopsis

Shuli Chai  1 , Junhua Chen  1 , Xiaolan Yue  1 , Chenlin Li  1 , Qiang Zhang  1 , Víctor Resco de Dios  1   2 , Yinan Yao  1 , Wenrong Tan  1
Affiliations

Interaction of BES1 and LBD37 transcription factors modulates brassinosteroid-regulated root forging response under low nitrogen in arabidopsis

Shuli Chai et al. Front Plant Sci. .

Abstract

Brassinosteriod (BR) plays important roles in regulation of plant growth, development and environmental responses. BR signaling regulates multiple biological processes through controlling the activity of BES1/BZR1 regulators. Apart from the roles in the promotion of plant growth, BR is also involved in regulation of the root foraging response under low nitrogen, however how BR signaling regulate this process remains unclear. Here we show that BES1 and LBD37 antagonistically regulate root foraging response under low nitrogen conditions. Both the transcriptional level and dephosphorylated level of BES1, is significant induced by low nitrogen, predominantly in root. Phenotypic analysis showed that BES1 gain-of-function mutant or BES1 overexpression transgenic plants exhibits progressive outgrowth of lateral root in response to low nitrogen and BES1 negatively regulates repressors of nitrate signaling pathway and positively regulates several key genes required for NO3 - uptake and signaling. In contrast, BES1 knock-down mutant BES1-RNAi exhibited a dramatical reduction of lateral root elongation in response to low N. Furthermore, we identified a BES1 interacting protein, LBD37, which is a negative repressor of N availability signals. Our results showed that BES1 can inhibit LBD37 transcriptional repression on N-responsive genes. Our results thus demonstrated that BES1-LBD37 module acts critical nodes to integrate BR signaling and nitrogen signaling to modulate the root forging response at LN condition.

Keywords: BES1 transcription factor; LBD37; brassinosteriods; low nitrogen; root forging response.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
LN induces transcript level and dephosphorylation of BES1. (A) The relative expression of BES1 transcripts in the indicated tissues of wide-type plants (Col-0). 5-d-old Col-0 seedlings that grown on 1/2MS medium were transferred to medium with 0.5mM KNO3 (LN) or 10mM KNO3 (HN) for 5 weeks and then the different tissues (leaves, root, shoot and flower) were collected for gene expression analyses. The relative gene expression levels were presented as values relative to that of leaf sample. (B) Regulation of BES1 expression paten by nitrogen supply. Col-0 seedlings were grown on medium with various NO3 − concentrations (10, 100, 500, 1,000, 3,000, 5000, 7000 and 10000 μM) for 7 days and the BES1 transcript levels in root tissues were analyzed by qRT-PCR. The relative gene expression levels were presented as values relative to that of the sample from 10000 μM NO3 −. (C, D) Time courses of BES1 expression in response to N availability. 7day-old Col-0 Seedlings grown on medium were treated by LN (C) or HN (D) for indicated times. The transcript levels of BES1 were assessed in the roots by qRT-PCR. (E) Immunoblots showing the abundance of BES1 proteins in the root tissues of 1-week-old seedlings treated at HN (top panel) or LN (bottom panel) for the indicated time periods. BES1 was detected by western blot using anti-BES1 antibody. ACTIN2 was used as a loading control. pBES1 represents phosphorylated BES1. At least three times were repeated. (F) Quantitative results for Immunoblots results in (E). The initial ratio of pBES1/BES1 of each treatment were set as ‘1’. In all gene expression analyses, ACTIN2 was used as an internal standard. Data are presented as the mean ± SD (n = 3). In all results, different lowercase letters represent statistically significant differences (p < 0.05, two-way ANOVA).
Figure 2
Figure 2
BES1 promotes the lateral root growth and development at LN. (A) Seedling phenotypes of Col-0, BES1-RNAi, bes1-D and 35S:BES1-GFP. 5-day-old seedlings were precultured on medium containing 10 mM NO3 - and then transferred to HN medium or LN medium for another 9 days for quantification of primary root length (B), average lateral root length (C), and lateral root number (D). Error bars indicate the s.d. (n = 10-15). Different Lowercase letters above the bars indicate statistically significant differences between samples (p < 0.05, two-way ANOVA). Scale bars, 1cm.
Figure 3
Figure 3
Expression of nitrate signaling repressors, LBD37/38/39 (A-C), and LN-responsive genes NRT1.1 (D), NRT2.1 (E) and ANR1 (F) in the roots of 7-day-old Col-0, BES1-RNAi, bes1-D and 35S:BES1-GFP at LN. The expression levels of genes in the Col-0 seedlings were set to 1.00. Error bars represent s.d. of three biological repeats. Different Lowercase letters above the bars indicate statistically significant differences between samples (p < 0.05, one-way ANOVA).
Figure 4
Figure 4
ChIP-qPCR showing BES1 binds to BRRE and E-box of LBD37/38/39 promoters in a BR- and LN-enhanced manner. (A-C) Promoter analysis of LBD37/38/39 promoters (top panels) and ChIP-qPCR (bottom panels). The E-box elements were showed as red circles and BRRE elements were showed as blue circles in the LBD37/38/39 promoters. P1-P5 represent the perfect E-box or BRRE sites. The numbers indicate relative position of the E-box or BRRE elements in the respective promoters relative to the ATG start codon. The ChIP assays were performed with chromatin prepared from 14-d-old Col-0 and 35S:BES1-GFP transgenic plants. The enrichment of DNA was calculated as the ratio between IP and Input, and TA3 was used as internal control. The enrichment level of Col-0 was set to 1.00. (D) The ChIP-qPCR assays were performed with chromatin prepared from Col-0 and BES1-RNAi plants using an anti-BES1 antibody-coupled Agarose beads (IP). The enrichment of DNA was calculated as the ratio between IP and Input, and TA3 was used as internal control. The enrichment level of BES1-RNAi was set to 1.00. (E) BR promotes the binding affinity of BES1 to LBDs promoters. 12-day-old Col-0 and 35S:BES1-GFP seedlings with BL or BRZ treatment for 2 days were used to ChIP-qPCR assays using anti-GFP-Agarose beads. The enrichment of DNA was calculated as the ratio between IP and Input, and TA3 was used as internal control. The enrichment level of Col-0 was set to 1.00. (F) LN enhances the binding of BES1 to the promoters of LBDs. 7-old-day Col-0 and 35S:BES1-GFP seedlings grown on normal medium were transferred to HN or LN medium for another 7 days and then the roots of these plants were collected for ChIP-qPCR assays using anti-GFP agarose. The enrichment of DNA was calculated as the ratio between IP and Input, and TA3 was used as internal control. The enrichment level of BES1-RNAi was set to 1.00. In the above results, error bars represent standard deviations of three biological repeats and Different Lowercase letters above the bars indicate statistically significant differences between samples (p < 0.05, two-way ANOVA).
Figure 5
Figure 5
BES1 interacts with LBD37. (A) Yeast two hybrid assays. The pGADT7- (shown as GAD) and pGBKT7- (shown as GBK) derivative constructs were shown on the left side. The interaction was selected on the Leu, Trp, His, and Ade dropout medium (-LWHA). (B) HIS pull-down assay. *represents non specific band. BES1 was fused with HIS tag, LBD37 was fused with MBP tag. After coincubation of HIS-BES1 and MBP-LBD37, the proteins were immunoprecipitated with HIS agarose beads. The pull-down protein was detected with anti-MBP antibody. MBP was used a negative control. (C) Split-Luc assays showing BES1 interacting with LBD37 in vivo. BES1 and LBD37 were fused with nLUC and cLUC of luciferase, respectively. Empty vectors were used as negative control. (D) Co-immunoprecipitation (Co-IP) assays. Total proteins from 35S:FLAG-HA or 35S:FALAG-HA-LBD37 plants were immunoprecipitated using anti-HA beads and detected by immunoblotting with anti GFP or anti BES1 antibody, respectively. The input of proteins for immunoprecipitation was shown on left panel.
Figure 6
Figure 6
BES1 inhibits LBD37 binding to its target gene. (A) EMSA showing BES1 inhibits the DNA binding ability of LBD37. The top panel showed the DNA sequences used as DNA probe containing the normal or mutated form of LOB site (GCGGCG). The bottom panel showing LBD37 bind to NRT2.1 promoter and addition of BES1 with LBD37 suppresses the DNA binding ability of LBD37. (B) ChIP-qPCR assay showing BES1 inhibits LBD37 binding to LOB site in vivo. The wild-type (Col-0), 35S:FLAG-HA-LBD37/Col-0, 35S:FLAG-HA-LBD37/bes1-D and 35S:FLAG-HA-LBD37/BES1-RNAi were used to prepare chromatin and ChIP assay with anti-HA Agarose beads. The ChIP products were used to detect promoters containing LOB site. error bars represent standard deviations of three biological repeats and different Lowercase letters above the bars indicate statistically significant differences between samples (p < 0.05, two-way ANOVA). (C) The transient transcriptional assay showing BES1 inhibits the binding activity of LBD37 on NRT2.1 promoter. The top panel showed the structures of pNRT2.1:Luc and two effector constructs. BES1, LBD37 and REN are driven by 35S promoter. The bottom panel showed the LUC/REN ratio calculated by pNRT2.1:Luc relative to the REN activity. Error bars represent SDs of three biological repeats. Different Lowercase letters above the bars indicate statistically significant differences (p < 0.05) determined by one-way ANOVA.
Figure 7
Figure 7
BES1 suppresses the inhibitory effect of LBD37 on lateral root growth in LN. (A) Seedling phenotypes of 35S:FLAG-HA-LBD37/Col-0, 35S:FLAG-HA-LBD37/bes1-D and 35S:FLAG-HA-LBD37/BES1-RNAi plants. 5-day-old seedlings were precultured on medium containing 10 mM NO3 - and then transferred to HN medium or LN medium for another 9 days for quantification of primary root length (B), average lateral root (LR) length (C), and lateral root (LR) number (D). Error bars indicate the s.d. (n=10-15). Different Lowercase letters above the bars indicate statistically significant differences between samples (p < 0.05, two-way ANOVA). (E-F) Expression analysis of LN-responsive genes NRT1.1 (E), NRT2.1 (F) and ANR1 (G) in the roots of 7-day-old Col-0, 35S:FLAG-HA-LBD37/Col-0, 35S:FLAG-HA-LBD37/bes1-D and 35S:FLAG-HA-LBD37/BES1-RNAi plants at LN. The expression levels of genes in the Col-0 seedlings were set to 1.00. Error bars represent s.d. of three biological repeats. Different Lowercase letters above the bars indicate statistically significant differences between samples (p < 0.05, one-way ANOVA).
Figure 8
Figure 8
Proposed model to illustrate how BES1-LBDs cascade integrates BR and LN response to precisely fine-tune lateral root growth. Under HN conditions, high concentration of nitrate inhibits BR level and promotes the phosphorylation of BES1 (inactivation) through an unknown mechanism. Inactivated BES1 failed to interact with LBD37/38/39 or suppresses their expression, resulting in binding of LDBs to LN-responsive genes promoters and reduction of their transcript level and thus inhibitory of lateral root growth. At mild nitrogen deficiency, LN induces the accumulation of BES1 transcript and dephosphorylation via promoting BR biosynthesis. Activated BES1 can suppress the expression and DNA binding ability of LBDs, and thus enhance the expression of LN-responsive genes, which stimulate lateral root growth and development in response to LN, commonly described as nitrogen foraging response.
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