Function of histone H2B monoubiquitination in transcriptional regulation of auxin biosynthesis in Arabidopsis

The auxin IAA is a vital plant hormone in controlling growth and development, but our knowledge about its complicated biosynthetic pathways and molecular regulation are still limited and fragmentary. cytokinin induced root waving 2 (ckrw2) was isolated as one of the auxin-deficient mutants in a large-scale forward genetic screen aiming to find more genes functioning in auxin homeostasis and/or its regulation. Here we show that CKRW2 is identical to Histone Monoubiquitination 1 (HUB1), a gene encoding an E3 ligase required for histone H2B monoubiquitination (H2Bub1) in Arabidopsis. In addition to pleiotropic defects in growth and development, loss of CKRW2/HUB1 function also led to typical auxin-deficient phenotypes in roots, which was associated with significantly lower expression levels of several functional auxin synthetic genes, namely TRP2/TSB1, WEI7/ASB1, YUC7 and AMI1. Corresponding defects in H2Bub1 were detected in the coding regions of these genes by chromatin immunoprecipitation (ChIP) analysis, indicating the involvement of H2Bub1 in regulating auxin biosynthesis. Importantly, application of exogenous cytokinin (CK) could stimulate CKRW2/HUB1 expression, providing an epigenetic avenue for CK to regulate the auxin homeostasis. Our results reveal a previously unknown mechanism for regulating auxin biosynthesis via HUB1/2-mediated H2Bub1 at the chromatin level. Li Zhang et al. characterize ckrw2, cytokinin-induced root waving 2, as a mutant form of HUB1 in Arabidopsis, the gene required for histone H2B monoubiquitination. This study implicates the involvement of H2Bub1 in regulating auxin biosynthesis.

Results and discussion ckrw2 is an auxin-deficient mutant. To uncover more genes functioning in auxin biosynthesis or homeostasis, we previously established an effective genetic screening protocol for isolating auxin-deficient mutants by using CK-induced root curling (ckrc) or root waving (ckrw) as a phenotypic marker, in which the ckrw2 mutant was isolated as one of the so-called group III ckrw mutants 48 . When grown on the medium containing 0.01 μM trans-zeatin (tZ), ckrw2 mutant displayed a root waving phenotype and had a significantly reduced endogenous IAA level 48 . In addition to a number of pleiotropic abnormalities in leaves, seeds, root hair, apical hook, cutin, petals, and flowering time (Supplementary Fig. 1 a-l), typical low-auxin phenotypes, such as the reduced root length, smaller meristematic zone, shorter mature epidermal cell length ( Supplementary Fig. 1m-p) and weaker gravitropic response, were observed, which could be rescued by exogenous auxins 48 (Fig. 1a, b). In line with these, the mutant had weaker Dr5:GUS/GFP expression 49 or brighter DII-VENUS 50 fluorescence (Fig. 1c, d, Supplementary Fig. 2) in the transgenic root tips, indicating a lower auxin activity that was most likely caused by the endogenous auxin deficiency.
CKRW2 gene encodes a functional E3 ubiquitin ligase for histone H2Bub1. As ckrw2 was isolated from a mutant pool generated by T-DNA tagging 51 we initially did Tail-polymerase chain reaction (PCR) amplification, finding a T-DNA flanking sequence located between AT5G25425-AT5G25430, which showed no genetic linkage to ckrw2 mutation 48 . However, mapbased cloning combined with whole-genome resequencing (WGRS) identified a G > A substitution in the coding region of AT2G44950/HUB1, altering the tryptophan (aa 91) codon (TGG) to a stop codon (TAG) (Fig. 2a) in this gene. Both genetic allelic analysis (Fig. 2b) and the full rescue of the defective ckrw2 phenotypes ( Fig. 2c; Supplementary Fig. 3) by the fused HUB1::YFP-HUB1 confirmed the At2g44950/HUB1 identity of CKRW2 gene.
CKRW2/HUB1 activates the transcription of TSB1, WEI7, AMI1, and YUC7 through H2Bub1. To investigate how ckrw2 mutation affected auxin homeostasis, we measured the expression of a number of known auxin biosynthesis genes 2 by qRT-PCR ( Fig. 3a and Supplementary Fig. 6), detecting significant reductions in the expression levels of TRP2/TSB1, WEI7/ASB1, YUC7, and AMI1 (Fig. 3a), which functioning at distinct steps in the complex tryptophan/auxin biosynthetic pathways, either upstream of L-Trp biosynthesis (ASB1/WEI7 and TSB1/TRP2) 10,13 , or downstream of it in the IPA pathway (YUC7) or the proposed IAM pathway (AMI1) 2,59-62 . Subsequent ChIP analysis detected a significantly lower amount of H2Bub1-associated DNA fragment in the coding but not 5′ upstream promoter or untranscribed regions of the four affected genes in ckrw2 mutant (Fig. 3b, c), which is a prominent feature of histone H2Bub1 modification in affecting gene activity in the process of transcriptional elongation. These data demonstrate that YUC7, TSB1, WEI7, and AMI1 in the auxin biosynthesis pathways are targeted by HUB1-mediated H2Bub1.
To clarify the functional roles of each of the four affected genes in HUB1/2-mediated regulation on auxin homeostasis, we did the mutant analysis. Among their loss of function mutants, tsb1 and wei7 had slightly more obvious auxin-deficient phenotypes of ckrw2-like curling/waving primary roots with a reduced length, but yuc7 and ami1 had not ( Supplementary Fig. 8a-c), suggesting that WEI7 and TSB1 are the two major functional genes in H2Bub1-mediated regulation on auxin biosynthesis. These two genes, encoding the β-subunit of anthranilate synthetase (WEI7/ ASB1) complex and tryptophan synthase β (TSB1), respectively, are required for L-Trp biosynthesis 8 , and their roles in auxin biosynthesis 10,63 and/or root waving 64 have been confirmed. Consisting with that, ckrw2 tsb1 double mutant displayed very similar or the same phenotype of tsb1 single mutant (Supplementary Fig. 8a-c). Moreover, like wei7 and tsb1, L-TRP can rescue ckrw2, but not ckrc1 in Dr5:GUS expression and plant growth analyses ( Fig. 4c; Supplementary Fig. 9), indicating that CKRW2 affects auxin homeostasis through regulating WEI7/ ASB1 and TSB1 for L-Trp biosynthesis.
The expression of CKRW2 are induced by CK. The above results promoted us to study how the regulation of auxin biosynthesis by CK was related to H2Bub1. Some mechanisms have been revealed for CK-mediated regulation on auxin production, mostly via transcriptional factors 31,65,66 . The qRT-PCR results (Fig. 4a) and both of GUS staining to detect the pCKRW2:GUS expression ( Supplementary Fig. 8d) and YFP fluorescence intensity to detect the pHUB1::YFP-HUB1 expression (Supplementary Fig. 8e) showed that tZ treatment can stimulate HUB1/ CKRW2 expression and increase the HUB1 protein level ( Fig. 4b and Supplementary Fig. 10), leading to an increase of H2Bub1 activity. Consequently, the expression of TSB1 and WEI7 were significantly upregulated, which was not observed in ckrw2 mutant (Fig. 4d), revealing that this upregulation depends on CKRW2/HUB1 function.
In summary, our present studies reveal a mechanism at the chromatin level via H2Bub1 to control transcription of auxin biosynthesis genes. In this process, H2B proteins in the chromatin wrapped by the DNAs of auxin biosynthesis genes of WEI7 and TSB1 can be monoubiquitinated by HUB1/2 heterotetramer after the recruitment of UBC1/2 37 , activating the transcriptional elongation of these genes 33 . Significantly, such an epigenetic    3 Reduced expression of auxin synthesis genes in ckrw2 and analysis of H2Bub1 at these loci. a Relative transcription levels of TSB1, WEI7, AMI1, and YUC7 genes in roots. ACTIN8 was used as an internal control. b Diagram representing the genomic structure and regions analyzed by ChIP assays, arrows indicate ATG start codon sites, and bars labeled "a" or "b" represent regions amplified by RT-qPCR in ChIP analysis. c H2Bub1 deposition at specific loci. LCRa and FLCP4 were used as a negative and positive control, respectively (shown in Supplementary Fig. 7). Data are presented as mean ± SD, three independent biological experiments, the asterisk indicates a significant difference based on Student's t test with ** P < 0.01, 0.01 < * P < 0.05. ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-01733-x mechanism via H2Bub1 can be used by CK as an effective way to regulate auxin biosynthesis through up-regulating HUB1/2 expression.

Methods
Plant material and growth conditions. The conditions of germination and growth, as previously described 48 , at 25°C with a 16 h light/8 h dark photoperiod. For growth analyses, seedlings were grown on vertical MS (Murashige and Skoog) plates with 1.1% w/v agar supplemented with 10 g/L sucrose for 7 days.
To generate ckrw2/DR5: GUS and ckrw2 tsb1 mutants, the ckrw2 mutant was crossed with DR5: GUS and tsb1 mutant, respectively, and double homozygous mutants were obtained from the F2 generation.
Phenotype characterization. The degree of root curling/waving (DC) was calculated by dividing the distance between the two ends of seedlings' roots (L 0 ) by the length of their roots (L) 48 . Primary root length was measured after grown vertically on MS plates for 7 days 18,48 .
For biochemical complementation experiments, seedlings were grown on a medium containing 0.01 μM tZ with auxin (0.01 μM 2, 4-D), and phenotypic observation and statistics were performed after 7 days of vertical cultivation.
For the L-TRP experiment, the 7-day-old seedlings were transferred to MS plates with or without 0.25 mM L-TRP and cultured for 2 weeks, and then the phenotype was observed 67 .  Effects of 1 µM tZ on CKRW2/HUB1 expression and on the protein levels of YFP-HUB1 and H2Bub1. H2B was used as a loading control. c DR5:GUS activity showing the rescuing effect of 3 μM L-TRP on wei7 and ckrw2, but bot on ckrc1. Bar = 50 µm. d Comparison of relative expression levels of TSB1 and WEI7 genes between WT and ckrw2 mutant after tZ treatment. Data are presented as mean ± SD, three independent experiments, the asterisk b indicates a significant difference based on Student's t test ( ** P < 0.01, 0.05 > * P > 0.01), and the letters d indicate a significant difference at P < 0.05, according to ANOVA followed by Tukey's multiple comparison tests. ACTIN8 was used as an internal control (a, d). method using Agrobacterium tumefaciens (GV3101) 68 . The seeds of the transformants were stratified in 4°C for 3 days, sterilized with 0.1% mercuric chloride, washed with sterilized water, and then isolated on MS medium containing 25 mg/L hygromycin B. The seedlings were transferred to the soil until maturity.
For confocal microscopic analyses, 7-day-old seedlings were treated in propidium iodide (PI) solution (10 μg/mL) for 5 min (time can be adjusted according to the pre-experiment), then washed three times with ddH 2 O, and visualized at 600-640 nm for PI and 500-560 nm for green fluorescent protein (GFP)/VENUS on a confocal microscope (TCS SP8, Leica, Germany). The DR5: GUS /DR5:GFP/DII-VENUS signal intensity of the root tip containing the GUS/ GFP/VENUS signal (approximately to the first 200 µm from the root tip) was quantified by measuring the mean gray value with ImageJ 69 .
For detecting the effect of CK on HUB1, seedlings were grown on MS medium for 7 days and then transferred to a liquid medium containing 1 µM tZ for 6 h. And then GUS staining and fluorescence observation were performed.
RNA extraction and quantitative real-time PCR. RNA was isolated using Trizol (No. B511321, Sangon Biotech) and reverse-transcribed using a reverse transcription kit (RR047, TAKARA). Quantitative RT-PCR was performed in a Real-time System (Bio-RAD CFX96, America) using TB Green (RR820A, Takara), with primers listed in Supplementary Data 2. The auxin synthesis gene expression analysis was carried out using the primary roots of the seedling grown on the MS medium for 7 days.
Protein extraction and immunoblot analysis. In order to detect the protein levels of HUB1 and H2Bub1, 7-day-old seedlings of pHUB1::YFP-HUB1 and WT were treated with tZ for a different time, respectively. For protein extraction and immunoblot analysis, a previously used experimental procedure was followed 70 . H2B was used as a loading control. The immunoblot analysis was carried out using an anti-H2B antibody (ab1790, Abcam) at a concentration of 0.1 μg/mL, anti-H2Bub1 antibody (MM-0029, Medimabs) at a concentration of 3-5 μg per sample, and an anti-GFP antibody (M20004, Abmart) at a concentration of 0.2 μg/mL. The signal was detected by a chemiluminescent horseradish peroxidase substrate system (No. C500044, Sangon).
Chromatin immunoprecipitation (ChIP) assays. ChIP assays were performed as previously described 71 using 7-day-old seedlings, which were grown on MS medium. In brief, the seedlings were vacuum cross-linked in 1% formaldehyde for 10 min, then 0.125 M glycine was added to the vacuum for 5 min to stop the crosslinking. To obtain 200-1000 bp DNA fragments, sonicate chromatin solution 5 times (5 s on, 15 s off in each time) by 50% power. Chromatin was immunoprecipitated using a specific anti-H2Bub1 antibody (MM-0029, Medimabs) and then specific protein A-agarose (11418033001, Roche). After the IP complex was pulled down and washed, the DNA was reverse cross-linked and then extracted using the phenol/chloroform method. The ChIP experiment used an equal amount of sample and protein A-agarose without antibody as a control. The ChIP DNA was finally analyzed by qPCR with three independent biological replicates.
Statistics and reproducibility. All results are expressed as the means ± standard deviation. The numbers of samples and replicates of experiments were shown as mentioned in the figure legends. Comparisons between groups were determined using Student's t test (significant difference at 0.01 < * P < 0.05, ** P < 0.01, *** P < 0.001) or ANOVA followed by Tukey's multiple comparison test (significant difference at P < 0.05). All data were analyzed using GraphPad Prism 7 software.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
The nucleotide sequence of CKRW2 was submitted to GenBank, and the accession number is BankIt2414347 ckrw2 MW431056. All other source data are included in the article as supplementary data 1-2. Uncropped scans of Western blots are shown in Supplementary Information. The unique biological materials of ckrw2, ckrw2/Dr5:GUS, ckrw2/DII-VENUS are available upon request to our lab.
Received: 15 May 2020; Accepted: 13 January 2021; Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/.