ARABIDOPSIS HOMOLOG OF TRITHORAX1 impacts lateral root development by epigenetic regulation of targets involved in root system architecture

Abstract

Developmental processes are regulated at multiple levels, including the epigenetic level. Among the epigenetic factors, histone H3 lysine 4 (H3K4) methyltransferases contribute to active transcription of target genes, and here, we explored how the H3K4 methyltransferase ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) affects Arabidopsis thaliana lateral root (LR) primordium (LRP) morphogenesis. We examined LR development in a loss-of-function null mutant (atx1-1) and a mutant affected in the ATX1 catalytic domain (atx1setm) through bright-field and long-term time-lapse confocal microscopy, transcriptomics, and chromatin immunoprecipitation. LRP morphogenesis in both mutants was severely abnormal, resulting from altered principal growth directions, and was accompanied by extended cell cycle durations and slower transitions between LRP stages. Among the differentially expressed genes downregulated in atx1setm, the most enriched Gene Ontology categories were cell wall organization and H₂O₂ metabolism, the latter of which included PEROXIDASE35 (PRX35). The LRP morphogenesis abnormalities were similar in prx35 and atx1 mutants. Both the deposition of H3K4me3 at the PRX35 promoter and the PRX35 expression in the atx1setm mutant were significantly reduced. Our results reveal a link between LR development and a redox homeostasis controlled by ATX1 at epigenetic level by maintaining active transcription of PRX35 and thereby impacting root system formation.

Keywords: cell cycle; epigenetic regulation; lateral roots; live imaging; methyltransferase activity; morphogenesis; reactive oxygen species; transcriptomics.

Fig. 1

Histone methyltransferase activity of ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) is essential for Arabidopsis thaliana root development. (a) Wild‐type (Wassilewskija (Ws)), atx1‐1, and atx1‐1; ATX1‐setm (atx1setm) seedlings at 8 d after germination (dag). Bars, 5 mm. (b) Primary root growth dynamics of Ws, atx1‐1, and atx1setm. Values are means ± SD (n = 25–31). Combined data of three independent experiments are shown; asterisks indicate statistical differences between each of the mutants and the Ws and among the two mutant alleles (*, P < 0.001, one‐way ANOVA).

Fig. 2

Quantitative analysis of Arabidopsis thaliana lateral root (LR) formation in the atx1‐1 and atx1setm mutants in comparison with the wild‐type (Wassilewskija (Ws)) in seedlings 8 d after germination. Lateral root primordia (LRPs) were analyzed within branching and LR formation zones, depicted at the bottom. All lateral root initiation (LRI) events were compared separately for a subsample of atx1setm plants that formed LRs and for a subsample in which LRs did not emerge. All other data include atx1setm subsample with LRs. Only statistically significant differences are indicated for each parameter using pairwise comparison among different genotypes by the Holm–Sidak method at ***, P < 0.001 and **, P < 0.01. Mean values (magenta) are shown; boxes span from 25 to 75% percentiles, and whiskers range from a minimum to a maximum value. Combined data of three independent experiments are shown: n ranges from 26 to 28 seedlings.

Fig. 3

ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) is required for correct timing during lateral root (LR) primordium (LRP) morphogenesis in Arabidopsis thaliana. (a) Representative images of LRPs in the wild type (Wassilewskija (Ws)), atx1‐1, and atx1setm mutants. Bar, 20 μm; all panels are at the same magnification. (b) Percentage of abnormal LRPs and LRs in Ws, atx1‐1, and atx1setm seedlings at 8 d after germination. Combined data of three independent experiments; mean ± SD, total n range from 26 to 28 seedlings. (c–e) Analysis of cell proliferation dynamics in Ws and atx1‐1, both transformed with the pTCTP1::3x mVENUS construct, using time‐lapse experiments. (c) Principal growth directions (PGDs) were analyzed in Ws and atx1‐1 mutant LRPs over a 37‐h period after the time‐lapse experiment started. During this time, depicted LRPs reached Stages VII and III for Ws and atx1‐1, respectively. The progeny of each cell was followed from Stage I LRPs, and related color‐coded clones are shown. Yellowish arrows indicate the PGDs of each clone, and black arrows indicate the average PGD of all the clones. Note that the latter in atx1‐1 is not perpendicular to the parent root growth axis, as in Ws. (d) Analysis of cell cycle duration in the developing LRPs in Ws and the atx1‐1 mutant. Data show means ± SD. Individual values are shown. A statistical significance (***) is at P < 0.001, Mann–Whitney rank sum test; wild‐type n = 80 cells from three LRPs, developed from Stages I to VII; each LRP was from different seedlings; in atx1‐1, n = 46 cells from four LRPs developed from Stages I to V; each LRP was from a different seedling. (e) Analysis of LRP development showing the dynamics of cell number increase at each developmental stage. The data in (d) and (e) were extracted by tracking the progeny of each cell in the central portion of the master cell file from each LRP: n = 78 cells from three LRPs in Ws and 44 cells from four LRPs in the atx1‐1 mutant. Time‐lapse experiments started at Stage I LRPs and spanned 72 h for the mutant; cell tracking for Ws was performed only until Stage VII that ranged between 34 and 40 h from the beginning of the experiment.

Fig. 4

Transcriptomic analysis of the atx1setm mutant of Arabidopsis thaliana. (a) Volcano plot displaying upregulated (blue) and downregulated (red) genes in the atx1setm mutant compared with the wild type (Ws); fold change ≥ 3, P‐value ≤ 0.0001. (b) Gene Ontology (GO) enrichment analysis of downregulated genes; circle size is proportional to the number, or count, of occurrences for each biological process and filled with a color scale according to the −Log₁₀ P‐value of the enrichment, which is given on the X‐axis. (c) Reads per kilobase per million mapped reads (RPKM) Z‐score of genes relevant for root system architecture. Genes reported as directly related to LRP development are indicated with an asterisk. Each column corresponds to a biological replicate of the indicated genotype. (d) RPKM Z‐score of genes differentially expressed in the atx1setm mutant, which were previously reported as being preferentially expressed in the pericycle (Parizot et al., 2012).

Fig. 5

Phenotype of the prx35‐2 mutant of Arabidopsis thaliana. (a) Primary root growth of wild‐type (Columbia‐0 (Col‐0)) and prx35‐2 seedlings. Mean ± SE; * indicates statistically significant difference for root lengths at P = 0.020 and P = 0.049, Student's t‐test, three and 8 d after germination, respectively. (b) Root apical meristem (RAM) length in Col‐0 and prx35‐2 seedlings. Two independent experiments. n = 21–22. Mean ± SD, * indicates a statistically significant difference at P = 0.008 (Student's t‐test). (c) Quantitative analysis of lateral root (LR) formation in Col‐0 and prx35‐2 seedlings. In all cases, P > 0.05, Student's t‐test; all data and mean values are shown, boxes span from 25 to 75% percentiles, and whiskers range from a minimum to a maximum value. (d) Percentage of lateral root primordia (LRPs) by stage in Col‐0 and the prx35‐2 mutant in the LR formation zone. Two independent experiments, n = 19–20. Mean ± SE. No statistically significant difference was observed (P > 0.05, Student's t‐test). All results are for seedlings at 8 d after germination.

Fig. 6

Lateral root (LR) primordium (LRP) morphogenesis in the prx35‐2 mutant of Arabidopsis thaliana. (a) Percentage of abnormal LRPs in the LR formation zone wild‐type (Columbia‐0 (Col‐0)) and prx35‐2 seedlings treated or not with 2 mM H₂O₂. Seedlings at 5 d after germination were transferred to a medium supplemented with 2 mM H₂O₂ where they grew for three additional days. Two independent experiments were performed; n = 19–22. (b) LRPs in the Col‐0. (c–e) Abnormalities in LRP formation in the prx35‐2 mutant. All results are for seedlings at 8 d after germination. Bar, 20 μm (the same for (b–e)). (f) Cladogram of some members of the A. thaliana peroxidase protein family related to LR development.

Fig. 7

ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) promotes PRX35 expression in Arabidopsis thaliana. (a) Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) quantification of transcript abundance in the wild type (WT) (Wassilewskija (Ws) and Columbia‐0 (Col‐0)), atx1setm mutant, and 35S::ATX1overexpressing (ATX1OE) lines in roots at 8 d after germination. Mean data ± SD are from two biological replicates with three technical replicates. Significant difference was determined by Student's t‐test (**, P = 0.001; *, P = 0.002). (b) Schematic diagram of PRX35 and PCR amplicons used for chromatin immunoprecipitation (ChIP)‐qPCR. TSS: transcription start site; amplicon positions are indicated as F + R (for forward and reverse primers of each amplicon). (c) Histone methylation profile. ChIP assays to determine the presence or absence of the histone H3 lysine‐4 trimethylation mark (H3K4me3) at three different regions within the PRX35 locus. Data are means ± SD. Each ChIP experiment was performed for two biological replicates (each with two technical replicates). Statistical significance (****) was determined by a Brown–Forsythe test (P‐value < 0.0001).