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The plant specific Histone Lysine Demethylase MtPKDM9B mediates the root nodule symbiosis by controlling H3K27me3 levels and expression of symbiotic genes | bioRxiv
Under nitrogen limiting conditions, legume plants interact with nitrogen fixing bacteria known as rhizobia, resulting in the formation of a new organ, the nodule. This process is accompanied by dramatic changes in gene expression, which operate at different levels. A previous study revealed that histone methylation is differentially modulated during nodulation. However, the histone methyl transferases and demethylases involved in this modulation have not been characterized. In this study we report the identification of the Medicago truncatula putative histone lysine demethylase MtPKDM9B, which is subject to alternative splicing (AS), and the differential modulation of AS variants at translational level during nodule symbiosis. Knockdown of MtPKDM9B impaired infection by rhizobia, nodule development, bacterial viability and the expression of the leghemoglobin coding gene MtLHB1. MtPKDM9B is the putative ortholog of Arabidopsis EARLY FLOWERING 6 (ELF6/AtPKDM9B) gene involved in the removal of the repressive mark H2K27me3. A combination of ChIP-seq and RNA-seq experiments revealed that MtPKDM9B is required for demethylation of H3K27me3 in regions nearby or contained within gene bodies of symbiotic genes and the upregulation of the cognate mRNAs in response to rhizobia, including those encoding the putative ubiquitin ligase MtPUB2, the MYB transcription factor MtMYB040 and the auxin conjugating enzyme MtGH3 (Gretchen Hagen 3). Our findings illustrate how AS and translational regulation of this plant specific histone lysine demethylase contributes to the removal of the repressive mark H3K27me3, promoting transcriptional activation of symbiotic genes required for the formation of functional nitrogen fixing nodules.
![Modified Fig. 8 | The OsRAP2.4h–PIP2 module plays a conserved role in regulating hypoxia tolerance and plant growth.
a, Seed germination of Zhonghua 11 (ZH11) and OsRAP2.4h knockout lines (Os2.4h-1 and Os2.4h-2) under normal (Air) or hypoxia (0.1% [v/v] O2) for 7 days.
b, The coleoptile length of the genotypes as presented in (a). Data are presented as means ± SD ( n = 3 independent experiments, each with 7 plants per genotype). Different letters without prime and with one prime indicate significant differences among genotypes under normal (Air) and hypoxia, respectively, at P < 0.05 (one -way ANOVA with Tukey ’s HSD test).
c, Growth phenotypes of 7-week-old ZH11, Osrap2.4h-1, and Osrap2.4h-2 under normal growth conditions.
d, The plant heights for the genotypes as presented in (c). Data are presented as means ± SD (n = 3 biological replicates). Different letters indicate significant differences among genotypes at P < 0.05 (one -way ANOVA with Tukey ’s HSD test).
e, RT-qPCR analysis of aquaporin genes OsPIP2;1 and OsPIP2;4 in 2-week-old ZH11, Osrap2.4h-1, and Osrap2.4h-2 plants. Transcripts were normalized to levels of ACTIN. The experiments were repeated three times with similar results. Data are presented as means ± SD (n = 3 independent experiments, each with three technical replicates). Different letters indicate significant differences among genotypes at P < 0.05 (one-way ANOVA with Tukey’s HSD test). Exact P values and 95% confidence intervals for all comparisons are provided in Source Data.](https://cdn.bsky.app/img/feed_thumbnail/plain/did:plc:tfidqyziwn7mqcunvhghjcno/bafkreignx6hblmdzagufxe5fgckhgyyxr3tohwkkd3chjmvi3hix7fck5y)
![Modified Fig. 2 | RAP2.4h is a key regulator balancing hypoxia tolerance and plant growth.
d, Phenotypes of 1-week -old WT, rap2.4h, OE-1, and OE-2 seedlings subjected to hypoxia (0.1% [v/v] O2) or normoxia (Air) in the dark for 7 h and then incubated in normoxic conditions for 4-d recovery.
e–f, Green cotyledon percentages (e) and relative chlorophyll contents (f) for the genotypes as presented in (d). For each genotype, chlorophyll content in hypoxia-treated seedlings was normalized to that of its own dark control (set to 100%). Data are presented as means ± SD ( n = 3 independent experiments, each with 5 seedlings per genotype).
g, Growth phenotypes of 5-week-old (top) and 6-week-old (bottom) WT, rap2.4h, OE-1, and OE-2 plants.
h, The number of rosette leaves in WT, rap2.4h, OE-1, and OE-2 plants at bolting. Data are presented as means ± SD (n = 3 independent experiments, each with 9 plants per genotype).
i, Phenotypes of the sixth leaf (top) of 5-week-old and main stem (bottom) of 7-week-old WT, rap2.4h, OE-1, and OE-2 plants. The statistics of leaf areas (cm2) and width of stems (mm) are shown below. Data are presented as means ± SD (n = 3 independent experiments, each with 3 plants per genotype). Different letters indicate significant differences among different genotypes at P < 0.05 (one-way ANOVA with Tukey’s HSD test). Exact P values and 95% confidence intervals for all comparisons are provided in Source Data.](https://cdn.bsky.app/img/feed_thumbnail/plain/did:plc:tfidqyziwn7mqcunvhghjcno/bafkreien4qdx3sxajcnjixn6jm7hc3ac5w3hmnweiygvpynbqukavitkku)
