One to rule both: shared histone deacetylases regulate Medicago root and nodule development

To form a mutualistic relationship with nitrogen-fixing rhizobacteria, legumes produce symbiotic organs called nodules. Nodulation requires two steps: it starts with bacterial infection structure formation at root hairs, which is followed by nodule organogenesis. Plant-derived flavonoids in the rhizosphere can attract some rhizobia, which in turn secrete compounds called nodulation (Nod) factors (Mergaert et al., 2020). Perception of Nod factors by root hairs triggers the formation of an infection pocket and infection thread that rhizobia colonize. The infection thread moves through the entire root hair and promotes root nodule organogenesis.

Nodule development and lateral root development share extensive morphological and transcriptional features and both processes are well coordinated by plants through hierarchical regulatory networks (Schiessl et al., 2019; Soyano et al., 2019). Although years of work have identified many elements in both regulatory networks, such as signaling pathways, transcription factors, and microRNAs, gaps remain (Soyano et al., 2019; Hoang et al., 2020). In particular, one recent study has identified a shared transcription factor functioning in transcriptional reprogramming during both nodule symbiosis and lateral root development, suggesting at least a partially shared hierarchical regulatory network may exist (Soyano et al., 2019).

Post-translational modification at the N-terminus of histone tails to regulate chromatin remodeling can activate or repress gene expression. As an example, the removal of acetylation from core histone tails can switch chromatin from gene activation-associated “open” to gene repression-associated “closed” status. Acetylation and deacetylation of histones are catalyzed by so-called chromatin remodelers, histone acetyltransferases, and histone deacetylases. In plants, histone acetylation dynamics play crucial roles in gene regulation in multiple developmental processes and in response to abiotic and biotic stresses (Liu et al., 2014). Whether chromatin remodelers that regulate root development also function in nodule formation is less well understood.

In this issue of Plant Physiology, Li et al. (2021) show plant-specific histone deacetylases regulate nodule and lateral root development in the legume Medicago (Medicago truncatula), which contains three histone deacetylases (MtHDTs). The authors first analyzed the phylogenetic relationship of HDTs from several dicots and rice (Oryza sativa) and inferred MtHDTs may regulate root development, as do their homologs. The authors observed the three MtHDTs are all present in the root meristem and elongation zone by analyzing the expression of GFP-MtHDT. Knocking down expression of these genes by RNAi significantly reduced the root meristem size and activity, which confirmed MtHDTs regulate root development.

The authors then showed MtHDT transcripts and protein products are present in the nodule meristem and infection zone by RNA in situ hybridization and subcellular localization analyses (Figure 1A), suggesting MtHDTs may function in nodule development. To functionally characterize MtHDTs, the authors generated a nodule-specific RNAiconstruct to target all three MtHDT transcripts and used a promoter with activity in nodule meristem and infection zone to drive the RNAi constructs. Compared with empty vector control nodules, the RNAi nodules showed dramatically reduced MtHDT transcript levels. The nodules were also fewer in number, smaller in size, and their shape was altered from elongated to spherical at 21 d post-inoculation (dpi) (Figure 1B and 1C). In addition to nodule morphological changes, knock-down of MtHDTs also reduced the number of cell layers, gene expression levels, and rhizobial colonization in the nodule meristem. Thus, these RNAi experiments provide solid evidence that MtHDTs function in nodule meristem.

Figure 1.

Histone deacetylases regulate root and nodule development in M. truncatula. A, Subcellular localization of pMtHDT2::GFP::HDT2 in M. truncatula nodules, showing expression in the meristem and infection zones. B and C, Nodule morphology under light microscopy of control empty vector (B) (ENOD12-EV) and (C) MtHDT RNAi, showing RNAi nodules are rounder and have fewer cell layers derived from the meristem. Nodule meristem zone (M), infection zone (I), and fixation zone (F) are labeled. Scale bars = 100 µM. (Adapted from Figure 2B, Figure 3D, and 3E; Li et al. [2021]).

Using young nodule primordia-specific promoters to drive expression of the MtHDT RNAi constructs, the authors showed MtHDTs are involved in nodule primordia development. They observed development of most nodule primordia was blocked at early stages 5 dpi, which resulted in reduced nodule number and morphological changes at 21 dpi. Visualization of cell division by staining developing nodule primordia with 5-ethynyl-29-deoxy-deoxy-uridine (Edu) revealed reduced mitotic activity in young primordia cells. Furthermore, expression pattern analysis revealed MtHDT2 transcripts are present in endodermal cells before cell division, suggesting expression of MtHDTs is required for nodule primordium development. Therefore, the authors revealed the regulatory role of MtHDTs in the root meristem, nodule meristem, and nodule primordium by a combination of expression pattern analysis and RNAi experiments.

How do MtHDTs control both root and nodule development? Generally, HDTs are involved in transcription repression. Transcription analysis of RNAi nodules with reduced MtHDT levels revealed 49 differentially expressed genes from the nodule meristem and infection zone, of which 33 genes were upregulated and 16 genes were downregulated. Gene ontology analysis showed most downregulated genes are stress-responsive genes, including genes encoding oxidoreductases and involved in jasmonate signaling, terpenoid biosynthesis, and flavonoid pathways. However, only the target HDTs overlapped between differentially expressed gene sets identified from MtHDTs RNAi nodules and Arabidopsis (Arabidopsis thaliana) RNAi roots, suggesting HDTs in Medicago nodules and Arabidopsis roots target different gene sets.

The shared epigenetic regulatory element identified in this work together with other regulators suggests a partially shared hierarchical regulatory network in legumes to rule both root and nodule development. Understanding the complete set of regulatory elements required for nodule symbiosis could bring us closer to engineering nitrogen-fixing nodules in crops.