Novel players in the AP2-miR172 regulatory network for common bean nodulation

Abstract

The intricate regulatory network for floral organogenesis in plants that includes AP2/ERF, SPL and AGL transcription factors, miR172 and miR156 along with other components is well documented, though its complexity and size keep increasing. The miR172/AP2 node was recently proposed as essential regulator in the legume-rhizobia nitrogen-fixing symbiosis. Research from our group contributed to demonstrate the control of common bean (Phaseolus vulgaris) nodulation by miR172c/AP2-1, however no other components of such regulatory network have been reported. Here we propose AGLs as new protagonists in the regulation of common bean nodulation and discuss the relevance of future deeper analysis of the complex AP2 regulatory network for nodule organogenesis in legumes.

The transcription factor (TF) superfamily APETALA2/Ethylene Responsive Factor (AP2/ERF) is conserved among the plant kingdom. Members of this large TF family, that regulate plant development and response to stress, have the characteristic AP2/ERF domain (IPR001471, www.ebi.ac.uk/interpro/) that consists of 60-70 amino acids and is involved in DNA binding.^1,2 Since 1994, Jofuku et al.³ reported the relevant role of AP2 in the control of flower development in Arabidopsis. The AP2 gene is part of a complex regulatory network that includes the microRNA172 (miR172) which promotes flowering by repressing AP2 through translation inhibition or mRNA cleavage.^4-6 Transcription of MIR172 genes is activated by SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) TF, that are targets miR156.⁷ In addition, both MIR156 and MIR172 promoters have binding sites for AP2 that acts as a dual transcriptional regulator activating MIR156 and repressing MIR172, thus constituting a negative feedback loop in the complex and robust floral organogenesis networks.⁸

The key regulatory role of miR172/AP2 in legume nodulation during the rhizobia nitrogen-fixation symbiosis has been reported for soybean (Glycine max) and common bean (Phaseolus vulgaris).^9-11 We have recently demonstrated that in common bean miR172c and its target gene AP2-1 (Phvul.005G138300) are essential regulators for rhizobial infection and nodulation through the regulation of the expression of early nodulation and autoregulation-of-nodulation (AON) genes. In addition we proposed that AP2-1 is a positive regulator of nodule senescence genes and thus it is silenced, through miR172c-induced target cleavage, in mature nodules.¹¹ Though these studies,^9-11 suggest that miR172 and their corresponding AP2 targets are part of complex regulatory networks in legume nodules, it is essential to decipher other yet unknown network components. We have analyzed SPLs as possible transcription regulators of common bean MIR172 genes, but only negative results were obtained.¹¹ In this work we propose other regulators involved in miR172/AP2-1 regulatory network for common bean nodulation and discuss the importance for their deeper future analysis

We used CLOVER,¹² to analyze the promoter sequence of each of the 6 P. vulgaris MIR172 genes,¹¹ in order to identify additional transcriptional regulators. We found that AGL (AGAMOUS-LIKE PROTEIN) TF binding sites were statistically overrepresented in these promoters. In addition, a similar analysis was performed to the promoters of genes previously identified as highly expressed in P. vulgaris mature nodules, these genes co-express with those from the purine and ureide biosynthetic pathways.¹³ Ureides are the main product from fixed-nitrogen assimilation in warm season legumes and the main nitrogen supply for plant nutrition. Notably, among other TF binding sites, AGL sites were statistically over-represented in the promoters of common bean nodule-enhanced genes.

The AGL TF family, also known as MADS (MINICHROMOSOME MAINTENANCE1, AGAMOUS, DEFICIENS and SERUM RESPONSE FACTOR) in other eukaryotes, has been widely studied for the regulation of flower organogenesis.¹⁴ Some members of this family, such as AP1, AP3, SOC, AG and FUL, are indispensable for the correct floral formation and may interact with members from the AP2/ERF superfamily, such as AP2 or TOE.^15,16 AGLs, such as AGL15 and SOC1, have been included as part of the complex AP2 network for floral transition and floral development in Arabidopsis.⁸

On this basis, we consider interesting to study AGLs from common bean as a new players of nodule organogenesis networks, something that, to our knowledge, has not been yet analyzed for legumes. Ninety-one genes were annotated as AGL or MADS TF genes in the P. vulgaris genome (www.phytozome.net/commonbean.php, v1.0).¹⁷ According to the P. vulgaris Gene Expression Atlas (Pv GEA),¹³ only 21 (out of 91) AGL genes were expressed in at least one root or nodule tissue sample; these are shown in Figure. 1. According to the common bean expression analysis among different tissues reported by O'Rourke et al.,¹³ 8 of the AGL genes shown in Figure. 1 were significantly up regulated in nodules and/or roots as compared to aerial tissues

Figure 1.

Heatmap of the P. vulgaris AGL genes that are expressed in root/nodule tissues. Values of expression were extracted from the Pv GEA, tissues were analyzed as reported in O'Rourke et al. ¹³. Gene IDs are from www.phytozome.net/commonbean.php. Left: Expression patterns as Z-score. The color scale represents the expression distribution, red indicates high expression, and black indicates the mean of expression and green indicates low expression. Right: Differential expression (up regulation) in root and/or nodule as compared to other tissues, based in data from the Pv GEA.¹³

This subset of 21 common bean AGL genes were carefully annotated based in their sequence similarity to Arabidopsis AGL genes, following the annotation from The Arabidopsis Information Resource (TAIR, www.arabidopsis.org) as shown in Table 1. In addition, Table 1 includes information from TAIR about the expression in roots tissues of the Arabidopsis AGL genes that are similar to the root/nodule common bean AGL genes. Notably, most of these genes showed expression in one or more root-tissue samples from Arabidopsis (Table1), something that correlates with their tissue expression in common bean (Fig. 1). In order to gain insight into the potential roles of AGL TF in the control of the symbiotic interaction between legumes and nitrogen fixing bacteria we searched for the orthologous genes, to those analyzed from common bean, in soybean (Glycine max), a legume closely related to P. vulgaris.¹⁷ As shown in Table 1, we identified 17 soybean genes orthologous to common bean AGL genes expressed in root or nodule. The data from the RNA-seq Atlas of G. max.¹⁸ revealed that 13 ortholog AGL genes expressed highest in underground tissues (Table 1). Thus, the data on soybean gene expression, shown in Table 1, support the hypothesis of AGL genes as regulators of the legume /rhizobia symbiosis

Table 1.

Annotation of P. vulgaris root/nodule AGL genes and their expression in A. thaliana and G. max (soybean) tissues. The P. vulgaris gene annotation was based in that for Arabidopsis (TAIR, www.arabidopsis.org). The expression (TAIR) in one or more root tissue samples of the Arabidopsis orthologues of each AGL common bean gene, is indicated. The soybean AGL genes identified as orthologues of the common bean genes are indicated as well as the plant tissue where each gene presents its highest expression value, according to data from the RNA-seq Atlas of G. max.¹⁸

Phaseolus vulgaris	Arabidopsis thaliana		Glycine max
Gene	Gene	Expression in root tissues	Gene	Highest expression in
Phvul.002G143800	AGL17	root epidermis, root, root cap, root elongation zone	Glyma.09G201700	root
Phvul.002G143900	ANR1	root	—	—
Phvul.002G147600	SVP	root	Glyma.02G041500	root
Phvul.002G212400	TT16	—	—	—
Phvul.002G212500	SVP	root	Glyma.08G068200	young leaf
Phvul.002G215500	ANR1	root	Glyma.08G065400	root
Phvul.003G182700	FUL	—	Glyma.05G018800	root
Phvul.003G213600	AGL19	root, lateral root cap	Glyma.17G132700	root
Phvul.004G123500	AGL13	root	Glyma.16G200700	nodule
Phvul.005G036700	AGL26	root	Glyma.08G152700	young leaf
Phvul.006G200300	AGL17	root epidermis, root, root cap, root elongation zone	Glyma.13G255200	root
Phvul.006G200400	ANR1	root	—	—
Phvul.007G065100	AGL13	root	Glyma.10G240900	root
Phvul.008G027800	CAL	root	Glyma.08G250800	root
Phvul.008G073800	SOC1	root	Glyma.09G266200	root
Phvul.008G183700	AGL21	lateral root primordium, primary root tip, central root cap of primary root, root endodermis, primary root apical meristem, root procambium, root, root stele	Glyma.14G183800	root
Phvul.009G037300	AGL24	—	Glyma.06G095700	root
Phvul.009G203400	FUL	—	Glyma.06G205800	pod shell 10DAF
Phvul.010G088000	SOC1	root	Glyma.07G080900	young leaf
Phvul.010G088100	XAL2	root	—	—
Phvul.011G005800	XAL1	non-hair root epidermal cell, root vascular system, primary root differentiation zone, root, root stele	Glyma.12G005000	root

AGL21 is expressed in several different Arabidopsis root tissues (Table1). This gene is crucial for lateral root development; its expression is affected by phytohormones and in response to stresses. Arabidopsis plants overexpressing AGL21 produce more and longer lateral roots. AGL21 has been implicated in auxin accumulation in lateral root primordia.¹⁹ The common bean AGL21 (Phvul.008G183700) is upregulated in roots vs pods, seeds and stems and it is also expressed in nodules (Fig.1). The soybean AGL21 (Glyma.14G183800) shows its highest expression in roots (Table 1). It is known that the auxin/cytokinin ratio is strictly controlled and plays an important role in nodule development during the legume-rhizobia symbiosis. Auxins plays (at least) a dual role during nodulation: in the early stages auxin transport inhibition might result in a reduced auxin/cytokinin ratio to allow cell division to start and later divisions are inhibited by super optimal auxin levels.²⁰ Thus, it is conceivable that the root/nodule expression of common bean AGL21 is related to auxin regulation of the symbiotic process

XAL1 and XAL2 are other AGL's important for Arabidopsis root development. Arabidopsis mutants affected in any of these genes present shorter roots. XAL1 is important for the regulation of cell cycle.²¹ XAL2 controls auxin transport in the root and is important in the formation and maintenance of the root stem-cell niche.²² In agreement, the soybean XAL1 (Glyma.12G005000) gene shows its highest expression in roots.¹⁸

ANR1 (ARABIDOPSIS NITRATE REGULATED1) is expressed in Arabidopsis roots (Table 1), it is up-regulated upon nitrate starvation and in nitrate rich media it controls local proliferation of lateral roots.²³ Arabidopsis ANR1 is part of a nitrogen sensing network that include NLP7 (NIN-LIKE PROTEIN 7).^23,24 The NIN (NODULE INCEPTION) TF family was first identified in Lotus japonicus,²⁵ now it is known that NIN is crucial for the initiation of nodule formation in different legumes. Recently it was shown that L. japonicus NIN directly transcribes the CLE root signal genes, involved in the AON.²⁶ We identified 3 copies of P. vulgaris ANR1 genes that are expressed in roots and nodules and one soybean ortholog (Glyma.08G065400) with highest expression in roots (Table 1, Fig. 1). Our previous report indicates a positive regulation of common bean NIN and AP2-1 to RIC1 (RHIZOBIUM-INDUCED CLE PEPTIDE1) and suggested the involvement of these regulators in the AON in common bean.¹¹ On this basis and considering the relevance of nitrogen metabolism in legume nodules we propose that ANR1 as new player in the regulation of legume/rhizobia symbiosis

The regulatory network for flower organogenesis, composed by AP2/ERF, AGL and miRNA members, is still growing in size and complexity. However, only few players have been described in networks for nodule development. Recently, AP2 genes were described as protagonists in nodulation,^9-11 it seems most probable that these do not play alone. Here we propose AGL transcription regulators as another component of nodulation networks. Future work is required to demonstrate our proposal and to decipher the complete set of players from networks controlling the intricate nodule organogenesis during the symbiotic nitrogen fixation, a most relevant process for sustainable agriculture.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by Dirección General de Asuntos del Personal Académico / UNAM (grants no. PAPIIT: IN209710 and IN210814). LPI and BNF received a PhD studentship from Consejo Nacional de Ciencia y Tecnología, México (nos. 340334 and 351615, respectively).