The Mechanism of Legume-Specific SnRK1 in Regulating Symbiotic Nitrogen Fixation
Introduction: Unveiling the Energy Regulation in Symbiotic Nitrogen Fixation
Symbiotic nitrogen fixation (SNF) between legumes and rhizobia is a highly energy-consuming process. For every gram of nitrogen fixed, legumes typically consume 5.2 to 18.8 grams of carbon sources [1]. While this mutualistic relationship benefits both parties, the smooth progression of SNF relies on the continuous supply of carbon sources from legumes [2]. The discovery of legume-specific energy sensors like GmNAS1/GmNAP1 has provided theoretical support for designing crops to efficiently utilize carbon sources and enhance SNF [3]. However, the molecular mechanism by which legumes allocate energy and carbon sources to support high-energy-consuming SNF under nitrogen deficiency remains unclear [4]. Scientists have been eager to explore whether legumes possess unique energy sensors involved in this process.
SnRK1: A Key Player in Energy Regulation
Eukaryotic organisms, unlike prokaryotes, can efficiently and rapidly respond to nutritional stress, adjusting energy distribution to maintain normal growth while tolerating stress [5]. In mammals, the AMP-activated protein kinase (AMPK), plant sucrose non-fermenting-1-related protein kinase 1 (SnRK1), and yeast sucrose non-fermenting 1 (SNF1) are central to energy regulation, conservatively present in all eukaryotes. They integrate nutritional, energy, and stress signals to reprogram transcription and metabolism, balancing energy demands for growth and survival [6,7].
SnRK1 is a heterotrimeric complex composed of α, β, and γ subunits. The α subunit, the catalytic subunit, has an N-terminal kinase domain (KD), a C-terminal ubiquitin-associated domain (UBA), and a kinase-associated 1 domain (KA1) [8]. In different species, each subunit of the AMPK/SnRK1/SNF1 complex has multiple isoforms. From green algae to flowering plants, the types and numbers of SnRK1 subunits vary significantly, reflecting species-specific diversity [9]. In Arabidopsis, three SnRK1α subunits (α1, α2, α3) have been identified, with α3 being inactive [10,11].
Discovery of Legume-Specific SnRK1α4
Through evolutionary analysis of AMPK α subunits, a novel SnRK1α subunit, SnRK1α4, was identified in legumes, evolving as a distinct branch [12]. Analysis of Medicago truncatula SnRK1α subunits revealed that MtSnRK1α4 differs from classic MtSnRK1α1/α2 in domain structure; it lacks the UBA and KA1 domains in the C-terminus but retains a conserved Thr175 phosphorylation site in the kinase domain [12]. MtSnRK1α4 is highly expressed in nodules and positively regulates SNF. Combining biochemical experiments and genetic evidence, the mechanism by which MtSnRK1α4 promotes SNF was elucidated, and this work was published in Molecular Plant [12].
MtSnRK1α4: A Unique Regulator of Symbiotic Nitrogen Fixation
During the research, a novel SnRK1α subunit exclusive to legumes was identified. Phylogenetic analysis showed that SnRK1α4 genes in legumes are tandemly duplicated with SnRK1α2 on the same chromosome, likely resulting from whole-genome duplication events during legume evolution [13]. Unlike conserved MtSnRK1α1/α2, MtSnRK1α4 is highly expressed in nodules, particularly in the nitrogen-fixing zone. Overexpression of MtSnRK1α4 led to larger nodules and enhanced nitrogenase activity, indicating its role in promoting carbon-nitrogen exchange between legumes and rhizobia under nitrogen deficiency [12].
Activation of MtSnRK1α4 by MtDMI2
The activation of SnRK1 is regulated by phosphorylation of the α subunit. The key phosphorylation site Thr175 in MtSnRK1α4 is phosphorylated by the kinase MtDMI2, which was previously known for its role in nodulation signaling [14,15]. MtDMI2 is highly expressed during nitrogen fixation, and immunofluorescence confirmed its expression in infected and non-infected cells of nodules. Biochemical and genetic studies showed that MtDMI2 phosphorylates and activates MtSnRK1α4, providing an energy source for continuous SNF [12].
Downstream Targets of MtSnRK1α4: Sucrose Invertase and Malate Dehydrogenase
SnRK1 regulates metabolism by phosphorylating target enzymes. MtSnRK1α4 interacts with sucrose invertase (MtINV1), enhancing its activity to promote sucrose hydrolysis. Additionally, MtSnRK1α4 phosphorylates malate dehydrogenases (MtMDH1/2) at Ser242, increasing their activity to generate more malate, which serves as a carbon source for bacteroids [12].
Conclusion: Implications for Crop Improvement
This study identifies MtSnRK1α4 as a key regulator of SNF energy supply. Activated by MtDMI2, MtSnRK1α4 enhances sucrose hydrolysis via MtINV1 and malate production via MtMDH1/2, ensuring carbon source supply for bacteroids. The DMI2-SnRK1α4-MDH pathway provides a theoretical basis for engineering symbiotic nitrogen fixation in cereal crops.
Citations and References
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DOI
doi: 10.1360/TB-2023-0905
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