Critical role for uricase and xanthine dehydrogenase in soybean nitrogen fixation and nodule development

Abstract

De novo purine biosynthesis is required for the incorporation of fixed nitrogen in ureide exporting nodules, as formed on soybean [Glycine max (L.) Merr.] roots. However, in many cases, the enzymes involved in this pathway have been deduced strictly from genome annotations with little direct genetic evidence, such as mutant studies, to confirm their biochemical function or importance to nodule development. While efforts to develop large mutant collections of soybean are underway, research on this plant is still hampered by the inability to obtain mutations in any specific gene of interest. Using a forward genetic approach, as well as CRISPR/Cas9 gene editing via Agrobacterium rhizogenes‐mediated hairy root transformation, we identified and characterized the role of GmUOX (Uricase) and GmXDH (Xanthine Dehydrogenase) in nitrogen fixation and nodule development in soybean. The gmuox knockout soybean mutants displayed nitrogen deficiency chlorosis and early nodule senescence, as exemplified by the reduced nitrogenase (acetylene reduction) activity in nodules, the internal greenish‐white internal appearance of nodules, and diminished leghemoglobin production. In addition, gmuox1 nodules showed collapsed infected cells with degraded cytoplasm, aggregated bacteroids with no discernable symbiosome membranes, and increased formation of poly‐β‐hydroxybutyrate granules. Similarly, knockout gmxdh mutant nodules, generated with the CRISPR/Cas9 system, also exhibited early nodule senescence. These genetic studies confirm the critical role of the de novo purine metabolisms pathway not only in the incorporation of fixed nitrogen but also in the successful development of a functional, nitrogen‐fixing nodule. Furthermore, these studies demonstrate the great utility of the CRISPR/Cas9 system for studying root‐associated gene traits when coupled with hairy root transformation.

CRISPR/Cas9 in Planta Hairy Root Transformation: A Powerful Platform for Functional Analysis of Root Traits in Soybean

“…Both in vitro and in planta Agrobacterium inoculation techniques have been applied for transient expression of CRISPR/Cas9 reagents to functional analysis of different genes in soybean hairy roots. These genes are mainly involved in nitrogen fixation/nodule development [97][98][99][100] and the interaction of soybean with pathogens [101,102]. The complete list of recently reported functional analyses is presented in Table 1.…”

Linking the key physiological functions of essential micronutrients to their deficiency symptoms in plants

“…The development of functional nodules in N 2 fixing legumes that utilize the ureide pathway for nitrogen transport from the nodules to the shoot also depends on the molybdoenzyme XDH (Soltabayeva et al ., 2018; Nguyen et al ., 2021). This is the case because XDH is involved in the biosynthetic pathway of the ureides allantoin and allantoate.…”

Genome editing and chromosome engineering in plants

“…These publicly available cloning resources would greatly reduce the bottleneck of technical challenges and accelerate the testing and deployment of new tools. In this special focus issue, using the optimized CRISPR-Cas9 approach, Biswas et al (2022) and Nguyen et al (2021) characterized multiple novel genes involved in resistant starch in rice and N fixation and nodule development in soybean, respectively. In addition, Yoon et al (2022) applied CRISPR-Cas9 to develop double haploid technology in switchgrass that could significantly speed up its plant breeding processes.…”

“…(2022) and Nguyen et al. (2021) characterized multiple novel genes involved in resistant starch in rice and N fixation and nodule development in soybean, respectively. In addition, Yoon et al.…”