Legumes can utilize atmospheric nitrogen via symbiotic nitrogen fixation, but this process is inhibited by high soil inorganic nitrogen. So far, how high nitrogen inhibits N₂ fixation in mature nodules is still poorly understood. Here we construct a co-expression network in soybean nodule and find that a dynamic and reversible transcriptional network underlies the high N inhibition of N₂ fixation. Intriguingly, several NAC transcription factors (TFs), designated as Soybean Nitrogen Associated NAPs (SNAPs), are amongst the most connected hub TFs. The nodules of snap1/2/3/4 quadruple mutants show less sensitivity to the high nitrogen inhibition of nitrogenase activity and acceleration of senescence. Integrative analysis shows that these SNAP TFs largely influence the high nitrogen transcriptional response through direct regulation of a subnetwork of senescence-associated genes and transcriptional regulators. We propose that the SNAP-mediated transcriptional network may trigger nodule senescence in response to high nitrogen.

Fructose-1,6-bisphosphate aldolase (FBA) is an important catalytic enzyme in carbon metabolism and plays an important role in plant growth and development. Currently, the biological functions of FBA in soybean (Glycine max) remain unknown. In this study, we conducted research on FBA in soybean and identified 14 GmFBA genes. Among them, GmFBAc1 and GmFBAc2 are broadly expressed in different tissues. Double mutant lines of GmFBAc1 and GmFBAc2 were obtained by CRISPR-Cas9 gene editing technology. Compared with the wild type, the double-gene homozygous mutant gmfbac1gmfbac2 exhibited dwarf seedlings and narrow leaflets, indicating that GmFBAc1 and GmFBAc2 are critical for soybean growth and development. The gmfbac1gmfbac2 metabolomic analysis revealed that compared to the wild type, carbohydrate metabolism was reduced and amino acid metabolism was enhanced in gmfbac1gmfbac2 mutant leaves. Transcriptomic analysis showed that genes in IAA signaling and JA signaling were downregulated and upregulated, respectively. Our study demonstrates an important role of GmFBAc1 and GmFBAc2 in modulating carbon metabolism and phytohormone homeostasis.

Legumes have the capability to form nodules that facilitate symbiotic nitrogen fixation (SNF) with rhizobia. Given the substantial energy consumption during the process of SNF, legumes need to optimize nodule number in response to everchanging environmental scenarios. The TGACG BINDING FACTOR1/4 (TGA1/4) are key players in the basal immune response of plants. In this study, both β-glucuronidase staining and quantitative reverse transcription PCR (qRT-PCR) demonstrated that both GmbZIP4a and GmbZIP4b are inducible upon rhizobial inoculation. To investigate their roles further, we constructed gmbzip4a/b double mutants using CRISPR/Cas9 system. Nodulation assessments revealed that these double mutants displayed a reduction in the number of infection threads, which subsequently resulted in a decreased nodule number. However, the processes associated with nodule development including nodule fresh weight, structural characteristics, and nitrogenase activity, remained unaffected in the double mutants. Subsequent transcriptome analyses revealed that zeatin biosynthesis was downregulated in gmbzip4a/b mutants post rhizobial inoculation. Supporting these findings, genes associated with cytokinin (CTK) signaling pathway were upregulated in Williams 82 (Wm82), but this upregulation was not observed in the double mutants after rhizobial treatment. These results suggest that GmbZIP4a/b positively influences nodule formation by promoting the activation of the CTK signaling pathway during the early stages of nodule formation.

Certain insect larvae can biodegrade plastic into frass (excrement) that functions as a biofertilizer. We found that yellow mealworms and superworms fed on polystyrene or polyethylene produced frass enriched in all four major classes of plant hormones, i.e., auxins, cytokinins, jasmonates, and salicylates, compared with bran-fed controls. This suggests that plastic-derived frass could simultaneously promote plant growth and prime plant defenses, adding sustainable value to this waste-to-fertilizer pathway.