miR169c-NFYA-C-ENOD40 modulates nitrogen inhibitory effects in soybean nodulation.

Legume crops contribute a great portion of clean nitrogen (N) to agro-ecosystems through symbiotic N2 fixation in the nodule; however, the nodulation is always inhibited by high N availability which is known as the N inhibitory effect through largely unknown mechanisms. We functionally investigated miR169c-GmNFYA-C-GmENOD40 under multiple N conditions in soybean (Glycine max) (ENOD, Early Nodulin; NFYA, Nuclear Factor-Y Subunit A). We elucidated their regulatory roles in soybean nodulation through analyzing expression patterns, micro-messenger RNA (miRNA-mRNA) interactions, phenotypes of transgenic soybean plants and genetic interactions. We found that miR169c expression was induced by high N, whereas its target GmNFYA-C was preferentially expressed in nodules and induced by rhizobium inoculation. Overexpression of miR169c inhibited nodulation through targeting 3'-UTR of GmNFYA-C, whereas knockout miR169c through CRISPR-cas9 promoted nodulation. However, overexpression of GmNFYA-C promoted soybean nodulation through facilitating rhizobium infection and increasing the expression of symbiotic signaling gene GmENOD40. Besides, GmNFYA-C directly induced the expression of GmENOD40. In addition, overexpression of GmNFYA-C without the target site of miR169c partially attenuated the inhibitory effect of high N on soybean nodulation. We discovered a new regulatory pathway involving the miR169c-NFYA-C-ENOD40 module that regulates soybean nodulation in response to N availability. This pathway provides substantial new insights into the mechanisms underlying the N inhibitory effect on nodulation.

Application of an Improved 2-Dimensional High-Throughput Soybean Root Phenotyping Platform to Identify Novel Genetic Variants Regulating Root Architecture Traits.

Nutrient-efficient root system architecture (RSA) is becoming an important breeding objective for generating crop varieties with improved nutrient and water acquisition efficiency. Genetic variants shaping soybean RSA is key in improving nutrient and water acquisition. Here, we report on the use of an improved 2-dimensional high-throughput root phenotyping platform that minimizes background noise by imaging pouch-grown root systems submerged in water. We also developed a background image cleaning Python pipeline that computationally removes images of small pieces of debris and filter paper fibers, which can be erroneously quantified as root tips. This platform was used to phenotype root traits in 286 soybean lines genotyped with 5.4 million single-nucleotide polymorphisms. There was a substantially higher correlation in manually counted number of root tips with computationally quantified root tips (95% correlation), when the background was cleaned of nonroot materials compared to root images without the background corrected (79%). Improvements in our RSA phenotyping pipeline significantly reduced overestimation of the root traits influenced by the number of root tips. Genome-wide association studies conducted on the root phenotypic data and quantitative gene expression analysis of candidate genes resulted in the identification of 3 putative positive regulators of root system depth, total root length and surface area, and root system volume and surface area of thicker roots (DOF1-like zinc finger transcription factor, protein of unknown function, and C2H2 zinc finger protein). We also identified a putative negative regulator (gibberellin 20 oxidase 3) of the total number of lateral roots.

Constitutive basis of root system architecture: uncovering a promising trait for breeding nutrient- and drought-resilient crops.

Root system architecture (RSA) plays a pivotal role in efficient uptake of essential nutrients, such as phosphorous (P), nitrogen (N), and water. In soils with heterogeneous nutrient distribution, root plasticity can optimize acquisition and plant growth. Here, we present evidence that a constitutive RSA can confer benefits for sorghum grown under both sufficient and limiting growth conditions. Our studies, using P efficient SC103 and inefficient BTx635 sorghum cultivars, identified significant differences in root traits, with SC103 developing a larger root system with more and longer lateral roots, and enhanced shoot biomass, under both nutrient sufficient and deficient conditions. In addition to this constitutive attribute, under P deficiency, both cultivars exhibited an initial increase in lateral root development; however, SC103 still maintained the larger root biomass. Although N deficiency and drought stress inhibited both root and shoot growth, for both sorghum cultivars, SC103 again maintained the better performance. These findings reveal that SC103, a P efficient sorghum cultivar, also exhibited enhanced growth performance under N deficiency and drought. Our results provide evidence that this constitutive nature of RSA can provide an avenue for breeding nutrient- and drought-resilient crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00112-w.