Genome-wide identification and expression analyses of nitrate transporter family genes in wild soybean (Glycine soja).

Nitrate transporters (NRTs) are important channel proteins facilitating cross-membrane movement of small molecules like NO3- which is a critical nutrient for all life. However, the classification and evolution of nitrate transporters in the legume plants are still elusive. In this study, we surveyed the wild soybean (G. soja) genomic databases and identified 120 GsNRT1 and 5 GsNRT2 encoding genes. Phylogenetic analyses show that GsNRT1 subfamily is consisted of eight clades (NPF1 to NPF8), while GsNRT2 subfamily has only one clade. Gene chromosomal location and evolutionary historic analyses indicate that GsNRT genes are unevenly distributed on 19 out of 20 G. soja chromosomes and segmental duplications may take a major part in the expansion of GsNRT family. Investigations of gene structure and protein motif compositions suggest that GsNRT family members are highly conserved in structures of both gene and protein levels. In addition, we analyzed the spatial expression patterns of representative GsNRT genes and their responses to exogenous nitrogen and carbon supplies and different abiotic stresses. The qRT-PCR data indicated that 16 selected GsNRT genes showed various expression levels in the roots, stems, leaves, and pods of young G. soja plants, and these genes were regulated by not only nitrogen and carbohydrate nutrients but also NaCl, NaHCO3, abscisic acid (ABA), and salicylic acid (SA). These results suggest that GsNRT genes may be involved in the regulation of plant growth, development, and adaptation to environmental stresses, and the study will shed light on functional dissection of plant nitrate transporter proteins in the future.

Identification of novel interactors and potential phosphorylation substrates of GsSnRK1 from wild soybean (Glycine soja).

The plant sucrose nonfermenting kinase 1 (SnRK1) kinases play the central roles in the processes of energy balance, hormone perception, stress resistance, metabolism, growth, and development. However, the functions of these kinases are still elusive. In this study, we used GsSnRK1 of wild soybean as bait to perform library-scale screens by the means of yeast two-hybrid to identify its interacting proteins. The putative interactions were verified by yeast retransformation and ß-galactosidase assays, and the selected interactions were further confirmed in planta by bimolecular fluorescence complementation and biochemical Co-IP assays. Protein phosphorylation analyses were carried out by phos-tag assay and anti-phospho-(Ser/Thr) substrate antibodies. Finally, we obtained 24 GsSnRK1 interactors and several putative substrates that can be categorized into SnRK1 regulatory ß subunit, protein modification, biotic and abiotic stress-related, hormone perception and signalling, gene expression regulation, water and nitrogen transport, metabolism, and unknown proteins. Intriguingly, we first discovered that GsSnRK1 interacted with and phosphorylated the components of soybean nodulation and symbiotic nitrogen fixation. The interactions and potential functions of GsSnRK1 and its associated proteins were extensively discussed and analysed. This work provides plausible clues to elucidate the novel functions of SnRK1 in response to variable environmental, metabolic, and physiological requirements.