Molecular-based characterization and bioengineering of Sorghum bicolor to enhance iron deficiency tolerance in iron-limiting calcareous soils.

Plant biomass can significantly contribute to alternative energy sources. Sorghum bicolor is a promising plant for producing energy, but is susceptible to iron deficiency, which inhibits its cultivation in iron-limiting calcareous soils. The molecular basis for the susceptibility of sorghum to iron deficiency remains unclear. Here, we explored the sorghum genome to identify genes involved in iron uptake and translocation. Iron deficiency-responsive gene expression was comparable to that in other graminaceous plants. A nicotianamine synthase gene, SbNAS1, was induced in response to iron deficiency, and SbNAS1 showed enzyme activity. Sorghum secreted 2'-deoxymugineic acid and other phytosiderophores under iron deficiency, but their levels were relatively low. Intercropping of sorghum with barley or rice rescued iron deficiency symptoms of sorghum. To produce bioengineered sorghum with enhanced tolerance to iron deficiency, we introduced four cassettes into sorghum: 35S promoter-OsIRO2 for activation of iron acquisition-related gene expression, SbIRT1 promoter-Refre1/372 for enhanced ferric-chelate reductase activity, and barley IDS3, and HvNAS1 genomic fragments for enhanced production of phytosiderophores and nicotianamine. The resultant single sorghum line exhibited enhanced secretion of phytosiderophores, increased ferric-chelate reductase activity, and improved iron uptake and leaf greenness compared with non-transformants under iron-limiting conditions. Similar traits were also conferred to rice by introducing the four cassettes. Moreover, these rice lines showed similar or better tolerance in calcareous soils and increased grain iron accumulation compared with previous rice lines carrying two or three comparable cassettes. These results provide a molecular basis for the bioengineering of sorghum tolerant of low iron availability in calcareous soils.