Over-expression of NtHb1 encoding a non-symbiotic class 1 hemoglobin of tobacco enhances a tolerance to cadmium by decreasing NO (nitric oxide) and Cd levels in Nicotiana tabacum

• Tobacco gene NtHb1 encoding a hemoglobin was identified to confer Cd tolerance in yeast.
• NtHb1-expressing tobacco exhibited higher tolerance to Cd, but lower level of Cd.
• NO content of NtHb1-tobacco was lower than that of control in the presence of Cd.
• KO Arabidopsis of AtNR1 and AtNR2 showed higher Cd tolerance and reduced NO amounts.
• NtHb1 expression enhances Cd tolerance by reducing Cd and NO levels in plants.

Tobacco gene NtHb1 encoding a non-symbiotic class 1 hemoglobin was identified to confer cadmium (Cd) tolerance in yeast Saccharomyces cerevisiae by expressing cDNA library of Nicotiana tabacum in WT yeast Y800. To examine the role of NtHb1 in plants, NtHb1 was over-expressed in tobacco (N. tabacum). NtHb1-expressing tobacco exhibited higher tolerance to Cd, but lower levels of Cd compared with vector-expressing control tobacco. Since it was reported that Hb (hemoglobin) is involved in scavenging NO, we investigated NO level in control and NtHb1-expressing tobacco. Interestingly, NO content of NtHb1-tobacco was lower than that of control tobacco in the presence of Cd, while there is no significant difference in NO accumulations of two plants in the absence of Cd. Among Cd transporters, CAX3 (cation/proton exchanger) transcript level of NtHb1-tobacco in response to Cd was higher than control tobacco, but IRT1 (iron-responsive transporter) expression was not. In addition, knockoutArabidopsis of AtNR1 and AtNR2 encoding NO producing enzymes nitrate reductase 1 and nitrate reductase 2 respectively, displayed enhanced Cd tolerance and reduced NO amounts in comparison with control Arabidopsis Col-0. Taken together, it is concluded that the increase of Hb by NtHb1 expression promotes Cd tolerance and reduces Cd accumulation by alleviating the level of NO induced by Cd in transgenic tobacco.