Supplementary material
Methods
Expression of OsNPSNs in transformed yeast cells
The yeast transformants harboring pFL-OsNPSN11, pFL-OsNPSN12, pFL-OsNPSN13 or pFL61, and wild type yeast cells, were cultured in YPD medium until the optical density (OD600) reached 1.0. Then, 50 µl cultures were transferred to 50ml YPD medium for further growth. When the OD600 was 2.0, the yeast cells were collected (Yu et al., 2005), and total RNA was extracted using Trizol reagent (Invitrogen). RT-PCR was conducted according to the methods described in Cloning of OsNPSNs (in Materials and methods).
Expression of OsNPSN11 in transgenic tobacco
Total RNA was extracted from the T0 transgenic tobacco with OsNPSN11 or wild type using Trizol reagent (Invitrogen). RT-PCR was conducted according to the methods described in Cloning of OsNPSNs (in Materials and methods). A 500bp PCR fragment of the constitutively expressed tobacco 18SrRNAgene 18S was amplified synchronously as a control. The primers for 18Swere:sense: 5’-TGGGATACCTGCCAGTAGTCAT-3’; antisense: 5’-CTGGATCCAATTACCAGACTCAA-3’.
Measurement of chlorophyll contents under oxidative stress
To evaluate the performance of the transgenic tobacco plants under oxidative stress, the seeds of T1 transgenic lines were germinated and grown in 1/2 Murashige and Skoog (MS) medium for three days, then transferred to the 1/2 MS medium plus 0,10 mM and 20 mM H2O2. After 15 days, the chlorophyll contents of each OsNPSN11 transgenic plant or wildtype plant were measured according to Zhang et al (2004).The experiment wasindependently repeated for three times and the standard errors (±SE) were measured.
Supplementary figures
Fig.S1 Expression of OsNPSNs in transformed yeast by RT-PCR assay. Lane 1, 4 and 7 represent wild type yeast; lane 2, 5 and 8 the transformantsharboring vector pFL61, and lane 3, 6, and 9 the yeast transformantsharboring pFL-OsNPSN11,pFL-OsNPSN12 and pFL-OsNPSN13 respectively. The corresponding ethidium bromide gel image shows the relative level of RNA loaded for each sample.
Fig.S2 Expression of OsNPSN11 in transformed tobacco by RT-PCR assay. WT represents wild type tobacco, and 5#, 15# and 32# the transgenic tobacco with 35S:OsNPSN11respectively.The 18S genewas used as the internal control.
Fig. S3Chlorophyll contents of 35S:OsNPSN11 transgenic and wild type tobacco plants under oxidative, osmotic and salt stresses. The seeds of T1 transgenic lines or wild type tobacco were germinated and grown in 1/2 Murashige and Skoog (MS) medium, and Seven-day-old seedlings transferred and grown for 15 daysvertically on 1/2 MS medium containing 0, 10 mM and 20 mM H2O2, respectively.
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