Supplementary Materials and methods

Plant growth conditions and physiological parameters analysis

Seeds from Camelina (Camelina sativa L., cv. Crantz.) were sown in wet rock wool media. Seven-day-old seedlings in rock-wool were transferred to a plastic box containing Hoagland’s solution with air pump and grown in a growth chamber with 16 h of illumination (about 60 µM m-2s-1) and 8 h of dark at 23±1◦C and 65% relative humidity. After 1 week (for gene analyses) and 2 weeks (for physiological tests), the plants were transferred to various stress condition treatments. For salt stress experiments, plants were treated with 0.05, 0.1, 0.15, or 0.2 M NaCl. For drought stress experiments, seedlings were transferred onto filter paper not containing water and exposed to air. Cold stress experiments were performed at 2◦C under the same growth conditions as control plants, except temperature.

To measure physiological parameters of Camelina under salt stress, leaf stomatal conductance and chlorophyll fluorescence were measured using a steady state diffusion porometer (Decagon Devices) and chlorophyll fluorescence measurement system (Opti Science), respectively. The third leaf of each Camelina plant was used, and measurements were performed between 11:00 AM to 12:00 PM.

Supplementary Data

Table S1. Gene specific primers of AtRCI2A, CsRCI2A, and CsRCI2E genes used for PCR.

Gene name / Primer sequence (5'-3') / Size / Temp.
(◦C)
cDNA
RT-PCR / AtRCI2A / F: ATGAGTACAGCTACTTTCGTTG / 165bp / 55
R: TCATTTGGTGAGGACATAAATG
CsRCI2A / F: ATGAGTACAGCTACTTTTG / 165bp / 55
R: TCATTTGGTGAGGACATAAA
CsRCI2E / F: ATGGCGAGCAACATGGAAGT / 222bp / 53
R: TCAAGCTGAGTTGAGTGGAG
CsTubulin / F: CACCTCAAGAGGGTCTCAGC / 300bp / 55
R: ACGTTCAGCATCTGCTCGTC
RACE-
PCR / CsRCI2A / 5': CAAGCAGCGTCAAAACCAAAC / 286bp / 64
3': GCTGTCCTCTTGCCTCCTCTTG / 465bp / 64

a

b Stomatal conductance

c Chlorophyll Fluorescence

Fig. S1. Physiological tests of Camelina under salinity stress. (a) Three-week-old Camelina plants after exposure to 0.05, 0.1, and 0.15 M NaCl for 5 days. (b) Measurement of stomatal conductance at day 2 and 4. (c) Measurement of chlorophyll fluorescence at day 2 and 4. Data represent the mean ±SE from five replicates.

Fig. S2. cDNA nucleotide and predicted protein sequences of CsRCI2A and CsRCI2Egenes . Predicted amino acid sequences are displayed under corresponding coding DNA sequences. CsRCI2A encodes a 54 amino acid peptide and CsRCI2E encodes a 73 amino acid peptide. Red boxes indicate the potential trans-membrane domains. Yellow box indicates the hydrophilic C-terminal tail domain. . Bold font indicates the start and stop codons.

Fig. S3. Time course of DiBAC4(3) fluorescence in wild type and various transgenic lines of Dpmps3 mutant yeast. Transgenic lines are identified in the graph by name of the gene construct carried. Names and structures of vector constructs for the transgenic years lines are presented in Figure 5a. Yeast lines were incubated with 1 µg DiBAC4(3) for 1 h and variance of fluorescence was detected during 10 min at 10-s intervals. After 10 min, decreased fluorescence units indicated hyperpolarization of the plasma membrane potential. The units values of the data were then smoothed using the 2D smoother algorithm of SigmaPlot 10.0.

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