Genetic indicators of iron limitation in wild populations of Thalassiosira oceanica from the northeast Pacific Ocean

Abstract

Assessing the iron (Fe) nutritional status of natural diatom populations has proven challenging as physiological and molecular responses can differ in diatoms of the same genus. We evaluated expression of genes encoding flavodoxin (FLDA1) and an Fe-starvation induced protein (ISIP3) as indicators of Fe limitation in the marine diatom Thalassiosira oceanica. The specificity of the response to Fe limitation was tested in cultures grown under Fe- and macronutrient-deficient conditions, as well as throughout the diurnal light cycle. Both genes showed a robust and specific response to Fe limitation in laboratory cultures and were detected in small volume samples collected from the northeast Pacific, demonstrating the sensitivity of this method. Overall, FLDA1 and ISIP3 expression was inversely related to Fe concentrations and offered insight into the Fe nutritional health of T. oceanica in the field. As T. oceanica is a species tolerant to low Fe, indications of Fe limitation in T. oceanica populations may serve as a proxy for severe Fe stress in the overall diatom community. At two shallow coastal locations, FLD1A and ISIP3 expression revealed Fe stress in areas where dissolved Fe concentrations were high, demonstrating that this approach may be powerful for identifying regions where Fe supply may not be biologically available.

Figure 1

Relative transcript abundances (transcript abundances normalized to actin) for FLDA1 (a) and ISIP3 (b) in T. oceanica cultures grown under Fe limitation (−Fe), replete (+Fe), and following Fe readdition to Fe-limited cultures (−Fe+Fe). 14:10 indicates cultures grown on a 14:10 light:dark cycle and represents the initial time (T0) for the Fe readdition. Error bars represent s.d. for 12 biological replicates for the 14:10 replete samples, 22 biological replicates for the 14:10 Fe-limited samples and biological triplicates for both the 24 h light and Fe feedback experiments (+2Hr and +4Hr). Single asterisks (*) indicate a significant increase over replete expression (P<0.001, one-way ANOVA). Double asterisks (**) indicate a significant increase over replete expression and both Fe readdition timepoints (P<0.001, one-way ANOVA).

Figure 2

FLDA1 (a) and ISIP3 (b) RTA (normalized to actin) in T. oceanica cultures grown under macronutrient- and Fe-limiting conditions. Error bars represent the s.d. from biological triplicates for nitrogen (−N), phosphorus (−P) and silica (−Si) limited cultures, 22 biological replicates for Fe-limited (−Fe) cultures and 12 biological replicates for replete (Re) cultures. Double asterisks (**) indicate significant difference in expression relative to all other treatments (P<0.001, one-way ANOVA). Single asterisks (*) indicate significant difference in expression relative to replete cultures (P<0.05, one-way ANOVA).

Figure 3

FLDA1 (a) and ISIP3 (b) RTA in T. oceanica semicontinuous batch cultures grown in Fe replete (+Fe) and Fe-limited (−Fe) media under a 14:10 h light:dark cycle. Error bars represent the s.d. of biological replicates. Black and white boxes along the x-axis indicate dark and light periods, respectively. Daily mean values of expression in Fe-limited cells were significantly higher than replete cells for both genes (P<0.001, one-way ANOVA).

Figure 4

Northeast Pacific sample collection sites used in this study. Shading on the plot indicates bottom depth. Plot was generated using Ocean Data View (Schlitzer, 2012).

Figure 5

(a.) Surface chlorophyll a values at stations sampled in the northeast Pacific. (b.) T. oceanica FLDA1 expression (RTA) and dissolved Fe (dFe) concentrations from the corresponding stations. (c.) ISIP3 expression (RTA) and dissolved Fe (dFe) concentrations from the corresponding stations. Physically proximal stations are displayed adjacently on the X-axis. The solid lines in b and c represent an expression level one order of magnitude higher than replete values in laboratory culture experiments. The highest nighttime replete value was used for ISIP3. Asterisks (*) denote samples taken during nighttime hours, where ISIP3 values may be elevated as a result of diel fluctuations. Error bars represent the s.d. of triplicate determinations. Dissolved Fe data previously presented in Chappell et al. (2013).