Physiological performance of transplastomic tobacco plants overexpressing aquaporin AQP1 in chloroplast membranes

Abstract

The leaf mesophyll CO2 conductance and the concentration of CO2 within the chloroplast are major factors affecting photosynthetic performance. Previous studies have shown that the aquaporin NtAQP1 (which localizes to the plasma membrane and chloroplast inner envelope membrane) is involved in CO2 permeability in the chloroplast. Levels of NtAQP1 in plants genetically engineered to overexpress the protein correlated positively with leaf mesophyll CO2 conductance and photosynthetic rate. In these studies, the nuclear transformation method used led to changes in NtAQP1 levels in the plasma membrane and the chloroplast inner envelope membrane. In the present work, NtAQP1 levels were increased up to 16-fold in the chloroplast membranes alone by the overexpression of NtAQP1 from the plastid genome. Despite the high NtAQP1 levels achieved, transplastomic plants showed lower photosynthetic rates than wild-type plants. This result was associated with lower Rubisco maximum carboxylation rate and ribulose 1,5-bisphosphate regeneration. Transplastomic plants showed reduced mesophyll CO2 conductance but no changes in chloroplast CO2 concentration. The absence of differences in chloroplast CO2 concentration was associated with the lower CO2 fixation activity of the transplastomic plants. These findings suggest that non-functional pores of recombinant NtAQP1 may be produced in the chloroplast inner envelope membrane.

Fig. 1.

Integration of Nicotiana tabacum AQP1 into the tobacco chloroplast genome. (A) Map of the wild-type (WT) and AQP1-transformed plastid (pt) genomes. The transgenes were targeted to the intergenic region between trnI and trnA. The selectable marker gene aadA (encoding aminoglycoside 3ʹ-adenylyltransferase) was driven by the 16S ribosomal RNA operon promoter (Prrn). AQP1 was driven by the psbA promoter and 5ʹ-untranslated region (PpsbA). Arrows within boxes show the direction of transcription. Numbers below each ptDNA indicate the predicted size of hybridizing fragments when total DNA was digested with BamHI. A 0.8 kb fragment of the targeting region for homologous recombination was used as a probe (P1) for Southern blot analysis. TpsbA, 3ʹ-untranslated region of the psbA gene. (B) Southern blot analysis of two independent lines (1 and 2) for each transformation cassette.

Fig. 2.

Analysis of AQP1 expression in wild-type (WT) and AQP1 and TicAQP1 transplastomic plants. (A) Northern blot analysis of leaf samples. The expected transcript sizes of the mono- and dicistrons originating from different promoters are indicated below the map of the transformed plastid genome. The 515 bp AQP1 sequence (P2) was used as a probe. A 10 μg aliquot of total RNA was loaded per well. Ethidium bromide-stained rRNA was used to assess loading. (B) Western blot analysis of total protein from leaf samples (two independent lines for each construction). The lower panel was overexposed to show the 30 kDa AQP1 monomer, which was not detected in the upper panel. A 30 μg aliquot of protein was loaded per well. (C) Western blot analysis of proteins extracted from the plasma membrane. A 3 μg aliquot of protein was loaded per well. The positions and sizes of molecular weight protein standards are indicated. The blots in B and C were detected using anti-NtAQP1 as the primary antibody.

Fig. 3.

Localization of AQP1 in the thylakoid and envelope membranes. Envelope, thylakoid, and stroma fractions were isolated from wild-type (WT), AQP1, and TicAQP1 leaves, and separated by SDS-PAGE. Samples of 2, 20, and 30 μg of protein from the envelope, thylakoid, and stroma, respectively, were loaded per well. Representative western blots performed with antibodies to AQP1, the inner-membrane Tic40 protein, the thylakoid membrane-specific LHC chlorophyll a/b binding protein 1 (Lhcb1), and the stroma-specific ADP-glucose pyrophosphorylase (AGPase) are shown. Asterisks indicate the positions of monomer (*), dimer (**), trimer (***), and tetramer (****) AQP1.

Fig. 4.

(A) Net photosynthesis, (A_N), (B) maximum carboxylation velocity of Rubisco (V_Cmax), (C) maximum electron transport rate contributing to ribulose 1,5-bisphosphate regeneration (J_max), (D) stomatal conductance (g_s), (E) mesophyll conductance (g_m), and (F) ¹³C isotope discrimination (Δ) of wild-type (WT) and AQP1 and TicAQP1 transplastomic plants. Representative data from two independent experiments are presented. Values are means ±SE (n=7). Different letters indicate significantly different values (ANOVA, P<0.05).

Fig. 5.

(A) Intercellular CO₂ concentration (C_i), (B) chloroplast CO₂ concentration (C_c), (C) and C_c/C_i ratio of wild-type (WT) and AQP1 and TicAQP1 transplastomic plants. Representative data from two independent experiments are presented. Values are means ±SE (n=7). Different letters indicate statistically different values (ANOVA, P<0.05).

Fig. 6.

Changes in chloroplast ultrastructure due to AQP1 overexpression from the plastid genome. Transmission electron microscopic images of chloroplasts from (A) wild-type (WT), (B) AQP1, and (C) TicAQP1 plants. (D–F) Detail of the thylakoids from (D) WT, (E) AQP1, and (F) TicAQP1 plants. (G–I) Detail of the chloroplast envelope (delineated by two arrowheads) in (G) WT, (H) AQP1, and (I) TicAQP1 plants. C, cytosol; s, starch granule; st, stroma; v, vacuole. Scale bar=1 μm.