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. 2003 Aug;132(4):2116-25.
doi: 10.1104/pp.103.021717.

Thylakoid-bound ascorbate peroxidase mutant exhibits impaired electron transport and photosynthetic activity

Cristian H Danna  1 , Carlos G Bartoli, Francisco Sacco, Lorena R Ingala, Guillermo E Santa-María, Juan J Guiamet, Rodolfo A Ugalde
Affiliations

Thylakoid-bound ascorbate peroxidase mutant exhibits impaired electron transport and photosynthetic activity

Cristian H Danna et al. Plant Physiol. 2003 Aug.

Abstract

In chloroplasts, stromal and thylakoid-bound ascorbate peroxidases (tAPX) play a major role in the removal of H(2)O(2) produced during photosynthesis. Here, we report that hexaploid wheat (Triticum aestivum) expresses three homeologous tAPX genes (TaAPX-6A, TaAPX-6B, and TaAPX-6D) mapping on group-6 chromosomes. The tAPX activity of a mutant line lacking TaAPX-6B was 40% lower than that of the wild type. When grown at high-light intensity photosystem II electron transfer, photosynthetic activity and biomass accumulation were significantly reduced in this mutant, suggesting that tAPX activity is essential for photosynthesis. Despite the reduced tAPX activity, mutant plants did not exhibit oxidative damage probably due to the reduced photochemical activity. This might be the result of a compensating mechanism to prevent oxidative damage having as a consequence a decrease in growth of the tAPX mutant plants.

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Figures

Figure 1.
Figure 1.
Sequence alignments of 3′-UTRs of tAPX cDNAs from wheat and proteins from different plant species. A, Alignment among 3′-UTRs of tAPX cDNAs from wheat. Nucleotide homology is boxed. Primers for each cDNA are indicated by bold type. The sense primer of TaAPX-6B is located over the coding region, hence, not shown (see “Materials and Methods”). Polyadenylation signals are underlined. B, Alignment among TaAPX-6B and other thylakoid-bound APX proteins from dicot species. Conserved amino acids are shown by a shadow box. Asterisks indicate essential amino acid residues for protein activity. Putative thylakoid membrane anchor domains are underlined.
Figure 2.
Figure 2.
Chromosomal location of tAPX genes. A, PCR amplification of tAPX genes from wheat genomic DNA obtained from R-SV8 (normal hexaploid genome), Nuli6A (a NuliA-TetraD genome), and Nuli6D (a NuliD-TetraA genome) plants. C, Negative PCR control. B, TaAPX-6B DNA gel-blot hybridization on genomic DNA from R-SV8 and S-SV8 plants digested with EcoRI or HindIII. C, TaAPX-6B DNA gel-blot hybridization on 10 μg of genomic DNA from rice (Oryza sativa), maize (Zea mays), and barley (Hordeum vulgare), digested with Hin-dIII. Results are representative of two independent experiments.
Figure 3.
Figure 3.
Expression of tAPX genes. A, Reverse transcriptase (RT)-PCR amplification of tAPX genes (TaAPX-6A, TaAPX-6B, and TaAPX-6D) from leaves of R-SV8 and S-SV8 plants. C, Negative PCR control. B, RT-PCR amplification of tAPX genes from different organs of R-SV8 plants. C, Negative PCR control; A, anthers; G, gynoecium; Ra, floral rachis; P, glumes and paleas; S, foliar sheaths; B, leaf blades; and Ro, roots. Results are representative of two independent experiments.
Figure 4.
Figure 4.
Semiquantitative RT-PCR analysis of TaAPX-6B mRNA accumulation in R-SV8 plants. A, Analysis of steady-state mRNA accumulation was carried out with plants grown in the dark (D), at 50 to 100 μmol photons m-2 s-1 (LL), or at 700 to 1,000 μmol photons m-2 s-1 (HL). B, Analysis of the short-term mRNA accumulation in plants suddenly exposed to 1,800 μmol photons m-2 s-1 (EL) combined with the application of MV for 4 h. In this case, plants were previously grown at 200 to 400 μmol photons m-2 s-1. From left to right, a base-two serial dilution of 100 and 10 ng of total cDNA was used as template for the amplification of TaAPX-6B and Actin, respectively. Asterisks indicate the last dilution at which TaAPX-6B was detected. Actin was detected up to the fifth cDNA dilution in every sample. Results are representative of two independent experiments.
Figure 5.
Figure 5.
APX activity in R-SV8 and S-SV8 plants. A, Foliar APX activity in R-SV8 (white bars) and S-SV8 (black bars) 2-week-old plants grown in the dark (D), at 50 to 100 μmol photons m-2 s-1 (LL), and at 700 to 1,000 μmol photons m-2 s-1 (HL). Bars are mean values ± se of three independent experiments, each one consisting of 10 replicates. B, Thylakoid-bound APX activity in HL-grown R-SV8 (white bars) and S-SV8 plants (black bars). Bars are mean values ± se of two experiments, each one consisting of three replicates. C, Foliar APX activity in R-SV8 (left panels) and S-SV8 plants (right panel) suddenly exposed to 1,800 μmol photons m-2 s-1 (white bars) and MV (black bars). Plants were previously grown at 200 to 400 μmol photons m-2 s-1. Bars are mean values ± se of two independent experiments, each one consisting of 10 replicates. Horizontal lines indicate APX activity at time 0. Asterisks indicate significant differences (t test, P < 0.05) between EL and MV treatments for a given genotype.
Figure 6.
Figure 6.
PFD-dependent growth of parental and mutant plants. A, Leaf area of R-SV8 (white bars) and S-SV8 plants (black bars) grown at 200, 400, or 800 μmol photons m-2 s-1. B, Dry weight of second leaf stage R-SV8 and S-SV8 plants grown at 200, 400, or 800 μmol photons m-2 s-1. Bars are mean values ± se, corresponding to three independent experiments, each one consisting of four replicates (plants). Asterisks indicate significant differences between genotypes (t test, P < 0.05).
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