Molecular physiological analysis of the two plastidic ATP/ADP transporters from Arabidopsis

Abstract

Arabidopsis (Arabidopsis thaliana) possesses two isoforms of plastidic ATP/ADP transporters (AtNTT1 and AtNTT2) exhibiting similar biochemical properties. To analyze the function of both isoforms on the molecular level, we examined the expression pattern of both genes by northern-blot analysis and promoter-beta-glucuronidase fusions. AtNTT1 represents a sugar-induced gene mainly expressed in stem and roots, whereas AtNTT2 is expressed in several Arabidopsis tissues with highest accumulation in developing roots and young cotyledons. Developing lipid-storing seeds hardly contained AtNTT1 or -2 transcripts. The absence of a functional AtNTT1 gene affected plant development only slightly, whereas AtNTT2T-DNA, AtNTT1-2T-DNA, and RNA interference (RNAi) plants showed retarded plant development, mainly characterized by a reduced ability to generate primary roots and a delayed chlorophyll accumulation in seedlings. Electron microscopic examination of chloroplast substructure also revealed an impaired formation of thylakoids in RNAi seedlings. Moreover, RNAi- and AtNTT1-2T-DNA plants showed reduced accumulation of the nuclear-encoded protein CP24 during deetiolation. Under short-day conditions reduced plastidic ATP import capacity correlates with a substantially reduced plant growth rate. This effect is absent under long-day conditions, strikingly indicating that nocturnal ATP import into chloroplasts is important. Plastidic ATP/ADP transport activity exerts significant control on lipid synthesis in developing Arabidopsis seeds. In total we made the surprising observation that plastidic ATP/ADP transport activity is not required to pass through the complete plant life cycle. However, plastidic ATP/ADP-transporter activity is required for both an undisturbed development of young tissues and a controlled cellular metabolism in mature leaves.

Figure 1.

Expression analysis of AtNTT-genes. Transcripts of AtNTT1 and AtNTT2 were detected by northern-blot analysis. For this, total RNA was isolated, separated by electrophoresis, and transferred to a nylon membrane. Ethidium bromide (EtBr) staining reveals equal RNA loading. A, Specificity of AtNTT1 and AtNTT2 probes. AtNTT1 and AtNTT2 cDNA was spotted onto nylon membranes (10–1,000 pg) and subsequently hybridized with an AtNTT1- or AtNTT2-specific probe, respectively. B, Tissue-specific mRNA accumulation. Total RNA (10 μg) was isolated from Arabidopsis tissues (roots, leaves, stems, flowers, and siliques) and hybridized with gene-specific probes. C, Alterations of AtCAB-, AtNTT1-, and AtNTT2-mRNA-levels during the first 6 d of development. The upper pictures represent the corresponding plant phenotypes. Total RNA was extracted from whole seedlings. Blots were hybridized with an AtCAB-, AtNTT1-, or AtNTT2-specific probe. D, Effects of sugar feeding on mRNA accumulation in source leaf discs. Source leaf discs were floated on either water (control), or 100 mm Glc or Suc. Samples were taken at the indicated time points and blots were hybridized with gene-specific probes.

Figure 2.

Histochemical localization of GUS expression under the control of either the AtNTT1 or AtNTT2 promoter in Arabidopsis seedlings and mature tissues. A, Promoter-GUS activity in developing seedlings. Seeds were sown on Murashige and Skoog agar plates and harvested after 1, 2, 3, and 6 d after imbibition and analyzed for GUS activity according to standard protocols. B, Promoter-GUS activity in source leaves. Rosette leaves were harvested from plants, grown under short-day conditions, and GUS stained. C, GUS expression in flowers at different developmental stages.

Figure 3.

Molecular characterization of homozygous AtNTT1 knockout mutants. A, Analysis of the AtNTT1-T-DNA-insertion line Salk_013530 (designated AtNTT1∷T-DNA). The insertion in AtNTT1∷T-DNA is localized in the first exon. The primers used for PCR analysis are marked as arrows. Primer NTT1/1 was chosen from the AtNTT1-promoter region, primer NTT1/2 from the 3′-untranslated region; SALK_LB-primer from the left border of the T-DNA. B, PCR analysis on genomic DNA of wild-type (WT) and homozygous AtNTT1∷T-DNA mutants. C, RT-PCR analysis of the expression of the AtNTT1 genes in wild-type and in AtNTT1∷T-DNA mutant plants. cDNA was isolated from rosette leaves. Actin PCR reveals correct PCR conditions. D, Northern-blot analysis of wild-type and AtNTT1∷T-DNA mutant plants. Total RNA was extracted from rosette leaves. EtBr staining revealed equal RNA loading. Blots were hybridized with AtNTT1 or AtNTT2 specific probes, respectively.

Figure 4.

Molecular characterization of homozygous AtNTT2 knockout mutants. A, Analysis of the AtNTT2-T-DNA-insertion line Garlic_288_E08.b.1a.Lb3FA (designated AtNTT2∷T-DNA). The insertion in AtNTT2∷T-DNA is localized in the second exon. The primers used for PCR analysis are marked as arrows. Primer NTT2/4 was chosen from the AtNTT2-promoter region, primer NTT2/2 from the 3′-untranslated region, and GARLIC_LB-primer from the left border of the T-DNA. B, PCR analysis on genomic DNA of wild-type (WT) and homozygous AtNTT2∷T-DNA mutants. C, RT-PCR analysis of the expression of the AtNTT2-genes in wild-type and in AtNTT2∷T-DNA mutant plants. cDNA was isolated from rosette leaves. Actin PCR revealed correct PCR conditions. D, Northern-blot analysis of wild-type and AtNTT2∷T-DNA mutant plants. Total RNA was extracted from rosette leaves. EtBr staining revealed equal RNA loading. Blots were hybridized with AtNTT1 or AtNTT2 specific probes, respectively.

Figure 5.

Molecular characterization of the double-knockout mutant (designated AtNTT1-2∷T-DNA) and RNAi mutant. A, Amplification of AtNTT1- and AtNTT2-specific PCR products. B, Identification of the T-DNA in the AtNTT1 and AtNTT2 gene in the double-knockout mutant. C, Structure of the RNAi construct. A 418-bp fragment from AtNTT1cDNA was cloned in sense and antisense orientation into the pHANNIBALL vector. D, Northern-blot analysis of wild-type plants and different RNAi-lines. Five independent RNAi-lines were tested. Total RNA was extracted from rosette leaves. EtBr staining shows equal RNA loading. Blots were hybridized with AtNTT1 or AtNTT2 specific probes, respectively.

Figure 6.

Root growth analysis during the first 24 h of germination. Arabidopsis seeds were sown on Murashige and Skoog agar plates. Twenty-four hours after imbibition the number of rooted seedlings was counted. A, Phenotype of wild-type and transgenic seedlings after 24 h on Murashige and Skoog agar plates. B, Number of rooted seedlings after 24 h. Per experiment, 30 seedlings were counted. Data represent the mean of two independent experiments.

Figure 7.

Growth analysis, chlorophyll quantification, and chloroplast ultrastructure analysis from wild-type and transgenic plants. A, Growth analysis of 5-d-old wild-type and transgenic plants, grown in a climate-controlled chamber on soil under ambient conditions. B, Chlorophyll content in wild-type and transgenic seedlings. Data correspond to plants shown in A. Per measurement, 40 seedlings without root tissue were harvested. The data represent the mean of two independent experiments. sd less than 6% of the given mean. C, Chloroplast ultrastructure analyzed by transmission electron microscopy. Chloroplast substructure has been determined on 5-d-old wild-type or RNAi (line 10) seedlings, grown under short-day conditions.

Figure 8.

Chlorophyll content and accumulation of nuclear-encoded CP24 protein in wild-type and mutant plants. Wild-type and transgenic seedlings were germinated for 6 d in the dark, and etiolated seedlings were subsequently illuminated for up to 24 h (at 100 μmol quanta m² s⁻¹). A, Change in the chlorophyll content during illumination. For each measurement, 0.1-g seedlings without root tissue were harvested (wild type, black bar; RNAi, gray bar; AtNTT1-2∷T-DNA, white bar). B, Accumulation of chlorophyll-binding protein CP24 during illumination. Western-blot analysis was carried out using a polyclonal antiserum raised against the purified CP24 protein.

Figure 9.

RT-PCR expression analysis of genes coding for various plastidic glycolytic enzymes and different plastidic phosphate transporters. Wild-type and RNAi seedlings were germinated for 6 d in the dark and subsequently illuminated for 8 h. mRNA was extracted from seedlings without root tissue and converted to cDNA by RT. Gene-specific primers were chosen to amplify about 500-bp fragments coding for plastidic phosphoglycerate kinases 1 and 2 (AtPGK1, AtPGK2); pyruvate kinase 1, 2, and 3 (AtPK1, AtPK2, AtPK3); plastidic triose phosphate- (AtTPT); Glc-6-phosphate- (AtGPT); xylulose-5-phosphate/phosphate translocator (AtXPT); or phosphoenolpyruvate/phosphate transporters 1 and 2 (AtPPT1, AtPPT2). AtActin is given as control.

Figure 10.

Growth analysis of wild-type and null mutants. Plants were grown on soil for 50 d in a climate-controlled growth chamber at 22°C and 100 μmol quanta m² s⁻¹. A, Growth pattern under short-day conditions (10 h light). B, Growth pattern under long-day conditions (16 h light).

Figure 11.

Seed quality analysis of wild-type and transgenic plants. Plants were grown for 35 d on soil under short-day conditions. Subsequently the light phase was prolonged to induce flowering (16 h light) until the life cycle was completed. Seeds from wild-type, AtNTT1∷T-DNA, AtNTT2∷T-DNA, AtNTT1-2∷T-DNA, and RNAi were collected, and seed weight (A), lipid content (B), and protein content (C) in dry seeds were quantified. Data represent the mean of three independent experiments.