Decreased Expression Of A Gene Caused By A T-DNA Insertion In ...

An aladin knockout mutant is defective in photosynthesis

ALADIN (ALacrima Achalasia aDrenal Insufficiency Neurologic disorder) is an evolutionally conserved nucleoporin that is a member of the nuclear pore complex (NPC) in higher animals and plants [18, 19]. In humans, mutations in the ALADIN gene lead to a rare autosomal recessive disorder called the triple A syndrome [20, 21]. However, mice lacking a functional ALADIN gene do not exhibit this phenotype and are indistinguishable phenotype from wild-type mice [22, 23], suggesting a species-specific function for ALADIN. In this study, a single line knockout mutant from the FLAG T-DNA Versailles INRA collection was used to examine the physiological role of ALADIN in Arabidopsis (Fig 1A). Another ALADIN T-DNA knockout mutant (SALK_148848 line) was available, but any plants had no T-DNA insertion in ALADIN gene. Genomic sequencing of the T-DNA flanking region confirmed that the aladin-1 mutant (FLAG_453B04) had a T-DNA insertion in the middle of the ALADIN gene (S1 Fig). RT-PCR revealed that the mutant accumulated no full-length ALADIN transcript (Fig 1B) but a truncated transcript (S2 Fig).

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Fig 1. Isolation of the aladin-1 mutant.

(A) Schematic representation of the ALADIN gene. A T-DNA insertion site in the FLAG_453B04 line is shown. The orientation of the left border sequence is indicated by an arrow. (B) RT-PCR analysis of ALADIN transcription in the aladin-1 mutant. ACTIN2 (ACT2) was used as a loading control. (C-D), Three-week- (C) and five-week- (D) old wild-type (WT) and aladin-1 plants.

https://doi.org/10.1371/journal.pone.0147911.g001

The aladin-1 mutant exhibited pleiotropic growth defects at various developmental stages. Compared to the wild type, the mutant showed extremely stunted growth (Fig 1C), including dwarfism (Fig 1D), and a shorter root length in seedlings (Fig 2A). The shorter root phenotype was partially rescued by exogenous sucrose (Fig 2A and 2B), suggesting an impairment of carbon metabolism in the mutant. Mutant leaves were pale green in color and contained less chlorophyll than wild type (Fig 2C). These results indicated that the aladin-1 mutant was significantly impaired in chloroplast development and photosynthesis. Next, electron microscopic analysis was performed to examine the ultrastructure of the chloroplasts. Chloroplasts in epidermal cotyledon cells were smaller and had a more irregular shape in the mutant than in wild type (Fig 2D, upper panels). Furthermore, in mutant cells, thylakoid membranes were highly fragmented and vesicle-like structures were apparent (Fig 2D, lower panels). The maximum quantum efficiency of photosystem II was 55% lower in the mutant than in wild type (Fig 2E). These results indicated that the aladin-1 mutation hindered the normal development of the thylakoid membrane and led to a loss of photosynthetic activity.

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Fig 2. The aladin-1 mutant exhibits low photosynthetic activity.

(A) Seven-day-old wild-type (WT) and aladin-1 seedlings grown on MS plates with (+ sucrose) or without (- sucrose) sucrose. (B) Root length of 10-day-old WT and aladin-1 seedlings grown on MS plates with (+ sucrose) or without (- sucrose) sucrose. Mean ± standard deviation for n > 20 (Student’s t-test, *P < 0.001). (C) Quantification of chlorophyll a (chl a), chlorophyll b (chl b), and total chlorophyll (total chl) in mature WT and aladin-1 leaves. Mean ± standard deviation for n > 3 (Student’s t-test, *P < 0.001). (D) Electron micrographs of mesophyll cells (upper) and chloroplasts (lower) in mature leaves from 14-day-old WT and aladin-1 plants. c, chloroplast; v, vacuole. (E) Maximum quantum yield of photosysytem II (Fv/Fm).

https://doi.org/10.1371/journal.pone.0147911.g002

Accumulation of chloroplast proteins was investigated by SDS-PAGE and immunoblot of lysates from mature leaves. Accumulation of RuBisCO subunits and PSBA (photosystem II reaction center protein A) was significantly lower in the mutant than in wild type (Fig 3A and 3B). By contrast, another nuclear-encoded chloroplast protein, PGL35 (plastoglobulin 35 kDa), was present at similar levels in wild type and the mutant (Fig 3B). Transcription levels of the genes encoding the RuBisCO subunits and PSBA were indistinguishable between the wild type and the mutant (Fig 3C). These results suggested that aladin-1 was impaired in the accumulation of some chloroplast proteins needed for proper chloroplast function.

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Fig 3. Translation of chloroplast proteins is significantly impaired in aladin-1.

(A-B) Protein extracts from 7-day-old wild-type (WT) and aladin-1 seedlings were subjected to SDS-PAGE followed by either Coomassie Brilliant Blue staining (A) or immunoblotting (B) with anti-RuBisCO large subunit (RBCL), RuBisCO small subunit (RBCS), light-harvesting chlorophyll a/b binding protein (LHCP), photosystem II reaction center protein A (PSBA), plastoglobulin 35 kDa (PGL35), or binding protein (BiP) antibodies. Molecular masses are indicated on Fig 3A (kDa). Technical duplicate was done: two independent extractions of protein (A) and mRNA (B) were subjected to two lanes on each panel. (C) RT-PCR analysis of RBCL, RBCS, PSBA, and ACT2 transcription in WT and aladin-1. Technical duplicate was done: two independent extractions of mRNA were subjected to two lanes on each panel.

https://doi.org/10.1371/journal.pone.0147911.g003