Figure 1.
abo8 mutants are hypersensitive to ABA in seed germination and root growth.
A. The root growth phenotype of abo8 mutants on MS medium or MS medium supplemented with different concentrations of ABA. Four-day-old seedlings grown on MS were transferred to MS medium containing 0, 10, or 30 µM ABA for 5 days before they were photographed. Bars = 5 mm. B. Statistical analysis of the root length in different growing times. C. Relative root growth. Root length is expressed relative to that of the wild type or abo8 mutants without ABA. In (B) and (C), three independent experiments were done with similar results, each with three biological repeats. Five roots from one plate were measured for each repeat. Values are means ±SE, n = 3. **: P<0.01. D. Seed germination greening of abo8 mutants after 7 days on MS medium supplemented with 0.0, 0.3, or 0.5 µM ABA. Bars = 2 mm. E. Statistical analysis of seed germination greening rate in (D). Three independent experiments were done, each with three replicates (from three different plates). About 24 seeds were counted for each plate. Values are means ±SE, n = 3. **: P<0.01.
Figure 2.
A. ABO8 was mapped to chromosome 4. A point mutation from G447 to A447 (counting from the first putative ATG in AT4G11690) was identified in abo8-1. A T-DNA insertion line SALK_025470 (abo8-2, T-DNA is inserted in position +358) was obtained from the Arabidopsis Biological Resource Center. B. RT-PCR analysis of ABO8 transcripts in abo8-2 using primers flanking the T-DNA insertion. Expression of the ACTIN2 gene was used as control. C. The protein structure of ABO8. SP: signal peptide; PPR: pentatricopeptide repeat. D. Genetic analysis of abo8-1 and abo8-2 for sensitivity of root growth to ABA. Four-day-old seedlings grown on MS medium were transferred to MS medium containing 0 or 30 µM ABA. abo8-1 abo8-2: F1 seedlings of abo8-1 crossed with abo8-2. Bars = 5 mm. E. Seed germination greening analysis of abo8-1 complemented with an ABO8 promoter driving ABO8 cDNA fused with GFP cDNA (ProABO8:ABO8-GFP) on MS medium containing 0.0 or 0.3 µM ABA. Bars = 5 mm. F. Statistical analysis of seed germination greening rate of the complemented abo8-1 in (E). Three independent experiments were done, each with three replicates (from three different plates). About 24 seeds were counted for each plate. Values are means ±SE, n = 3. **: P<0.01. G. Roots of abo8-1 complemented with ProABO8:ABO8-GFP. Four-day-old seedlings were transferred to MS medium containing 0 or 30 µM ABA. Bars = 5 mm. H. Statistical analysis of root length in (G). Root length is expressed relative to that of the wild type or abo8-1 without ABA. Three independent experiments were done with similar results, each with three biological repeats. Five roots from one plate were measured for each repeat. Values are means ±SE, n = 3. **: P<0.01.
Figure 3.
The expression and localization of ABO8.
A. The expression pattern of ProABO8:GUS in the whole seedling and roots. a, a seedling, bar = 1 mm; b, a primary root, bar = 50 µm; c, an amplified primary root tip, bar = 20 µm; d, a lateral root primordium, bars = 50 µm. B. The expression of ProABO8:ABO8-GFP in a lateral root (a, left) and a lateral root primordium (b, right). Bars = 50 µm. C. The expression of ProABO8:ABO8-GFP in a primary root. Bars = 50 µm. D. Co-subcellular localization of ABO8-GFP with Mito-Tracker. Bars = 10 µm. E. Expression of ABO8 is reduced by ABA treatment. Seedlings were treated with or without 30 µM ABA for 8 h, and total RNAs were used for qRT-PCR. ACTIN2 was used as a control. Three independent experiments were done with similar results, each with three biological replicates. Results shown are from one experiment. Values are means ±SE, n = 3. **: P<0.01.
Figure 4.
ABO8 regulates the splicing of NAD4 intron 3.
A. Northern blot analyses of the expression of different subunits in complex I. Only NAD4 showed the splicing defects in abo8-1. B. qRT-PCR analysis of different fragments of the NAD4 transcript. A pair of primers covering different fragments or different introns was used for qRT-PCR. Total RNAs from 10-day-old seedlings were used for qRT-PCR. Three independent experiments were done with similar results, each with three biological repeats. Values are means ±SE from one experiment. **: P<0.01.
Figure 5.
abo8-1 accumulates more ROS than the wild type in root tips.
A. The activity of complex I in the wild type (WT) and abo8-1. Crude mitochondria membrane protein extracted from 5-day-old seedlings treated with or without 50 µM ABA for 24 h were separated by Blue Native-PAGE. Coomassie staining (left) indicates protein complexes after electrophoresis; Complex I activity was preformed by in-gel staining with NADH and NBT (nitroblue tetrazolium) for the NADH dehydrogenase activity of Complex I. B. ATP content in WT and abo8-1 plants. 5-day-old WT and abo8-1 seedlings treated with or without 50 µM ABA for 24 h were harvested for measurement of ATP content. FW, fresh weight. Means with different letters are significantly different at P<0.01. C. Fluorescence analysis of mitochondrial cpYFP in primary root tips of the wild type and abo8-1 with or without ABA treatment. Bars = 50 µm. D. cpYFP intensity as determined with AxioVision Rel. 4.8 software. E. DAB staining for H2O2 in primary root tips of the wild type and abo8-1 with or without ABA treatment. Bars = 50 µm. F. DAB staining intensity as determined with Adobe Photoshop 3.0 software. G. DCFH-DA staining for H2O2 in primary root tips of the wild type and abo8-1 with or without ABA treatment. Bars = 50 µm. H. DCFH-DA staining intensity as determined with AxioVision Rel. 4.8 software. I. NBT staining for superoxide in primary root tips of the wild type and abo8-1 with or without ABA treatment. Bars = 50 µm. J. NBT staining intensity as determined with Adobe Photoshop 3.0 software. Three independent experiments were done with similar results, each with three replicates, and each replicate with 15–20 roots. Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01. K. Relative expression levels of AOX1a in seedlings treated with or without 50 µM ABA for 5 h in liquid MS. Three independent experiments were done with similar results, each with three biological replicates. Results shown are from one experiment. Values are means ±SE, n = 3. Means with different letters are significantly different at P<0.01.
Figure 6.
abo8 mutation decreases the activity of the root meristem.
A. The meristem zone (white line), elongation zone (blue line), and differentiation zone (green line) with 0 or 30 µM ABA treatment. Bars = 100 µm. Each image was made by joining several photographs of the same root. B. Meristem cell number of the wilt type, abo8-1 and abo8-2 in different times after seed germination (DAG). C. The meristem zone length, cell number, and cell length with 0 or 30 µM ABA treatment. D. The elongation zone length, cell number, and cell length with 0 or 30 µM ABA treatment. E. The differentiation zone length, cell number, and cell length with 0 or 30 µM ABA treatment. In C–E, two experiments were done with similar results, each with three repeats, each repeat with 10 roots. Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01. F. Size of the amplified root meristem in the wild type and abo8-1 with and without ABA treatment. Bars = 50 µm. G. The expression of ProCYCB1;1:GUS in the wild type and abo8-1 with or without 30 µM ABA treatment. Bars = 50 µm.
Figure 7.
Addition of GSH partially recovers the ABA-hypersensitive phenotypes of abo8-1 mutants.
A. Germination greening of wild-type and abo8-1 seeds on MS medium or MS medium containing 0.3 µM ABA, 0.3 µM ABA plus 100 µM GSH, or 0.3 µM ABA plus 300 µM GSH. Bars = 5 mm. B. Statistical analysis of the seed germination greening rate in (A). Three independent experiments were done with similar results, each with three replicates. About 20 seeds were counted from one plate as one replicate. Values are means ±SE from one experiment. **: P<0.01. C. Roots of wild-type and abo8-1 seedlings grown on MS medium or MS medium containing 30 µM ABA, 30 µM ABA plus 100 µM GSH, or 30 µM ABA plus 300 µM GSH. The red line indicates the starting point of root growth after transfer. Bars = 5 mm. D. Statistical analysis of root growth in (C). Root growth is expressed relative to that of the wild type or abo8-1 in MS medium without supplement. Three independent experiments were done with similar results, each with three replicates. Eight roots were measured from one plate as one replicate. Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01. E. Root meristem zones of seedlings grown on MS medium or MS medium containing 30 µM ABA, 30 µM ABA plus 100 µM GSH, or 30 µM ABA plus 300 µM GSH. Bars = 50 µm. F. Statistical analysis of the MZ cell number in (E). Three independent experiments were done with similar results, each with three repeats, and each repeat with 15–20 roots. Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01.
Figure 8.
Expression of DR5, IAA2, PIN1, PIN2, and AUX1 is altered in the abo8-1 mutant.
A. The expression of ProDR5:GUS in roots of seedlings grown on MS medium or MS medium containing 30 µM ABA, 300 µM GSH, or 30 µM ABA plus 300 µM GSH for 5 days. Bars = 50 µm. B. The expression of ProDR5:GUS in 5-day old seedlings treated in liquid MS medium without or with 1 µM 2,4-D, 5 µM IAA, and 10 µM NAA, or 30 nM NAA for 18 h. Bars = 50 µm. C. The expression of ProDR5:GUS in 5-day old seedlings treated in MS liquid medium without or with 300 µM GSH, 5 µM IAA, or 5 µM IAA plus 300 µM GSH for 18 h. Bars = 50 µm. D. The expression of ProIAA2:GUS with and without ABA treatment. Bars = 50 µm. E. The expression of ProPIN1:PIN1-GFP with and without ABA treatment. Bars = 50 µm. F. The fluorescence of PIN1-GFP in (E). Fluorescence is expressed relative to that of the wild type without ABA. Three experiments were done with similar results, each experiment with three repeats (each repeat with 15 roots). Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01 for a and b and at P<0.05 for c and d. G. The expression of ProPIN2:PIN2-GFP with and without ABA treatment. Bars = 50 µm. H. The fluorescence of PIN2-GFP in (G). Fluorescence is expressed relative to that of the wild type without ABA. Three experiments were done with similar results, each experiment with three repeats (each repeat with 15 roots). Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01 except for c and d at P<0.05. I. The expression of ProAUX1:AUX1-YFP with or without ABA treatment. Bars = 50 µm. J. The fluorescence of AUX1-YFP in (I). Fluorescence is expressed relative to that of the wild type without ABA. Three experiments were done with similar results, each experiment with three repeats (each repeat with 15 roots). Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01 except for b and c at P<0.05. K. The abo8-1 contained less auxin than wild type. 7-day seedlings were used for auxin contents measurement. Each value is the mean ± SD (n = 4). *: P<0.05.
Figure 9.
The abo8 mutation affects the expression of PLT1 and PLT2.
A. The expression of ProPLT1:PLT1-YFP on MS medium or MS medium supplemented with ABA, GSH, or ABA plus GSH. Bars = 50 µm. B. The fluorescence of PLT1-YFP in (A). Fluorescence is expressed relative to that of the wild type in MS without supplement. Three independent experiments were done with similar results, each with three replicates. In each replicate, 8–15 roots were measured from one plate. Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01, except for c and d, d and e, at P<0.05. C. The expression of ProPLT1:CFP on MS medium or MS medium supplemented with ABA, GSH, or ABA plus GSH. Bars = 50 µm. D. The relative fluorescence of ProPLT1:CFP in (E). Fluorescence is expressed relative to that of the wild type in MS without supplement. Three independent experiments were done with similar results, each with three replicates. In each replicate, 8–15 roots were measured from one plate. Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01, except for a and b, c and d, at P<0.05. E. The expression of ProPLT2:PLT2-YFP on MS medium or MS medium supplemented with ABA, GSH, or ABA plus GSH. Bars = 50 µm. F. The relative fluorescence of PLT2-YFP in (C). Fluorescence is expressed relative to that of the wild type in MS without supplement. Three independent experiments were done with similar results, each with three replicates. In each replicate, 8–15 roots were measured from one plate. Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.05.
Figure 10.
Genetic analysis of ABO8 with PLT1 and PLT2.
A. The roots of the wild type Col-0, Ws, plt1-4, plt2-2, abo8-1, abo8-1 plt1-4, and abo8-1 plt2-2 grown on MS medium or MS medium supplemented with different concentrations of ABA, 300 µM GSH or 30 µM ABA plus 300 µM GSH for 5 days. Red line indicates the root tip positions after 5-day seedlings were just transferred onto different medium. Bars = 5 mm. B–E. The root growth and relative root growth of different plants in (A). Relative root growth is expressed to that of the wild type or mutants on MS without ABA. About 15 roots from three plates were measured in each experiment, and three experiments were done with similar results. Values are means ±SE, n = 3. Means with different letters are significantly different at P<0.01. F. The length of the root meristem (red line) of different genotypes on MS medium or MS medium supplemented with 30 µM ABA, 300 µM GSH or 30 µM ABA plus 300 µM GSH. Bars = 50 µm. G. The MZ cell number in (F). Three independent experiments were done with similar results, and each experiment had three replicates. About 20 roots were measured for each replicate. Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01.
Figure 11.
The sensitivity of abo8-1 roots to ABA is partially rescued by the expression of the inducible Pro35S:PLT2-GR.
A. The length of the root meristem (red line) of wild-type (carrying Pro35S:PLT2-GR) and abo8-1 (carrying Pro35S:PLT2-GR) seedlings on MS medium or MS medium supplemented with DEX, ABA, or ABA plus DEX. Bars = 50 µm. B. Root meristem cell number of the wild type (Col-0) and abo8-1 in (A). Three independent experiments were done with similar results, and each experiment had three replicates. About 20 roots were measured for each replicate. Values are means ±SE from one experiment. Means with different letters are significantly different at P<0.01.
Figure 12.
abo8 mutation delays the distal stem cell (DSC) differentiation.
A. Differentiation status of DSC in 5-day-old seedlings on MS medium or MS medium with 300 µM GSH. DSC (green arrowheads) are characterized as cells between the QC (pink triangle) and the starch containing differentiated columella cells. Bars = 20 µm. B. Quantitative evaluation of DSC layers in A. About 30 roots were examined for each biological repeat, three experiments were done with similar results. Values are means ±SE from one experiment. Means in different letters are P<0.01. C. The expression of DSC marker J2341 in roots of the wild type and abo8-1 mutants. Bars = 20 µm. D. A proposed model for ABO8 in root growth. ABO8 is downreguated by ABA. ABO8 negatively modulates mitochondria ROS, and ROS negatively modulate auxin level and signaling to control PLTs expression. It is likely that ROS also directly regulate PLT1 as posttranscriptional level.