<i>abo8</i> mutation decreases the activity of the root meristem.

<p>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, <i>abo8-1</i> and <i>abo8-2</i> 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 <i>abo8-1</i> with and without ABA treatment. Bars = 50 µm. G. The expression of <i>ProCYCB1;1:GUS</i> in the wild type and <i>abo8-1</i> with or without 30 µM ABA treatment. Bars = 50 µm.</p

ABO8 regulates the splicing of <i>NAD4</i> intron 3.

<p>A. Northern blot analyses of the expression of different subunits in complex I. Only <i>NAD4</i> showed the splicing defects in <i>abo8-1</i>. B. qRT-PCR analysis of different fragments of the <i>NAD4</i> 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.</p

The expression and localization of ABO8.

<p>A. The expression pattern of <i>ProABO8:GUS</i> 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 <i>ProABO8:ABO8-GFP</i> in a lateral root (a, left) and a lateral root primordium (b, right). Bars = 50 µm. C. The expression of <i>ProABO8:ABO8-GFP</i> in a primary root. Bars = 50 µm. D. Co-subcellular localization of ABO8-GFP with Mito-Tracker. Bars = 10 µm. E. Expression of <i>ABO8</i> 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. <i>ACTIN2</i> 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.</p

ABA-Mediated ROS in Mitochondria Regulate Root Meristem Activity by Controlling <i>PLETHORA</i> Expression in <i>Arabidopsis</i>

<div><p>Although research has determined that reactive oxygen species (ROS) function as signaling molecules in plant development, the molecular mechanism by which ROS regulate plant growth is not well known. An <i><u>ab</u></i>a <i><u>o</u></i>verly sensitive mutant, <i>abo8-1</i>, which is defective in a pentatricopeptide repeat (PPR) protein responsible for the splicing of <i>NAD4</i> intron 3 in mitochondrial complex I, accumulates more ROS in root tips than the wild type, and the ROS accumulation is further enhanced by ABA treatment. The <i>ABO8</i> mutation reduces root meristem activity, which can be enhanced by ABA treatment and reversibly recovered by addition of certain concentrations of the reducing agent GSH. As indicated by low <i>ProDR5:GUS</i> expression, auxin accumulation/signaling was reduced in <i>abo8-1</i>. We also found that ABA inhibits the expression of <i>PLETHORA1</i> (<i>PLT1</i>) and <i>PLT2</i>, and that root growth is more sensitive to ABA in the <i>plt1</i> and <i>plt2</i> mutants than in the wild type. The expression of <i>PLT1</i> and <i>PLT2</i> is significantly reduced in the <i>abo8-1</i> mutant. Overexpression of PLT2 in an inducible system can largely rescue root apical meristem (RAM)-defective phenotype of <i>abo8-1</i> with and without ABA treatment. These results suggest that ABA-promoted ROS in the mitochondria of root tips are important retrograde signals that regulate root meristem activity by controlling auxin accumulation/signaling and <i>PLT</i> expression in <i>Arabidopsis</i>.</p></div

Genetic analysis of <i>ABO8</i> with <i>PLT1</i> and <i>PLT2</i>.

<p>A. The roots of the wild type Col-0, Ws, <i>plt1-4</i>, <i>plt2-2</i>, <i>abo8-1</i>, <i>abo8-1 plt1-4</i>, and <i>abo8-1 plt2-2</i> 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.</p

<i>abo8</i> mutation delays the distal stem cell (DSC) differentiation.

<p>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 <i>abo8-1</i> 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.</p

Map-based cloning of <i>ABO8</i>.

<p>A. <i>ABO8</i> was mapped to chromosome 4. A point mutation from G447 to A447 (counting from the first putative ATG in <i>AT4G11690</i>) was identified in <i>abo8-1</i>. A T-DNA insertion line SALK_025470 (<i>abo8-2</i>, T-DNA is inserted in position +358) was obtained from the Arabidopsis Biological Resource Center. B. RT-PCR analysis of <i>ABO8</i> transcripts in <i>abo8-2</i> using primers flanking the T-DNA insertion. Expression of the <i>ACTIN2</i> gene was used as control. C. The protein structure of ABO8. SP: signal peptide; PPR: pentatricopeptide repeat. D. Genetic analysis of <i>abo8-1</i> and <i>abo8-2</i> 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. <i>abo8-1 abo8-2</i>: F1 seedlings of <i>abo8-1</i> crossed with <i>abo8-2</i>. Bars = 5 mm. E. Seed germination greening analysis of <i>abo8-1</i> complemented with an <i>ABO8</i> promoter driving <i>ABO8</i> cDNA fused with <i>GFP</i> cDNA (<i>ProABO8:ABO8-GFP</i>) 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 <i>abo8-1</i> 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 <i>abo8-1</i> complemented with <i>ProABO8:ABO8-GFP</i>. 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 <i>abo8-1</i> 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.</p

Expression of <i>DR5</i>, <i>IAA2</i>, <i>PIN1</i>, <i>PIN2</i>, and <i>AUX1</i> is altered in the <i>abo8-1</i> mutant.

<p>A. The expression of <i>ProDR5:GUS</i> 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 <i>ProDR5:GUS</i> 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 <i>ProDR5:GUS</i> 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 <i>ProIAA2:GUS</i> with and without ABA treatment. Bars = 50 µm. E. The expression of <i>ProPIN1:PIN1-GFP</i> 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 <i>ProPIN2:PIN2-GFP</i> 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 <i>ProAUX1:AUX1-YFP</i> 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 <i>abo8-1</i> 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.</p

<i>abo8-1</i> accumulates more ROS than the wild type in root tips.

<p>A. The activity of complex I in the wild type (WT) and <i>abo8-1</i>. 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 <i>abo8-1</i> plants. 5-day-old WT and <i>abo8-1</i> 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 <i>abo8-1</i> 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 <i>abo8-1</i> 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 <i>abo8-1</i> 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 <i>abo8-1</i> 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 <i>AOX1a</i> 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.</p

<i>abo8</i> mutants are hypersensitive to ABA in seed germination and root growth.

<p>A. The root growth phenotype of <i>abo8</i> 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 <i>abo8</i> 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 <i>abo8</i> 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.</p