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. 2018 Aug 15;37(16):e99134.
doi: 10.15252/embj.201899134. Epub 2018 Jul 16.

Acidic cell elongation drives cell differentiation in the Arabidopsis root

Elena Pacifici  1 , Riccardo Di Mambro  2 , Raffaele Dello Ioio  1 , Paolo Costantino  1   3 , Sabrina Sabatini  4
Affiliations

Acidic cell elongation drives cell differentiation in the Arabidopsis root

Elena Pacifici et al. EMBO J. .

Abstract

In multicellular systems, the control of cell size is fundamental in regulating the development and growth of the different organs and of the whole organism. In most systems, major changes in cell size can be observed during differentiation processes where cells change their volume to adapt their shape to their final function. How relevant changes in cell volume are in driving the differentiation program is a long-standing fundamental question in developmental biology. In the Arabidopsis root meristem, characteristic changes in the size of the distal meristematic cells identify cells that initiated the differentiation program. Here, we show that changes in cell size are essential for the initial steps of cell differentiation and that these changes depend on the concomitant activation by the plant hormone cytokinin of the EXPAs proteins and the AHA1 and AHA2 proton pumps. These findings identify a growth module that builds on a synergy between cytokinin-dependent pH modification and wall remodeling to drive differentiation through the mechanical control of cell walls.

Keywords: cell differentiation; cell wall acidification; cell wall expansion; cytokinin; root meristem.

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Figures

Figure 1
Figure 1. EXPA1 positively controls the initiation of meristematic cell differentiation
  1. A

    Confocal microscopy images of WT and expa1 root tips. Changes in cell size at the cortex transition boundary (TB) in WT root are shown in the insert. The TB is a tissue‐specific boundary where divisions cease and cells start to differentiate. The zone encompassing the TB of the different cell tissues represents the TZ. Blue and white arrowheads indicate the QC and the cortex TB, respectively.

  2. B

    Root meristem cell number of WT and expa1 mutant (n = 60. ***P < 0.001; Student's t‐test).

  3. C

    Seedling of WT and expa1 mutant.

  4. D

    Root length quantification of WT and expa1 mutant (n = 15. ***P < 0.001; Student's t‐test).

  5. E

    Confocal images of WT and expa1 root tip. White asterisk indicates the QC, yellow arrowheads indicate the first detectable root hair, and light blue arrowheads mark the first appearance of differentiated tracheids.

  6. F, G

    Measurements of tracheids (F) and root hair (G) distance from the QC in WT and expa1 root tips (n = 10. ***P < 0.001; Student's t‐test).

  7. H

    Measurement of cell length of newly differentiated cortex cells of WT and expa1 roots (n = 15).

  8. I

    Measurement of cell area of newly differentiated cortex cells of WT and expa1 roots (n = 15).

  9. J

    Confocal microscopy images of roots expressing the pEXPA1::ER‐GFP construct in WT (n = 20), in arr1‐4 (n = 15) mutant backgrounds and of pEXPA1::ER‐GFP root upon cytokinin treatments (n = 15), respectively. pEXPA1::ER‐GFP‐specific expression at the epidermis TB is shown in the insert.

  10. K

    qRT–PCR analysis of EXPA1 mRNA levels in the root tip of WT, arr1‐4, and cytokinin‐treated WT and arr1‐4 roots (**P < 0.01, ***P < 0.001, n.s. corresponds to not significant; Student's t‐test; three technical replicates performed on two independent RNA batches).

  11. L

    qRT–PCR analysis of EXPA1 mRNA levels in the root tip of 35S::ARR1ΔDDK:GR plants upon dexamethasone (Dex) treatments. The value for the control mock‐treated WT was set to 1, and the relative values are reported (**P < 0.01; Student's t‐test; three technical replicates performed on two independent RNA batches).

Data information: In (A‐L), experiments were performed on seedlings at 5 dpg. Error bars indicate SD.
Figure EV1
Figure EV1. EXPA1,EXPA10,EXPA14, and EXPA15 control cell differentiation initiation at the TZ
  1. A

    DIC microscopy images of WT, expa15;expa10, expa14;expa10, and expa15;expa14 root tips.

  2. B

    Root meristem cell number of the WT, expa10, expa14, and expa15 single mutants (n = 50. n.s. corresponds to not significant; Student's t‐test).

  3. C, D

    Measurement of root meristem cell number (n = 50) (C) and root length (n = 15) (D) of expa15;expa10, expa14;expa10, and expa15;expa14 double mutants (***P < 0.001; Student's t‐test).

  4. E

    DIC microscopy images of CYCB1;1:GUS (n = 20) and CYCB1;1:GUS;expa1 (n = 20) roots.

  5. F

    Confocal microscopy images of RCH2::GFP (n = 10) and RCH2::GFP;expa1 (n = 30) roots. Note changes in TZ position in the expa1 mutant correspond to changes in CYCB1;1:GUS and RCH2::GFP domains.

  6. G

    Measurement of root meristem cell number of WT, expa1, and expa1;EXPA1‐GFP (n = 50. ***P < 0.001, n.s. corresponds to not significant; Student's t‐test)

Data information: In (A–G), experiments were performed on seedlings at 5 dpg. Blue and white arrowheads indicate the QC and the cortex TB, respectively. Error bars indicate SD.
Figure EV2
Figure EV2. Low pH‐activated EXPA10, EXPA14, and EXPA15 proteins are expressed at the TZ and control cell differentiation
  1. A

    EXPA1 fold enrichment deriving from ChIP‐qPCR analysis performed on immunoprecipitated chromatin of pARR1::ARR1:GFP roots (***P < 0.001; Student's t‐test; two technical replicates performed on two independent DNA batches).

  2. B–D

    DIC microscopy images of WT roots expressing pEXPA10‐GUS (B), pEXPA14‐GUS (C), and pEXPA15‐GUS (D) constructs, respectively.

  3. E

    qRT–PCR analysis of EXPA10, EXPA14, and EXPA15 mRNA levels in the root tip of WT plants upon cytokinin treatment (***P < 0.001; Student's t‐test; three technical replicates performed on two independent RNA batches).

  4. F

    EXPA10, EXPA14, and EXPA15 fold enrichment deriving from ChIP‐qPCR analysis performed on immunoprecipitated chromatin of pARR1::ARR1:GFP roots. (Two technical replicates performed on two independent DNA batches)

  5. G, H

    Measurements of tracheids (G) and root hairs (H) distance from the QC in WT and UBQ10‐EXPA1 root tips grown on standard (5.8) or acidic (4.0) pH. (n = 10. **P < 0.01, ***P < 0.001; Student's t‐test).

  6. I

    Confocal microscopy images of WT (n = 10) root tips grown on standard (5.8) pH and of UBQ10‐EXPA10 (n = 15), UBQ10‐EXPA14 (n = 20), and UBQ10‐EXPA15 (n = 15) root tips grown on standard (5.8) or acidic (4.0) pH.

Data information: In (A–I), experiments were performed on seedlings at 5 dpg. Blue and white arrowheads indicate the QC and the cortex TB, respectively. Error bars indicate SD.
Figure 2
Figure 2. Activated EXPA1 protein induces cell differentiation revealing its function in vivo

  1. Confocal microscopy images of WT and UBQ10‐EXPA1 root tips grown on standard (5.8) or acidic (4.0) pH. Blue and white arrowheads indicate the QC and the cortex TB, respectively.

  2. Stem cell niche expressing the QC‐specific marker (QC25) of WT and UBQ10‐EXPA1 root tips grown on standard (5.8) or acidic (4.0) pH. Lugol staining highlights differentiated columella cells. Violet arrowheads indicate columella stem cells. Asterisk indicates the presumptive position of QC cells in UBQ10‐EXPA1 root grown on pH 4.

  3. Meristem cell number of WT and UBQ10‐EXPA1 grown on standard and acidic pH (n = 30. ***P < 0.001, n.s. corresponds to not significant; Student's t‐test).

  4. Measurement of cell length of newly differentiated cortex cells of WT and UBQ10‐EXPA1 root tips grown on standard or acidic pH (n = 15; three replicates).

  5. Measurement of cell area of newly differentiated cortex cells of WT and UBQ10‐EXPA1 root tips grown on standard or acidic pH (n = 15; three replicates).

Data information: In (A–E), experiments were performed on seedlings at 5 dpg. In (C–E), error bars indicate SD.
Figure 3
Figure 3. AHA1 and AHA2 control meristematic cell differentiation initiation
  1. A–C

    qRT–PCR analysis of AHA1 and AHA2 mRNA levels in WT root tip upon cytokinin treatment (A) in the root tip of arr1‐4 mutant (B) and in the root tip of 35S::ARR1ΔDDK:GR plants upon Dex induction (C) (**P < 0.001; Student's t‐test; three technical replicates performed on two independent RNA batches).

  2. D, E

    Relative fluorescence quantification of roots depicted in (F) and (G), respectively (n = 10. ***P < 0.001; Student's t‐test).

  3. F, G

    Confocal microscopy images of WT (n = 20) and arr1‐4 (n = 20) roots expressing the AHA1‐GFP construct (F) and the AHA2‐GFP construct (G).

  4. H, I

    Root meristem cell number (n = 60) (H) and root length (n = 20) (I) of WT and aha1‐8/AHA1;aha2‐4/AHA2 mutant (***P < 0.001; Student's t‐test).

  5. J

    Confocal microscopy images of WT and aha1‐8/AHA1;aha2‐4/AHA2 roots meristem. Blue and white arrowheads indicate the QC and the cortex TB, respectively.

  6. K

    Seedlings of WT and the aha1‐8/AHA1;aha2‐4/AHA2 mutant.

Data information: In (A–K), experiments were performed on seedlings at 5 dpg. Error bars indicate SD.
Figure EV3
Figure EV3. AHA1‐ and AHA2‐dependent apoplastic acidification at the TZ induces cell differentiation

  1. AHA1 and AHA2 fold enrichment deriving from ChIP‐qPCR analysis performed on immunoprecipitated chromatin of pARR1::ARR1:GFP roots. (***P < 0.001; Student's t‐test; two technical replicates performed on two independent DNA batches). Error bars indicate SD.

  2. Measurements of meristem cell number of aha1‐8 and aha2‐4 mutants. (n = 15. n.s. corresponds to not significant; Student's t‐test). Error bars indicate SD.

  3. Medium acidification through proton extrusion from WT, aha1‐8, aha2‐4, and aha1‐8/AHA1;aha2‐4/AHA2 seedlings, 16 h after transfer onto fresh medium containing pH indicator. (n = 4; three replicates). Colored bar indicates pH values.

  4. Proposed model: cytokinin, via ARR1, controls both apoplastic acidification (via positive regulation of AHA1 and AHA2) and cell wall (brown lines) loosening (via positive regulation of EXPA1). As a result, at the TZ, cells expand and initiate the differentiation program.

Data information: In (A–C), experiments were performed on seedlings at 5 dpg.
Figure 4
Figure 4. AHA1‐ and AHA2‐dependent apoplast acidification activates EXPA1 and induces cell differentiation

  1. DIC microscopy images of WT and expa1 roots expressing the pUBQ10::AHA2:GR construct treated and untreated with Dex. Blue and black arrowheads indicate the QC and the cortex TB, respectively.

  2. Measurement of meristem cell number of roots depicted in a (n = 30. For statistical analysis, untreated pUBQ10::AHA2:GR was set as reference. ***P < 0.001, n.s. corresponds to not significant; Student's t‐test).

  3. Measurement of cell length of newly differentiated cortex cells of root depicted in (A) (n = 15).

  4. Measurement of cell area of newly differentiated cortex cells of root depicted in (A) (n = 15).

Data information: In (A–D), experiments were performed on seedlings at 5 dpg. Error bars indicate SD.
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