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. 2012 Jun 19;109(25):10101-6.
doi: 10.1073/pnas.1205726109. Epub 2012 Jun 4.

Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin

Sadaf Naseer  1 , Yuree Lee, Catherine Lapierre, Rochus Franke, Christiane Nawrath, Niko Geldner
Affiliations

Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin

Sadaf Naseer et al. Proc Natl Acad Sci U S A. .

Abstract

Casparian strips are ring-like cell-wall modifications in the root endodermis of vascular plants. Their presence generates a paracellular barrier, analogous to animal tight junctions, that is thought to be crucial for selective nutrient uptake, exclusion of pathogens, and many other processes. Despite their importance, the chemical nature of Casparian strips has remained a matter of debate, confounding further molecular analysis. Suberin, lignin, lignin-like polymers, or both, have been claimed to make up Casparian strips. Here we show that, in Arabidopsis, suberin is produced much too late to take part in Casparian strip formation. In addition, we have generated plants devoid of any detectable suberin, which still establish functional Casparian strips. In contrast, manipulating lignin biosynthesis abrogates Casparian strip formation. Finally, monolignol feeding and lignin-specific chemical analysis indicates the presence of archetypal lignin in Casparian strips. Our findings establish the chemical nature of the primary root-diffusion barrier in Arabidopsis and enable a mechanistic dissection of the formation of Casparian strips, which are an independent way of generating tight junctions in eukaryotes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Lignin, but not suberin stains, correlate with the appearance of the endodermal diffusion barrier. (A) Penetration of PI into the stele is blocked at 14.2 ± 0.6 endodermal cells after onset of elongation. (B) Dot-like appearance of Casparian strip formation at 11.7 ± 0.9 endodermal cells as visualized by green autofluorescence after clearing. (C) Fluorol yellow staining reveals the presence of lamellar suberin on the cellular surface of endodermal cells at 37.5 ± 2.6 endodermal cells. (Scale bars, 20 μm.) (D) Quantification of A–C shows that appearance of green autofluorescence correlates well with block of PI uptake; Fluorol yellow signal appears much later. (E) Root schematic showing the different root zones and stages of endodermal differentiation as inferred from A–D. Stele (st), endodermis (en), cortex (ct), epidermis (ep). A–D: n ≥ 20 roots counted per condition. “Onset of elongation” was defined as the zone where an endodermal cell was clearly more than twice its width.
Fig. 2.
Fig. 2.
Suberin biosynthetic genes are turned on after Casparian strip formation. Endodermis-specific Promoter::GUS fusion activity of (A) pCASP1::NLS-GFP-GUS, (B) pASFT::NLS-GFP-GUS, (C) pCYP86B1::NLS-GFP-GUS, (D) pDAISY::NLS-GFP-GUS, (E) pFAR1::NLS-GFP-GUS, (F) pHORST::NLS-GFP-GUS, (G) pGPAT5::NLS-GFP-GUS, (H) pKCR1::NLS-GFP-GUS; asterisks mark the start of GUS expression. n = 16 roots counted. (I) Quantifiation of the cellular distance from the meristem at which onset of GUS expression is observed. Appearance of all but one suberin biosynthetic reporter gene coincided well with appearance of Fluorol yellow signals but not with appearance of green autofluorescence. (J and K) Arrowheads point to patchy GUS-expression pattern (J), which matches closely the patterns observed with Fluorol yellow stains (K) (Scale bars, 50 μm.)
Fig. 3.
Fig. 3.
Suberin degradation has no effect on the formation of functional Casparian strips. (A) Fluorol yellow staining reveals significant delay in the appearance of suberin lamellae formation in horst-1 and horst-3 insertion lines compared with wild-type (wt); (B) horst mutants do not affect formation of Casparian strips, visualized by autofluorescence. (C) Establishment of a functional diffusion barrier, visualized by PI, is also not affected in horst mutants. (D) No Fluorol yellow signal observed in the pCASP1::CDEF1 transgenic line, compared with wild-type seedlings; asterisks show the presence (wt, Left) and lack of Fluorol yellow signals in endodermis (Right). (Scale bar, 100 μm.) (E) Casparian strip autofluorescence is not affected by suberin degradation in pCASP1::CDEF1 transgenic line. (F) PI stainings also shows no difference in the formation of a functional diffusion barrier between pCASP1::CDEF1 and wild-type. n = 16 roots counted. (Scale bars E and F, 20 μm.) (G) Quantification of data in D–F. Stele (st), endodermis (en), cortex (ct), epidermis (ep), not applicable (n.a).
Fig. 4.
Fig. 4.
Interference with monolignol biosynthesis abrogates Casparian strip formation. (A) Schematic representation of a seedling explaining how continued root growth after lignin inhibitor treatment results in an apparent “upward shift” of Casparian strip appearance when the cellular distance to the meristem is counted after 24 h. (B–D) Autofluorescence after clearing shows the suppression of Casparian strip formation in seedlings treated for 24 h with lignin biosynthesis inhibitor (PA) (C), compared with the control (B). This effect is complemented by the exogenous application of two monolignols: 20 μM of each coniferyl alcohol and sinapyl alcohol, which allows for the formation of functional Casparian strips (D). (E) Quantification of B–D shows that in the control samples green autofluorescent signal appears around three cells, whereas PA treatment results in the apparent upward-shift of autofluorescent signal to 16 cells, and monolignols complement this inhibitor induced-effect. Signals appear around four cells; close to the control value (F–H) PA also blocks the establishment of the diffusion barrier in newly forming cells (G) compared with the control (F), and this effect is also complemented by monolignol addition (H). (I) Quantification of F–H shows that PA treatment also shifts the block of PI uptake to 21 cells compared with the control samples where PI penetration is blocked around 6 cells. Monolignol addition complements this inhibitor-induced effect and block of PI uptake again appears around 6 cells, matching with the control samples. Arrowheads points to the seventh endodermal cell after onset of elongation. (J) Genetic interference using multiple insertion mutants (ccr1;cad4;cad5;f5h1;f5h2) of lignin biosynthetic genes reveals a delay in the formation of the diffusion barrier, visualized by PI. In a population of quadruple homozygous (cad4;cad5;f5h1;f5h2), segregating for ccr1, a delay in the formation of the diffusion barrier is observed in the quadruple mutants, which is further increased in the quintuple mutant. Wild-type (Col): n = 60; quadruple mutant (cad4;cad5;f5h1;f5h2 with CCR1 either CCR1/CCR1 or CCR1/ccr1): n = 227 and the quintuple mutant (cad4,cad5,f5h1,f5h2;ccr1): n = 41. Stele (st), endodermis (en), cortex (ct), epidermis (ep). B–D and F–H: n = 20 roots counted. (Scale bars, 20 μm.)
Fig. 5.
Fig. 5.
Casparian strips are made of lignin or a closely related, lignin-like polymer. (A) Autofluorescence after clearing shows the appearance of Casparian strip formation (dot-like) and the protoxylem formation. (B) PI staining shows functional diffusion barrier. (C) Autofluorescence after clearing shows only the dot-like appearance of Casparian strips but no protoxylem formation in ahp6-1 mutant treated with 10 nM of the cytokinin benzyl-adenine (ahp6+ck). (D) PI staining confirms presence of a functional diffusion barrier. (Scale bars, 20 μm.) (E and F) Similar presence and relative abundance of thioacidolysis monomers specifically released from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin units and from lignin coniferaldehyde end-groups is observed in wt (E) and ahp6+ck root tips (F). Total lignin monomers released by thioacidolysis are 208 ± 49 nmol/g for wt and 449 ± 48 nmol/g for ahp6+ck root tips. Asterisks mark the presence of xylem vessels in wild-type; arrowheads point to the dot-like structures of the Casparian strips in wt and ahp6-1. Stele (st), endodermis (en), cortex (ct), epidermis (ep), wild-type (wt), Cytokinin (ck).
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