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First published online 3 July 2008
doi: 10.1242/dev.019349

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ACAULIS5 controls Arabidopsis xylem specification through the prevention of premature cell death

Luis Muñiz^1,*,, Eugenio G. Minguet^2,*, Sunil Kumar Singh^1,*, Edouard Pesquet^1,, Francisco Vera-Sirera², Charleen L. Moreau-Courtois¹, Juan Carbonell², Miguel A. Blázquez^2, and Hannele Tuominen¹

¹ Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 90187 Umeå, Sweden.
² Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Universidad Politécnica de Valencia, Avda de los Naranjos s/n, 46022 Valencia, Spain.

View larger version (103K): [in this window] [in a new window]   Fig. 1. ACL5 is specifically expressed in the xylem vessel elements. (A-E) In situ hybridization of ACL5. Sections were taken from the junction between the silique and the pedicel (A,B) and the basal part of the inflorescence stem (C-E), and analyzed using antisense (A,C,E) or sense (B,D) probes for the ACL5 gene. (A,B) Longitudinal sections; (C-E) an area of the stem with the vascular bundles in the transverse plane. The sections hybridized with the sense probe showed sometimes dark coloration of the vascular tissues (B) but not the positive purple precipitate derived from the chromogenic substrate (A,C,E). (F-H) Histochemical β-glucuronidase (GUS) staining of transgenic ProACL5:GUS seedlings. Transverse sections were taken from the inflorescence stem (one vascular bundle shown in F), the hypocotyl of a 4-day-old seedling (G) and the hypocotyl of a 1.5-month-old seedling (H). do, developing ovary; pc, procambium; ph, phloem; sx, secondary xylem; v, vessel element; vc, vascular cambium; xp, xylem parenchyma. Asterisks indicate the protoxylem poles (G). Scale bars: 20 µm in E-G; 50 µm in C,D,H; 100 µm in A,B.

View larger version (71K): [in this window] [in a new window]   Fig. 2. Xylem development is severely distorted in the acl5 mutant. (A-H) General anatomy of acl5 and wild-type hypocotyls was examined by light microscopy in transverse sections (A,B,E-H) and in longitudinal sections (C,D) from seedlings grown for seven (A-D), 13 (E,F) or 35 days (G,H). (I) A confocal image of a representative vessel element from wild type. (J) A confocal image of a representative vessel element from acl5. mx, metaxylem; px, protoxylem; sx, secondary xylem; vc, vascular cambium. Scale bars: 50 µm in A-F,H; 100 µm in G.

View larger version (93K): [in this window] [in a new window]   Fig. 3. The vacuole collapses early during vessel maturation in acl5. (A-D) Xylem anatomy and cell morphology of the wild-type. (E-H) Xylem anatomy and cell morphology of acl5. An overview of the xylem tissues (A,E) reveals the absence of fiber differentiation in acl5. Individual vessel elements are shown during early maturation (B,F), moderate maturation (C,G) and late maturation (D,H). All panels represent electron microscopy images of transverse sections from the hypocotyls of 3-week-old plants. The central vacuole is absent in maturing (i.e. secondary cell wall depositing) vessel elements of acl5, even at the earliest stage of maturation (F). f, fiber; cv, central vacuole; n, nucleus; v, a vessel element that is either living or undergoing cell death. The arrows indicate presence of secondary cell walls. The asterisks indicate dead, autolyzed vessel elements. Scale bars: 10 µm in A,E; 2 µm in B-D,F-H.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Xylem vessel elements of the acl5 mutant are small and simple in structure. Two-month-old hypocotyls of acl5 and wild-type plants were macerated and their vessel element morphology was investigated using light microscopy. (A) Proportion of the different types of vessel elements. (B) Length of the vessel elements. (C) Width of the vessel elements. More than 200 cells were examined from three plants of each genotype. Data were compared (B,C) using a Welch corrected t-test (***P<0.005; acl5 versus wild-type), and presented as average±s.e.m.

View larger version (104K): [in this window] [in a new window]   Fig. 5. Expression of an auxin and a cell death marker is altered in acl5. (A,B) Histochemical GUS staining of 7-day-old ProDR5:GUS (A) and acl5 ProDR5:GUS (B) seedlings grown in vitro. ProDR5:GUS expression is present in the developing vessel elements of acl5 hypocotyl but not in the wild type. (C,D) Histochemical GUS staining in 7-day-old ProXCP2:GUS (C) and in acl5 ProXCP2:GUS (D) seedlings grown in vitro. ProXCP2:GUS activity in the immature vessel elements (arrowheads) of acl5 is indicative of an early onset of the vessel cell death program. Scale bar: 50 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Exogenous spermine modifies tracheary element differentiation in Zinnia elegans xylogenic cell cultures. (A) Tracheary element (TE) differentiation efficiency, expressed as the number of TEs as a percentage of all cells, in response to 50-200 µM spermine 84 and 168 hours after the initiation of the cell culture. (B) Proportion of the different types of TEs at 168 hours in response to 50-200 µM spermine. (C) The size of the TEs (±s.e.m.) after 168 hours in response to 50-200 µM spermine. Statistics are presented (C) for each treatment compared with the previous treatment, using a Kruskal-Wallis test (***P<0.005). (D) Typical TEs after 168 hours without the addition of spermine. (E) Typical TEs 168 hours after the addition of 200 µM spermine. Scale bar: 50 µm in D,E.

View larger version (49K): [in this window] [in a new window]   Fig. 7. Expression of ProACL5:DT-A alters plant growth and xylem development. (A) The general phenotype of 1-month-old wild-type, ProACL5:DT-A heterozygous line 4, ProACL5:DT-A homozygous line 4 and acl5 seedlings. (B-D) Resin-embedded transverse sections of 2-month-old acl5 (B), ProACL5:DT-A homozygous line 4 (C) and wild-type (D) hypocotyls stained with Toluidine Blue. (E-G) Appearance of xylem elements after maceration of the hypocotyls of 2-month-old acl5 (E), ProACL5:DT-A homozygous line 4 (F) and wild type (G). Asterisks indicate the presence of xylem fibers in the wild-type (G). (H-Q) Expression of ProACL5:GUS in wild-type (H-J), ProACL5:DT-A homozygous line 4 (K-N) and acl5 seedlings (O-Q). Histochemical GUS staining is shown for hypocotyls of whole mounts (H,J,K,M,N,O,Q) and transverse sections of resin-embedded hypocotyls (I,L,P). Xylem differentiation was delayed in ProACL5:DT-A seedlings, and comparisons were therefore made between 3-day-old wild-type and acl5 and 6-day-old ProACL5:DT-A in vitro grown seedlings. The arrows indicate expression of ProACL5:GUS and therefore DT-A toxin production in the incipient vessel elements (with first signs of secondary cell wall deposition in cell corners) of the ProACL5:DT-A seedlings (L). sx, secondary xylem; vc, vascular cambium. Scale bars: 20 µm in I,J,L,M,N,P,Q; 50 µm in E,F,G,H,K,O; 100 µm in B,C; 200 µm in D.

View larger version (28K): [in this window] [in a new window]   Fig. 8. ProACL5:DT-A expressing plants show acl5-like xylem specification. (A) Proportion of the different types of the vessel elements in the hypocotyls of wild-type, acl5 and ProACL5:DT-A homozygous lines 4 and 5. (B) The length and width of individual xylem vessel elements in the hypocotyls of the different genotypes. More than 200 cells were scored from macerated hypocotyls of three 2-month-old plants for each genotype. Data are presented as average±s.e.m. ***P<0.005, as compared with the wild type using a Kruskal-Wallis test.

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