QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00966

This Article Summary Full Text Full Text (PDF) Supplemental Figure Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ryser, U. Articles by Keller, B. Search for Related Content PubMed PubMed Citation Articles by Ryser, U. Articles by Keller, B. Social Bookmarking                         What's this?

A new structural element containing glycine-rich proteins and rhamnogalacturonan I in the protoxylem of seed plants

¹ University of Fribourg, Biology Department, Plant Biology, Fribourg, Switzerland
² University of Zürich, Institute of Plant Biology, Zürich Switzerland

View larger version (88K): [in a new window]   Fig. 1. 3D reconstruction of PX in different regions of soybean hypocotyls. (A-E) Apical region of the hypocotyl. (A) PX with ring-shaped, lignified SW (red fluorescence) and GRPCWM (green fluorescence) interconnecting the lignified SW. (B) PX with a helical, lignified SW. The attachments of the GRPCWM are clearly visible and are often somewhat broadened along the lignified SW thickening (arrow). (C) Two PX. The younger one, with closely spaced SW, does not yet contain GRPCWM. The older one is characterized by more widely spaced SW and the typical strands of the GRPCWM. The wall between the two is also clearly fluorescent (arrows). (D) The ends of two PX joined together end-by-end. The arrows indicate the GRP-containing wall connecting these two cells with a third PX (not retained in the preparation). The same type of wall, seen here in front view, is seen in (C) in side view. The ends of the two PX are outlined with a hatched line. (E) Rabbit serum control: no green fluorescence is observed. Ethidium bromide stains the SW and (very faintly) the GRPCWM. Notice the small protuberances on the SW in the direction of the longitudinal axis of the hypocotyl (arrows). (F) Middle region of the hypocotyl: PX with strongly elongated helical SW. Notice the stress-fibre-like appearance of the GRPCWM and the attachments between the GRPCWM and the SW (arrows). In contrast to the apical region of the hypocotyl, where the GRPCWM spans only the distance between adjacent SW, long, continuous filaments or sheets are typically observed in the mature regions of the hypocotyl (arrowheads). (G-I) Basal region of the hypocotyl. New MX are differentiating but the structure of the PX remains much the same as in the middle region of the hypocotyl. (G) Three stretched helical SW are visible to the left and a newly formed PX with a flat helical SW to the right. Notice the GRPCWM along the helical SW of this PX. Arrows indicate a faintly stained cell corner. (H) Overview, showing (from left to right) a MX (m) and a series of younger to older PX. The older elements are characterized by passively elongated helical SW and intense staining with GRP1.8 antibodies. Little staining is observed in the MX, with the exception of a cell corner adjacent to the youngest PX (arrow). (I) Rabbit serum control: no green fluorescence is observed. Ethidium bromide stains the SW and, faintly, the GRPCWM (arrows). Bars, 10 µm.

View larger version (42K): [in a new window]   Fig. 2. Schematic representation of the 3D arrangement of GRPCWM in the protoxylem of soybean hypocotyls. (A) Differentiating PX at the top of the hypocotyl. (B) Mature protoxylem in the middle of the hypocotyl. Green shows GRP-containing structures. Red shows lignified secondary wall thickenings. Differentiation of the cells occurred from left to right. Therefore, the oldest protoxylem element is located at the left and the metaxylem element (m) at the right. A comparison between the two oldest PX in (A,B) shows their passive elongation and the continued deposition of GRPCWM in the dead cells. The nonextensible metaxylem elements do not contain GRP, except in the cell corners shared with protoxylem elements.

View larger version (22K): [in a new window]   Fig. 3. Soybean PX fragment after prolonged homogenization. Some typical medium-sized fragments from the apical region of the hypocotyl are shown in (A-C). (A) Two lignified rings are interconnected with GRPCWM. A separation between the SW and the GRPCWM occurs preferentially near the lignified SW. However, some GRPCWM remains associated with the lignified SW (arrows), indicating that the SW and the GRPCWM are linked very tightly together. (B) The GRPCWM between two PX is still associated with a ring-shaped SW. Two other rings are missing but their original position remains visible (arrows). (C) Compression of the GRPCWM was sometimes observed between helical SW (arrowheads), whereas, at the end of the fragment, the GRPCWM is present in an extended form (arrow). The compression of the GRPCWM is explained by a shortening of the helical SW. Bars, 10 µm (A,B); 5 µm (C).

View larger version (53K): [in a new window]   Fig. 4. Enzymatic digestion of isolated soybean PX. Isolated PX with widely spaced ring-shaped SW are compared after different treatments. (A,D) Buffer control. (B,E) Cellulase. (C,F) Proteinase K. (A-C) Phase contrast microscopy; arrows indicate GRPCWM. (D-F) Epifluorescence microscopy of the same PX as shown in (A-C). The arrows indicating GRPCWM are located at the same positions as in (A-C). The ring-shaped SW remain connected after all three treatments. However, the intensity of the fluorescence is reduced after the treatment with proteinase K. Bars, 20 µm.

View larger version (74K): [in a new window]   Fig. 5. Cytochemistry of the GRPCWM of soybean PX. (A-D,H,I) Apical region of hypocotyls. (E-G) Middle region of hypocotyls. (A) Differential interference contrast microscopy, Coomassie-Blue staining. The GRP-containing filaments of the PX are strongly stained (arrow). With this technique, an optical section is obtained. Therefore, not all GRP-containing filaments are visible in a single plane of focus. A similar PX is shown in Fig. 1A after staining with a GRP1.8 probe. (B) Calcofluor stains cell-wall fragments of different cell types (arrowheads) and the lignified SW of PX. Filaments corresponding to GRPCWM (arrow) are only weakly stained with Calcofluor. (C) JIM5 monoclonal antibodies consistently stain the ends of PX (arrow), thus staining components not being stained with GRP1.8 antibodies, as well as the cell corners (arrowheads) commonly stained with GRP1.8 antibodies. (D) Control preparation, treated with 500 mM NaCl inhibiting specifically the staining with JIM5. (E) JIM5 monoclonal antibody. Two mature PX are shown, together with a differentiating reticulated tracheary element. The SW of the latter are only weakly stained with ethidium bromide, indicating that lignification of the SW is not yet complete. The cross-wall between the reticulated tracheary element and the younger PX is stained with JIM5 antibodies but not the cross-wall between the younger and the older PX. The GRPCWM is only occasionally stained (arrow) in the younger PX, whereas the older PX with the elongated helical SW remains almost unstained. Arrowheads indicate a faint staining of the cell corners of the reticulated xylem element. (F) The ends of two immature MX are clearly stained with JIM 5 monoclonal antibodies (arrows), as are some cell corners (arrowheads). (G) Enlarged view of the zone of contact between two MX. A thin region between the two MX is stained with JIM5. (H) No staining of the isolated PX was observed with the CCRC-M1 monoclonal antibody, which specifically stains xyloglucan. Therefore, this antibody can serve as a control for the CCRC-M2 monoclonal antibody staining RG-I. (I) PX with helical SW stained with CCRC-M2. The GRPCWM between two PX (arrowheads) and in the cell corners is stained. CCRC-M2 stains the same structures as the GRP1.8 probe in the protoxylem. In addition, cell-wall fragments of other cell types present in the preparation are also stained (arrow). Bars, (A) 5 µm; (B-I) 10 µm.

View larger version (82K): [in a new window]   Fig. 6. GRPCWM in the protoxylem of other seed plants. (A-I) Confocal 3D reconstruction of the protoxylem of different species after staining with bean GRP1.8 antibodies. (J) Differential interference contrast, Coomassie-Blue staining. (A) Arabidopsis thaliana hypocotyl. The GRP network is restricted to the protoxylem, as in soybean. The ring-shaped SW (arrow) is tilted, probably owing to the collapse of the PX. Arrowheads, helical SW; m, metaxylem. (B) Nicotiana tabacum hypocotyl. Two ring-shaped SW are interconnected by a helical SW. (C) Leucojum vernum, floral axis. SW in the form of squares, interconnected by four filaments of GRPCWM. (D) Galanthus nivalis floral axis. GRPCWM is associated with ring-shaped SW. Arrowheads indicate the position of missing ring-shaped SW. The GRPCWM between two adjacent PX is indicated by arrows. One of the partners was removed by the isolation procedure. (E) G. nivalis floral axis. GRPCWM (arrow) interconnects the ring-shaped SW of two PX. Side view of a situation similar to (D). (F) G. nivalis floral axis. Rabbit serum control. As in soybean, the GRPCWM is faintly stained with ethidium bromide. (G) Pinus sylvestris cotyledon. Thin filaments of GRPCWM interconnect the SW. (H) Picea abies hypocotyl. Anastomosing SW are interconnected by GRPCWM. (I) P. abies hypocotyl. Rabbit serum control. As shown for other species the GRPCWM is faintly stained with ethidium bromide. (J) P. abies hypocotyl. Differential interference contrast microscopy. Arrows, filaments stained with Coomassie Blue. Bars, 10 µm.

View larger version (52K): [in a new window]   Fig. 7. Analysis of GRP-related sequences from seed plants. (A) Amino acid alignment of the N-terminal region of proteins structurally similar to bean GRP1.8. The first 40 amino acids were used to perform multiple alignments. (B) Classification of plant proteins structurally similar to GRP1.8. The classification was based on multiple protein sequence alignments of the first 40 amino acids of proteins. The bootstrap values for the five main families are indicated at the nodes of the tree. N-terminal amino acid sequences were aligned with PILEUP and analysed with the neighbour-joining method.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter