Xyloglucan endotransglycosylases have a function during the formation of secondary cell walls of vascular tissues

Abstract

Xyloglucan transglycosylases (XETs) have been implicated in many aspects of cell wall biosynthesis, but their function in vascular tissues, in general, and in the formation of secondary walls, in particular, is less well understood. Using an in situ XET activity assay in poplar stems, we have demonstrated XET activity in xylem and phloem fibers at the stage of secondary wall formation. Immunolocalization of fucosylated xylogucan with CCRC-M1 antibodies showed that levels of this species increased at the border between the primary and secondary wall layers at the time of secondary wall deposition. Furthermore, one of the most abundant XET isoforms in secondary vascular tissues (PttXET16A) was cloned and immunolocalized to fibers at the stage of secondary wall formation. Together, these data strongly suggest that XET has a previously unreported role in restructuring primary walls at the time when secondary wall layers are deposited, probably creating and reinforcing the connections between the primary and secondary wall layers. We also observed that xylogucan is incorporated at a high level in the inner layer of nacreous walls of mature sieve tube elements.

Figure 1.

Localization of XET Activity in Poplar Stem Tissues. Fresh stem sections were incubated with rhodamine-labeled XG oligosaccharides. Sites of label incorporation indicate the presence of XG donor and active XET enzyme. (A) Cross-section of the stem. Most XET activity is in the cambium, its recent derivatives, and collenchyma cells in the cortex. Activity also can be seen in developing phloem fibers. (B) Control section heated for 10 min at 90°C before incubation with the substrate. (C) Closeup of cambium and phloem. XET activity is seen only early during sieve tube development, whereas it is detected at all developmental stages in the phloem ray cells. (D) High-magnification image of the cambium and its derivatives. XET activity is seen in both fusiform and ray cell initials and at the early stages of sieve tube development. In the radial expansion zone of the developing xylem, the activity clearly is decreased compared with that in the cambium. Note the separation of adjacent radial cell files caused by the differential extraction of radial walls by formic acid (arrowhead). (E) Phloem fibers. XET activity is found in developing fibers during secondary wall deposition as well as in surrounding parenchyma cells. (F) Differentiating xylem. Cells are labeled faintly in the radial expansion phase and strongly during the early stages of secondary wall deposition. Note that there is more activity associated with the fibers than with the vessel elements. C, cambium; Co, cortex; Col, collenchyma; DS, developing sieve tube cells; F, fiber; Ph, phloem; PhF, phloem fibers; Ph Ra, phloem ray; R, radial wall; Ra, ray; RE, radial expansion zone; SW, secondary wall formation zone; T, tangential wall; V, vessel element; X, xylem. Bars = 100 μm in (A) and (B), 20 μm in (C) and (E), and 10 μm in (D) and (F).

Figure 2.

Localization of XG in Secondary Vascular Tissues by Indirect Immunofluorescence Using the Monoclonal Antibody CCRC-M1. Localization was performed in either resin-embedded tissue ([A] to [C]) or in nonembedded tissue ([D] to [F]). (A) Overview of the XG localization pattern in the stem. XG is detected in cell walls of the cambial cells, developing xylem, and phloem. It also is seen in the phloem rays, developing phloem fibers, and the cortex. (B) Control section incubated with CCRC-M1 antibodies saturated with Rubus XG. (C) Closeup of the cambium and phloem. XG labeling is particularly strong in the walls of the sieve tube elements. (D) Closeup of the cambium and its recent derivatives. XG is detected in cambial cells and in developing phloem sieve tubes. Note the reduction of labeling in the radial expansion zone. (E) Phloem fibers. Note the presence of the label in fibers in which secondary wall is being deposited. (F) Developing secondary xylem. Note the strong labeling of walls and cytoplasm in fibers undergoing secondary wall thickening. Less label is detected in the radial expansion zone and in developing vessel elements. All labels are as in Figure 1. Bars = 50 μm (A) and (B) and 20 μm in (C) to (F).

Figure 3.

Transmission Electron Microscopy Immunolocalization of XG in the Walls of Developing Xylem Fibers Using the Monoclonal Antibody CCRC-M1. (A) to (D) Tangential walls of fibers in the meristematic stage (A), the radial expansion stage (B), the early secondary wall deposition stage (C), and the almost mature stage (D). Note the accumulation of the label at the primary/S1 layer boundary. Bar in (D) = 2 μm for (A) to (D). (E) Quantification of the label in tangential walls of developing fibers. Scores were taken from 10 random locations of tangential walls in 10 different cells for each stage. Bars indicate standard errors.

Figure 4.

Transmission Electron Microscopy Immunolocalization of XG in Cambial Region Cells Using the Monoclonal Antibody CCRC-M1. (A) Phloem fibers depositing secondary wall layers. Note the accumulation of label in the primary wall layer. (B) Cambial fusiform initials. Note the presence of label in both radial and tangential walls. (C) Late secondary wall deposition stage in xylem fibers and vessels. Note that the XG label is associated with fibers but not with the vessel element. (D) Comparison of the nacreous sieve tube wall with companion cell and parenchyma cell walls. XG is abundant in the inner nacreous layer. CC, companion cell; F, fiber; PP, phloem parenchyma; R, radial wall; ST, sieve tube; T, tangential wall; V, vessel. Bars = 2 μm.

Figure 5.

Structure of PttXET16A Coding Sequence.

Figure 6.

Phylogenetic Tree of XET Genes. Relationships among Arabidopsis XET genes, poplar PttXET16A, and its relatives in other species: NtXET1 (tobacco), BRU1 (soybean), and VaEXT (adzuki bean). Arabidopsis genes were named according to Yokoyama and Nishitani (2001a). The scale bar represents 0.1 substitutions per site. 1, 2, and 3 represent subfamily number.

Figure 7.

RNA Gel Blot Analysis of PttXET16A Expression in Vegetative Tissues and Organs of Poplar. (A) Detection of PttXET16A transcript in 10 μg of total RNA extracted from the tissues indicated in the drawing above the blot. PttXET16A is expressed most strongly in mature stem segments with secondary growth, in root tips, and in young roots. In the mature stem, it is expressed most strongly in the phloem/cambium and in the differentiating xylem fractions. A, apical bud; ST1, young expanding stem; L1, young expanding leaf; ST2, stem at the onset of secondary growth; L2, leaf at the end of expansion; Co, cortex and periderm; Ph+C, secondary phloem and cambium; X, secondary xylem; L3, mature leaf; R2, young roots from lateral root formation zone; R1, root tips with elongation zone. (B) Cross-reactivity of the PttXET16A probe to its closest homologs, PttXET16B and PttXET16C.

Figure 8.

Protein Gel Blot Analysis of PttXET16A Expression in the Developing Secondary Xylem and Phloem of Poplar. Thirty micrograms of proteins soluble in low-salinity buffer, from both phloem/cambium (P) and xylem (X) fractions, were separated on an SDS–polyacrylamide gel under reducing conditions, electroblotted onto a polyvinylidene difluoride membrane, and probed with rabbit antibody raised against the recombinant PttXET16A protein. No signal was detected with preimmune serum from the same animals (data not shown).

Figure 9.

Immunolocalization of PttXET16A in Secondary Vascular Tissues of Poplar. Localization was performed in either resin-embedded tissue ([A] to [C], [E], and [F]) or in nonembedded tissue (D). (A) Overview of the localization pattern. The strongest signals are found in the phloem and rays, and weaker signals are present in the cambium, developing xylem, phloem fibers, and cortex. (B) Negative control. Antibodies were saturated with the recombinant protein before labeling. (C) Closeup of the phloem and cambium. The most intense label is found in the sieve tubes. (D) Cambial region. High-intensity label is found in nonembedded material in the walls and cytoplasm of cambial fusiform and ray initials, developing phloem sieve tubes, and xylem fibers. Less PttXET16A label is detected in the radial expansion zone and developing vessel elements. (E) Phloem fibers. Label is evident in cells undergoing secondary wall thickening. (F) Secondary xylem at the final differentiation stages. Signal is seen in the cytoplasm fibers and ray cells in the early stages of secondary wall formation. All labels are as in Figure 1. Bars = 50 μm in (A) and (B), 20 μm in (C) and (E), and 10 μm in (D) and (F).

Figure 10.

Overview of Cambial Region Tissues. Developmental zones are color coded: C, cambial zone; DP, developing phloem; Ph, conducting phloem; RE, radial expansion zone; SW, secondary wall formation zone in which successive S1, S2, and S3 layers of secondary wall are deposited. R, radial wall; T, tangential wall; V, vessel element.