A Glycosyltransferase from Nicotiana alata Pollen Mediates Synthesis of a Linear (1,5)-α-L-Arabinan When Expressed in Arabidopsis

Abstract

The walls of Nicotiana alata pollen tubes contain a linear arabinan composed of (1,5)-α-linked arabinofuranose residues. Although generally found as a side chain on the backbone of the pectic polysaccharide rhamnogalacturonan I, the arabinan in N. alata pollen tubes is considered free, as there is no detectable rhamnogalacturonan I in these walls. Carbohydrate-specific antibodies detected arabinan epitopes at the tip and along the shank of N. alata pollen tubes that are predominantly part of the primary layer of the bilayered wall. A sequence related to ARABINAN DEFICIENT1 (AtARAD1), a presumed arabinan arabinosyltransferase from Arabidopsis (Arabidopsis thaliana), was identified by searching an N alata pollen transcriptome. Transcripts for this ARAD1-like sequence, which we have named N. alata ARABINAN DEFICIENT-LIKE1 (NaARADL1), accumulate in various tissues, most abundantly in the pollen grain and tube, and encode a protein that is a type II membrane protein with its catalytic carboxyl terminus located in the Golgi lumen. The NaARADL1 protein can form homodimers when transiently expressed in Nicotiana benthamiana leaves and heterodimers when coexpressed with AtARAD1 The expression of NaARADL1 in Arabidopsis led to plants with more arabinan in their walls and that also exuded a guttation fluid rich in arabinan. Chemical and enzymatic characterization of the guttation fluid showed that a soluble, linear α-(1,5)-arabinan was the most abundant polymer present. These results are consistent with NaARADL1 having an arabinan (1,5)-α-arabinosyltransferase activity.

Figure 1.

Distribution of arabinan in the walls of N. alata pollen tubes grown in vitro. Arabinan epitopes were detected (green) with the monoclonal antibodies LM6 and LM13. Pollen tubes were counterstained with FM4-64 (red). A to D show 4-h (A and B) and 16-h (C and D) pollen tube shank (A and C) and tip (B and D) regions labeled with LM6. E to H show 4-h (E and F) and 16-h (G and H) pollen tube shank (E and G) and tip (F and H) regions labeled with LM13. I shows a cross section of a 4-h pollen tube shank imaged with immunogold transmission electron microscopy labeled with the monoclonal antibody LM13. PW, Fibrillar primary wall; SW, electron-lucent secondary wall. Bars = 5 µm.

Figure 2.

Phylogenetic tree of GT 47 subclade B from selected flowering and nonflowering plants. The phylogenetic tree shows relationships among 38 AtARAD1-like protein sequences from representative bryophytes (P. patens), lycopods (S. moellendorffii), and angiosperms (rice, maize, tomato, potato, and Arabidopsis) and the N. alata sequence NaARADL1. Numbers indicate Bayesian posterior probability support for the node. Less supported nodes (posterior probability < 0.9) have been collapsed.

Figure 3.

Histochemical staining of pNaARADL1:GUS-expressing Arabidopsis plants. Seedlings 7 d after germination (DAG) show GUS staining in roots, particularly at the root tip (A), and the tip of the developing leaf and its basal organ boundary (B). Seedlings 14 DAG show GUS activity along the margins of the leaves (arrows; C). In fully expanded leaves, GUS accumulation is limited to the hydathodes (D). GUS also accumulates between the axils of secondary shoots (E), at the junction of the pedicel with the stem (arrows; F), and at the base of the sepals in the intersepal zone (arrowheads; F and G). Expression is also evident in the developing ovules (G) and developing anther (I), including the tapetum (tp), pollen grains, and pollen tubes growing through the style, as viewed in a longitudinal section under dark-field optics. Expression is also present in the nectaries (J). Black bars = 1 mm; white bars = 250 µm.

Figure 4.

Subcellular locations of fluorescently tagged versions of NaARADL1 in transiently transformed N. alata pollen tubes (A–D) and N. benthamiana leaves (E–H). A to D, Imaging of N. alata pollen grains transiently cotransformed with pLAT52:NaARADL1-CERULEAN (green) and the Golgi marker rat sialyltransferase translationally fused to dsRED (magenta). The merged and bright-field images of this cell also are shown. E to H, Imaging of N. benthamiana leaf cotransformed with the NaARADL1-VENUS translational fusion (yellow), the mCHERRY fluorescent Golgi apparatus marker soybean α-(1,2)-MANNOSIDASE I (magneta), and the endoplasmic reticulum (ER) marker SP-WAK2-mCERULEAN-HDEL (cyan). The merged image is also shown. Bars = 10 µm.

Figure 5.

GO-PROMTO analysis showing that NaARADL1 is a type II membrane protein located in the Golgi apparatus. A and B, Coexpression of either the cytosolic reporters VN/VC-TMD (A) or the luminal Golgi apparatus reporters TMD-VN/VC (B). C and D, No fluorescence was detected when NaARADL1-VN or NaARADL1-VC was coexpressed with the cytosolic reporters VC-TMD (C) or VN-TMD (D). E and F, A positive signal was detected when either NaARADL1-VN or NaARADL1-VC was coexpressed with the Golgi apparatus luminal reporter TMD-VC (E) or TMD-VN (F), indicating that the C terminus of NaARADL1 is located in the lumen. Bar = 10 μm.

Figure 6.

BiFC analysis showing that NaARADL1 forms homodimers and can heterodimerize with AtARAD1. A, Coexpression of the C-terminal VN or VC fusions of NaMUR3. B to D, Expression of NaARADL1-VN and NaMUR3-VC (B), NaMUR3-VN and NaARADL1-VC (C), or NaARADL1-VN and NaARADL1-VC (D). E and F, Negative controls including AtARAD1-VN/VC (E) and NaMUR3-VC/VN (F). G to I, Coexpression of AtARAD1-VN and AtARAD1-VC (G) or NaARADL1-VN/VC with AtARAD1-VC/VN (H and I). Bar = 10 μm.

Figure 7.

MALDI-TOF-MS analysis of endoarabinase-digested guttation fluid from plants expressing 35S:NaARADL1-VENUS. Endoarabinase generated arabinosyl oligosaccharides released from the sugar beet control (A) and guttation fluid collected from 35S:NaARADL1-VENUS-expressing plants (B). a.u., Arbitrary units.