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. 2017 Jan;173(1):183-205.
doi: 10.1104/pp.16.01385. Epub 2016 Nov 9.

KNS4/UPEX1: A Type II Arabinogalactan β-(1,3)-Galactosyltransferase Required for Pollen Exine Development

Toshiya Suzuki  1   2 , Joan Oñate Narciso  1   2 , Wei Zeng  1   2 , Allison van de Meene  1   2 , Masayuki Yasutomi  1   2 , Shunsuke Takemura  1   2 , Edwin R Lampugnani  1   2 , Monika S Doblin  1   2 , Antony Bacic  1   2 , Sumie Ishiguro  3   4
Affiliations

KNS4/UPEX1: A Type II Arabinogalactan β-(1,3)-Galactosyltransferase Required for Pollen Exine Development

Toshiya Suzuki et al. Plant Physiol. 2017 Jan.

Abstract

Pollen exine is essential for protection from the environment of the male gametes of seed-producing plants, but its assembly and composition remain poorly understood. We previously characterized Arabidopsis (Arabidopsis thaliana) mutants with abnormal pollen exine structure and morphology that we named kaonashi (kns). Here we describe the identification of the causal gene of kns4 that was found to be a member of the CAZy glycosyltransferase 31 gene family, identical to UNEVEN PATTERN OF EXINE1, and the biochemical characterization of the encoded protein. The characteristic exine phenotype in the kns4 mutant is related to an abnormality of the primexine matrix laid on the surface of developing microspores. Using light microscopy with a combination of type II arabinogalactan (AG) antibodies and staining with the arabinogalactan-protein (AGP)-specific β-Glc Yariv reagent, we show that the levels of AGPs in the kns4 microspore primexine are considerably diminished, and their location differs from that of wild type, as does the distribution of pectin labeling. Furthermore, kns4 mutants exhibit reduced fertility as indicated by shorter fruit lengths and lower seed set compared to the wild type, confirming that KNS4 is critical for pollen viability and development. KNS4 was heterologously expressed in Nicotiana benthamiana, and was shown to possess β-(1,3)-galactosyltransferase activity responsible for the synthesis of AG glycans that are present on both AGPs and/or the pectic polysaccharide rhamnogalacturonan I. These data demonstrate that defects in AGP/pectic glycans, caused by disruption of KNS4 function, impact pollen development and viability in Arabidopsis.

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Figures

Figure 1.
Figure 1.
Pollen phenotypes of kns4 mutants. A to F, Surface view of wild-type (Ler; A–C) and kns4-1 (D–F) pollen grains by SEM. Arrow in D indicates cell wall debris adhered to pollen grains. G, Schematic structure of At1g33430/KNS4. The shorter splice variant, At1g33430.1, is depicted, as only this form is detected in flower buds. Rectangles represent exons [noncoding exon 1 (nc1) and coding exons 1 to 6], and coding region and 5′ and 3′ UTRs are indicated with white, black, and gray, respectively. The thick black line represents noncoding sequence/introns. The positions of point mutation (kns4-1) and T-DNA insertions (kns4-2 and kns4-3) as well as the SALK and SAIL line numbers are indicated. Gray arrows represent the position of primers used in the RT-PCR experiment shown in Fig. 4A. H to J, Surface view of kns4-2 (H and I) and kns4-3 (J) pollen grains by SEM. K, Rescued pollen phenotype of the kns4-1 mutant after transformation with the ProKNS4:KNS4 construct. Scale bars = 20 μm in (A, D, H, J, and K), 10 μm in (B, E, and I), and 2 μm in (C and F).
Figure 2.
Figure 2.
Light and TEM micrographs reveal defects in pollen development in kns4-2 mutants. A to L, Light micrographs of longitudinal sections of resin-embedded anthers of wild-type (Col-0; A–F) and kns4-2 (G–L) plants stained with toluidine blue. Developmental stages (stages 8L to 12L) of anthers are indicated along with the developmental stage of pollen development in parentheses. Asterisks in (J) and (K) indicate swollen and vacuolated tapetal cells in the mutant. Arrows and arrowheads in (L) indicate cell wall debris and crushed pollen grains, respectively. M to U, TEM images of wild-type (Col-0; M to P) and kns4-2 (Q to U) anthers. M and Q, Primexine layers and developing exine in tetrads at stage 9M. (N, O, R, S, and T), Longitudinal section of stage 12E anthers containing unicellular microspores. (P) and (U), Exine and pollen coat of pollen grains (tricellular stage) at stage 12L. Arrows in (R) show walls between tapetal cells. Arrowheads in (S) show granules containing sporopollenin deposited on the surface of tapetal cell wall. Ba, Bacula; CW, callosic wall; Ex, exine; In, intine; MS, microspore; PBa, probacula; PC, pollen coat; PG, pollen grain; PM, plasma membrane; PMC, pollen mother cell; Td, tetrad; Te, tectum; Tp, tapetal cell. Scale bars = 10 μm in (A–L), 5 μm in (N and R), and 0.5 μm in (M, O, P, Q, S, T, and U).
Figure 3.
Figure 3.
Abnormal sporopollenin deposition and delayed callose degradation in kns4-2. A to F, Calcofluor white staining of anther cross sections in wild type (Col-0; A to C), and kns4-2 (D–F) observed by fluorescence microscopy. Sections of stage 9L (newborn unicellular; A), stage 12E (unicellular; B and D), stage 12M (bicellular; C and E), and open flower (stage 14, tricellular; F) are shown. Cell walls containing cellulose and callose are stained whitish blue, and autofluorescence of sporopollenin is yellow. White and red arrows in (D) indicate boundary between tapetal cells and microspores and the walls separating adjacent tapetal cells, respectively. White arrows in (F) indicate tapetal wall remnant onto which sporopollenin is deposited. G and H, Aggregates of microspores (black arrows) squeezed out from kns4-2 anthers at stage 12E were observed with transmitted light (G) and fluorescence microscopy (H) after aniline blue staining; (H) shows the framed area in (G). I to L, Aniline blue staining of anther cross sections in wild-type (Col-0; I and J) and kns4-2 (K and L) observed by CLSM. Stages are 9M (tetrad; I and K) and 12E (unicellular; J and L). Green and magenta channels show fluorescence of aniline blue and sporopollenin autofluorescence, respectively. Dotted lines indicate the position of the tapetum. Arrowhead in (L) indicates callose in walls that remained in late unicellular stage. Ex, Exine; In, intine; Lo, locular space; MS, microspore; PG, pollen grain; Td, tetrad; Tp, tapetal cell. Scale bars = 50 μm in (A– F) and H, 200 μm in (G), 20 μm in (I–L).
Figure 4.
Figure 4.
KNS4 is specifically expressed in tapetal cells in young anthers. A, RT-PCR analysis of KNS4 using RNA extracted from rosette leaves, inflorescence stems, roots, inflorescence apices, open flowers, and fruits (siliques) of wild-type Arabidopsis (Col-0), and inflorescence apices of kns4-2 and kns4-3 mutants. Leftmost lane shows the PCR product amplified from Col-0 genomic DNA (gDNA) with the same primer set. ACT2 was used as a control. Black and gray triangles indicate cDNA- and gDNA-derived bands, respectively. B, ProKNS4-GUS expression in flower buds. C to F, ProKNS4-GFP expression in anthers from stages 8 to 10/11 observed by CLSM. Green and magenta channels show GFP signal and chlorophyll autofluorescence, respectively. G, Expression levels of KNS4 in anthers at various flower developmental stages (stages 8 and 9E, 9M and 9L, 10/11, 12E, 12M, and 12L) measured by qRT-PCR. Bars show relative average expression values (stage 8/9E = 1) of triplicate samples ± sd. H to Q, In situ hybridization using either an antisense (H–L) or sense (M–Q) probe for KNS4. Cross sections of paraffin-embedded anthers from stages 8 to 10/11 were examined. Scale bars = 1 mm in B, 100 μm in (C–F), and 50 μm in (H–Q).
Figure 5.
Figure 5.
Enzymatic activity of heterologously expressed KNS4 in microsomal membranes from Nicotiana leaf. A and B, RP-HPLC profiles of GalT enzyme assay products with KNS4-expressing MMs in the reaction mixture (B) and control experiment with VENUS-expressing MMs (A). Numbers above peaks indicate the number of Gal residues. C and D, Treatment of the KNS4 reaction products with either SGalase1 (C) or β-galactosidase (D). E, ESI-MS full scan spectrum of KNS4 GalT enzyme assay products. Peaks corresponding to β-Gal1-5-NBD are labeled 1 to 5, respectively. Yellow circles represent Gal residues.
Figure 6.
Figure 6.
Distribution of JIM8- and MAC204-epitope labeling of AGPs in microsporocytes and microspores. Cross sections of resin-embedded anthers of wild type (Col-0; A–D and I–L) and kns4-2 (E–H and M–P) from stages 8 to 12M were incubated with the JIM8 (A–H) and the MAC204 (I–P) antibodies, and subsequently with Alexa Fluor 488-labeled secondary antibody. Fluorescence of Alexa Fluor 488 (green) and autofluorescence (magenta) were separately captured by epifluorescence microscopy and overlaid. Magenta arrowheads indicate the signal in the primary cell wall of the PMC, tetrad, and tapetal-cell walls facing the locule. Yellow arrow indicates the signal in the exine cavities. Insets in (B) and (F) are enlargements of a microspore in a tetrad. White arrowhead indicates the signal in primexine. En, Endothecium; Ep, epidermis; ML, middle layer cell; MS, microspore; PMC, pollen mother cell; Td, tetrad; Tp, tapetal cell. Bars = 20 μm.
Figure 7.
Figure 7.
Distribution of MAC204-epitope labeling of AGPs on the surface of developing microspores. A to F, Median optical sections of wild type (Col-0; A–C) and kns4-2 (D–F) microspores isolated from stages 9L, 10/11, and 12E anthers after labeling with the MAC204 antibody and subsequently with Alexa Fluor 546-labeled secondary antibody. Exine was visualized by auramine O staining. Specimens were observed by CLSM in which magenta and green channels show fluorescence of auramine O and Alexa Fluor 546, respectively. G to N, Enlarged optical sections of wild-type (G–J) and kns4-2 (K–N) microspores isolated from stages 9L (G, H, K, and L) and 12E (I, J, M, and N) anthers. (G, I, K, and M) are median optical sections of microspores showing the exine in longitudinal cross section. Blue, white, and yellow arrowheads indicate the top and bottom boundary of the AGP layer, and the top of representative bacula, respectively. (H, L, and N) are tangential optical sections almost at the microspore surface. J, Tangential optical section that transversely cuts baculae. White and red arrowheads indicate the position of representative baculae and lacunae, respectively. Bars = 5 μm in (A–F), and 1 μm in (G–N).
Figure 8.
Figure 8.
Yariv dye reagent staining of microspores (stage 12M) isolated from flower buds of wild type and kns4 mutants. Microspores from wild type (Col-0; A–C), kns4-2 (D–F), and kns4-3 (G–I) were independently stained either with α-Gal Yariv (A, D, and G), β-Glc Yariv (B, E, and H), or left unstained (C, F, and I) and viewed with light microscopy. Positive staining is observed as a reddish-orange color. Bars = 20 µm.
Figure 9.
Figure 9.
Fluorescence micrographs of the distribution of JIM7- and JIM5-epitope labeling of pectins within anthers. Cross sections of resin-embedded anthers of wild type (Col-0; A–D and I–L) and kns4-2 (E–H and M–P) from stages 8 to 12M were incubated with either JIM7 (A to H) or JIM5 antibody (I to P), and subsequently with Alexa Fluor 488-labeled secondary antibody. Fluorescence of Alexa Fluor 488 (green) and autofluorescence (magenta) were captured separately by epifluorescence microscopy and overlaid. Note that the exposure time for capturing JIM5 signals is longer than that for JIM7, resulting in relatively higher background in JIM5 images. En, Endothecium; Ep, epidermis; ML, middle layer cell; MS, microspore; PMC, pollen mother cell; Td, tetrad; Tp, tapetal cell. Bar = 20 μm.
Figure 10.
Figure 10.
Fluorescence micrographs of the distribution of JIM7- and JIM5-epitope labeled pectins on the surface of isolated tetrads and developing microspores. A to D, Median optical sections of JIM7-labeled tetrads isolated from stage 9M anthers (A and C) and microspores isolated from stage 10/11 anthers (B and D), respectively. Exine was visualized by auramine O staining. Specimens were observed by CLSM in which magenta and green channels show fluorescence of auramine O and Alexa Fluor 546, respectively. Wild type (A and B) and kns4-2 (C and D) are compared. E to L, Median optical sections of JIM5-labeled tetrads (E and I) isolated from stage 9M anthers and microspores (F–H and J–L isolated from anthers at indicated stages. Wild type (Col-0; E–H) and kns4-2 (I–L) are compared. M to T, Enlarged optical sections of JIM5-labeled microspores. Median optical sections showing exine longitudinal sections (M, O, Q, and S) and tangential optical sections that cut the surface of microspore (N, P, R, and T) or that cut baculae transversely (O). Microspores were isolated from stage 9L (M, N, Q, and R) and stage 12E (O, P, S, and T) anthers. Wild type (M–P) and kns4-2 (Q–T) are compared. Blue, white, and yellow arrowheads indicate top and bottom boundary of the pectin layer, and the top of representative bacula, respectively. a, aperture. Bars = 5 μm in (A–L), and 1 μm in (M–T).
Figure 11.
Figure 11.
kns4 has defects in sexual reproduction. A to C, Morphological phenotypes of fully elongated fruit length (A) and seed set (number of seeds per upper and lower half of fruit; B and C) were observed and quantified. Error bars indicate SD. *P < 0.0001 in a Student’s t-test, wild type (Col-0) compared to kns4-2 and kns4-3 in (A) and (B). D, Reciprocal crosses between wild type and kns4-2. Fruits 7 d after pollination are shown. Dissection of fruits from kns4-2-pollinated wild-type pistils revealed formation of fewer seeds (black arrows) and many unfertilized ovules (white arrowheads), indicating lower probability of ovule fertilization by kns4 pollen. E, wild-type, or kns4-2 pollen tubes growing in vivo in manually pollinated wild-type pistils stained with aniline blue after 24 h. Staining appears as a bright light blue color. Scale bar = 2 mm in (C), 500 μm in (D) and 5 mm in (E). L, lower; U, upper.
Figure 12.
Figure 12.
Viability of wild-type and kns4-2 pollen grains. A to E, Staining of wild-type (Col-0) and kns4-2 pollen grains with Alexander dye (A and C, respectively) and DAPI (B and D, respectively). A few aborted pollen grains are observed in the kns4-2 pollen population (inset in C). The numbers in (B) and (D) show the number of nuclei in each pollen grain; (E) shows the percentage of wild-type and kns4-2 pollen grains with 0, 1, 2, or 3 nuclei. F and G, Images of self-pollinated pistils in wild-type (Col-0; F) and kns4-2 (G) flowers. Pollen adhesion is more obvious on the wild-type stigma (red arrowheads). H, Lengths of wild-type (Col-0) and kns4-2 pollen tubes grown in vitro for 16 h as represented by a bar-and-whiskers plot (n = 28). *P < 0.01 (Student’s t-test).
Figure 13.
Figure 13.
Schematic model of the function of microspore primexine in exine development. On the surface of wild-type microspores just released from the tetrad (stage 9L), the tectum of developing exine is sandwiched between pectin and AGP. The top view indicates AGPs lie on the outer surface of the tectum and display a similar reticulate pattern, and pectins show an internal distribution. As microspores develop, AGPs and sporopollenin supplied by tapetal cells are deposited into the primexine and exine, respectively. By stage 12E, the AGP layer laid down over the pectins becomes thicker, lifts the tectum up, and extends the baculae, resulting in exine thickening. AGPs fill the exine cavities except for baculae and lacunae, whereas pectins remain in the lacunae. In kns4, the AGP and pectin layers are incompletely formed at stage 9L. Subsequent deposition of AGP and sporopollenin is reduced and the exine remains thin at stage 12E.
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