PECTIN METHYLESTERASE48 is involved in Arabidopsis pollen grain germination.

65 Germination of pollen grains is a crucial step in plant reproduction. However, the molecular 66 mechanisms involved remain unclear. We investigated the role of PECTIN 67 METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in 68 Arabidopsis thaliana pollen. A combination of functional genomics, gene expression, in vivo 69 and in vitro pollen germination, immunolabeling and biochemical analyses was used on wild- 70 type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the 71 male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. 72 Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and 73 germination in vitro and in vivo . Moreover, numerous pollen grains showed two tips emerging 74 instead of one in the wild-type. Immunolabeling and FT-IR analyses showed that the degree 75 of methylesterification of the homogalacturonan (HG) was higher in pme48-/- pollen grains. 76 In contrast, the PME activity was lower in pme48-/- partly due to a reduction of PME48 77 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme48- 78 /- with the optimum germination medium supplemented with 2.5 mM calcium chloride 79 suggesting that in the wild-type pollen, the weakly methylesterified HG is a source of Ca 2+ 80 necessary for pollen germination. Although pollen specific PMEs are traditionally associated 81 with pollen tube elongation, this study provides strong evidence that PME48 impacts the 82 mechanical properties of the intine wall during maturation of the pollen grain which, in turn, 83 influence pollen grain germination. Here, we report the study of a homozygous knock-down mutant for AtPME48 during imbibition and germination of pollen grains and pollen tube growth. We have investigated in vitro and in vivo pollen germination, pollen tube morphology and growth as well as the level 161 of PME activity and the degree of methylesterification of the HG by immunolabeling and FT- 162 IR in mutant and wild-type plants. Our results show that the group 1 PME48, the second most 163 expressed PME gene in pollen, plays a major role in remodeling the HG of the intine cell wall 164 during Arabidopsis pollen grain maturation resulting, after rehydration, in a normal pollen 165 grain germination.

shank. Back from the tip, the inner layer is mainly composed of callose, a (1,3)-β-glucan, 1 0 2 which is not detectable in the tip region in normal growth conditions (Ferguson et al., 1998;1 0 3 Derksen et al., 2002;Parre and Geitmann, 2005b;Dardelle et al., 2010;Chebli et al., 2012). Moreover, callose is also deposited periodically within the pollen tube to form plugs that  In eudicot species, pectins constitute a major portion of the primary cell wall. Pectins In order to assess further the biochemical differences between the wild-type and pme48-/-, we 2 7 7 investigated the total PME activity in pollen grains by using enzymatic assays. The data 2 7 8 showed a 50 % reduction of the total PME activity in pme48-/-pollen grains compared to the 2 7 9 wild-type (Fig. 5A). Moreover, zymogram after isoelectrofocusing (IEF) of total proteins 2 8 0 extracted from pollen grains revealed a disappearance of a diffuse band in the pI range 2 8 1 between 8.2 and 9 in pme48-/- (Fig. 5B) that may correspond at least in part to PME48 which 2 8 2 has a predicted pI of 8.3. Two spots, at pI ranging from 9.8 to 10.2 and from 9.5 to 9.7, in the was higher in pme48-/-pollen grains. The DM of the HG was consistently higher in the 2 9 0 pme48-/-(32.5 ± 1.7 %) compared to the wild type (13.4 ± 1.1 %) pollen grains (Fig. 5D). As 2 9 1 de-esterified HG binds more Ca 2+ than esterified HG, our data may suggest that HGs in the 2 9 2 intine wall of pme48-/-are less associated with Ca 2+ compared to wild-type pollen grains. To assess if the germination defect of pme48-/-pollen grains was related to the possible lower 2 9 5 Ca 2+ sink in the intine wall due to the higher DM of the HG, the optimum culture medium 2 9 6 containing 5 mM CaCl 2 was supplemented with 2.5 mM CaCl 2 to reach a final concentration 2 9 7 of 7.5 mM or with the Ca 2+ chelator, EDTA. In the presence of 1 mM EDTA, none of the 2 9 8 wild-type and pme48-/-pollen grains germinated, even after 24h of culture (n>1,000) (Fig. 6A-B). In the supplemented medium with Ca 2+ , pme48-/-pollen grain germination rates were 1 4 restored reaching the levels of the ones observed with wild-type pollen grains (n>1,000). The 6A-B). Moreover, the speed of imbibition of pme48-/-pollen grains was as fast as the wild-3 0 3 type pollen grains (Fig. 6C). In addition, a significant reduction of the abnormal phenotypes 3 0 4 such as burst tubes (from 33% to 13%) (Fig. 3F, 6D) and the double-tipped tubes (from 18% 3 0 5 to 2%) (Fig. 3G, 6E) was observed in pme48-/-pollen grains cultured in the supplemented 3 0 6 GM. Finally, in the CaCl 2 supplemented medium, the rates of burst tubes increased in wild-3 0 7 type pollen grains (32%) (Fig. 6D) compared to that observed when pollen grains were grown 3 0 8 in the optimum GM (~10%) (Fig. 3F). The regulation of the DM of HG has been implicated in many aspects of plant development predicted PME genes in the genome of Arabidopsis, 14 are specifically expressed in the male 2007). Two of them have already been studied using functional genomics approaches. The 3 2 3 first one is VANGUARD1 (At2g47040), the alteration of which led to unstable pollen tubes 3 1 5 and retarded growth of the pollen tube in the style and transmitting tract, resulting in a 3 2 5 significant reduction of male fertility (Jiang et al., 2005). The second pollen specific PME 3 2 6 characterized to date is PPME1 (At1g69940) (Tian et al., 2006). The homozygous mutant 3 2 7 atppme1 displayed reduced growth and irregular shape of pollen tube grown in vitro. In the 3 2 8 present study, we show that the alteration of the expression of PME48, the second most 3 2 9 expressed PMEs in pollen grains, results in a strong delay in imbibition and in germination 3 3 0 both in vivo and in vitro as well as altered phenotypes with abnormal rates of burst tubes and 3 3 1 two pollen tube tips emerging from the same pollen grain. The phenotype is rescued by  These data suggest that PME48 is mainly involved in the remodeling of the intine cell wall simulations (Chebli and Geitmann, 2007;Zerzour et al., 2009;Fayant et al., 2010). The more 3 4 0 abundant highly methylesterified HG in the intine wall of pme48-/-pollen grains and at the tip  intine. In many species such as Nicotiana tabacum (Li et al., 1995), Arabidopsis thaliana HGs, the latest being more abundant. The almost absent labelling of weakly methylesterified 3 5 2 HG with JIM5 at the pollen mother cell and tetrad stages (Rhee and Somerville, 1998) but its 3 5 3 strong detection in the intine at the late microspore stage and mature dry Arabidopsis pollen Golgi apparatus and may be secreted under a highly methylesterified form and then processed The early de-methylesterification of HG by PME48 during the pollen formation and pollen grain hydration (Fig. 7 ). The DM of HG can affect the water holding capacity of the  methylesterified HG increased compared to the wild-type suggesting that i) β -galactosidase 3 7 5 may be required for PME activity (Western et al., 2001;Walker et al., 2011) and ii) PME DM of the HG in the intine of pme48-/-is almost 2.5-fold higher than in the wild-type. Second, upon the block-wise action of PMEs, de-esterified blocks of HG chains can be cross- the unesterified HG in the maturing pollen grain may act as a reservoir of Ca 2+ that will be 3 8 5 used later during rehydration and germination (Fig. 7 ). The reduction of PME activity in prepare the future protrusion of the pollen tube tip by weakening the intine wall ( Fig. 7 b ).

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Recently, Rafińska et al., (2014) suggested that the cell wall of the female tissues can also act 3 9 9 as a reservoir of Ca 2+ in order to ensure correct germination and pollen tube growth in Larix However, we cannot rule out that PMEs originating from the stigma may also participate in   Although pollen specific PMEs are traditionally associated with pollen tube 4 3 0 elongation, the present study provides substantial evidence that PMEs, especially PME48, are phase with 60% humidity with daily watering. Valencia, CA) following the instructions of the supplier. the accumulated fluorescence signal to cross a threshold above the background) were acquired 4 6 0 with the LightCycler 480 software (Roche) using the second derivative maximum method.

7 0
PCR was performed on the cDNA generated from total RNA extracted from 6 h-old pollen 4 7 1 tubes grown in vitro. Primers used for the RT-PCR are in bold and underlined in Figure S1D.  were then cleared with several washes in 70% ethanol. The promoter of the PME48 gene (At5g07410) was PCR-amplified from a plasmid construct In vitro pollen tube growth 5 0 0 Pollen grains were grown in vitro in a liquid medium according to the method described by with ImageJ (n=126 and n=172 for the wild type and pme48-/-pollen tubes, respectively from 5 2 2 4 independent experiments).

2 3
Pollen grains were also cultured in the GM supplemented with 1 mM EDTA or 2.5 In vivo pollen tube growth 5 2 7 Emasculated mature flowers from wild-type plants were hand-pollinated with wild-type or Viability test and DAPI staining 5 3 7 The viability of pollen grains was assessed using fluorescein diacetate (FDA) dissolved in 5 3 8 acetone at 10 mg mL -1 and stored at -20°C. Prior to each experiment, FDA was diluted in a 5 3 9 10% sucrose solution to a final concentration of 0.2 mg mL -1 . Hydrated pollen grains were

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The nuclei of the pollen grains were stained with 10 µg mL -1 DAPI (4',6-diamidino-2- Immunolocalization was performed by cell surface labelling as previously described by serum albumin as a standard. PME activity was measured using the alcool oxidase method 6 1 0 according to Klavons and Bennett (1986). One IU of PME activity induces the production of Isoelectric focalisation (IEF) and zymogram were performed as described by Paynel et al. according to Bertheau et al. (1984). The gel was incubated for 1 h at 25 °C and the de- Data were statistically treated using the graphpad software (www.graphpad.com).  Table S1. List of primer pairs used for the qRT-PCR analyses.  2 8 Figure S4. Estimation of the growth speed in liquid medium of wild-type (black bars) and pme48-/-(grey bars) pollen tubes.

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Movie S1. Time-lapse imaging of germination and tube growth of wild-type pollen grains. Wageningen University (The Netherlands) for the gift of the pPLV06 plasmid. Arabinogalactan-proteins in root and pollen tube cells: distribution and functional thaliana is associated with persistence of pectic polysaccharides of the pollen mother wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the improves the ultrastructural preservation of in vivo grown lily pollen tubes.    Table S1. List of primer pairs used for the qRT-PCR analyses.