BcMF27, A Pectin Methylesterase Gene, Regulates Pollen Development and Pollen Tube Growth in Brassica Campestris

Functional pollen grains are an essential ingredient of successful reproduction in owering plants and are protected by outer walls. Pectin methylesterases (PMEs) modify pectin, a structural component of pollen intine. However, there are few studies on PMEs. Articial microRNA (amiRNA) and overexpression technology was performed to investigate the function of pollen-specic PME gene, BcMF27, in pollen development. Knockdown of BcMF27 led to pollen wall collapse, 20% of which unknown material adhered to. Wall-collapsed pollen had abnormally thick intine outside of the germinal furrows. A portion of the cytoplasm was degraded in the remaining pollen with unknown material on the wall, in addition to a thick intine. Overexpression of BcMF27 resulted in 66.67% pollen wall disruption, causing an abnormally thick intine. In addition, functional interruption of BcMF27 gave rise to pollen tubes twisted in vitro. Taken together, BcMF27 contributes to the intine morphogenesis during pollen development and stabilizes pollen tube elongation. This research can promote knowledge of PMEs function and the molecular mechanism in pollen wall construction.


Introduction
Pollen wall is the outer structure of pollen grain, protecting the male gametophyte from physical and biological stress. The pollen wall plays an important role in pollen development, the key procedure of the angiosperm life cycle (Zhang et al. 2016). The elaborately organized pollen wall consists of the exine and intine. The major component of the exine is sporopollenin. The intine comprises pectin, structural proteins, and micro brillar cellulose (Huang et al. 2009). Pectin is a structurally covalently linked heteropolysaccharide (Mohnen 2008). PMEs (EC 3.1.1.11) catalyze demethylation of HG at the C-6 to release the free negatively charged carboxyl group and reduce the degree of methylesteri cation (DM) (Pelloux et al. 2007). De-esteri ed pectin molecules link to each other by calcium ions to form an "egg-box" supermolecular gel (Yoneda et al. 2010). The pectic gel is then integrated into the wall to strengthen wall rigidity (Roger et al. 2001). Therefore, the demethylation of pectin by PMEs affects pollen development by participating in wall construction.
PMEs exist ubiquitously in the whole plant life cycle (Pelloux et al. 2007) and have important biological signi cance in plant growth and development, including pollen development, cell elongation, stem morphogenesis, cell adhesion and separation, seed coat mucilage extrusion, abiotic stress, and biological stress (Micheli 2001 (Jiang 2005;Tian et al. 2006;Chen and Ye 2007), which have been con rmed to affect pollen tube growth. Moreover, Francis et al. (2006) observed that QRT1 was involved in the separation of tetrad. AtPME48 regulated pollen germination (Leroux et al. 2015). BcMF23a contributed to pollen development and pollen tube growth (Yue et al. 2018a). BcPME37c in uenced pollen intine formation (Xiong et al. 2019). However, studies on the biological function of PMEs, particularly in pollen development, still remain limited.
Previously, BcMF27 from B. campestris was identi ed as a putative pectin methylesterase gene. BcMF27 was expressed signi cantly in mature pollen and pollinated pistils (Yue et al. 2018b). In this study, arti cial microRNA (amiRNA) and overexpression technology was used to further determine the biological function of BcMF27. Knockdown and overexpression of BcMF27 led to intine formation outside of the germinal furrows, pollen morphology abortion, and pollen tube twisting. According to these results, it is suggested that BcMF27 is necessary for pollen development and pollen tube growth by participating in pollen intine and pollen tube wall, respectively.

Construction of vectors and plant transformation
The amiRNA sequence was designed according to the procedure in the Web MicroRNA Designer (WMD3-Web MicroRNA Designer; 5′-TAAGCAACATACACTGCGCGA-3′), and miR164a was used as the gene backbone. The incorporated sequence was integrated into the binary vector pCAMBIA1301 between XbaI and HindIII with a constitutive CaMV35S promoter. The cDNA sequence of BcMF27 (Yue et al. 2018b) was cloned and inserted into the same vector between the restriction sites. Then, amiRNA, overexpression, and empty vectors were induced in B. campestris ssp. chinensis var. parachinensis by Agrobacterium tumefaciens to obtain transgenic lines according to Yu et al. (2004). Transgenic plants were cultivated in an illumination incubator at 22°C with a photoperiod of 16 h light/8 h dark.

Detection of positive transgenic plants
To identify the transformed lines, PCR was performed with genomic DNA extracted from fresh young leaves using the primers 5′-CCAGGCTTTACACTTTATGC-3′ and 5′-GCGATTAAGTTGGGTAACGC-3′. Total RNA of the whole in orescence from positive plants was extracted by the Trizol ® reagent (Invitrogen, Carlsbad, CA, USA) and then used to synthesize the rst-strand cDNA with the PrimerScript RT reagent kit (TaKaRa, Japan). Real-time RT-PCR was performed with the SYBR Premix Ex Taq Kit (TaKaRa, Japan) using a Bio-Rad CFX96 Real-time PCR Detection System (Bio-Rad, USA). UBC10 was used as a control. The primer sets were as follows: BcMF27F 5′-ATGGCGTTTCAGGATTTCGAC AA-3′ and BcMF27R 5′-TCACGCATCATAAAGACCAAGC-3′; and UBC10F 5′-GGGTCCT ACAGACAGTCCTTAC-3′ and UBC10R 5′-ATGGAACACCTTCGTCCTAAA-3′. Three biological repeats were performed. The 2 −ΔΔCt method was used to analyze the relative expression levels of BcMF27 (Livak and Schmittgen 2001).

Pollen microscopy observation
Scanning electron microscopy (SEM) of mature pollen and transmission electron microscopy (TEM) of pollen during pollen development were performed according to Lin et al. (2014). Semi-thin anther sections (2 μm) from anthers collected during pollen development were stained by dimethyl blue and photographed with a uorescent microscope (Leica, Germany). The pollen abortion percentage was analyzed.

Pollen germination in vitro
Pollen germination in vitro was performed according to Lin et al. (2014). Pollen tubes were observed and photographed with a uorescent microscope. The rate of pollen germination and percentage of abnormal pollen tubes were calculated. Three biological repeats were performed.

Up-and downregulation of BcMF27 resulted in pollen abnormality
To determine the function in pollen development, amiRNA and overexpression vectors of BcMF27 were constructed (Fig. 1a, b) and induced in B. campestris ssp. chinensis var. parachinensis. Positive transgenic lines were con rmed by PCR analysis and named BcMF27-amiR and BcMF27 OE (Fig. 1c). The BcMF27 transcripts from the in orescence of BcMF27-amiR and BcMF27 OE plants were detected by realtime RT-PCR (Fig. 1d). The results showed that the expression of BcMF27 decreased in the in orescence of BcMF27-amiR1-3 plants and increased in the in orescence of BcMF27 OE -1-3 plants.
No difference was observed in vegetative growth, ower organs, and fertility among BcMF27-amiR and BcMF27 OE and control transgenic plants (data not shown). However, SEM indicated that the pollen of BcMF27-amiR and BcMF27 OE plants was defective (Fig. 2). Of pollen from BcMF27-amiR plants, 93.56% possessed collapsed walls (Fig. 2b, e, f, h). The surface of 29% of abnormal pollen walls locally accumulated unknown material (Fig. 2e). Of BcMF27 OE pollen, 66.67% had collapsed walls (Fig. 2c, g, i). Pollen from control plants had an ellipsoid shape and normal reticular structure, and the deformity rate was only 13.75% (Fig. 2a, d, h, i).
To further detail BcMF27-amiR and BcMF27 OE pollen development, TEM was performed (Fig. 4). BcMF27-amiR pollen development normally proceeded to the uninucleate stage ( Fig. 4f-h, r) in accordance with the control (Fig. 4a-c, q). At the binuclear stage, the intine thickened normally at germinal furrow regions in control pollen (Fig. 4d, t), but 95.45% of BcMF27-amiR pollen intines formed abnormally outside germinal furrows (Fig. 4i, u). Furthermore, 36.36% of BcMF27-amiR pollen with an additional germinal aperture displayed partial cytoplasm degradation and unknown material on the surface of pollen (Fig. 4j, x) and the remaining defective pollen still included four germinal furrows (Fig. 4p, z), while the control pollen contained a normal organized wall with three germinal furrows and a dense cytoplasm (Fig. 4e, w) at the trinucleate stage. BcMF27 OE pollen showed normal development from the pollen mother cell stage to the binuclear pollen stage (Fig. 4k-n, s, v), whereas 52% of pollen exhibited aberrant intine deposition outside the germinal furrow regions at the mature pollen stage (Fig. 4o, y).

Functional disruption of BcMF27 caused morphologically unstable pollen tubes
In BcMF27-amiR and BcMF27 OE transgenic plants, pollen germination in vitro was checked to determine the effect of BcMF27 expression imbalance on pollen germination and pollen tube growth (Fig. 5). The average germination rates of BcMF27-amiR and BcMF27 OE pollen were 74.95% and 71.86%, respectively.

Discussion
The elaborately decorated pollen wall protects pollen from stress and consists of the exine and intine. Pectin is a structural component of the intine and is involved in the modi cation of wall characteristics. PMEs hydrolyze pectin and demethylate pectin molecules linked to each other by calcium ion bonds; they are then integrated into the intine to strengthen wall rigidity. Therefore, PMEs have an essential function in pollen wall construction and pollen development. Yue et al. (2018a) observed that knockdown of BcMF23a caused shape deformities and defective intine construction in pollen. Therefore, it was inferred that BcMF23a in uenced pollen development via intine formation. BcMF27, the characteristic pollen-speci c pectin methylesterase, was remarkably expressed in mature pollen (Yue et al. 2018b). BcMF27-amiR and BcMF27 OE mature pollen walls collapsed and the intine thickened abnormally outside of the germinal apertures, which was similar to the phenotype of bcmf23a pollen. In addition, in 36.36% of BcMF27-amiR pollen, the cytoplasm partially disappeared, which also occurred in bcmf23a pollen. Thus, it was estimated that the imbalance of BcMF27 expression led to abnormal intine formation, which further affected pollen morphology construction.
The pollen tube is an extension of the pollen intine and grows rapidly in the pistil ( Many pollen-speci c PME genes have demonstrated an important function in pollen germination and pollen tube growth (Kim et al. 2020). AtPME48 belongs to the pollen-speci c PME gene (Leroux et al. 2015). Functional interruption of AtPME48 resulted in the appearance of two pollen tubes in vitro, delayed growth of pollen tubes in the pistil, and increased the DM of pme48 pollen grains. It was concluded that AtPME48 affected pollen germination by in uencing the formation of the pollen intine. VGD1 was also expressed in pollen and the pollen tube (Jiang 2005). VGD1 knockout caused the pollen tube to burst in vitro, and pollen tube growth in vivo was postponed, causing the reduction of PME activity in the pollen tube, although the pollen grain displayed normal morphology. ZmGa1P and ZmPME10-1, two pollenspeci c PMEs, assembled to modulate pectin esteri cation in pollen tube wall, which affected pollen tube growth (Zhang et al. 2018). Similarly, BcMF27 was speci cally expressed in pollen and pollen tubes, and down-and upregulation of BcMF27 led the tube to be signi cantly twisted. Therefore, according to these results, disruption of BcMF27 function reduced PME activity in the pollen tube, causing depressed rigidity in the tube wall, which destabilized pollen tube growth. The functional interruption of BcMF27 and BcMF23a led to intine construction abnormality, pollen morphology deformity, and pollen tube growth instability (Yue et al. 2018a). However, the transcript of BcMF27 appeared remarkably in pollen tubes, as well as mature pollen, while BcMF23a was expressed in pollen but not in the pollen tube (Lin et al. 2017;Yue et al. 2018a). Consequently, BcMF27 was involved in intine formation to in uence pollen development and contributed to pollen tube growth by modifying pollen tube walls.

Declarations
Author contribution statement JC and YY conceived and designed the research. YY conducted the experiments, analyzed the data, and wrote the manuscript. JC revised the manuscript. All authors read and approved the nal manuscript.