Comparison of transverse sections of pollen development in the control and <i>bcmf26a/b</i> anthers.

<p>(A to E) Semi-thin sections of anthers from the control plants. (F to J) Semi-thin sections of anthers from <i>bcmf26a/b</i> transformed plants. The pollen at the (A and F) pollen mother cell stage, (B and G) tetrad stage, (C and H) uninucleate stage, (D and I) binucleate microscope stage, (E and J) trinucleate microscope stage were observed. No obvious difference in pollen mother cell stage and tetrad stage was observed. The differences in pollen morphology between the control and <i>bcmf26a/b</i> were observed from the uninucleate microscope stage to the trinucleate microscope stage. aP, aborted pollen; E, epidermis; En, endothecium; ML, middle layer; Msp, microspore; P, pollen; Tp, tapetum; Tds, tetrads; Bm, binucleate microscope; MP, mature pollen. Scale bars = 50 μm.</p

Pollen morphologies of the <i>bcmf26a/b</i> and control lines.

<p>(A and B) Alexander staining of pollen grains from the control plants and <i>bcmf26a/b</i> plants. (A) Mature pollen grains of the control plants were bright red upon staining with Alexander solution, whereas (B) the nonviable pollen grains from <i>bcmf26a/b</i> were blue-green. (C–F) DAPI staining observation of pollen grains from the (C and D) control plants and <i>bcmf26a/b</i> plants underfluorescence and (D and F) bright-field microscopy. (E and F) Mature pollen grains of control plants contained normal sperm nucleus and vegetative nuclei. Whereas, (C and E) the nuclei were absent in <i>bcmf26a/b</i> nonviable pollen grains. (G–N) SEM observation of pollen grains from control plants and <i>bcmf26a/b</i> plants. (G and H) (G and H) Mature pollen grains of control plants were uniformly spheroid and had finely reticulate ornamentation on their surface; whereas, (I–N) the irregular pollen grains from <i>bcmf26a/b</i> exhibited abnormal pollen wall and germinal furrows. Scale bars of A—F = 50 μm.</p

<i>BcMF26a</i> and <i>BcMF26b</i> Are Duplicated Polygalacturonase Genes with Divergent Expression Patterns and Functions in Pollen Development and Pollen Tube Formation in <i>Brassica campestris</i>

<div><p>Polygalacturonase (PG) is one of the cell wall hydrolytic enzymes involving in pectin degradation. A comparison of two highly conserved duplicated PG genes, namely, <i>Brassica campestris Male Fertility 26a</i> (<i>BcMF26a</i>) and <i>BcMF26b</i>, revealed the different features of their expression patterns and functions. We found that these two genes were orthologous genes of <i>At4g33440</i>, and they originated from a chromosomal segmental duplication. Although structurally similar, their regulatory and intron sequences largely diverged. QRT-PCR analysis showed that the expression level of <i>BcMF26b </i>was higher than that of <i>BcMF26a</i> in almost all the tested organs and tissues in <i>Brassica campestris</i>. Promoter activity analysis showed that, at reproductive development stages, <i>BcMF26b</i> promoter was active in tapetum, pollen grains, and pistils, whereas <i>BcMF26a</i> promoter was only active in pistils. In the subcellular localization experiment, BcMF26a and BcMF26b proteins could be localized to the cell wall. When the two genes were co-inhibited, pollen intine was formed abnormally and pollen tubes could not grow or stretch. Moreover, the knockout mutants of <i>At4g33440</i> delayed the growth of pollen tubes. Therefore, <i>BcMF26a/b</i> can participate in the construction of pollen wall by modulating intine information and <i>BcMF26b</i> may play a major role in co-inhibiting transformed plants.</p></div

Sequence characterization and phylogenetic tree analysis.

<p>(A) The phylogenetic trees of PG genes from <i>Brassica campestris</i> and <i>Arabidopsis thaliana</i> genomes were generated using the neighbor-joining (NJ) method with 1000 bootstrap repeats (part of data displayed). <i>At4g33440</i>, <i>BcMF26a</i>, and <i>BcMF26b</i> are indicated by solid circle and triangles, respectively. (B) <i>BcMF26a</i>, <i>BcMF26b</i>, <i>At4g33440</i>, and their flanking regions representing ~45 kb of chromosomes are drawn to scale. Collinear conserved blocks are identified. The black solid lines indicate noncolinear chromosome fragments. The dotted lines represent the other regions of the chromosomes, which are not drawn to scale. The positions of <i>BcMF26a</i>, <i>BcMF26b</i>, and their orthologous gene <i>At4g33440</i> in <i>A</i>. <i>thaliana</i> are labeled with blue, red and green, respectively. Segmental chromosomal duplication and rearrangement are shown. (C) Phylogenetic tree constructed based on the amino acid sequence of <i>BcMF26a</i>, <i>BcMF26b</i>, <i>At4g33440</i>, and 35 PG genes from different plant species in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131173#pone.0131173.s010" target="_blank">S2 Table</a> by NJ method. Confidence values from the bootstrap test (1000 replicates) are indicated by the numbers on the tree. The genes were clustered into six clades (Clade A to Clade F). <i>BcMF26a</i>, <i>BcMF26b</i>, and <i>At4g33440</i> are grouped in Clade E.</p

QRT-PCR analysis of <i>BcMF26a and BcMF26b</i> in different tissues and organs of <i>Brassica campestris</i>.

<p>(A) Relative expression patterns of <i>BcMF26a</i> and <i>BcMF26b</i> in roots (R), stems (St), leaves (L), inflorescences (Inf), and siliques (Si). Both <i>BcMF26a</i> and <i>BcMF26b</i> could express in all the five tested tissues. The transcript level of <i>BcMF26b</i> was higher than that of <i>BcMF26a</i>. (B) The relative expression levels of <i>BcMF26a</i> and <i>BcMF26b</i> in flower buds of ‘<i>Bcajh97-01A/B</i>’. The relative expression level of <i>BcMF26b</i> was much higher than that of <i>BcMF26a</i> in all the flower buds examined. The relative expression levels of both genes were much higher in buds of ‘<i>Bcajh97-01B</i>’ than those of ‘<i>Bcajh97-01A</i>’, except B3. A1–A5 and B1–B5 indicate flower buds at five developmental stages (Stage I to Stage V), namely, pollen mother cell stage, tetrad stage, uninucleate microspore stage, binucleate microspore stage, and mature pollen stage. (C) Relative expression levels of <i>BcMF26a</i> and <i>BcMF26b</i> in separate flower organs of B5, including sepals (Se), petals (Pe), stamens (Sta), and pistils (Pi). <i>BcMF26b</i> mainly expressed in stamens and pistils. The relative expression level of <i>BcMF26b</i> was much higher than that of <i>BcMF26a</i>. Standard errors for three independent experiments are shown.</p

Subcellular localization of BcMF26a-GFP and BcMF26b-GFP fusion proteins in onion epidermal cells.

<p>(A, C, and E) Fluorescence images of plasmolyzed cells transformed with pFGC–BcMF26a: GFP, pFGC–BcMF26b: GFP, and pFGC: GFP, respectively. (B, D and F) Bright field image of the corresponding onion epidermal cells. (A and C) The fluorescence signal of the target proteins could be observed in the cytoplasm, cell wall, and space between the membrane and cell wall. (E) The fluorescence in the control cell could only be observed in the cytoplasm. Scale bars = 50 μm.</p

Analysis of the <i>BcMF26a/b</i> mRNA levels in the inflorescences of the <i>bcmf26a/b</i> and control lines using qRT-PCR analysis.

<p>The expression levels of <i>BcMF26a/b</i> in the inflorescences of <i>bcmf26a/b</i> lines were significantly less than that of the empty vector pCAMBIA1301-transformed plants (control). <i>UBC-10</i> was used as an internal control. Standard errors for three independent experiments are shown.</p

Spatial expression pattern of proBcMF26b: GUS-GFP.

<p>(A–M) Histochemical GUS assays of the T₂<i>Arabidopsis</i> transformed with pBGWFS7.0–proBcMF26b: GUS-GFP vector. (A–E) GUS assays in a four-day-old seedling. The expression signals were detected in all parts of the seedling, except for a weaker staining in (B) the shoot apical meristem. (F–M) GUS assays in inflorescence of 35-day-old <i>Arabidopsis</i>. GUS expression could be observed in (F) pedicels, in (G and H) anthers at the early stage of flower buds development, and in (K–M) pistils and filaments at the late stages of flower buds development. (N–P) Transverse section of flower buds at the (N) pollen mother cell stage, (O) binucleate microspore stage and (P) mature pollen stage, GUS activity was observed in anthers at the pollen mother cell stage, as well as in the tapetum and pollen grains at the binucleate microspore stage and mature pollen stage. Scale bars of N–P are 50 μm.</p

Transmission electron micrographs of microspores from the control plants and <i>bcmf26a/b</i> lines.

<p>Pollen at the (A and F) uninucleate microscope stage, (B and G) binucleate stage, and (C and H) mature pollen stage of the control plants and <i>bcmf26a/b</i> plants were observed. (A–C) The control pollen grains showed normal morphology with three germinal furrows and a normal development process of the pollen wall; the pollen intine (D) outside and (E) inside the germinal furrow region developed normally. (F–O) The pollen grains from the <i>bcmf26a/b</i> lines showed two kinds of deficient phenotype. One ultimately produced (F–H) vacuolated pollen grains with disordered formation of the intine. The pollen intine (I) disappeared in some regions, while the intine was (J) abnormally thick in other regions. The other kind of deficient phenotype was the formation of (K–M) swollen pollen grains. The development of pollen cytoplasmic inclusions was abnormal. Remarkable thickening (N) outside and (O) inside the germinal furrow regions was observed, compared with the control pollen. Ba, baculum; Ex, exine; Gf, germinal furrow; In, intine; N, nucleus; SC, sperm cell; Te, tectum.</p

Viability percentage and irregular percentage of pollen grains in <i>bcmf26a/b</i> and control lines.

<p>Nearly half of pollen grains were nonviable in the <i>bcmf26a/b</i> plants, whereas the percentage was only 1.01% in the control. Approximately 38.9%–71.1% of the pollen grains were irregular in the <i>bcmf26a/b</i> plants, whereas ~3.75% was irregular in the control. Standard errors are shown.</p