Auxin production in diploid microsporocytes is necessary and sufficient for early stages of pollen development

<div><p>Gametophytic development in Arabidopsis depends on nutrients and cell wall materials from sporophytic cells. However, it is not clear whether hormones and signaling molecules from sporophytic tissues are also required for gametophytic development. Herein, we show that auxin produced by the flavin monooxygenases YUC2 and YUC6 in the sporophytic microsporocytes is essential for early stages of pollen development. The first asymmetric mitotic division (PMI) of haploid microspores is the earliest event in male gametophyte development. Microspore development in <i>yuc2yuc6</i> double mutants arrests before PMI and consequently <i>yuc2yuc6</i> fail to produce viable pollens. Our genetic analyses reveal that <i>YUC2</i> and <i>YUC6</i> act as sporophytic genes for pollen formation. We further show that ectopic production of auxin in tapetum, which provides nutrients for pollen development, fails to rescue the sterile phenotypes of <i>yuc2yuc6</i>. In contrast, production of auxin in either microsporocytes or microspores rescued the defects of pollen development in <i>yuc2yuc6</i> double mutants. Our results demonstrate that local auxin biosynthesis in sporophytic microsporocytic cells and microspore controls male gametophyte development during the generation transition from sporophyte to male gametophyte.</p></div

Localized auxin production in tapetum did not rescue the pollen defects in <i>yuc2yuc6</i> mutants.

<p>(A) The green color indicates the expression pattern of the <i>A9</i> promoter. Msp, Microsporocytes; Mp, microspores; Bc, Bicellular pollen; Tc, Tricellular pollen. The <i>A9</i> promoter cloned from Arabidopsis is used to drive <i>YUC2-GFP</i> expression specifically in tapetum cells from stages 6 to 9 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007397#pgen.1007397.ref061" target="_blank">61</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007397#pgen.1007397.ref062" target="_blank">62</a>]. (B) Morphology of adult shoots (Bars = 2cm). (C) Alexander staining (Bars = 100 μm) and DAPI staining (Bars = 10 μm) of Pro<i>A9</i>:<i>YUC2-GFP</i> (<i>yuc2yuc6</i>) anthers and pollens. Note that the sterility phenotype and pollen defects were not rescued in Pro<i>A9</i>:<i>YUC2-GFP</i> (<i>yuc2yuc6</i>) transgenic plants. TS, Tricellular Stage; BS, Bicellular Stage; US; Unicellular Stage. (D) In situ hybridization of GFP in Pro<i>A9</i>:<i>YUC2-GFP</i> (<i>yuc2yuc6</i>) transgenic plants (Bars = 20 μm). (E) Fluorescence images (Bars = 100 μm) of the YUC2-GFP fusion protein in anthers from <i>yuc2yuc6</i> transformed with Pro<i>A9</i>:<i>YUC2-GFP</i>. In Pro<i>A9</i>: <i>YUC2-GFP</i> anther, GFP is significantly expressed in tapetum cell of microsporocyte stage and early microspore stage.</p

The early stages of male gametogenesis were defective in the <i>yuc2yuc6</i> double mutants.

<p>(A-J) DAPI staining of pollens from wild type (A-E) and <i>yuc2yuc6</i> (F-J) at different developmental stages. Microspores from anthers at (A and F) stage 8; (B and G) stage 9; (C and H) stage 10 were shown. Some of the <i>yuc2yuc6</i> microspores were degenerated and had little DAPI staining (H right). (D and I) pollens at the bicellular stage. Note that the nuclei of <i>yuc2yuc6</i> were arrested at the unicellular state or degenerated and could not form two nuclei as in the wild type (I). (E and J) Pollen at the tricellular stage. The <i>yuc2yuc6</i> pollen grains lacked genomic DNA (J) while WT pollen showed strong DAPI staining (E). (K) Quantitative analysis and comparison of pollen defects between wild type and <i>yuc2yuc6</i> (n>600 for each stage). Uc, Unicellular pollen; Mn, microspore nucleus; Gc, generative cell; Vn, vegetative nucleus; Sc, sperm cell. Bc, Bicellular pollen; Tc, Tricellular pollen; De, Degenerated pollen. Bars = 5 μm.</p

DII-VENUS for visualizing auxin-induced Aux/IAA degradation during pollen development.

<p>(A) Fluorescence of pro<i>MSP1</i>:<i>mDII</i> control and pro<i>MSP1</i>:<i>DII</i> plants. <i>MSP1</i> promoter (pro<i>MSP1</i>) is a microspore-specific promoter. E: early, L: late. (B) Quantitative representation of the relative VENUS fluorescence intensity from nucleus of stage 10 and stage 11 pollen. The fluorescence intensity was measured using ImageJ software. Data shown in B represent mean with SD based on more than 20 pollen grains for each group (set as 1 for stage 11 pollen from pro<i>MSP1</i>:<i>DII</i>). The symbol * indicates where the difference between pro<i>MSP1</i>:<i>DII</i> and pro<i>MSP1</i>:<i>mDII</i> at corresponding stage is significant in statistic test (<i>p</i>-value<0.05). Note that the fluorescence signals of DII in stage 10 and stage 11 were much weaker than that in mDII microspores. Bars = 7 μm.</p

Effects of mutations in <i>YUC2</i> and <i>YUC6</i> on male transmission frequency.

<p>Effects of mutations in <i>YUC2</i> and <i>YUC6</i> on male transmission frequency.</p

The expression patterns of DR5:GFP in the anther of <i>yuc2yuc6</i>.

<p>(A-B) Comparison of the auxin reporter <i>DR5</i>:<i>GFP</i> signal in anthers of wild type (A) and <i>yuc2yuc6</i> (B) at different developmental stages. Reporter signal in anthers observed in wild type from stages 9 to 12 is completely absent in <i>yuc2yuc6</i>. Bars = 100 μm.</p