Modulation of Auxin Levels in Pollen Grains Affects Stamen Development and Anther Dehiscence in Arabidopsis

Abstract

Auxin regulates diverse aspects of flower development in plants, such as differentiation of the apical meristem, elongation of the stamen, and maturation of anthers and pollen. It is known that auxin accumulates in pollen, but little information regarding the biological relevance of auxin in this tissue at different times of development is available. In this work, we manipulated the amount of free auxin specifically in developing pollen, using transgenic Arabidopsis lines that express the bacterial indole-3-acetic acid-lysine synthetase (iaaL) gene driven by a collection of pollen-specific promoters. The iaaL gene codes for an indole-3-acetic acid-lysine synthetase that catalyzes the conversion of free auxin into inactive indole-3-acetyl-l-lysine. The transgenic lines showed several abnormalities, including the absence of short stamina, a diminished seed set, aberrant pollen tubes, and perturbations in the synchronization of anther dehiscence and stamina development. This article describes the importance of auxin accumulation in pollen and its role in stamina and anther development.

Keywords: anther; auxin; dehiscence; iaaL; pollen.

Figure 1

Auxin signaling is activated in pollen grain. Pollen from plants carrying the auxin transcriptional reporter DR5:β-glucuronidase (GUS) were evaluated. Histological GUS staining was observed during different stages of pollen development. UNM: uninucled microspore; BPG: bicellular pollen grain; TPG: tricellular pollen grain; MPG: mature pollen grain (upper panels, [14]). The corresponding floral development stages following the staging described by Bowman et al. (1994) [17] are shown (numbers, lower panels). Tapetum cells and pollen are indicated by asterisks and arrowheads, respectively. Scalebar: 20 μm.

Figure 2

Flowers of transgenic plants expressing the indole-3-acetic acid-lysine synthetase (iaaL) gene controlled by floral tissue-specific promoters show a decrease in the expression of the auxin sensitive reporter DR5:GUS during different pollen developmental stages. (A) GUS activity was evaluated on inflorescences (left panels, scalebar: 20 mm), flowers (central panels, scalebar: 1 mm), and pollen grains (right panels, scalebar: 20 μm). The control plants DR5:GUS (upper panels) and the double transgenic plants carrying the DR5:GUS construct and the different promoters controlling the expression of the iaaL gene are shown: early pollen promoter (_pEPP:iaaL); two intermediate pollen promoters (_pIPP:iaaL; _pIPP2:iaaL), and late pollen promoter (_pLPP:iaaL). (B) The expression of transgenic constructs was evaluated with RT-PCR using RNA from pollen of DR5:GUS and DR5:GUS/_pIPP2:iaaL plants. The expressions of iaaL and GUS genes were evaluated. The EF1a gene was used as housekeeping gene. Gene promoter names are indicated in parentheses.

Figure 3

Transgenic _pIPP2:iaaL plants show a decrease in auxin content during the intermediate floral developmental stage. Different flowers stages were collected from plants DR5:GUS (black diamonds) and DR5:GUS/_pIPP2:iaaL (white circles) to evaluate the content of auxin (pmol IAA/g FW) using gas chromatography–tandem mass spectrometry analysis (GC–MS/MS). Floral stages are indicated (stage I: 7–8, stage II: 9–10, and stage III: 11–13). Asterisks indicate significant differences comparing the auxin levels of DR5:GUS/_pIPP2:iaaL and the control DR5:GUS transgenic line according to a two-way ANOVA and Bonferroni post test (** p < 0.01, n = 3).

Figure 4

Flowers from transgenic DR5:GUS/IPP2:iaaL plants with diminished auxin content show loss of short stamina. (A) Representative flowers of DR5:GUS and DR5:GUS/_pIPP2:iaaL plants (scalebar: 1 mm). (B) Quantification of the number of stamina per flower was performed in 200 flowers per genotype. The results were grouped as flowers with five (white boxes) or six stamina (gray boxes). Asterisks indicate significant differences comparing number of stamina of both DR5:GUS and DR5:GUS/_pIPP2:iaaL genotypes according to a Fisher’s exact test (p < 0,01, n = 200).

Figure 5

The pollen viability and the morphology of the pollen tube are affected in flowers of transgenic plants with a reduction in the auxin content. (A) Pollen viability analysis. Pollen from the DR5:GUS and DR5:GUS/_pIPP2:iaaL transgenic plants was stained with Alexander staining, and was then observed and counted using microscopy techniques. The percentages of dead and live pollen are represented as black and white bars, respectively. (B) Representative images of pollen germination assays from DR5:GUS and DR5:GUS/_pIPP2:iaaL plants. Pollen was germinated according to the conditions described in the methods section. The arrowhead indicates the aberrant balloon-shaped tip. Scalebar: 20 μm. The data were analyzed using a Fisher’s exact test comparing the genotypes. No significant differences were found (p = 0.0594, n = 50).

Figure 6

The reduction in auxin content in plants DR5:GUS/_pIPP2:iaaL affects the number of seeds per silique. Quantification of seed content was performed in DR5:GUS/_pIPP2:iaaL plants and the control DR5:GUS plants. Pictures of representative siliques are shown (A, scalebar: 5μm) and the number of seeds per silique was quantified. (B) Analysis was performed in 20 siliques of each genotype. Asterisks represent statistical differences between the genotypes according to an unpaired t-test (p < 0.001).

Figure 7

Reduction in flower auxin content via the expression of _pIPP2:iaaL gene construct produces altered phenotypes in the dehiscence synchronization. (A) Pictures of DR5:GUS/_pIPP2:iaaL flowers with different problems of dehiscence synchronization. A DR5:GUS flower is shown as a control. The arrowheads indicate different dehiscence problems as early dehiscence (black arrowheads), indehiscent anthers (gray arrowheads), and desynchronized dehiscence (white arrowheads). (B) Different defects in dehiscence synchronization were observed and quantified in DR5:GUS/_pIPP2:iaaL and control DR5:GUS plants. The data are expressed as the percentage of normal flowers (black box), flowers with indehiscence (dotted white box), flowers with desynchronized dehiscence (dashed white box), and flowers with early dehiscence (white box). A total of 200 flowers per genotype were analyzed. Asterisks indicate significant differences between genotypes according to a Fisher’s exact test (p < 0.001).