Intracellular auxin transport in pollen

Abstract

Cellular auxin homeostasis is controlled at many levels that include auxin biosynthesis, auxin metabolism, and auxin transport. In addition to intercellular auxin transport, auxin homeostasis is modulated by auxin flow through the endoplasmic reticulum (ER). PIN5, a member of the auxin efflux facilitators PIN protein family, was the first protein to be characterized as an intracellular auxin transporter. We demonstrated that PIN8, the closest member of the PIN family to PIN5, represents another ER-residing auxin transporter. PIN8 is specifically expressed in the male gametophyte and is located in the ER. By combining genetic, physiological, cellular and biochemical data we demonstrated a role for PIN8 in intracellular auxin homeostasis. Although our investigation shed light on intracellular auxin transport in pollen, the physiological function of PIN8 still remains to be elucidated. Here we discuss our data taking in consideration other recent findings.

Keywords: :

• PIN8
• PILS5
• PIN5
• auxin
• ER
• pollen
• transport
• homeostasis
• Arabidopsis thaliana

This article refers to:

The phytohormone auxin plays a key role in coordinating many aspects of plant development. Thus far, the role of auxin in microgametogenesis remains poorly understood. It is known that pollen and anthers accumulate high levels of auxin,^1,2 and that auxin is important for pollen maturation, filament elongation and anther dehiscence,³ but how auxin works in developing and germinating pollen grains is unknown.

We analyzed publically available pollen-specific microarrays data⁴ and highlighted several auxin related genes probably involved in male gametophyte development.⁵ We found that PIN8, a member of the auxin efflux facilitator protein family, was highly expressed during pollen development. This observation was confirmed by promoter expression analysis and qRT-PCR assays. We also showed that PIN8 resides in the ER of pollen grains and germinated pollen tubes, where it is involved in intracellular auxin homeostasis.

Despite the specific expression pattern of PIN8 during microgametogenesis, pin8 pollen of T-DNA insertion lines developed normally. Pollen from these lines was fully competent for germination and fertilization resulted in normal seed set.⁵ We hypothesized that the absence of an obvious morphological phenotype could be due to a fine tuning activity performed by PIN8 at the ER or to the involvement of compensating mechanisms. More recently, Ding et al.⁶ reported that pin8 mutation resulted in aborted or misshaped pollen grains, though at very low frequencies. In addition, abnormal pollen germination rates, reduced transmission capacity and a decreased density of the rough ER in 10–15% of the pin8 pollen grains were observed.⁶ Although it remains to be clarified how PIN8 regulates all these different aspects, collectively the described phenotypes indicated a role for PIN8 in male gametophyte development. This study also revealed some interplay between PIN8 and PIN5, the other ER-localized member of the PIN family, in pollen. Pin5 loss of function mutants displayed the same percentage of morphologically defected pollen grains as pin8 and pin5pin8 double mutants could rescue the pollen morphology defects observed in single mutants.⁶ These results are quite astonishing considering the mutual exclusive expression domain of the two genes, with PIN5 being broadly expressed in sporophytic tissues⁷ but not expressed at significant levels in the male gametophyte. In contrast, PIN8 is expressed only in the male gametophyte.^4,5,8 How such a cooperation between sporophytic and gametophytic tissues can be mediated by PIN5 and PIN8 and how the lack of the sporophytic PIN5 during pollen development can rescue pollen-specific phenotypes has not been explained by Ding et al.⁶ By combining the analysis of pin5 and pin8 single and double mutants and the PIN5 and PIN8 single and double overexpression lines, an antagonistic/compensatory activity of the two proteins has been suggested.⁶ PIN5 is considered to mediate the auxin transport from the cytosol into the lumen of the ER,⁷ thus an antagonistic action of PIN8 would imply either an active auxin transport activity performed by PIN8 from the ER to the cytosol or a PIN8-mediated negative regulation of the PIN5 activity.

Although both hypotheses could account for the observed antagonistic effects of PIN5 and PIN8 in the double overexpression lines, the compensatory effect of pin5 mutation in pin5pin8 pollen remains to be explained. The fact that the double pin5pin8 mutation resulted in wild type-like pollen instead of causing a stronger phenotype, suggests that other mechanisms regulating auxin homeostasis in pollen could account for the low penetrance of the observed phenotypes in the single mutants.

Recently, a novel putative auxin carrier family has been characterized.⁹ PILS represent a distinct protein family which consists of seven PIN-likes proteins, sharing a similar topology with short loop PINs. The cellular localization of several family members was investigated, including PILS5, which were localized to the ER.⁹ This observation suggests that PILS proteins are involved in the regulation of intracellular auxin homeostasis, like PIN5 and PIN8. We wondered whether some PILSs were expressed in pollen and re-analyzed available pollen transcriptomic data.⁴ The analysis revealed that of the five PILS genes present on the ATH1 Arachip, PILS5 is significantly upregulated in pollen. A further comparison of transcriptomic data^8,10 showed that PILS5 is widely expressed during pollen development, from bicellular to mature pollen⁸ as well as during pollen germination.¹⁰ This expression pattern suggests a potential new player, which besides PIN8, may be involved in the regulation of auxin transport in the male gametophyte. A comparison of the reported phenotypes for PIN8⁵ and PILS5⁹ gain of function mutants revealed common features. Particularly, p35S:PIN8-VEN and p35S:PILS5-GFP plants showed a hyposensitive root growth on auxin supplemented medium and a reduced auxin response, as assayed by DR5::GUS staining and qRT-PCR analysis of auxin induced genes. Both phenotypes are indicative of a negative effect of PIN8 and PILS5 on nuclear auxin signaling. Additionally, root hair specific expression of PILS5 resulted in an inhibitory effect on cell length.⁹ Likewise, root hair specific expression of PIN8 resulted in shorter root hairs while PIN5 expression promoted the root hair length.¹¹ Perhaps, the activity of PILS5 could account for the low penetrance of the observed phenotypes in pin8 pollen. Further studies will unveil if the functions of PILS5 and PIN8 are interconnected, and how their activities at the ER are influencing the pollen functionality.

Acknowledgments

This work was supported by the DFG-SFB 592, the Excellence Initiative of the German Federal and State Governments (EXC 294), Bundesministerium für Forschung und Technik (BMBF, SYSBRA 0315329B), Deutsches Zentrum für Luft und Raumfahrt (DLR 50WB1022), the Freiburg Initiative for Systems Biology (FRISYS) and the European Union Framework 6 Program (AUTOSCREEN, LSHG-CT-2007–037897).