The endoplasmic reticulum localized PIN8 is a pollen‐specific auxin carrier involved in intracellular auxin homeostasis

Abstract

SUMMARYThe plant hormone auxin is a mobile signal which affects nuclear transcription by regulating the stability of auxin/indole-3-acetic acid (IAA) repressor proteins. Auxin is transported polarly from cell to cell by auxin efflux proteins of the PIN family, but it is not as yet clear how auxin levels are regulated within cells and how access of auxin to the nucleus may be controlled. The Arabidopsis genome contains eight PINs, encoding proteins with a similar membrane topology. While five of the PINs are typically targeted polarly to the plasma membranes, the smallest members of the family, PIN5 and PIN8, seem to be located not at the plasma membrane but in endomembranes. Here we demonstrate by electron microscopy analysis that PIN8, which is specifically expressed in pollen, resides in the endoplasmic reticulum and that it remains internally localized during pollen tube growth. Transgenic Arabidopsis and tobacco plants were generated overexpressing or ectopically expressing functional PIN8, and its role in control of auxin homeostasis was studied. PIN8 ectopic expression resulted in strong auxin-related phenotypes. The severity of phenotypes depended on PIN8 protein levels, suggesting a rate-limiting activity for PIN8. The observed phenotypes correlated with elevated levels of free IAA and ester-conjugated IAA. Activation of the auxin-regulated synthetic DR5 promoter and of auxin response genes was strongly repressed in seedlings overexpressing PIN8 when exposed to 1-naphthalene acetic acid. Thus, our data show a functional role for endoplasmic reticulum-localized PIN8 and suggest a mechanism whereby PIN8 controls auxin thresholds and access of auxin to the nucleus, thereby regulating auxindependent transcriptional activity.

Comprehensive Tissue-Specific Transcriptome Analysis Reveals Distinct Regulatory Programs during Early Tomato Fruit Development

“…The influx carrier LIKE-AUX2 and the plasma membrane-targeted efflux carriers PIN1, PIN3, PIN4, PIN7, and PIN9 displayed the highest expression, which peaked in either the funiculus or placenta at 4 DPA. In contrast, PIN transporters thought to localize to the endomembrane system (Mravec et al, 2009;Dal Bosco et al, 2012) showed a divergent expression profile. Specifically, PIN5 was highly expressed in endosperm, whereas PILS2 and PILS5, PIN-LIKE proteins found in the endoplasmic reticulum in Arabidopsis (Barbez et al, 2012), showed highest expression in the pericarp of 4-DPA fruit (Fig.…”

Regulation of Auxin Homeostasis and Gradients in Arabidopsis Roots through the Formation of the Indole-3-Acetic Acid Catabolite 2-Oxindole-3-Acetic Acid

“…The AUX1/LAX and some PIN proteins are auxin influx and efflux carriers, respectively, and the polar localization of these transporters in specific cells is critical for differential auxin distribution during plant development. PIN5, PIN6, and PIN8 and the newly discovered PILS proteins are believed to be involved in the regulation of intracellular auxin homeostasis (Mravec et al, 2009;Barbez et al, 2012;Dal Bosco et al, 2012;Ding et al, 2012). The ATP-dependent ABCB transporters do not show the same polarized localization in the cells, but data indicate that they are likely to be important both for long-distance auxin transport and for local auxin gradient and auxin maxima/minima formation (Zazímalová et al, 2010).…”

Intracellular auxin transport in pollen

“…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 PIN8 5 and PILS5 9 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.…”

“…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.…”

“…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.…”