Functionally different PIN proteins control auxin flux during bulbil development in Agave tequilana

Abstract

In Agave tequilana, reproductive failure or inadequate flower development stimulates the formation of vegetative bulbils at the bracteoles, ensuring survival in a hostile environment. Little is known about the signals that trigger this probably unique phenomenon in agave species. Here we report that auxin plays a central role in bulbil development and show that the localization of PIN1-related proteins is consistent with altered auxin transport during this process. Analysis of agave transcriptome data led to the identification of the A. tequilana orthologue of PIN1 (denoted AtqPIN1) and a second closely related gene from a distinct clade reported as 'Sister of PIN1' (denoted AtqSoPIN1). Quantitative real-time reverse transcription-PCR (RT-qPCR) analysis showed different patterns of expression for each gene during bulbil formation, and heterologous expression of the A. tequilana PIN1 and SoPIN1 genes in Arabidopsis thaliana confirmed functional differences between these genes. Although no free auxin was detected in induced pedicel samples, changes in the levels of auxin precursors were observed. Taken as a whole, the data support the model that AtqPIN1 and AtqSoPIN1 have co-ordinated but distinct functions in relation to auxin transport during the initial stages of bulbil formation.

Keywords: Agave tequilana; PIN proteins.; auxin flux; bulbil; development; gene expression; immunolocalization.

Fig. 1.

Removal of flower buds leads to formation of vegetative or floral structures at bracteoles in A. tequilana. (A) Unopened floral buds. (B) Pedicel tissue following removal of buds. (C) Vegetative bulbils and floral structures formed at bracteoles. (D–G) Normal flower morphology. (D) Completely normal flower; (E) normal carpelar organization shown in cross-section; (F) normal anther; (G) normal pistil. (H–K) Morphology of floral structures formed at bracteoles. (H) Completely distorted flower; (I) distorted carpelar organization; (J) distorted anther; (K) stigma-like structure. Scales are 2.0mm. (This figure is available in colour at JXB online.)

Fig. 2.

Immunolocalization of agave PIN protein in pedicel and S5 bulbil tissue. (A) Bisected bud before sectioning. (B) Example of pedicel tissue with floral bud removed; the box indicates the region surrounding the bracteoles on which immunolocalization of PIN was carried out; brackets indicate pedicel tissue. (C) Immunolocalization of PIN protein using an anti-PIN1a antibody against maize PIN1a protein, in pedicel tissue where bud has been removed as shown in (B). An asterisk (*) indicates the cut surface. (D) Enlarged image showing PIN location in the lower periphery of cells; circles indicate individual cells outside vascular tissue; arrows indicate PIN localization in individual cells within the vascular tissue. Scale bar in (C) is 100 μm. (E) Light microscopy and (F) PIN immunolocalization images of untreated pedicel tissue (box in B), underlying the region where a floral bud was removed at the S5 stage of bulbil induction, ×20 magnification, scale bar=50 μm. (This figure is available in colour at JXB online.)

Fig. 3.

Exogenous application of NPA and IAA to the cut surface of pedicel tissue following removal of floral buds. (A–C) Agarve tequilana 10 d after bud removal. (A) Lanolin-only control; (B) NPA; (C) IAA. (D–F) Agarve desmettiana 10 d after bud removal. (D) Lanolin-only control; (E) NPA; (F) IAA. Meristems are indicated with arrows. Bars are 0.5cm. (This figure is available in colour at JXB online.)

Fig. 4.

Dendrogram showing the relationship between PIN1 amino acid sequences from monocotyledonous and dicotyledonous species and A. tequilana. The agave sequences are boxed. PIN1 and ‘Sister of PIN1’ (SoPIN1) clades containing dicotyledonous or monocotyledonous species are indicated.

Fig. 5.

RT-qPCR expression profiles of agave PIN1 genes in pedicel tissue at different stages of bulbil formation in the presence or absence of NPA or IAA. (A and C) AtqPIN1. (B and D) AtqSoPIN1. S0–S5, stages of bulbil formation; C-, application of lanolin-only control; N-, application of NPA; I-, application of IAA.

Fig. 6.

Quantification of free and conjugated forms of IAA and IAA precursors (TRP and TRA) by UHPLC-Q-TOF MS in different stages of bulbil formation in A. tequilana. S0–S5: different stages of bulbil formation; DW, dry weight. Leaf and root samples are included as controls.

Fig. 7.

Heterologous expression of A. tequilana PIN1 and SoPIN1 genes in A. thaliana. (A) Gravitropic responses of DR5::GUS A. thaliana seedlings expressing AtqPIN1 or AtqSoPIN1 under control of the 35S promoter and DR5::GUS control. (B) Auxin localization based on GUS-stained tissues of DR5::GUS A. thaliana plants expressing AtqPIN1 or AtqSoPIN1 under control of the 35S promoter and DR5::GUS control. (This figure is available in colour at JXB online.)

Fig. 8.

Model for the role of PIN1-mediated auxin flux in induction of secondary structures at the bracteoles in A. tequilana following removal of floral buds. (A) Auxin suppresses secondary structure formation. (a) A normal auxin gradient from the flower bud suppresses formation of secondary structures at the bracteoles. (b) Under natural light conditions, removal of flower buds leads to a drop in auxin flux in pedicel vascular tissue, stimulating the development of new meristems and vegetative bulbils at the bracteoles. (c) Under reduced light conditions, removal of flower buds leads to a drop in auxin flux in pedicel vascular tissue, stimulating the development of a mixture of determinate floral meristems producing non-viable floral structures and indeterminate meristems producing vegetative bulbils at the bracteoles. (B) Putative role of agave PIN1 proteins in bulbil formation. (a) Polarized AtqPIN1 is present in vascular tissue, leading to a downward auxin flux from the flower bud. (b) Removal of the flower bud interrupts the auxin flux. AtqPIN1 expression decreases and AtqSoPIN1 expression increases, allowing auxin to accumulate close to the bracteoles where new meristems will form (dots). (c) AtqSoPIN1 allows accumulation of auxin at the SAM of developing bulbil meristems and AtqPIN1 allows downward auxin flux and formation of vascular tissue. (This figure is available in colour at JXB online.)