Andrea L. Skirpan

Auxin plays a fundamental role in organogenesis in plants. Multiple pathways for auxin biosynthesis have been proposed, but none of the predicted pathways are completely understood. Here, we report the positional cloning and characterization of the vanishing tassel2 (vt2) gene of maize (Zea mays). Phylogenetic analyses indicate that vt2 is a co-ortholog of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1), which converts Trp to indole-3-pyruvic acid in one of four hypothesized Trp-dependent auxin biosynthesis pathways. Unlike single mutations in TAA1, which cause subtle morphological phenotypes in Arabidopsis thaliana, vt2 mutants have dramatic effects on vegetative and reproductive development. vt2 mutants share many similarities with sparse inflorescence1 (spi1) mutants in maize. spi1 is proposed to encode an enzyme in the tryptamine pathway for Trp-dependent auxin biosynthesis, although this biochemical activity has recently been questioned. Surprisingly, spi1 vt2 double mutants had only a slightly more severe phenotype than vt2 single mutants. Furthermore, both spi1 and vt2 single mutants exhibited a reduction in free auxin levels, but the spi1 vt2 double mutants did not have a further reduction compared with vt2 single mutants. Therefore, both spi1 and vt2 function in auxin biosynthesis in maize, possibly in the same pathway rather than independently as previously proposed.

Although pollen tube growth is essential for plant fertilization and reproductive success, the regulators of the actin-related growth machinery and the cytosolic Ca2+ gradient thought to determine how these cells elongate remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators; however, the relevant phospholipase(s) have not been characterized. Therefore, we cloned cDNA for a pollen-expressed phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-cleaving phospholipase C (PLC) from Petunia inflata, named Pet PLC1. Expressing a catalytically inactive form of Pet PLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton, and delocalization of growth at the tube tip. These phenotypes were suppressed by depolymerizing actin with low concentrations of latrunculin B, suggesting that a critical site of action of Pet PLC1 is in regulating actin structure at the growing tip. A green fluorescent protein (GFP) fusion to Pet PLC1 caused enrichment in regions of the apical plasma membrane not undergoing rapid expansion, whereas a GFP fusion to the PtdInsP2 binding domain of mammalian PLC delta1 caused enrichment in apical regions depleted in PLC. Thus, Pet PLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell.

Organogenesis in plants is controlled by meristems. Axillary meristems, which give rise to branches and flowers, play a critical role in plant architecture and reproduction. Maize (Zea mays) and rice (Oryza sativa) have additional types of axillary meristems in the inflorescence compared to Arabidopsis (Arabidopsis thaliana) and thus provide an excellent model system to study axillary meristem initiation. Previously, we characterized the barren inflorescence2 (bif2) mutant in maize and showed that bif2 plays a key role in axillary meristem and lateral primordia initiation in the inflorescence. In this article, we cloned bif2 by transposon tagging. Isolation of bif2-like genes from seven other grasses, along with phylogenetic analysis, showed that bif2 is a co-ortholog of PINOID (PID), which regulates auxin transport in Arabidopsis. Expression analysis showed that bif2 is expressed in all axillary meristems and lateral primordia during inflorescence and vegetative development in maize and rice. Further phenotypic analysis of bif2 mutants in maize illustrates additional roles of bif2 during vegetative development. We propose that bif2/PID sequence and expression are conserved between grasses and Arabidopsis, attesting to the important role they play in development. We provide further support that bif2, and by analogy PID, is required for initiation of both axillary meristems and lateral primordia.

Polar auxin transport, mediated by the PIN-FORMED (PIN) class of auxin efflux carriers, controls organ initiation in plants. In maize, BARREN INFLORESCENCE2 (BIF2) encodes a serine/threonine protein kinase co-orthologous to PINOID (PID), which regulates the subcellular localization of AtPIN1 in Arabidopsis. We show that BIF2 phosphorylates ZmPIN1a, a maize homolog of AtPIN1, in vitro and regulates ZmPIN1a subcellular localization in vivo, similar to the role of PID in Arabidopsis. In addition, bif2 mutant inflorescences have lower auxin levels later in development. We propose that BIF2 regulates auxin transport through direct regulation of ZmPIN1a during maize inflorescence development.