The gynoecium is the most complex floral organ, designed to protect the ovules and ensure their fertilization. Correct patterning and tissue specification in the developing gynoecium involves the concerted action of a host of genetic factors. In addition, apical-basal patterning into different domains, stigma and style, ovary and gynophore, appears to depend on the establishment and maintenance of asymmetric auxin distribution, with an auxin maximum at the apex. Here, we show that a small subfamily of the B3 transcription factor superfamily, the NGATHA (NGA) genes, act redundantly to specify style development in a dosage-dependent manner. Characterization of the NGA gene family is based on an analysis of the activation-tagged mutant named tower-of-pisa1 (top1), which was found to overexpress NGA3. Quadruple nga mutants completely lack style and stigma development. This mutant phenotype is likely caused by a failure to activate two auxin biosynthetic enzymes, YUCCA2 and YUCCA4, in the apical gynoecium domain. The NGA mutant phenotypes are similar to those caused by multiple combinations of mutations in STYLISH1 (STY1) and additional members of its family. NGA3/TOP1 and STY1 share almost identical patterns of expression, but they do not appear to regulate each other at the transcriptional level. Strong synergistic phenotypes are observed when nga3/top1 and sty1 mutants are combined. Furthermore, constitutive expression of both NGA3/TOP1 and STY1 induces the conversion of the ovary into style tissue. Taken together, these data suggest that the NGA and STY factors act cooperatively to promote style specification, in part by directing YUCCA-mediated auxin synthesis in the apical gynoecium domain.

In Arabidopsis, interplay between nuclear auxin perception and trans-cellular polar auxin transport determines the transcriptional auxin response. In brevis radix (brx) mutants, this response is impaired, probably indirectly because of disturbed crosstalk between the auxin and brassinosteroid pathways. Here we provide evidence that BRX protein is plasma membrane-associated, but translocates to the nucleus upon auxin treatment to modulate cellular growth, possibly in conjunction with NGATHA class B3 domain-type transcription factors. Application of the polar auxin transport inhibitor naphthalene phthalamic acid (NPA) resulted in increased BRX abundance at the plasma membrane. Thus, nuclear translocation of BRX could depend on cellular auxin concentration or on auxin flux. Supporting this idea, NPA treatment of wild-type roots phenocopied the brx root meristem phenotype. Moreover, BRX is constitutively turned over by the proteasome pathway in the nucleus. However, a stabilized C-terminal BRX fragment significantly rescued the brx root growth phenotype and triggered a hypocotyl gain-of-function phenotype, similar to strong overexpressors of full length BRX. Therefore, although BRX activity is required in the nucleus, excess activity interferes with normal development. Finally, similar to the PIN-FORMED 1 (PIN1) auxin efflux carrier, BRX is polarly localized in vascular cells and subject to endocytic recycling. Expression of BRX under control of the PIN1 promoter fully rescued the brx short root phenotype, suggesting that the two genes act in the same tissues. Collectively, our results suggest that BRX might provide a contextual readout to synchronize cellular growth with the auxin concentration gradient across the root tip.

The four NGATHA genes (NGA) form a small subfamily within the large family of B3-domain transcription factors of Arabidopsis thaliana. NGA genes act redundantly to direct the development of the apical tissues of the gynoecium, the style, and the stigma. Previous studies indicate that NGA genes could exert this function at least partially by directing the synthesis of auxin at the distal end of the developing gynoecium through the upregulation of two different YUCCA genes, which encode flavin monooxygenases involved in auxin biosynthesis. We have compared three developing pistil transcriptome data sets from wildtype, nga quadruple mutants, and a 35S::NGA3 line. The differentially expressed genes showed a significant enrichment for auxin-related genes, supporting the idea of NGA genes as major regulators of auxin accumulation and distribution within the developing gynoecium. We have introduced reporter lines for several of these differentially expressed genes involved in synthesis, transport and response to auxin in NGA gain- and loss-of-function backgrounds. We present here a detailed map of the response of these reporters to NGA misregulation that could help to clarify the role of NGA in auxin-mediated gynoecium morphogenesis. Our data point to a very reduced auxin synthesis in the developing apical gynoecium of nga mutants, likely responsible for the lack of DR5rev::GFP reporter activity observed in these mutants. In addition, NGA altered activity affects the expression of protein kinases that regulate the cellular localization of auxin efflux regulators, and thus likely impact auxin transport. Finally, protein accumulation in pistils of several ARFs was differentially affected by nga mutations or NGA overexpression, suggesting that these accumulation patterns depend not only on auxin distribution but could be also regulated by transcriptional networks involving NGA factors.

The NGATHA (NGA) clade of transcription factors (TFs) forms a small subfamily of four members in Arabidopsis thaliana. NGA genes act redundantly to direct the development of apical tissues in the gynoecium, where they have been shown to be essential for style and stigma specification. In addition, NGA genes have a more general role in controlling lateral organ growth. The four NGA genes in Arabidopsis are expressed in very similar domains, although little is known about the nature of their putative regulators. Here, we have identified a conserved region within the four NGA promoters that we have used as a bait to screen a yeast library, aiming to identify such NGA regulators. Three members of the TCP family of TFs, named after the founding factors TEOSINTE BRANCHED 1, CYCLOIDEA and PROLIFERATING CELL FACTOR 1 AND 2), were recovered from this screening, of which two [TCP2 and TCP3, members of the CINCINNATA (CIN) family of TCP genes (CIN-TCP) subclade] were shown to activate the NGA3 promoter in planta. We provide evidence that support that CIN-TCP genes are true regulators of NGA gene expression, and that part of the CIN-TCP role in leaf development is mediated by NGA upregulation. Moreover, we have found that this TCP-NGA regulatory interaction is likely conserved in angiosperms, including important crop species, for which the regulation of leaf development is a target for biotechnological improvement.