An ancient and conserved function for Armadillo-related proteins in the control of spore and seed germination by abscisic acid.

Armadillo-related proteins regulate development throughout eukaryotic kingdoms. In the flowering plant Arabidopsis thaliana, Armadillo-related ARABIDILLO proteins promote multicellular root branching. ARABIDILLO homologues exist throughout land plants, including early-diverging species lacking true roots, suggesting that early-evolving ARABIDILLOs had additional biological roles. Here we investigated, using molecular genetics, the conservation and diversification of ARABIDILLO protein function in plants separated by c. 450 million years of evolution. We demonstrate that ARABIDILLO homologues in the moss Physcomitrella patens regulate a previously undiscovered inhibitory effect of abscisic acid (ABA) on spore germination. Furthermore, we show that A. thaliana ARABIDILLOs function similarly during seed germination. Early-diverging ARABIDILLO homologues from both P. patens and the lycophyte Selaginella moellendorffii can substitute for ARABIDILLO function during A. thaliana root development and seed germination. We conclude that (1) ABA was co-opted early in plant evolution to regulate functionally analogous processes in spore- and seed-producing plants and (2) plant ARABIDILLO germination functions were co-opted early into both gametophyte and sporophyte, with a specific rooting function evolving later in the land plant lineage.

The decision to germinate is regulated by divergent molecular networks in spores and seeds.

Dispersal is a key step in land plant life cycles, usually via formation of spores or seeds. Regulation of spore- or seed-germination allows control over the timing of transition from one generation to the next, enabling plant dispersal. A combination of environmental and genetic factors determines when seed germination occurs. Endogenous hormones mediate this decision in response to the environment. Less is known about how spore germination is controlled in earlier-evolving nonseed plants. Here, we present an in-depth analysis of the environmental and hormonal regulation of spore germination in the model bryophyte Physcomitrella patens (Aphanoregma patens). Our data suggest that the environmental signals regulating germination are conserved, but also that downstream hormone integration pathways mediating these responses in seeds were acquired after the evolution of the bryophyte lineage. Moreover, the role of abscisic acid and diterpenes (gibberellins) in germination assumed much greater importance as land plant evolution progressed. We conclude that the endogenous hormone signalling networks mediating germination in response to the environment may have evolved independently in spores and seeds. This paves the way for future research about how the mechanisms of plant dispersal on land evolved.

AtMYB93 is a novel negative regulator of lateral root development in Arabidopsis.

Plant root system plasticity is critical for survival in changing environmental conditions. One important aspect of root architecture is lateral root development, a complex process regulated by hormone, environmental and protein signalling pathways. Here we show, using molecular genetic approaches, that the MYB transcription factor AtMYB93 is a novel negative regulator of lateral root development in Arabidopsis. We identify AtMYB93 as an interaction partner of the lateral-root-promoting ARABIDILLO proteins. Atmyb93 mutants have faster lateral root developmental progression and enhanced lateral root densities, while AtMYB93-overexpressing lines display the opposite phenotype. AtMYB93 is expressed strongly, specifically and transiently in the endodermal cells overlying early lateral root primordia and is additionally induced by auxin in the basal meristem of the primary root. Furthermore, Atmyb93 mutant lateral root development is insensitive to auxin, indicating that AtMYB93 is required for normal auxin responses during lateral root development. We propose that AtMYB93 is part of a novel auxin-induced negative feedback loop stimulated in a select few endodermal cells early during lateral root development, ensuring that lateral roots only develop when absolutely required. Putative AtMYB93 homologues are detected throughout flowering plants and represent promising targets for manipulating root systems in diverse crop species.

ARABIDILLO gene homologues in basal land plants: species-specific gene duplication and likely functional redundancy.

ARABIDILLO proteins regulate multicellular root development in Arabidopsis thaliana. Conserved ARABIDILLO homologues are present throughout land plants, even in early-evolving plants that do not possess complex root architecture, suggesting that ARABIDILLO genes have additional functions. Here, we have cloned and characterised ARABIDILLO gene homologues from two early-evolving land plants, the bryophyte Physcomitrella patens and the lycophyte Selaginella moellendorffii. We show that two of the PHYSCODILLO genes (PHYSCODILLO1A and -1B) exist as a tail-to-tail tandem array of two almost identical 12 kb sequences, while a third related gene (PHYSCODILLO2) is located elsewhere in the Physcomitrella genome. Physcomitrella possesses a very low percentage of tandemly arrayed genes compared with the later-evolving plants whose genomes have been sequenced to date. Thus, PHYSCODILLO1A and -1B genes represent a relatively unusual gene arrangement. PHYSCODILLO promoters are active largely in the haploid gametophyte, with additional activity at the foot of the sporophyte. The pattern of promoter activity is uniform in filamentous and leafy tissues, suggesting pleiotropic gene functions and likely functional redundancy: the latter possibility is confirmed by the lack of discernible phenotype in a physcodillo2 deletion mutant. Interestingly, the pattern of PHYSCODILLO promoter activity in female reproductive organs is strikingly similar to that of an Arabidopsis homologue, suggesting co-option of some PHYSCODILLO functions or regulation into both the sporophyte and gametophyte. In conclusion, our work identifies and characterises some of the earliest-evolving land plant ARABIDILLO homologues. We confirm that all land plant ARABIDILLO genes arose from a single common ancestor and suggest that PHYSCODILLO proteins have novel and pleiotropic functions, some of which may be conserved in later-evolving plants.

ARABIDILLO proteins have a novel and conserved domain structure important for the regulation of their stability.

ARABIDILLO proteins are F-box-Armadillo (ARM) proteins that regulate root branching in Arabidopsis. Many F-box proteins in plants, yeast and mammals are unstable. In plants, the mechanism for this instability has not been fully investigated. Here, we show that a conserved family of plant ARABIDILLO-related proteins has a unique domain structure consisting of an F-box and leucine-rich repeats (LRRs) followed by ARM-repeats. The LRRs are similar to those found in other plant and animal F-box proteins, including cell cycle proteins and hormone receptors. We demonstrate that the LRRs are required for ARABIDILLO1 function in vivo. ARABIDILLO1 protein is unstable: we show that ARABIDILLO1 protein is associated with ubiquitin and is turned over by the proteasome. Both the F-box and LRR regions of ARABIDILLO1 appear to enable this turnover to occur. Application of known lateral root-regulating signals has no effect on ARABIDILLO1 stability. In addition, plants that lack or overexpress ARABIDILLO proteins respond normally to known lateral root-regulating signals. Thus, we suggest that the signal(s) regulating ARABIDILLO stability in vivo may be either highly specific or novel. The structural conservation between ARABIDILLOs and other plant and animal F-box proteins suggests that the stability of other F-box proteins may be controlled by similar mechanisms.