Evolutionarily conserved hierarchical gene regulatory networks for plant salt stress response.

Plant cells constantly alter their gene expression profiles to respond to environmental fluctuations. These continuous adjustments are regulated by multi-hierarchical networks of transcription factors. To understand how such gene regulatory networks (GRNs) have stabilized evolutionarily while allowing for species-specific responses, we compare the GRNs underlying salt response in the early-diverging and late-diverging plants Marchantia polymorpha and Arabidopsis thaliana. Salt-responsive GRNs, constructed on the basis of the temporal transcriptional patterns in the two species, share common trans-regulators but exhibit an evolutionary divergence in cis-regulatory sequences and in the overall network sizes. In both species, WRKY-family transcription factors and their feedback loops serve as central nodes in salt-responsive GRNs. The divergent cis-regulatory sequences of WRKY-target genes are probably associated with the expansion in network size, linking salt stress to tissue-specific developmental and physiological responses. The WRKY modules and highly linked WRKY feedback loops have been preserved widely in other plants, including rice, while keeping their binding-motif sequences mutable. Together, the conserved trans-regulators and the quickly evolving cis-regulatory sequences allow salt-responsive GRNs to adapt over a long evolutionary timescale while maintaining some consistent regulatory structure. This strategy may benefit plants as they adapt to changing environments.

Crosstalk between heterotrimeric G protein-coupled signaling pathways and WRKY transcription factors modulating plant responses to suboptimal micronutrient conditions.

Nutrient stresses induce foliar chlorosis and growth defects. Here we propose heterotrimeric G proteins as signaling mediators of various nutrient stresses, through meta-analyses of >20 transcriptomic data sets associated with nutrient stresses or G protein mutations. Systematic comparison of transcriptomic data yielded 104 genes regulated by G protein subunits under common nutrient stresses: 69 genes under Gß subunit (AGB1) control and 35 genes under Gα subunit (GPA1) control. Quantitative real-time PCR experiments validate that several transcription factors and metal transporters changed in expression level under suboptimal iron, zinc, and/or copper concentrations, while being misregulated in the Arabidopsis Gß-null (agb1) mutant. The agb1 mutant had altered metal ion profiles and exhibited severe growth arrest under zinc stress, and aberrant root waving under iron and zinc stresses, while the Gα-null mutation attenuated leaf chlorosis under iron deficiency in both Arabidopsis and rice. Our transcriptional network analysis inferred computationally that WRKY-family transcription factors mediate the AGB1-dependent nutrient responses. As corroborating evidence of our inference, ectopic expression of WRKY25 or WRKY33 rescued the zinc stress phenotypes and the expression of zinc transporters in the agb1-2 background. These results, together with Gene Ontology analyses, suggest two contrasting roles for G protein-coupled signaling pathways in micronutrient stress responses: one enhancing general stress tolerance and the other modulating ion homeostasis through WRKY transcriptional regulatory networks. In addition, tolerance to iron stress in the rice Gα mutant provides an inroad to improve nutrient stress tolerance of agricultural crops by manipulating G protein signaling.

The RopGEF KARAPPO Is Essential for the Initiation of Vegetative Reproduction in Marchantia polymorpha.

Many plants can reproduce vegetatively, producing clonal progeny from vegetative cells; however, little is known about the molecular mechanisms underlying this process. Liverwort (Marchantia polymorpha), a basal land plant, propagates asexually via gemmae, which are clonal plantlets formed in gemma cups on the dorsal side of the vegetative thallus [1]. The initial stage of gemma development involves elongation and asymmetric divisions of a specific type of epidermal cell, called a gemma initial, which forms on the floor of the gemma cup [2, 3]. To investigate the regulatory mechanism underlying gemma development, we focused on two allelic mutants in which no gemma initial formed; these mutants were named karappo, meaning "empty." We used whole-genome sequencing of both mutants and molecular genetic analysis to identify the causal gene, KARAPPO (KAR), which encodes a ROP guanine nucleotide exchange factor (RopGEF) carrying a plant-specific ROP nucleotide exchanger (PRONE) catalytic domain. In vitro GEF assays showed that the full-length KAR protein and the PRONE domain have significant GEF activity toward MpROP, the only ROP GTPase in M. polymorpha. Moreover, genetic complementation experiments showed a significant role for the N- and C-terminal variable regions in gemma development. Our investigation demonstrates an essential role for KAR/RopGEF in the initiation of plantlet development from a differentiated cell, which may involve cell-polarity formation and subsequent asymmetric cell division via activation of ROP signaling, implying a similar developmental mechanism in vegetative reproduction of various land plants.

Structural Basis for pH-mediated Regulation of F-actin Severing by Gelsolin Domain 1.

Six-domain gelsolin regulates actin structural dynamics through its abilities to sever, cap and uncap F-actin. These activities are modulated by various cellular parameters like Ca2+ and pH. Until now, only the molecular activation mechanism of gelsolin by Ca2+ has been understood relatively well. The fragment comprising the first domain and six residues from the linker region into the second domain has been shown to be similar to the full-length protein in F-actin severing activity in the absence of Ca2+ at pH 5. To understand how this gelsolin fragment is activated for F-actin severing by lowering pH, we solved its NMR structures at both pH 7.3 and 5 in the absence of Ca2+ and measured the pKa values of acidic amino acid residues and histidine residues. The overall structure and dynamics of the fragment are not affected significantly by pH. Nevertheless, local structural changes caused by protonation of His29 and Asp109 result in the activation on lowering the pH, and protonation of His151 directly effects filament binding since it resides in the gelsolin/actin interface. Mutagenesis studies support that His29, Asp109 and His151 play important roles in the pH-dependent severing activity of the gelsolin fragment.

Talin determines the nanoscale architecture of focal adhesions.

Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.