Pleiotropic and nonredundant effects of an auxin importer in Setaria and maize.

Directional transport of auxin is critical for inflorescence and floral development in flowering plants, but the role of auxin influx carriers (AUX1 proteins) has been largely overlooked. Taking advantage of available AUX1 mutants in green millet (Setaria viridis) and maize (Zea mays), we uncover previously unreported aspects of plant development that are affected by auxin influx, including higher order branches in the inflorescence, stigma branch number, glume (floral bract) development, and plant fertility. However, disruption of auxin flux does not affect all parts of the plant, with little obvious effect on inflorescence meristem size, time to flowering, and anther morphology. In double mutant studies in maize, disruptions of ZmAUX1 also affect vegetative development. A green fluorescent protein (GFP)-tagged construct of the Setaria AUX1 protein Sparse Panicle1 (SPP1) under its native promoter showed that SPP1 localizes to the plasma membrane of outer tissue layers in both roots and inflorescences, and accumulates specifically in inflorescence branch meristems, consistent with the mutant phenotype and expected auxin maxima. RNA-seq analysis indicated that most gene expression modules are conserved between mutant and wild-type plants, with only a few hundred genes differentially expressed in spp1 inflorescences. Using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, we disrupted SPP1 and the other four AUX1 homologs in S. viridis. SPP1 has a larger effect on inflorescence development than the others, although all contribute to plant height, tiller formation, and leaf and root development. The AUX1 importers are thus not fully redundant in S. viridis. Our detailed phenotypic characterization plus a stable GFP-tagged line offer tools for future dissection of the function of auxin influx proteins.

ROS-mediated plasmodesmal regulation requires a network of an Arabidopsis receptor-like kinase, calmodulin-like proteins, and callose synthases.

Plasmodesmata (PD) play a critical role in symplasmic communication, coordinating plant activities related to growth & development, and environmental stress responses. Most developmental and environmental stress signals induce reactive oxygen species (ROS)-mediated signaling in the apoplast that causes PD closure by callose deposition. Although the apoplastic ROS signals are primarily perceived at the plasma membrane (PM) by receptor-like kinases (RLKs), such components involved in PD regulation are not yet known. Here, we show that an Arabidopsis NOVEL CYS-RICH RECEPTOR KINASE (NCRK), a PD-localized protein, is required for plasmodesmal callose deposition in response to ROS stress. We identified the involvement of NCRK in callose accumulation at PD channels in either basal level or ROS-dependent manner. Loss-of-function mutant (ncrk) of NCRK induces impaired callose accumulation at the PD under the ROS stress resembling a phenotype of the PD-regulating GLUCAN SYNTHASE-LIKE 4 (gsl4) knock-out plant. The overexpression of transgenic NCRK can complement the callose and the PD permeability phenotypes of ncrk mutants but not kinase-inactive NCRK variants or Cys-mutant NCRK, in which Cys residues were mutated in Cys-rich repeat ectodomain. Interestingly, NCRK mediates plasmodesmal permeability in mechanical injury-mediated signaling pathways regulated by GSL4. Furthermore, we show that NCRK interacts with calmodulin-like protein 41 (CML41) and GSL4 in response to ROS stress. Altogether, our data indicate that NCRK functions as an upstream regulator of PD callose accumulation in response to ROS-mediated stress signaling pathways.