The LaCLE35 peptide modifies rootlet density and length in cluster roots of white lupin.

White lupin (lupinus albus L.) forms special bottlebrush-like root structures called cluster roots (CR) when phosphorus is low, to remobilise sparingly soluble phosphates in the soil. The molecular mechanisms that control the CR formation remain unknown. Root development in other plants is regulated by CLE  (CLAVATA3/ EMBRYO SURROUNDING REGION (ESR)-RELATED) peptides, which provide more precise control mechanisms than common phytohormones. This makes these peptides interesting candidates to be involved in CR formation, where fine tuning to environmental factors is required. In this study we present an analysis of CLE peptides in white lupin. The peptides LaCLE35 (RGVHy PSGANPLHN) and LaCLE55 (RRVHy PSCHy PDPLHN) reduced root growth and altered CR in hydroponically cultured white lupins. We demonstrate that rootlet density and rootlet length were locally, but not systemically, impaired by exogenously applied CLE35. The peptide was identified in the xylem sap. The inhibitory effect of CLE35 on root growth was attributed to arrested cell elongation in root tips. Taken together, CLE peptides affect both rootlet density and rootlet length, which are two critical factors for CR formation, and may be involved in fine tuning this peculiar root structure that is present in a few crops and many Proteaceae species, under low phosphorus availability.

Nuclear localization of Arabidopsis HD-Zip IV transcription factor GLABRA is driven by Importin α.

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor (TF) from Arabidopsis, is a developmental regulator of specialized cell types in the epidermis. GL2 contains a monopartite nuclear localization sequence (NLS) that is conserved in most HD-Zip IV members across the plants. We demonstrate that NLS mutations affect nuclear transport and result in a loss-of-function phenotypes. NLS fusions to EYFP show that it is sufficient for nuclear localization in roots and trichomes. Despite partial overlap of the NLS with the homeodomain, genetic dissection indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plants followed by MS-based proteomics identified Importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Cytosolic yeast two-hybrid assays and co-immunoprecipitation experiments with recombinant proteins verified NLS-dependent interactions between GL2 and several IMPα isoforms. IMPα triple mutants (impα-1,2,3) exhibit abnormal trichome formation and defects in GL2 nuclear localization in trichomes, consistent tissue-specific and redundant functions of IMPα isoforms. Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 in Arabidopsis, a process that is critical for cell-type differentiation of the epidermis.

Transceptor NRT1.1 and receptor-kinase QSK1 complex controls PM H+-ATPase activity under low nitrate.

NRT1.1, a nitrate transceptor, plays an important role in nitrate binding, sensing, and nitrate-dependent lateral root (LR) morphology. However, little is known about NRT1.1-mediated nitrate signaling transduction through plasma membrane (PM)-localized proteins. Through in-depth phosphoproteome profiling using membranes of Arabidopsis roots, we identified receptor kinase QSK1 and plasma membrane H+-ATPase AHA2 as potential downstream components of NRT1.1 signaling in a mild low-nitrate (LN)-dependent manner. QSK1, as a functional kinase and molecular link, physically interacts with NRT1.1 and AHA2 at LN and specifically phosphorylates AHA2 at S899. Importantly, we found that LN, not high nitrate (HN), induces formation of the NRT1.1-QSK1-AHA2 complex in order to repress the proton efflux into the apoplast by increased phosphorylation of AHA2 at S899. Loss of either NRT1.1 or QSK1 thus results in a higher T947/S899 phosphorylation ratio on AHA2, leading to enhanced pump activity and longer LRs under LN. Our results uncover a regulatory mechanism in which NRT1.1, under LN conditions, promotes coreceptor QSK1 phosphorylation and enhances the NRT1.1-QSK1 complex formation to transduce LN sensing to the PM H+-ATPase AHA2, controlling the phosphorylation ratio of activating and inhibitory phosphorylation sites on AHA2. This then results in altered proton pump activity, apoplast acidification, and regulation of NRT1.1-mediated LR growth.