Phototropin2 3'UTR overlaps with the AT5G58150 gene encoding an inactive RLK kinase.

BACKGROUND: This study examines the biological implications of an overlap between two sequences in the Arabidopsis genome, the 3'UTR of the PHOT2 gene and a putative AT5G58150 gene, encoded on the complementary strand. AT5G58150 is a probably inactive protein kinase that belongs to the transmembrane, leucine-rich repeat receptor-like kinase family. Phot2 is a membrane-bound UV/blue light photoreceptor kinase. Thus, both proteins share their cellular localization, on top of the proximity of their loci. RESULTS: The extent of the overlap between 3'UTR regions of AT5G58150 and PHOT2 was found to be 66 bp, using RACE PCR. Both the at5g58150 T-DNA SALK_093781C (with insertion in the promoter region) and 35S::AT5G58150-GFP lines overexpress the AT5G58150 gene. A detailed analysis did not reveal any substantial impact of PHOT2 or AT5G58150 on their mutual expression levels in different light and osmotic stress conditions. AT5G58150 is a plasma membrane protein, with no apparent kinase activity, as tested on several potential substrates. It appears not to form homodimers and it does not interact with PHOT2. Lines that overexpress AT5G58150 exhibit a greater reduction in lateral root density due to salt and osmotic stress than wild-type plants, which suggests that AT5G58150 may participate in root elongation and formation of lateral roots. In line with this, mass spectrometry analysis identified proteins with ATPase activity, which are involved in proton transport and cell elongation, as putative interactors of AT5G58150. Membrane kinases, including other members of the LRR RLK family and BSK kinases (positive regulators of brassinosteroid signalling), can also act as partners for AT5G58150. CONCLUSIONS: AT5G58150 is a membrane protein that does not exhibit measurable kinase activity, but is involved in signalling through interactions with other proteins. Based on the interactome and root architecture analysis, AT5G58150 may be involved in plant response to salt and osmotic stress and the formation of roots in Arabidopsis.

Arabidopsis Root Development Regulation by the Endogenous Folate Precursor, Para-Aminobenzoic Acid, via Modulation of the Root Cell Cycle.

The continuous growth of roots depends on their ability to maintain a balanced ratio between cell production and cell differentiation at the tip. This process is regulated by the hormonal balance of cytokinin and auxin. However, other important regulators, such as plant folates, also play a regulatory role. In this study, we investigated the impact of the folate precursor para-aminobenzoic acid (PABA) on root development. Using pharmacological, genetic, and imaging approaches, we show that the growth of Arabidopsis thaliana roots is repressed by either supplementing the growth medium with PABA or overexpressing the PABA synthesis gene GAT-ADCS. This is associated with a smaller root meristem consisting of fewer cells. Conversely, reducing the levels of free root endogenous PABA results in longer roots with extended meristems. We provide evidence that PABA represses Arabidopsis root growth in a folate-independent manner and likely acts through two mechanisms: (i) the G2/M transition of cell division in the root apical meristem and (ii) promoting premature cell differentiation in the transition zone. These data collectively suggest that PABA plays a role in Arabidopsis root growth at the intersection between cell division and cell differentiation.

Phototropin Interactions with SUMO Proteins.

The disruption of the sumoylation pathway affects processes controlled by the two phototropins (phots) of Arabidopsis thaliana, phot1 and phot2. Phots, plant UVA/blue light photoreceptors, regulate growth responses and fast movements aimed at optimizing photosynthesis, such as phototropism, chloroplast relocations and stomatal opening. Sumoylation is a posttranslational modification, consisting of the addition of a SUMO (SMALL UBIQUITIN-RELATED MODIFIER) protein to a lysine residue in the target protein. In addition to affecting the stability of proteins, it regulates their activity, interactions and subcellular localization. We examined physiological responses controlled by phots, phototropism and chloroplast movements, in sumoylation pathway mutants. Chloroplast accumulation in response to both continuous and pulse light was enhanced in the E3 ligase siz1 mutant, in a manner dependent on phot2. A significant decrease in phot2 protein abundance was observed in this mutant after blue light treatment both in seedlings and mature leaves. Using plant transient expression and yeast two-hybrid assays, we found that phots interacted with SUMO proteins mainly through their N-terminal parts, which contain the photosensory LOV domains. The covalent modification in phots by SUMO was verified using an Arabidopsis sumoylation system reconstituted in bacteria followed by the mass spectrometry analysis. Lys 297 was identified as the main target of SUMO3 in the phot2 molecule. Finally, sumoylation of phot2 was detected in Arabidopsis mature leaves upon light or heat stress treatment.

Root Gravitropism Is Regulated by a Crosstalk between para-Aminobenzoic Acid, Ethylene, and Auxin.

Plants respond to gravitational force through directional growth along the gravity vector. Although auxin is the central component of the root graviresponse, it works in concert with other plant hormones. Here, we show that the folate precursor para-aminobenzoic acid (PABA) is a key modulator of the auxin-ethylene interplay during root gravitropism in Arabidopsis (Arabidopsis thaliana). In gravistimulated roots, PABA promotes an asymmetric auxin response, which causes the asymmetric growth responsible for root curvature. This activity requires the auxin response transcription factors AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 as well as ethylene biosynthesis and signaling, indicating that PABA activity requires both auxin and ethylene pathways. Similar to ethylene, exogenous PABA reverses the agravitropic root growth of the auxin transport mutant pin-formed2 (pin2) and the auxin biosynthetic double mutant with loss of function of weak ethylene insensitive (wei) genes, wei8wei2, but not the pin2wei8wei2 triple mutant. This finding suggests that PABA regulates the ethylene-dependent reciprocal compensation between auxin transport and biosynthesis. Furthermore, manipulation of endogenous free PABA levels by modulating the expression of the gene encoding its glucosylation enzyme, UDP-GLYCOSYL TRANSFERASE75B1, impacts the root graviresponse, suggesting that endogenous free PABA levels may play a crucial role in modulating the auxin-ethylene cross talk necessary for root gravitropism.

Interplay of the two ancient metabolites auxin and MEcPP regulates adaptive growth.

The ancient morphoregulatory hormone auxin dynamically realigns dedicated cellular processes that shape plant growth under prevailing environmental conditions. However, the nature of the stress-responsive signal altering auxin homeostasis remains elusive. Here we establish that the evolutionarily conserved plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) controls adaptive growth by dual transcriptional and post-translational regulatory inputs that modulate auxin levels and distribution patterns in response to stress. We demonstrate that in vivo accumulation or exogenous application of MEcPP alters the expression of two auxin reporters, DR5:GFP and DII-VENUS, and reduces the abundance of the auxin-efflux carrier PIN-FORMED1 (PIN1) at the plasma membrane. However, pharmacological intervention with clathrin-mediated endocytosis blocks the PIN1 reduction. This study provides insight into the interplay between these two indispensable signaling metabolites by establishing the mode of MEcPP action in altering auxin homeostasis, and as such, positioning plastidial function as the primary driver of adaptive growth.

Characterization of auxin transporter PIN6 plasma membrane targeting reveals a function for PIN6 in plant bolting.

Auxin gradients are sustained by series of influx and efflux carriers whose subcellular localization is sensitive to both exogenous and endogenous factors. Recently the localization of the Arabidopsis thaliana auxin efflux carrier PIN-FORMED (PIN) 6 was reported to be tissue-specific and regulated through unknown mechanisms. Here, we used genetic, molecular and pharmacological approaches to characterize the molecular mechanism(s) controlling the subcellular localization of PIN6. PIN6 localizes to endomembrane domains in tissues with low PIN6 expression levels such as roots, but localizes at the plasma membrane (PM) in tissues with increased PIN6 expression such as the inflorescence stem and nectary glands. We provide evidence that this dual localization is controlled by PIN6 phosphorylation and demonstrate that PIN6 is phosphorylated by mitogen-activated protein kinases (MAPKs) MPK4 and MPK6. The analysis of transgenic plants expressing PIN6 at PM or in endomembrane domains reveals that PIN6 subcellular localization is critical for Arabidopsis inflorescence stem elongation post-flowering (bolting). In line with a role for PIN6 in plant bolting, inflorescence stems elongate faster in pin6 mutant plants than in wild-type plants. We propose that PIN6 subcellular localization is under the control of developmental signals acting on tissue-specific determinants controlling PIN6-expression levels and PIN6 phosphorylation.

Volatile signalling by sesquiterpenes from ectomycorrhizal fungi reprogrammes root architecture.

The mutualistic association of roots with ectomycorrhizal fungi promotes plant health and is a hallmark of boreal and temperate forests worldwide. In the pre-colonization phase, before direct contact, lateral root (LR) production is massively stimulated, yet little is known about the signals exchanged during this step. Here, we identify sesquiterpenes (SQTs) as biologically active agents emitted by Laccaria bicolor while interacting with Populus or Arabidopsis. We show that inhibition of fungal SQT production by lovastatin strongly reduces LR proliferation and that (-)-thujopsene, a low-abundance SQT, is sufficient to stimulate LR formation in the absence of the fungus. Further, we show that the ectomycorrhizal ascomycote, Cenococcum geophilum, which cannot synthesize SQTs, does not promote LRs. We propose that the LR-promoting SQT signal creates a win-win situation by enhancing the root surface area for plant nutrient uptake and by improving fungal access to plant-derived carbon via root exudates.