Single-cell-based system to monitor carrier driven cellular auxin homeostasis

Background: Abundance and distribution of the plant hormone auxin play important roles in plant development. Besides other metabolic processes, various auxin carriers control the cellular level of active auxin and, hence, are major regulators of cellular auxin homeostasis. Despite the developmental importance of auxin transporters, a simple medium-to-high throughput approach to assess carrier activities is still missing. Here we show that carrier driven depletion of cellular auxin correlates with reduced nuclear auxin signaling in tobacco Bright Yellow-2 (BY-2) cell cultures. Results: We developed an easy to use transient single-cell-based system to detect carrier activity. We use the relative changes in signaling output of the auxin responsive promoter element DR5 to indirectly visualize auxin carrier activity. The feasibility of the transient approach was demonstrated by pharmacological and genetic interference with auxin signaling and transport. As a proof of concept, we provide visual evidence that the prominent auxin transport proteins PIN-FORMED (PIN) 2 and PIN5 regulate cellular auxin homeostasis at the plasma membrane and endoplasmic reticulum (ER), respectively. Our data suggest that PIN2 and PIN5 have different sensitivities to the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Also the putative PIN-LIKES (PILS) auxin carrier activity at the ER is insensitive to NPA in our system, indicating that NPA blocks intercellular, but not intracellular auxin transport. Conclusions: This single-cell-based system is a useful tool by which the activity of putative auxin carriers, such as PINs, PILS and WALLS ARE THIN1 (WAT1), can be indirectly visualized in a medium-to-high throughput manner. Moreover, our single cell system might be useful to investigate also other hormonal signaling pathways, such as cytokinin

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

The PIN-FORMED (PIN) protein family of auxin transporters

The PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies

Cadmium and arsenic affect root development in Oryza sativa L. negatively interacting with auxin

Cadmium (Cd) and arsenic (As), non essential, but toxic, elements for animals and plants are frequently present in paddy fields. Oryza sativa L., a staple food for at least the half of world population, easily absorbs As and Cd by the root, and in this organ the pollutants evoke consistent damages, reducing/modifying the root system. Auxins are key hormones in regulating all developmental processes, including root organogenesis. Moreover, plants respond to environmental stresses, such as those caused by Cd and As, by changing levels and distribution of endogenous phytohormones. Even though the effects of Cd and As on the roots have been investigated in some species, it remains necessary to deepen the knowledge about the cross-talk between these toxic elements and auxin during root formation and development, in particular in agronomically important plants, such as rice. Hence, the research goal was to investigate the interactions between Cd and As, alone or combined, and auxin during the development of rice roots. To reach the aim, morphological, histological and histochemical analyses were carried out on seedlings, exposed or not to Cd and/or As, belonging to the wild type and transgenic lines useful for monitoring indole-3-acetic acid (IAA) localization, i.e., OsDR5:GUS, and IAA cellular influx and efflux, i.e., OsAUX1:GUS and OsPIN5b:GUS. Moreover, the transcript levels of the YUCCA2 and ASA2, IAA biosynthetic genes were also monitored in Cd and/or As exposed wild type seedlings. The results highlight that As and Cd affect cyto-histology and morphology of the roots. In particular, they alter the lateral root primordia organization and development with negative consequences on root system architecture. This is due to a disturbance of IAA biosynthesis and transport, as indicated by the altered expression of both ASA2 and YUCCA2 biosynthetic genes, and AUX1 and PIN5b transporter genes

Perturbation of indole-3-butyric acid homeostasis by the UDP-glucosyltransferase UGT74E2 modulates Arabidopsis architecture and water stress tolerance

Reactive oxygen species and redox signaling undergo synergistic and antagonistic interactions with phytohormones to regulate protective responses of plants against biotic and abiotic stresses. However, molecular insight into the nature of this crosstalk remains scarce. We demonstrate that the hydrogen peroxide–responsive UDP-glucosyltransferase UGT74E2 of Arabidopsis thaliana is involved in the modulation of plant architecture and water stress response through its activity toward the auxin indole-3-butyric acid (IBA). Biochemical characterization of recombinant UGT74E2 demonstrated that it strongly favors IBA as a substrate. Assessment of indole-3-acetic acid (IAA), IBA, and their conjugates in transgenic plants ectopically expressing UGT74E2 indicated that the catalytic specificity was maintained in planta. In these transgenic plants, not only were IBA-Glc concentrations increased, but also free IBA levels were elevated and the conjugated IAA pattern was modified. This perturbed IBA and IAA homeostasis was associated with architectural changes, including increased shoot branching and altered rosette shape, and resulted in significantly improved survival during drought and salt stress treatments. Hence, our results reveal that IBA and IBA-Glc are important regulators of morphological and physiological stress adaptation mechanisms and provide molecular evidence for the interplay between hydrogen peroxide and auxin homeostasis through the action of an IBA UGT

Role of the Arabidopsis PIN6 auxin transporter in auxin homeostasis and auxin-mediated development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.Christopher I. Cazzonelli, Marleen Vanstraelen, Sibu Simon, Kuide Yin, Ashley Carron-Arthur, Nazia Nisar, Gauri Tarle, Abby J. Cuttriss¤, Iain R. Searle, Eva Benkova, Ulrike Mathesius, Josette Masle, Jiří Friml, Barry J. Pogso

NHX antiporters regulate the pH of endoplasmic reticulum and auxin-mediated development

AtNHX5 and AtNHX6 are endosomal Na+,K+/H+ antiporters that are critical for growth and development in Arabidopsis, but the mechanism behind their action remains unknown. Here, we report that AtNHX5 and AtNHX6, functioning as H+ leak, control auxin homeostasis and auxin-mediated development. We found that nhx5 nhx6 exhibited growth variations of auxin-related defects. We further showed that nhx5 nhx6 was affected in auxin homeostasis. Genetic analysis showed that AtNHX5 and AtNHX6 were required for the function of the ER-localized auxin transporter PIN5. Although AtNHX5 and AtNHX6 were co-localized with PIN5 at ER, they did not interact directly. Instead, the conserved acidic residues in AtNHX5 and AtNHX6, which are essential for exchange activity, were required for PIN5 function. AtNHX5 and AtNHX6 regulated the pH in ER. Overall, AtNHX5 and AtNHX6 may regulate auxin transport across the ER via the pH gradient created by their transport activity. H+-leak pathway provides a fine-tuning mechanism that controls cellular auxin fluxes

Regulation of Local Auxin Metabolism during Soybean Nodule Development

Legume-rhizobia symbiosis leads to the development of secondary root organs called nodules. Rhizobia bacteria housed inside nodules assimilate atmospheric nitrogen and convert them into plant usable forms thereby reducing the need for fertilizer application in crop legumes like soybean. Nodule development is a coordinated process orchestrated by multiple plant hormones. In soybean, the auxin responsive gene expression was detected in nodule primordia and in the periphery of mature nodules, primarily in nodule vasculature. Auxin hypersensitivity reduces nodule formation in soybean and also polar auxin transport inhibition at the site of nodule development is not crucial for determinate nodule formation. Therefore, auxin distribution and sensitivity appear to be crucial for proper nodule development. However, the role of auxin metabolism in nodule development is unclear. Using global gene expression analysis, we have identified genes involved in the auxin metabolism that are specifically expressed in nodule tissues at early and mature stages. A cytochrome P450 oxidase gene, GmCYP83B1 was preferentially enriched in mature nodules and it was also identified to be a close ortholog of AtCYP83B1. Suppression of GmCYP83B1 expression through RNA interference (GmCYP83B1-RNAi) in soybean roots led to a significant reduction in nodule number and altered mature nodule morphology. Auxin accumulation was significantly higher in GmCYP83B1-RNAi nodules compared to vector control which suggested that suppression of GmCYP83B1 led to auxin accumulation which might have led to reduced nodule organogenesis and altered nodule development. Using the global gene expression data, we also identified three nodule-enriched genes encoding GRETCHEN HAGEN 3 (GH3) enzymes. Biochemical assays showed that the three GmGH3 enzymes can conjugate IAA with Asp for inactivation of free auxin levels. GmGH3-15 showed a broad substrate preference, especially with different forms of auxin. We hypothesized that these GH3s might maintain auxin homeostasis in soybean nodules. Promoter:GUS expression analysis indicated that GmGH3-14 acts primarily in the root epidermis and the nodule primordium where as GmGH3-15 might act in the vasculature. Silencing the expression of these GH3 genes in soybean composite plants led to altered nodule numbers, maturity, and size. Our results indicate that these GH3s are needed for proper nodule maturation in soybean, but the precise mechanism by which they regulate nodule development remains to be explained. Overall the results suggest that GmCYP83B1 and GmGH3 might act to regulate local auxin levels to direct proper soybean nodule development