Cell surface and intracellular auxin signalling for H+ fluxes in root growth.

Growth regulation tailors development in plants to their environment. A prominent example of this is the response to gravity, in which shoots bend up and roots bend down1. This paradox is based on opposite effects of the phytohormone auxin, which promotes cell expansion in shoots while inhibiting it in roots via a yet unknown cellular mechanism2. Here, by combining microfluidics, live imaging, genetic engineering and phosphoproteomics in Arabidopsis thaliana, we advance understanding of how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on rapid regulation of apoplastic pH, a causative determinant of growth. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H+-ATPases for apoplast acidification, while intracellular canonical auxin signalling promotes net cellular H+ influx, causing apoplast alkalinization. Simultaneous activation of these two counteracting mechanisms poises roots for rapid, fine-tuned growth modulation in navigating complex soil environments.

TMK-based cell-surface auxin signalling activates cell-wall acidification.

The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion1. The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H+-ATPase that pumps protons into the apoplast2, yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H+-ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced interactions between TMKs and H+-ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H+-ATPase and are required for auxin-induced H+-ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H+-ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling.

PIF4 enhances the expression of SAUR genes to promote growth in response to nitrate.

Nitrate supply is fundamental to support shoot growth and crop performance, but the associated increase in stem height exacerbates the risks of lodging and yield losses. Despite their significance for agriculture, the mechanisms involved in the promotion of stem growth by nitrate remain poorly understood. Here, we show that the elongation of the hypocotyl of Arabidopsis thaliana, used as a model, responds rapidly and persistently to upshifts in nitrate concentration, rather than to the nitrate level itself. The response occurred even in shoots dissected from their roots and required NITRATE TRANSPORTER 1.1 (NRT1.1) in the phosphorylated state (but not NRT1.1 nitrate transport capacity) and NIN-LIKE PROTEIN 7 (NLP7). Nitrate increased PHYTOCHROME INTERACTING FACTOR 4 (PIF4) nuclear abundance by posttranscriptional mechanisms that depended on NRT1.1 and phytochrome B. In response to nitrate, PIF4 enhanced the expression of numerous SMALL AUXIN-UP RNA (SAUR) genes in the hypocotyl. The growth response to nitrate required PIF4, positive and negative regulators of its activity, including AUXIN RESPONSE FACTORs, and SAURs. PIF4 integrates cues from the soil (nitrate) and aerial (shade) environments adjusting plant stature to facilitate access to light.

SAUR63 stimulates cell growth at the plasma membrane.

In plants, regulated cell expansion determines organ size and shape. Several members of the family of redundantly acting Small Auxin Up RNA (SAUR) proteins can stimulate plasma membrane (PM) H+-ATPase proton pumping activity by inhibiting PM-associated PP2C.D phosphatases, thereby increasing the PM electrochemical potential, acidifying the apoplast, and stimulating cell expansion. Similarly, Arabidopsis thaliana SAUR63 was able to increase growth of various organs, antagonize PP2C.D5 phosphatase, and increase H+-ATPase activity. Using a gain-of-function approach to bypass genetic redundancy, we dissected structural requirements for SAUR63 growth-promoting activity. The divergent N-terminal domain of SAUR63 has a predicted basic amphipathic α-helix and was able to drive partial PM association. Deletion of the N-terminal domain decreased PM association of a SAUR63 fusion protein, as well as decreasing protein level and eliminating growth-promoting activity. Conversely, forced PM association restored ability to promote H+-ATPase activity and cell expansion, indicating that SAUR63 is active when PM-associated. Lipid binding assays and perturbations of PM lipid composition indicate that the N-terminal domain can interact with PM anionic lipids. Mutations in the conserved SAUR domain also reduced PM association in root cells. Thus, both the N-terminal domain and the SAUR domain may cooperatively mediate the SAUR63 PM association required to promote growth.

Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase.

Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.

A plastidial retrograde signal potentiates biosynthesis of systemic stress response activators.

Plants employ an array of intricate and hierarchical signaling cascades to perceive and transduce informational cues to synchronize and tailor adaptive responses. Systemic stress response (SSR) is a recognized complex signaling and response network quintessential to plant's local and distal responses to environmental triggers; however, the identity of the initiating signals has remained fragmented. Here, we show that both biotic (aphids and viral pathogens) and abiotic (high light and wounding) stresses induce accumulation of the plastidial-retrograde-signaling metabolite methylerythritol cyclodiphosphate (MEcPP), leading to reduction of the phytohormone auxin and the subsequent decreased expression of the phosphatase PP2C.D1. This enables phosphorylation of mitogen-activated protein kinases 3/6 and the consequential induction of the downstream events ultimately, resulting in biosynthesis of the two SSR priming metabolites pipecolic acid and N-hydroxy-pipecolic acid. This work identifies plastids as a major initiation site, and the plastidial retrograde signal MEcPP as an initiator of a multicomponent signaling cascade potentiating the biosynthesis of SSR activators, in response to biotic and abiotic triggers.

SAUR proteins and PP2C.D phosphatases regulate H+-ATPases and K+ channels to control stomatal movements.

Activation of plasma membrane (PM) H+-ATPase activity is crucial in guard cells to promote light-stimulated stomatal opening, and in growing organs to promote cell expansion. In growing organs, SMALL AUXIN UP RNA (SAUR) proteins inhibit the PP2C.D2, PP2C.D5, and PP2C.D6 (PP2C.D2/5/6) phosphatases, thereby preventing dephosphorylation of the penultimate phosphothreonine of PM H+-ATPases and trapping them in the activated state to promote cell expansion. To elucidate whether SAUR-PP2C.D regulatory modules also affect reversible cell expansion, we examined stomatal apertures and conductances of Arabidopsis thaliana plants with altered SAUR or PP2C.D activity. Here, we report that the pp2c.d2/5/6 triple knockout mutant plants and plant lines overexpressing SAUR fusion proteins exhibit enhanced stomatal apertures and conductances. Reciprocally, saur56 saur60 double mutants, lacking two SAUR genes normally expressed in guard cells, displayed reduced apertures and conductances, as did plants overexpressing PP2C.D5. Although altered PM H+-ATPase activity contributes to these stomatal phenotypes, voltage clamp analysis showed significant changes also in K+ channel gating in lines with altered SAUR and PP2C.D function. Together, our findings demonstrate that SAUR and PP2C.D proteins act antagonistically to facilitate stomatal movements through a concerted targeting of both ATP-dependent H+ pumping and channel-mediated K+ transport.

BRASSINOSTEROID-SIGNALING KINASE 3, a plasma membrane-associated scaffold protein involved in early brassinosteroid signaling.

Brassinosteroids (BRs) are steroid hormones essential for plant growth and development. The BR signaling pathway has been studied in some detail, however, the functions of the BRASSINOSTEROID-SIGNALING KINASE (BSK) family proteins in the pathway have remained elusive. Through forward genetics, we identified five semi-dominant mutations in the BSK3 gene causing BSK3 loss-of-function and decreased BR responses. We therefore investigated the function of BSK3, a receptor-like cytoplasmic kinase, in BR signaling and plant growth and development. We find that BSK3 is anchored to the plasma membrane via N-myristoylation, which is required for its function in BR signaling. The N-terminal kinase domain is crucial for BSK3 function, and the C-terminal three tandem TPR motifs contribute to BSK3/BSK3 homodimer and BSK3/BSK1 heterodimer formation. Interestingly, the effects of BSK3 on BR responses are dose-dependent, depending on its protein levels. Our genetic studies indicate that kinase dead BSK3K86R protein partially rescues the bsk3-1 mutant phenotypes. BSK3 directly interacts with the BSK family proteins (BSK3 and BSK1), BRI1 receptor kinase, BSU1 phosphatase, and BIN2 kinase. BIN2 phosphorylation of BSK3 enhances BSK3/BSK3 homodimer and BSK3/BSK1 heterodimer formation, BSK3/BRI1 interaction, and BSK3/BSU1 interaction. Furthermore, we find that BSK3 upregulates BSU1 transcript and protein levels to activate BR signaling. BSK3 is broadly expressed and plays an important role in BR-mediated root growth, shoot growth, and organ separation. Together, our findings suggest that BSK3 may function as a scaffold protein to regulate BR signaling. The results of our studies provide new insights into early BR signaling mechanisms.

A subset of plasma membrane-localized PP2C.D phosphatases negatively regulate SAUR-mediated cell expansion in Arabidopsis.

The plant hormone auxin regulates numerous growth and developmental processes throughout the plant life cycle. One major function of auxin in plant growth and development is the regulation of cell expansion. Our previous studies have shown that SMALL AUXIN UP RNA (SAUR) proteins promote auxin-induced cell expansion via an acid growth mechanism. These proteins inhibit the PP2C.D family phosphatases to activate plasma membrane (PM) H+-ATPases and thereby promote cell expansion. However, the functions of individual PP2C.D phosphatases are poorly understood. Here, we investigated PP2C.D-mediated control of cell expansion and other aspects of plant growth and development. The nine PP2C.D family members exhibit distinct subcellular localization patterns. Our genetic findings demonstrate that the three plasma membrane-localized members, PP2C.D2, PP2C.D5, and PP2C.D6, are the major regulators of cell expansion. These phosphatases physically interact with SAUR19 and PM H+-ATPases, and inhibit cell expansion by dephosphorylating the penultimate threonine of PM H+-ATPases. PP2C.D genes are broadly expressed and are crucial for diverse plant growth and developmental processes, including apical hook development, phototropism, and organ growth. GFP-SAUR19 overexpression suppresses the growth defects conferred by PP2C.D5 overexpression, indicating that SAUR proteins antagonize the growth inhibition conferred by the plasma membrane-localized PP2C.D phosphatases. Auxin and high temperature upregulate the expression of some PP2C.D family members, which may provide an additional layer of regulation to prevent plant overgrowth. Our findings provide novel insights into auxin-induced cell expansion, and provide crucial loss-of-function genetic support for SAUR-PP2C.D regulatory modules controlling key aspects of plant growth.

Mutation of a Conserved Motif of PP2C.D Phosphatases Confers SAUR Immunity and Constitutive Activity.

The phytohormone auxin promotes the growth of plant shoots by stimulating cell expansion via plasma membrane (PM) H+-ATPase activation, which facilitates cell wall loosening and solute uptake. Mechanistic insight was recently obtained by demonstrating that auxin-induced SMALL AUXIN UP RNA (SAUR) proteins inhibit D-CLADE TYPE 2C PROTEIN PHOSPHATASE (PP2C.D) activity, thereby trapping PM H+-ATPases in the phosphorylated, activated state, but how SAURs bind PP2C.D proteins and inhibit their activity is unknown. Here, we identified a highly conserved motif near the C-terminal region of the PP2C.D catalytic domain that is required for SAUR binding in Arabidopsis (Arabidopsis thaliana). Missense mutations in this motif abolished SAUR binding but had no apparent effect on catalytic activity. Consequently, mutant PP2C.D proteins that could not bind SAURs exhibited constitutive activity, as they were immune to SAUR inhibition. In planta expression of SAUR-immune pp2c.d2 or pp2c.d5 derivatives conferred severe cell expansion defects and corresponding constitutively low levels of PM H+-ATPase phosphorylation. These growth defects were not alleviated by either auxin treatment or 35S:StrepII-SAUR19 coexpression. In contrast, a PM H+-ATPase gain-of-function mutation that results in a constitutively active H+ pump partially suppressed SAUR-immune pp2c.d5 phenotypes, demonstrating that impaired PM H+-ATPase function is largely responsible for the reduced growth of the SAUR-immune pp2c.d5 mutant. Together, these findings provide crucial genetic support for SAUR-PP2C.D regulation of cell expansion via modulation of PM H+-ATPase activity. Furthermore, SAUR-immune pp2c.d derivatives provide new genetic tools for elucidating SAUR and PP2C.D functions and manipulating plant organ growth.

Constitutive Expression of Arabidopsis SMALL AUXIN UP RNA19 (SAUR19) in Tomato Confers Auxin-Independent Hypocotyl Elongation.

The plant hormone indole-3-acetic acid (IAA or auxin) mediates the elongation growth of shoot tissues by promoting cell expansion. According to the acid growth theory proposed in the 1970s, auxin activates plasma membrane H+-ATPases (PM H+-ATPases) to facilitate cell expansion by both loosening the cell wall through acidification and promoting solute uptake. Mechanistically, however, this process is poorly understood. Recent findings in Arabidopsis (Arabidopsis thaliana) have demonstrated that auxin-induced SMALL AUXIN UP RNA (SAUR) genes promote elongation growth and play a key role in PM H+-ATPase activation by inhibiting PP2C.D family protein phosphatases. Here, we extend these findings by demonstrating that SAUR proteins also inhibit tomato PP2C.D family phosphatases and that AtSAUR19 overexpression in tomato (Solanum lycopersicum) confers the same suite of phenotypes as previously reported for Arabidopsis. Furthermore, we employ a custom image-based method for measuring hypocotyl segment elongation with high resolution and a method for measuring cell wall mechanical properties, to add mechanistic details to the emerging description of auxin-mediated cell expansion. We find that constitutive expression of GFP-AtSAUR19 bypasses the normal requirement of auxin for elongation growth by increasing the mechanical extensibility of excised hypocotyl segments. In contrast, hypocotyl segments overexpressing a PP2C.D phosphatase are specifically impaired in auxin-mediated elongation. The time courses of auxin-induced SAUR expression and auxin-dependent elongation growth were closely correlated. These findings indicate that induction of SAUR expression is sufficient to elicit auxin-mediated expansion growth by activating PM H+-ATPases to facilitate apoplast acidification and mechanical wall loosening.

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis.

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H+-ATPases (PM H+-ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H+-ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H+-ATPase function. We find that SAUR19 stimulates PM H+-ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H+-ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H+-ATPases, and negatively regulate PM H+-ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion.

Proteome Scale-Protein Turnover Analysis Using High Resolution Mass Spectrometric Data from Stable-Isotope Labeled Plants.

Protein turnover is an important aspect of the regulation of cellular processes for organisms when responding to developmental or environmental cues. The measurement of protein turnover in plants, in contrast to that of rapidly growing unicellular organismal cultures, is made more complicated by the high degree of amino acid recycling, resulting in significant transient isotope incorporation distributions that must be dealt with computationally for high throughput analysis to be practical. An algorithm in R, ProteinTurnover, was developed to calculate protein turnover with transient stable isotope incorporation distributions in a high throughput automated manner using high resolution MS and MS/MS proteomic analysis of stable isotopically labeled plant material. ProteinTurnover extracts isotopic distribution information from raw MS data for peptides identified by MS/MS from data sets of either isotopic label dilution or incorporation experiments. Variable isotopic incorporation distributions were modeled using binomial and beta-binomial distributions to deconvolute the natural abundance, newly synthesized/partial-labeled, and fully labeled peptide distributions. Maximum likelihood estimation was performed to calculate the distribution abundance proportion of old and newly synthesized peptides. The half-life or turnover rate of each peptide was calculated from changes in the distribution abundance proportions using nonlinear regression. We applied ProteinTurnover to obtain half-lives of proteins from enriched soluble and membrane fractions from Arabidopsis roots.

Target specificity among canonical nuclear poly(A) polymerases in plants modulates organ growth and pathogen response.

Polyadenylation of pre-mRNAs is critical for efficient nuclear export, stability, and translation of the mature mRNAs, and thus for gene expression. The bulk of pre-mRNAs are processed by canonical nuclear poly(A) polymerase (PAPS). Both vertebrate and higher-plant genomes encode more than one isoform of this enzyme, and these are coexpressed in different tissues. However, in neither case is it known whether the isoforms fulfill different functions or polyadenylate distinct subsets of pre-mRNAs. Here we show that the three canonical nuclear PAPS isoforms in Arabidopsis are functionally specialized owing to their evolutionarily divergent C-terminal domains. A strong loss-of-function mutation in PAPS1 causes a male gametophytic defect, whereas a weak allele leads to reduced leaf growth that results in part from a constitutive pathogen response. By contrast, plants lacking both PAPS2 and PAPS4 function are viable with wild-type leaf growth. Polyadenylation of SMALL AUXIN UP RNA (SAUR) mRNAs depends specifically on PAPS1 function. The resulting reduction in SAUR activity in paps1 mutants contributes to their reduced leaf growth, providing a causal link between polyadenylation of specific pre-mRNAs by a particular PAPS isoform and plant growth. This suggests the existence of an additional layer of regulation in plant and possibly vertebrate gene expression, whereby the relative activities of canonical nuclear PAPS isoforms control de novo synthesized poly(A) tail length and hence expression of specific subsets of mRNAs.

Phytochrome-interacting factor 4 (PIF4) regulates auxin biosynthesis at high temperature.

At high ambient temperature, plants display dramatic stem elongation in an adaptive response to heat. This response is mediated by elevated levels of the phytohormone auxin and requires auxin biosynthesis, signaling, and transport pathways. The mechanisms by which higher temperature results in greater auxin accumulation are unknown, however. A basic helix-loop-helix transcription factor, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), is also required for hypocotyl elongation in response to high temperature. PIF4 also acts redundantly with its homolog, PIF5, to regulate diurnal growth rhythms and elongation responses to the threat of vegetative shade. PIF4 activity is reportedly limited in part by binding to both the basic helix-loop-helix protein LONG HYPOCOTYL IN FAR RED 1 and the DELLA family of growth-repressing proteins. Despite the importance of PIF4 in integrating multiple environmental signals, the mechanisms by which PIF4 controls growth are unknown. Here we demonstrate that PIF4 regulates levels of auxin and the expression of key auxin biosynthesis genes at high temperature. We also identify a family of SMALL AUXIN UP RNA (SAUR) genes that are expressed at high temperature in a PIF4-dependent manner and promote elongation growth. Taken together, our results demonstrate direct molecular links among PIF4, auxin, and elongation growth at high temperature.

Xyloglucan deficiency leads to a reduction in turgor pressure and changes in cell wall properties, affecting early seedling establishment.

Xyloglucan is believed to play a significant role in cell wall mechanics of dicot plants. Surprisingly, Arabidopsis plants defective in xyloglucan biosynthesis exhibit nearly normal growth and development. We investigated a mutant line, cslc-Δ5, lacking activity in all five Arabidopsis cellulose synthase like-C (CSLC) genes responsible for xyloglucan backbone biosynthesis. We observed that this xyloglucan-deficient line exhibited reduced cellulose crystallinity and increased pectin levels, suggesting the existence of feedback mechanisms that regulate wall composition to compensate for the absence of xyloglucan. These alterations in cell wall composition in the xyloglucan-absent plants were further linked to a decrease in cell wall elastic modulus and rupture stress, as observed through atomic force microscopy (AFM) and extensometer-based techniques. This raised questions about how plants with such modified cell wall properties can maintain normal growth. Our investigation revealed two key factors contributing to this phenomenon. First, measurements of turgor pressure, a primary driver of plant growth, revealed that cslc-Δ5 plants have reduced turgor, preventing the compromised walls from bursting while still allowing growth to occur. Second, we discovered the conservation of elastic asymmetry (ratio of axial to transverse wall elasticity) in the mutant, suggesting an additional mechanism contributing to the maintenance of normal growth. This novel feedback mechanism between cell wall composition and mechanical properties, coupled with turgor pressure regulation, plays a central role in the control of plant growth and is critical for seedling establishment in a mechanically challenging environment by affecting shoot emergence and root penetration.

The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion.

The plant hormone auxin controls numerous aspects of plant growth and development by regulating the expression of hundreds of genes. SMALL AUXIN UP RNA (SAUR) genes comprise the largest family of auxin-responsive genes, but their function is unknown. Although prior studies have correlated the expression of some SAUR genes with auxin-mediated cell expansion, genetic evidence implicating SAURs in cell expansion has not been reported. The Arabidopsis SAUR19, SAUR20, SAUR21, SAUR22, SAUR23, and SAUR24 (SAUR19-24) genes encode a subgroup of closely related SAUR proteins. We demonstrate that these SAUR proteins are highly unstable in Arabidopsis. However, the addition of an N-terminal GFP or epitope tag dramatically increases the stability of SAUR proteins. Expression of these stabilized SAUR fusion proteins in Arabidopsis confers numerous auxin-related phenotypes indicative of increased and/or unregulated cell expansion, including increased hypocotyl and leaf size, defective apical hook maintenance, and altered tropic responses. Furthermore, seedlings expressing an artificial microRNA targeting multiple members of the SAUR19-24 subfamily exhibit short hypocotyls and reduced leaf size. Together, these findings demonstrate that SAUR19-24 function as positive effectors of cell expansion. This regulation may be achieved through the modulation of auxin transport, as SAUR gain-of-function and loss-of-function seedlings exhibit increased and reduced basipetal indole-3-acetic acid transport, respectively. Consistent with this possibility, SAUR19-24 proteins predominantly localize to the plasma membrane.

Arabidopsis PIS1 encodes the ABCG37 transporter of auxinic compounds including the auxin precursor indole-3-butyric acid.

Differential distribution of the plant hormone auxin within tissues mediates a variety of developmental processes. Cellular auxin levels are determined by metabolic processes including synthesis, degradation, and (de)conjugation, as well as by auxin transport across the plasma membrane. Whereas transport of free auxins such as naturally occurring indole-3-acetic acid (IAA) is well characterized, little is known about the transport of auxin precursors and metabolites. Here, we identify a mutation in the ABCG37 gene of Arabidopsis that causes the polar auxin transport inhibitor sensitive1 (pis1) phenotype manifested by hypersensitivity to auxinic compounds. ABCG37 encodes the pleiotropic drug resistance transporter that transports a range of synthetic auxinic compounds as well as the endogenous auxin precursor indole-3-butyric acid (IBA), but not free IAA. ABCG37 and its homolog ABCG36 act redundantly at outermost root plasma membranes and, unlike established IAA transporters from the PIN and ABCB families, transport IBA out of the cells. Our findings explore possible novel modes of regulating auxin homeostasis and plant development by means of directional transport of the auxin precursor IBA and presumably also other auxin metabolites.

Biphasic control of cell expansion by auxin coordinates etiolated seedling development.

Seedling emergence is critical for food security. It requires rapid hypocotyl elongation and apical hook formation, both of which are mediated by regulated cell expansion. How these events are coordinated in etiolated seedlings is unclear. Here, we show that biphasic control of cell expansion by the phytohormone auxin underlies this process. Shortly after germination, high auxin levels restrain elongation. This provides a temporal window for apical hook formation, involving a gravity-induced auxin maximum on the eventual concave side of the hook. This auxin maximum induces PP2C.D1 expression, leading to asymmetrical H+-ATPase activity across the hypocotyl that contributes to the differential cell elongation underlying hook development. Subsequently, auxin concentrations decline acropetally and switch from restraining to promoting elongation, thereby driving hypocotyl elongation. Our findings demonstrate how differential auxin concentrations throughout the hypocotyl coordinate etiolated development, leading to successful soil emergence.

Measuring the turnover rates of Arabidopsis proteins using deuterium oxide: an auxin signaling case study.

Rapid environmental responses in plants rely on endogenous signaling mechanisms, which in many cases are mediated by changes in protein turnover rates. It is therefore necessary to develop methods for measuring protein dynamics that monitor large sets of plant proteins to begin to apply a systems biology approach to the study of plant behavior. The use of stable isotope labeling strategies that are adaptable to proteomic methods is particularly attractive for this purpose. Here, we explore one example of such methods that is particularly suitable for plants at the seedling stage, where measurement of amino acid and protein turnover rates is accomplished using a heavy water labeling strategy. The method is backed by microarray evaluation to define its feasibility for specific experimental approaches, and the CULLIN-ASSOCIATED AND NEDDYLATION DISSOCIATED 1 (CAND1) and TRANSPORT INHIBITOR RESPONSE 1 (TIR1) proteins are used to illustrate the potential utility in understanding hormonal signaling regulation. These studies provide insight not only into the potential utility of the method, but also address possible areas of concern regarding the use of heavy water labeling during plant growth. These considerations suggest a prescription for specific experimental designs that minimize interference resulting from the induction of treatment-specific gene expression in the results obtained.