Acid Growth Theory Research Articles

Bending to auxin: fast acid growth for tropisms.

The phytohormone auxin is the major growth regulator governing tropic responses including gravitropism. Auxin build-up at the lower side of stimulated shoots promotes cell expansion, whereas in roots it inhibits growth, leading to upward shoot bending and downward root bending, respectively. Yet it remains an enigma how the same signal can trigger such opposite cellular responses. In this review, we discuss several recent unexpected insights into the mechanisms underlying auxin regulation of growth, challenging several existing models. We focus on the divergent mechanisms of apoplastic pH regulation in shoots and roots revisiting the classical Acid Growth Theory and discuss coordinated involvement of multiple auxin signaling pathways. From this emerges a more comprehensive, updated picture how auxin regulates growth.

TMK: A crucial piece of the acid growth puzzle

Plant cells have relatively high turgor pressure driving cell expansion, which is restricted by the cell wall. How plant cells grow in an intrinsically controlled manner is an intriguing question, and the phytohormone auxin (indole-3-acetic acid, IAA) plays a vital regulatory role. The auxin-mediated acid growth theory was proposed decades ago, whereas the underlying mechanisms were not fully elucidated. Plasma membrane (PM) H+-ATPases, also called Arabidopsis AUTOINHIBITED H+ ATPASEs (AHAs), are proton pumps majorly responsible for regulating cell growth in plants.

Some New Methodological and Conceptual Aspects of the "Acid Growth Theory" for the Auxin Action in Maize (Zea mays L.) Coleoptile Segments: Do Acid- and Auxin-Induced Rapid Growth Differ in Their Mechanisms?

Two arguments against the “acid growth theory” of auxin-induced growth were re-examined. First, the lack of a correlation between the IAA-induced growth and medium acidification, which is mainly due to the cuticle, which is a barrier for proton diffusion. Second, acid- and the IAA-induced growth are additive processes, which means that acid and the IAA act via different mechanisms. Here, growth, medium pH, and membrane potential (in some experiments) were simultaneously measured using non-abraded and non-peeled segments but with the incubation medium having access to their lumen. Using such an approach significantly enhances both the IAA-induced growth and proton extrusion (similar to that of abraded segments). Staining the cuticle on the outer and inner epidermis of the coleoptile segments showed that the cuticle architecture differs on both sides of the segments. The dose-response curves for the IAA-induced growth and proton extrusion were bell-shaped with the maximum at 10−4 M over 10 h. The kinetics of the IAA-induced hyperpolarisation was similar to that of the rapid phase of the IAA-induced growth. It is also proposed that the K+/H+ co-transporters are involved in acid-induced growth and that the combined effect of the K+ channels and K+/ H+ co-transporters is responsible for the IAA-induced growth. These findings support the “acid growth theory” of auxin action.

Rapid Auxin-Mediated Cell Expansion.

The promotive effect of auxin on shoot cell expansion provided the bioassay used to isolate this central plant hormone nearly a century ago. While the mechanisms underlying auxin perception and signaling to regulate transcription have largely been elucidated, how auxin controls cell expansion is only now attaining molecular-level definition. The good news is that the decades-old acid growth theory invoking plasma membrane H+-ATPase activation is still useful. The better news is that a mechanistic framework has emerged, wherein Small Auxin Up RNA (SAUR) proteins regulate protein phosphatases to control H+-ATPase activity. In this review, we focus on rapid auxin effects, their relationship to H+-ATPase activation and other transporters, and dependence on TIR1/AFB signaling. We also discuss how some observations, such as near-instantaneous effects on ion transport and root growth, do not fit into a single, comprehensive explanation of how auxin controls cell expansion, and where more research is warranted.

The SNARE SYP132 Fine-Tunes Proton Transporter Levels at the Plasma Membrane during Plant Growth.

The acid-growth theory was proposed over 40 years ago based on early observations that low apoplastic pH promotes growth in a pathway involving the plant hormone auxin ([Rayle and Cleland, 1992][1]; [Hager, 2003][2]; [Arsuffi and Braybrook, 2018][3]). Since this discovery, a clearer understanding of

How Does pH Fit in with Oscillating Polar Growth?

Polar growth in root hairs and pollen tubes is an excellent model for investigating plant cell size regulation. While linear plant growth is historically explained by the acid growth theory, which considers that auxin triggers apoplastic acidification by activating plasma membrane P-type H+-ATPases (AHAs) along with cell wall relaxation over long periods, the apoplastic pH (apopH) regulatory mechanisms are unknown for polar growth. Polar growth is a fast process mediated by rapid oscillations that repeat every ∼20-40s. In this review, we explore a reactive oxygen species (ROS)-dependent mechanism that could generate oscillating apopH gradients in a coordinated manner with growth and Ca2+ oscillations. We propose possible mechanisms by which apopH oscillations are coordinated with polar growth together with ROS and Ca2+ waves.

Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana

Plant cells are embedded within cell walls, which provide structural integrity, but also spatially constrain cells, and must therefore be modified to allow cellular expansion. The long-standing acid growth theory postulates that auxin triggers apoplast acidification, thereby activating cell wall-loosening enzymes that enable cell expansion in shoots. Interestingly, this model remains heavily debated in roots, because of both the complex role of auxin in plant development as well as technical limitations in investigating apoplastic pH at cellular resolution. Here, we introduce 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) as a suitable fluorescent pH indicator for assessing apoplastic pH, and thus acid growth, at a cellular resolution in Arabidopsis thaliana roots. Using HPTS, we demonstrate that cell wall acidification triggers cellular expansion, which is correlated with a preceding increase of auxin signaling. Reduction in auxin levels, perception, or signaling abolishes both the extracellular acidification and cellular expansion. These findings jointly suggest that endogenous auxin controls apoplastic acidification and the onset of cellular elongation in roots. In contrast, an endogenous or exogenous increase in auxin levels induces a transient alkalinization of the extracellular matrix, reducing cellular elongation. The receptor-like kinase FERONIA is required for this physiological process, which affects cellular root expansion during the gravitropic response. These findings pinpoint a complex, presumably concentration-dependent role for auxin in apoplastic pH regulation, steering the rate of root cell expansion and gravitropic response.

Recent research progress on acid-growth theory

Recent research progress on acid-growth theory

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.

Connecting Homogalacturonan-Type Pectin Remodeling to Acid Growth.

According to the 'acid growth theory', cell wall acidification controls cell elongation, therefore plant growth. This notably involves changes in cell wall mechanics through modifications of cell wall polysaccharide structure. Recently, advances in cell biology showed that changes in cell elongation rate can be mediated by the remodeling of pectins, and in particular of homogalacturonans (HGs). Their demethylesterification appears to be a key element controlling the chemistry and the rheology of the cell wall. We postulate that precise and dynamic modulation of extracellular pH plays a central role in the control of HG-modifying enzyme activities, and in particular those of pectin methylesterases and polygalacturonases. We propose that acid growth requires dynamic HG remodeling through the tight control of cell wall pH.

TIR1/AFB-Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls.

Despite being composed of immobile cells, plants reorient along directional stimuli. The hormone auxin is redistributed in stimulated organs leading to differential growth and bending. Auxin application triggers rapid cell wall acidification and elongation of aerial organs of plants, but the molecular players mediating these effects are still controversial. Here we use genetically-encoded pH and auxin signaling sensors, pharmacological and genetic manipulations available for Arabidopsis etiolated hypocotyls to clarify how auxin is perceived and the downstream growth executed. We show that auxin-induced acidification occurs by local activation of H+-ATPases, which in the context of gravity response is restricted to the lower organ side. This auxin-stimulated acidification and growth require TIR1/AFB-Aux/IAA nuclear auxin perception. In addition, auxin-induced gene transcription and specifically SAUR proteins are crucial downstream mediators of this growth. Our study provides strong experimental support for the acid growth theory and clarified the contribution of the upstream auxin perception mechanisms.

Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H+-ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H+-ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H+-ATPase activity by 77-155% (ATP hydrolysis), or 70-306% (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57% larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H+-ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana. However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium.

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

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.

In low transpiring conditions, nitrate and water fluxes for growth of B. napus plantlets correlate with changes in BnNrt2.1 and BnNrt1.1 transporter expression

We analyzed how changes in BnNrt nitrate transporter gene expression induced by nitrate are associated with morphological changes in plantlets and osmotic water flow for growth. We hypothesized that in a Petri dish system, reduction in transpiration should induce conditions where nitrate and water fluxes for growth depend directly on nitrate transporter activity and nitrate signaling. Rape seedlings growing on agar plates were supplied with increasing external K15NO3 concentrations from 0.05 to 20 mM. After 5 d of treatment, morphological switches in plantlet growth were observed between 0.5 and 5 mM nitrate supply. Root elongation was reduced by 50% while the cotyledon surface area was doubled. These morphological switches were strongly associated with increases in 15NO3- and water uptake rates as well as 15N and water allocation to the shoot. These switches were also highly correlated with the upregulation of BnNrt1.1 and BnNrt2.1 in the root. However, while root expression of BnNrt2.1 was correlated linearly with a shoot growth-associated increase in 15N and water uptake, BnNrt1.1 expression was correlated exponentially with both 15N and water accumulation. In low transpiring conditions, the tight control exercised by nitrate transporters on K15NO3 uptake and allocation clearly demonstrates that they modulated the nitrate-signaling cascade involved in cell growth and as a consequence, water uptake and allocation to the growing organs. Deciphering this signaling cascade in relation to acid growth theory seems to be the most important challenge for our understanding of the nitrate-signaling role in plant growth.

Auxin Activates the Plasma Membrane H+-ATPase by Phosphorylation during Hypocotyl Elongation in Arabidopsis

The phytohormone auxin is a major regulator of diverse aspects of plant growth and development. The ubiquitin-ligase complex SCF(TIR1/AFB) (for Skp1-Cul1-F-box protein), which includes the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) auxin receptor family, has recently been demonstrated to be critical for auxin-mediated transcriptional regulation. Early-phase auxin-induced hypocotyl elongation, on the other hand, has long been explained by the acid-growth theory, for which proton extrusion by the plasma membrane H(+)-ATPase is a functional prerequisite. However, the mechanism by which auxin mediates H(+)-ATPase activation has yet to be elucidated. Here, we present direct evidence for H(+)-ATPase activation in etiolated hypocotyls of Arabidopsis (Arabidopsis thaliana) by auxin through phosphorylation of the penultimate threonine during early-phase hypocotyl elongation. Application of the natural auxin indole-3-acetic acid (IAA) to endogenous auxin-depleted hypocotyl sections induced phosphorylation of the penultimate threonine of the H(+)-ATPase and increased H(+)-ATPase activity without altering the amount of the enzyme. Changes in both the phosphorylation level of H(+)-ATPase and IAA-induced elongation were similarly concentration dependent. Furthermore, IAA-induced H(+)-ATPase phosphorylation occurred in a tir1-1 afb2-3 double mutant, which is severely defective in auxin-mediated transcriptional regulation. In addition, α-(phenylethyl-2-one)-IAA, the auxin antagonist specific for the nuclear auxin receptor TIR1/AFBs, had no effect on IAA-induced H(+)-ATPase phosphorylation. These results suggest that the TIR1/AFB auxin receptor family is not involved in auxin-induced H(+)-ATPase phosphorylation. Our results define the activation mechanism of H(+)-ATPase by auxin during early-phase hypocotyl elongation; this is the long-sought-after mechanism that is central to the acid-growth theory.

Multivariate analyses of salt stress and metabolite sensing in auto‐ and heterotroph Chenopodium cell suspensions

Global warming increases plant salt stress via evaporation after irrigation, but how plant cells sense salt stress remains unknown. Here, we searched for correlation-based targets of salt stress sensing in Chenopodium rubrum cell suspension cultures. We proposed a linkage between the sensing of salt stress and the sensing of distinct metabolites. Consequently, we analysed various extracellular pH signals in autotroph and heterotroph cell suspensions. Our search included signals after 52 treatments: salt and osmotic stress, ion channel inhibitors (amiloride, quinidine), salt-sensing modulators (proline), amino acids, carboxylic acids and regulators (salicylic acid, 2,4-dichlorphenoxyacetic acid). Multivariate analyses revealed hirarchical clusters of signals and five principal components of extracellular proton flux. The principal component correlated with salt stress was an antagonism of γ-aminobutyric and salicylic acid, confirming involvement of acid-sensing ion channels (ASICs) in salt stress sensing. Proline, short non-substituted mono-carboxylic acids (C2-C6), lactic acid and amiloride characterised the four uncorrelated principal components of proton flux. The proline-associated principal component included an antagonism of 2,4-dichlorphenoxyacetic acid and a set of amino acids (hydrophobic, polar, acidic, basic). The five principal components captured 100% of variance of extracellular proton flux. Thus, a bias-free, functional high-throughput screening was established to extract new clusters of response elements and potential signalling pathways, and to serve as a core for quantitative meta-analysis in plant biology. The eigenvectors reorient research, associating proline with development instead of salt stress, and the proof of existence of multiple components of proton flux can help to resolve controversy about the acid growth theory.

Apoplastic pH and growth in expanding leaves of Vicia faba under salinity

Salinity affects water availability in the soil and subsequently the plant uptake capacity. Upon exposure to salt stress, leaf growth in monocot plants has been shown to be reduced instantaneously, followed by a gradual acclimation. The growth reactions are caused by an initial water deficit and an accompanied osmotic effect, followed by an IAA-induced sequestration of protons into the apoplast that increases leaf growth again as explained by the acid growth theory. In this study, we investigated the dynamics of growth reactions and apoplastic pH in leaves of the dicot Vicia faba in the presence of NaCl during the initiation of salt stress. Concurrent changes in apoplastic pH were detected by ratiometric fluorescence microscopy using the fluorescent dye fluorescein tetramethylrhodamine dextran. To elucidate the possible relation between the dynamics of leaf growth and apoplastic pH, results of the ratio imaging technique were combined with an in vivo growth analysis imaging approach. Leaf growth rate of V. faba was highest in the dusk and the early night phase; at this time a concomitant decrease of the apoplastic pH was observed. Under salinity, the apoplastic pH in leaves of V. faba increased with a simultaneous decrease of leaf growth towards increasing developmental stages, but with complex aberrations in the 24-h-leaf-growth pattern compared to control leaves. In conclusion, these results show that salt stress leads to an increase in apoplastic pH and to a declined leaf growth activity with complex 24-h-interactions of growth and pH in V. faba.

In vitro effect of different Na+/K+ ratios on plasma membrane H+-ATPase activity in maize and sugar beet shoot

Plant growth is impaired primarily by osmotic stress in the first phase of salt stress, whereas Na+ toxicity affects the plant growth mainly in the second phase. Salinity leads to increased Na+/K+ ratio and thus displacement of K+ by Na+ in the plant cell. Relatively higher cytosolic Na+ concentrations may have an effect on the activity of plasma membrane (PM) H+ -ATPase. A decreased PM-H+ -ATPase activity could increase the apoplastic pH. This process could limit the cell-wall extensibility and thus reduce growth according to the acid growth theory. To compare the effect of Na+ on PM H+ -ATPase activity in salt-sensitive maize (Zea mays L.) and salt-resistant sugar beet (Beta vulgaris L.) shoot, PM vesicles were isolated from growing shoots of both species and ATPase activity was determined by assaying the P(i) released by hydrolysis of ATP. The H+ pumping activity was measured as the quenching of acridine-orange absorbance. An increased Na+/K+ ratio decreased the PM H+ -ATPase activity in vesicles of maize as well as of sugar beet shoots. Nevertheless, the detrimental effect of increased Na+/K+ ratio was more severe in salt-sensitive maize compared to salt-resistant sugar beet. At 25 mM Na+ concentration, hydrolytic activity was not affected in sugar beet. However, a significant decrease in hydrolytic activity was observed in maize at pH 7. In maize and sugar beet, reduction in active H+ flux was 20% and 5% at 25 mM Na+ concentration in the assay, respectively. The active H+ flux was decreased to 80% and 60%, when 100 mM K+ were substituted by 100mM Na+. We conclude that PM H+ -ATPases of salt-resistant sugar beet and maize shoot are sensitive to higher concentration of Na+. However, sugar beet PM-H+ -ATPases are relatively efficient and may have constitutive resistance against lower concentration (25 mM) of Na+ as compared to that of salt-sensitive maize.