Experimental Procedures for Studying Skotomorphogenesis in Arabidopsis thaliana.

Seedlings grown in darkness exhibit distinct morphologies comparing with light-grown seedlings. Elongated hypocotyls, closed yellow cotyledons, and the formation of apical hooks are typical characteristics for etiolated seedlings, which are collectively named skotomorphogenesis. Various plant hormones and environmental factors are essential for maintaining skotomorphogenesis. Due to the diverse morphological outcomes in etiolated seedlings grown under different treatments, studies on skotomorphogenesis are of particular importance to reveal the molecular mechanisms underlying plant response to environmental cues. Here, we detailed experimental procedures to facilitate researchers who are investigating etiolation growth-related studies.

The miR396-GRFs Module Mediates the Prevention of Photo-oxidative Damage by Brassinosteroids during Seedling De-Etiolation in Arabidopsis.

The switch from dark- to light-mediated development is critical for the survival and growth of seedlings, but the underlying regulatory mechanisms are incomplete. Here, we show that the steroids phytohormone brassinosteroids play crucial roles during this developmental transition by regulating chlorophyll biosynthesis to promote greening of etiolated seedlings upon light exposure. Etiolated seedlings of the brassinosteroids-deficient det2-1 (de-etiolated2) mutant accumulated excess protochlorophyllide, resulting in photo-oxidative damage upon exposure to light. Conversely, the gain-of-function mutant bzr1-1D (brassinazole-resistant 1-1D) suppressed the protochlorophyllide accumulation of det2-1, thereby promoting greening of etiolated seedlings. Genetic analysis indicated that phytochrome-interacting factors (PIFs) were required for BZR1-mediated seedling greening. Furthermore, we reveal that GROWTH REGULATING FACTOR 7 (GRF7) and GRF8 are induced by BZR1 and PIF4 to repress chlorophyll biosynthesis and promote seedling greening. Suppression of GRFs function by overexpressing microRNA396a caused an accumulation of protochlorophyllide in the dark and severe photobleaching upon light exposure. Additionally, BZR1, PIF4, and GRF7 interact with each other and precisely regulate the expression of chlorophyll biosynthetic genes. Our findings reveal an essential role for BRs in promoting seedling development and survival during the initial emergence of seedlings from subterranean darkness into sunlight.

Light and Abscisic Acid Coordinately Regulate Greening of Seedlings.

The greening of etiolated seedlings is crucial for the growth and survival of plants. After reaching the soil surface and sunlight, etiolated seedlings integrate numerous environmental signals and internal cues to control the initiation and rate of greening thus to improve their survival and adaption. However, the underlying regulatory mechanisms by which light and phytohormones, such as abscisic acid (ABA), coordinately regulate greening of the etiolated seedlings is still unknown. In this study, we showed that Arabidopsis (Arabidopsis thaliana) DE-ETIOLATED1 (DET1), a key negative regulator of photomorphogenesis, positively regulated light-induced greening by repressing ABA responses. Upon irradiating etiolated seedlings with light, DET1 physically interacts with FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and subsequently associates to the promoter region of the FHY3 direct downstream target ABA INSENSITIVE5 (ABI5). Further, DET1 recruits HISTONE DEACETYLASE6 to the locus of the ABI5 promoter and reduces the enrichments of H3K27ac and H3K4me3 modification, thus subsequently repressing ABI5 expression and promoting the greening of etiolated seedlings. This study reveals the physiological and molecular function of DET1 and FHY3 in the greening of seedlings and provides insights into the regulatory mechanism by which plants integrate light and ABA signals to fine-tune early seedling establishment.

Molecular basis and potential applications of capsaicinoids and capsinoids against the elongation of etiolated wheat (Triticum aestivum L.) coleoptiles in foods.

Capsaicinoids and capsinoids from dietary peppers have promising sensory properties and bioactivity, but the molecular basis of their penetration mechanism through cell lipid bilayers and its relationship to their bioavailability as food constituents are still poorly understood. Herein, statistically significant linear and quadratic quantitative structure-activity relationships were constructed to derive the essential structural elements required for their bioactivity against the elongation of etiolated wheat coleoptiles that mainly occurs via penetration. The resultant optimal models had high predictivity and reliability (r2 > 0.825 and r2pred > 0.950), which elucidate the importance of steric structural elements. Besides, their mechanistic hypothesis and rational design strategy were proposed, and the correlation between this bioactivity and their food-sensory properties was supposed. Finally, the bioactivity of newly designed analogs with methyl terminals and/or conjugated CC links was screened. Hopefully, this work would benefit the better understanding of their penetration mechanism and facile identification of bioactive analogs for designing food/drug formulations.

Deetiolation Enhances Phototropism by Modulating NON-PHOTOTROPIC HYPOCOTYL3 Phosphorylation Status.

Phototropin (phot) receptor kinases play important roles in promoting plant growth by controlling light-capturing processes, such as phototropism. Phototropism is mediated through the action of NON-PHOTOTROPIC HYPOCOTYL3 (NPH3), which is dephosphorylated following phot activation. However, the functional significance of this early signaling event remains unclear. Here, we show that the onset of phototropism in dark-grown (etiolated) seedlings of Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) is enhanced by greening (deetiolation). Red and blue light were equally effective in promoting phototropism in Arabidopsis, consistent with our observations that deetiolation by phytochrome or cryptochrome was sufficient to enhance phototropism. Increased responsiveness did not result from an enhanced sensitivity to the phytohormone auxin, nor does it involve the phot-interacting protein, ROOT PHOTOTROPISM2. Instead, deetiolated seedlings showed attenuated levels of NPH3 dephosphorylation and diminished relocalization of NPH3 from the plasma membrane during phototropism. Likewise, etiolated seedlings that lack the PHYTOCHROME-INTERACTING FACTORS (PIFs) PIF1, PIF3, PIF4, and PIF5 displayed reduced NPH3 dephosphorylation and enhanced phototropism, consistent with their constitutive photomorphogenic phenotype in darkness. Phototropic enhancement could also be achieved in etiolated seedlings by lowering the light intensity to diminish NPH3 dephosphorylation. Thus, phototropism is enhanced following deetiolation through the modulation of a phosphorylation rheostat, which in turn sustains the activity of NPH3. We propose that this dynamic mode of regulation enables young seedlings to maximize their establishment under changing light conditions, depending on their photoautotrophic capacity.

Comparative adventitious root development in pre-etiolated and flooded Arabidopsis hypocotyls exposed to different auxins.

Adventitious roots (ARs) emerge from stems, leaves or hypocotyls, being strategic for clonal propagation. ARs may develop spontaneously, upon environmental stress or hormonal treatment. Auxins strongly influence AR development (ARD), depending on concentration and kind. However, the role of different types of auxin is rarely compared at the molecular level. Rooting triggered by light exposure and flooding was examined in intact etiolated Arabidopsis thaliana hypocotyls treated with distinct auxin types. Morphological aspects, rooting-related gene expression profiles, and IAA immunolocalization were recorded. NAA and 2,4-D effects were highly dose-dependent; at higher concentrations NAA inhibited root growth and 2,4-D promoted callus formation. NAA yielded the highest number of roots, but inhibited elongation. IAA increased the number of roots with less interference in elongation, yielding the best overall rooting response. IAA was localized close to the tissues of root origin. Auxin stimulated ARD was marked by increased expression of PIN1 and GH3.3. NAA treatment induced expression of CYCB1, GH3.6 and ARF8. These NAA-specific responses may be associated with the development of numerous shorter roots. In contrast, expression of the auxin action inhibitor IAA28 was induced by IAA. Increased PIN1 expression indicated the relevance of auxin efflux transport for focusing in target cells, whereas GH3.3 suggested tight control of auxin homeostasis. IAA28 increased expression during IAA-induced ARD differs from what was previously reported for lateral root development, pointing to yet another possible difference in the molecular programs of these two developmental processes.

EHB1 and AGD12, two calcium-dependent proteins affect gravitropism antagonistically in Arabidopsis thaliana.

The ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN (AGD) 12, a member of the ARF-GAP protein family, affects gravitropism in Arabidopsis thaliana. A loss-of-function mutant lacking AGD12 displayed diminished gravitropism in roots and hypocotyls indicating that both organs are affected by this regulator. AGD12 is structurally related to ENHANCED BENDING (EHB) 1, previously described as a negative effector of gravitropism. In contrast to agd12 mutants, ehb1 loss-of function seedlings displayed enhanced gravitropic bending. While EHB1 and AGD12 both possess a C-terminal C2/CaLB-domain, EHB1 lacks the N-terminal ARF-GAP domain present in AGD12. Subcellular localization analysis using Brefeldin A indicated that both proteins are elements of the trans Golgi network. Physiological analyses provided evidence that gravitropic signaling might operate via an antagonistic interaction of ARF-GAP (AGD12) and EHB1 in their Ca2+-activated states.

Jasmonic acid protects etiolated seedlings of Arabidopsis thaliana against herbivorous arthropods.

Seed predators can cause mass ingestion of larger seed populations. As well, herbivorous arthropods attempt to attack etiolated seedlings and chose the apical hook for ingestion, aimed at dropping the cotyledons for later consumption. Etiolated seedlings, as we show here, have established an efficient mechanism of protecting their Achilles' heel against these predators, however. Evidence is provided for a role of jasmonic acid (JA) in this largely uncharacterized plant-herbivore interaction during skotomorphogenesis and that this comprises the temporally and spatially tightly controlled synthesis of a cysteine protease inhibitors of the Kunitz family. Interestingly, the same Kunitz protease inhibitor was found to be expressed in flowers of Arabidopsis where endogenous JA levels are high for fertility. Because both the apical hook and inflorescences were preferred isopod targets in JA-deficient plants that could be rescued by exogenously administered JA, our data identify a JA-dependent mechanism of plant arthropod deterrence that is recalled in different organs and at quite different times of plant development.

Ethylene Regulates the Arabidopsis Microtubule-Associated Protein WAVE-DAMPENED2-LIKE5 in Etiolated Hypocotyl Elongation.

The phytohormone ethylene plays crucial roles in the negative regulation of plant etiolated hypocotyl elongation. The microtubule cytoskeleton also participates in hypocotyl cell growth. However, it remains unclear if ethylene signaling-mediated etiolated hypocotyl elongation involves the microtubule cytoskeleton. In this study, we functionally identified the previously uncharacterized microtubule-associated protein WAVE-DAMPENED2-LIKE5 (WDL5) as a microtubule-stabilizing protein that plays a positive role in ethylene-regulated etiolated hypocotyl cell elongation in Arabidopsis (Arabidopsis thaliana). ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, directly targets and up-regulates WDL5. Etiolated hypocotyls from a WDL5 loss-of-function mutant (wdl5-1) were more insensitive to 1-aminocyclopropane-1-carboxylic acid treatment than the wild type. Decreasing WDL5 expression partially rescued the shorter etiolated hypocotyl phenotype in the ethylene overproduction mutant eto1-1. Reorganization of cortical microtubules in etiolated hypocotyl cells from the wdl5-1 mutant was less sensitive to 1-aminocyclopropane-1-carboxylic acid treatment. These findings indicate that WDL5 is an important participant in ethylene signaling inhibition of etiolated hypocotyl growth. This study reveals a mechanism involved in the ethylene regulation of microtubules through WDL5 to inhibit etiolated hypocotyl cell elongation.

Multiple Interactions between Glucose and Brassinosteroid Signal Transduction Pathways in Arabidopsis Are Uncovered by Whole-Genome Transcriptional Profiling.

Brassinosteroid (BR) and glucose (Glc) regulate many common responses in plants. Here, we demonstrate that under etiolated growth conditions, extensive interdependence/overlap occurs between BR- and Glc-regulated gene expression as well as physiological responses. Glc could regulate the transcript level of 72% of BR-regulated genes at the whole-genome level, of which 58% of genes were affected synergistically and 42% of genes were regulated antagonistically. Presence of Glc along with BR in medium could affect BR induction/repression of 85% of BR-regulated genes. Glc could also regulate several genes involved in BR metabolism and signaling. Both BR and Glc coregulate a large number of genes involved in abiotic/biotic stress responses and growth and development. Physiologically, Glc and BR interact to regulate hypocotyl elongation growth of etiolated Arabidopsis (Arabidopsis thaliana) seedlings in a dose-dependent manner. Glc may interact with BR via a hexokinase1 (HXK1)-mediated pathway to regulate etiolated hypocotyl elongation. Brassinosteroid insensitive1 (BRI1) is epistatic to HXK1, as the Glc insensitive2bri1-6 double mutant displayed severe defects in hypocotyl elongation growth similar to its bri1-6 parent. Analysis of Glc and BR sensitivity in mutants defective in auxin response/signaling further suggested that Glc and BR signals may converge at S-phase kinase-associated protein1-Cullin-F-box-transport inhibitor response1/auxin-related f-box-auxin/indole-3-acetic acid-mediated auxin-signaling machinery to regulate etiolated hypocotyl elongation growth in Arabidopsis.

MAOHUZI6/ETHYLENE INSENSITIVE3-LIKE1 and ETHYLENE INSENSITIVE3-LIKE2 Regulate Ethylene Response of Roots and Coleoptiles and Negatively Affect Salt Tolerance in Rice.

Ethylene plays important roles in plant growth, development, and stress responses. The ethylene signaling pathway has been studied extensively, mainly in Arabidopsis (Arabidopsis thaliana). However, the molecular mechanism of ethylene signaling is largely unknown in rice (Oryza sativa). Previously, we have isolated a set of rice ethylene-response mutants. Here, we characterized the mutant maohuzi6 (mhz6). Through map-based cloning, we found that MHZ6 encodes ETHYLENE INSENSITIVE3-LIKE1 (OsEIL1), a rice homolog of ETHYLENE INSENSITIVE3 (EIN3), which is the master transcriptional regulator of ethylene signaling in Arabidopsis. Disruption of MHZ6/OsEIL1 caused ethylene insensitivity mainly in roots, whereas silencing of the closely related OsEIL2 led to ethylene insensitivity mainly in coleoptiles of etiolated seedlings. This organ-specific functional divergence is different from the functional features of EIN3 and EIL1, both of which mediate the incomplete ethylene responses of Arabidopsis etiolated seedlings. In Arabidopsis, EIN3 and EIL1 play positive roles in plant salt tolerance. In rice, however, lack of MHZ6/OsEIL1 or OsEIL2 functions improves salt tolerance, whereas the overexpressing lines exhibit salt hypersensitivity at the seedling stage, indicating that MHZ6/OsEIL1 and OsEIL2 negatively regulate salt tolerance in rice. Furthermore, this negative regulation by MHZ6/OsEIL1 and OsEIL2 in salt tolerance is likely attributable in part to the direct regulation of HIGH-AFFINITY K(+) TRANSPORTER2;1 expression and Na(+) uptake in roots. Additionally, MHZ6/OsEIL1 overexpression promotes grain size and thousand-grain weight. Together, our study provides insights for the functional diversification of MHZ6/OsEIL1 and OsEIL2 in ethylene response and finds a novel mode of ethylene-regulated salt stress response that could be helpful for engineering salt-tolerant crops.

Global analysis of the role of autophagy in cellular metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation.

Germination and early seedling establishment are developmental stages in which plants face limited nutrient supply as their photosynthesis mechanism is not yet active. For this reason, the plant must mobilize the nutrient reserves provided by the mother plant in order to facilitate growth. Autophagy is a catabolic process enabling the bulk degradation of cellular constituents in the vacuole. The autophagy mechanism is conserved among eukaryotes, and homologs of many autophagy-related (ATG) genes have been found in Arabidopsis thaliana. T-DNA insertion mutants (atg mutants) of these genes display higher sensitivity to various stresses, particularly nutrient starvation. However, the direct impact of autophagy on cellular metabolism has not been well studied. In this work, we used etiolated Arabidopsis seedlings as a model system for carbon starvation. atg mutant seedlings display delayed growth in response to carbon starvation compared with wild-type seedlings. High-throughput metabolomic, lipidomic, and proteomic analyses were performed, as well as extensive flux analyses, in order to decipher the underlying causes of the phenotype. Significant differences between atg mutants and wild-type plants have been demonstrated, suggesting global effects of autophagy on central metabolism during carbon starvation as well as severe energy deprivation, resulting in a morphological phenotype.

Defective copper transport in the copt5 mutant affects cadmium tolerance.

Cadmium toxicity interferes with essential metal homeostasis, which is a problem for both plant nutrition and the consumption of healthy food by humans. Copper uptake is performed by the members of the Arabidopsis high affinity copper transporter (COPT) family. One of the members, COPT5, is involved in copper recycling from the vacuole toward the cytosolic compartment. We show herein that copt5 mutants are more sensitive to cadmium stress than wild-type plants, as indicated by reduced growth. Exacerbated cadmium toxicity in copt5 mutants is due specifically to altered copper traffic through the COPT5 transporter. Three different processes which have been shown to affect cadmium tolerance are altered in copt5 mutants. First, ethylene biosynthesis diminishes under copper deficiency and, in the presence of cadmium, ethylene production diminishes further. Copper deficiency responses are also attenuated under cadmium treatment. Remarkably, while copt5 roots present higher oxidative stress toxicity symptoms than controls, aerial copt5 parts display lower oxidative stress, as seen by reduced cadmium delivery to shoots. Taken together, these results demonstrate that copper transport plays a key role in cadmium resistance, and suggest that oxidative stress triggers an NADPH oxidase-mediated signaling pathway, which contributes to cadmium translocation and basal plant resistance. The slightly lower cadmium levels that reach aerial parts in the copt5 mutants, irrespective of the copper content in the media, suggest a new biotechnological approach to minimize toxic cadmium entry into food chains.

Ultraviolet radiation induces stress in etiolated Landoltia punctata, as evidenced by the presence of alanine, a universal stress signal: a ¹5N NMR study.

Analysis with (15) N NMR revealed that alanine, a universal cellular stress signal, accumulates in etiolated duckweed plants exposed to 15-min pulsed UV light, but not in the absence of UV irradiation. The addition of 10 mm vitamin C, a radical scavenger, reduced alanine levels to zero, indicating the involvement of free radicals. Free D-alanine was detected in (15) N NMR analysis of the chiral amino acid content, using D-tartaric acid as solvent. The accumulation of D-alanine under stress conditions presents a new perspective on the biochemical processes taking place in prokaryote and eukaryote cells.

Signaling role of phospholipid hydroperoxide glutathione peroxidase (PHGPX) accompanying sensing of NaCl stress in etiolated sunflower seedling cotyledons.

Sunflower seedlings subjected to 120 mM NaCl stress exhibit high total peroxidase activity, differential expression of its isoforms and accumulation of lipid hydroperoxides. This coincides with high specific activity of phospholipid hydroperoxide glutathione peroxidase (PHGPX) in the 10,000g supernatant from the homogenates of 2-6 d old seedling cotyledons. An upregulation of PHGPX activity by NaCl is evident from Western blot analysis. Confocal laser scanning microscopic (CLSM) analysis of sections of cotyledons incubated with anti-GPX4 (PHGPX) antibody highlights an enhanced cytosolic accumulation of PHGPX, particularly around the secretory canals. Present work, thus, highlights sensing of NaCl stress in sunflower seedlings in relation with lipid hydroperoxide accumulation and its scavenging through an upregulation of PHGPX activity in the cotyledons.

Phototropin 1 and dim-blue light modulate the red light de-etiolation response.

Light signals regulate seedling morphological changes during de-etiolation through the coordinated actions of multiple light-sensing pathways. Previously we have shown that red-light-induced hypocotyl growth inhibition can be reversed by addition of dim blue light through the action of phototropin 1 (phot1). Here we further examine the fluence-rate relationships of this blue light effect in short-term (hours) and long-term (days) hypocotyl growth assays. The red stem-growth inhibition and blue promotion is a low-fluence rate response, and blue light delays or attenuates both the red light and far-red light responses. These de-etiolation responses include blue light reversal of red or far-red induced apical hook opening. This response also requires phot1. Cryptochromes (cry1 and cry2) are activated by higher blue light fluence-rates and override phot1's influence on hypocotyl growth promotion. Exogenous application of auxin transport inhibitor naphthylphthalamic acid abolished the blue light stem growth promotion in both hypocotyl growth and hook opening. Results from the genetic tests of this blue light effect in auxin transporter mutants, as well as phytochrome kinase substrate mutants indicated that aux1 may play a role in blue light reversal of red light response. Together, the phot1-mediated adjustment of phytochrome-regulated photomorphogenic events is most robust in dim blue light conditions and is likely modulated by auxin transport through its transporters.

Arabidopsis DE-ETIOLATED1 represses photomorphogenesis by positively regulating phytochrome-interacting factors in the dark.

Arabidopsis thaliana seedlings undergo photomorphogenic development even in darkness when the function of DE-ETIOLATED1 (DET1), a repressor of photomorphogenesis, is disrupted. However, the mechanism by which DET1 represses photomorphogenesis remains unclear. Our results indicate that DET1 directly interacts with a group of transcription factors known as the phytochrome-interacting factors (PIFs). Furthermore, our results suggest that DET1 positively regulates PIF protein levels primarily by stabilizing PIF proteins in the dark. Genetic analysis showed that each pif single mutant could enhance the det1-1 phenotype, and ectopic expression of each PIF in det1-1 partially suppressed the det1-1 phenotype, based on hypocotyl elongation and cotyledon opening angles observed in darkness. Genomic analysis also revealed that DET1 may modulate the expression of light-regulated genes to mediate photomorphogenesis partially through PIFs. The observed interaction and regulation between DET1 and PIFs not only reveal how DET1 represses photomorphogenesis, but also suggest a possible mechanism by which two groups of photomorphogenic repressors, CONSTITUTIVE PHOTOMORPHOGENESIS/DET/FUSCA and PIFs, work in concert to repress photomorphogenesis in darkness.

Abscisic acid suppresses hypocotyl elongation by dephosphorylating plasma membrane H(+)-ATPase in Arabidopsis thaliana.

Plasma membrane H(+)-ATPase is thought to mediate hypocotyl elongation, which is induced by the phytohormone auxin through the phosphorylation of the penultimate threonine of H(+)-ATPase. However, regulation of the H(+)-ATPase during hypocotyl elongation by other signals has not been elucidated. Hypocotyl elongation in etiolated seedlings of Arabidopsis thaliana was suppressed by the H(+)-ATPase inhibitors vanadate and erythrosine B, and was significantly reduced in aha2-5, which is a knockout mutant of the major H(+)-ATPase isoform in etiolated seedlings. Application of the phytohormone ABA to etiolated seedlings suppressed hypocotyl elongation within 30 min at the half-inhibitory concentration (4.2 µM), and induced dephosphorylation of the penultimate threonine of H(+)-ATPase without affecting the amount of H(+)-ATPase. Interestingly, an ABA-insensitive mutant, abi1-1, did not show ABA inhibition of hypocotyl elongation or ABA-induced dephosphorylation of H(+)-ATPase. This indicates that ABI1, which is an early ABA signaling component through the ABA receptor PYR/PYL/RCARs (pyrabactin resistance/pyrabactin resistance 1-like/regulatory component of ABA receptor), is involved in these responses. In addition, we found that the fungal toxin fusiccocin (FC), an H(+)-ATPase activator, induced hypocotyl elongation and phosphorylation of the penultimate threonine of H(+)-ATPase, and that FC-induced hypocotyl elongation and phosphorylation of H(+)-ATPase were significantly suppressed by ABA. Taken together, these results indicate that ABA has an antagonistic effect on hypocotyl elongation through, at least in part, dephosphorylation of H(+)-ATPase in etiolated seedlings.

Cessation of coleoptile elongation and loss of auxin sensitivity in developing rye seedlings: a quantitative proteomic analysis.

The use of the grass coleoptile for the elucidation of the mechanism of cell elongation is a legacy of the classic experiments of Charles Darwin, who described this organ in 1880 as a "reddish sheath". In this study we quantified the growth of intact, etiolated rye (Secale cereale L.) seedlings and selected 3-day-old (growing) vs. 4-day-old (pierced) coleoptiles for a comparative analysis. Upon emergence of the reddish primary leaf on day 4 after sowing, growth slowed down by 70% and the sensitivity of the coleoptile to auxin (Indole-3-acetic acid) was lost, but turgor pressure was maintained. A quantitative comparison of the proteome (microsomal- and cytoplasmic protein fractions, respectively), using the two-dimensional difference gel electrophoresis (2-D DIGE)-technique, revealed that at least 28 proteins (spots) were differentially up- or down-regulated more than 1.5-fold. Eight of these proteins were identified by reverse-phase liquid chromatography-electrospray tandem mass spectrometry. Cessation of coleoptile growth was associated with the down-regulation (- 81 %) of subunit E of the vacuolar H(+)-ATPase (V-ATPase) and the up-regulation of enzymes involved in lignification (phenylalanine ammonia lyase) and wounding responses (xylanase inhibitor; two lipoxygenases). We conclude that the degradation of the V-ATPases, electrogenic proton pumps on the tonoplast and the membranes of the Golgi- dependent secretory pathway, may be the cause for the cessation of growth in turgid coleoptiles and the associated loss of auxin sensitivity. However, the intracellular signals that cause these proteomic changes have not yet been identified.