Lincoln Taiz

Regulation of auxin transport by aminopeptidases and endogenous flavonoids.

Abstract. The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsisthaliana (L.) Heynh. seedlings. Moreover, amino acyl-β-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [3H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [14C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux.

Comparison of Metallothionein Gene Expression and Nonprotein Thiols in Ten Arabidopsis Ecotypes (Correlation with Copper Tolerance)

Seedlings of 10 Arabidopsis ecotypes were compared with respect to copper tolerance, expression of two metallothionein genes (MT1 and MT2), and nonprotein thiol levels. MT1 was uniformly expressed in all treatments, and MT2 was copper inducible in all 10 ecotypes. MT1 and MT2 mRNA levels were compared with various growth parameters for the 10 ecotypes in the presence of 40 [mu]M Cu2+. The best correlation (R = 0.99) was obtained between MT2 mRNA and the rate of root extension. MT2 mRNA levels also paralleled the recovery phase following inhibition by copper. Induction of MT2 mRNA was initiated at copper concentrations below the threshold for growth inhibition. In cross-induction experiments, Ag+, Cd2+, Zn2+, Ni2+, and heat shock all induced significant levels of MT2 gene expression, whereas Al3+ and salicylic acid did not. The correlation between copper tolerance and nonprotein thiol levels in the 10 ecotypes was not statistically significant. However, 2 ecotypes, Ws and Enkheim, previously shown to exhibit an acclimation response, had the highest levels of nonprotein thiols. We conclude that MT2 gene expression may be the primary determinant of ecotypic differences in the copper tolerance of nonpretreated Arabidopsis seedlings.

Identification, Purification, and Molecular Cloning of N-1-Naphthylphthalmic Acid-Binding Plasma Membrane-Associated Aminopeptidases from Arabidopsis

Polar transport of the plant hormone auxin is regulated at the cellular level by inhibition of efflux from a plasma membrane (PM) carrier. Binding of the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) to a regulatory site associated with the carrier has been characterized, but the NPA-binding protein(s) have not been identified. Experimental disparities between levels of high-affinity NPA binding and auxin transport inhibition can be explained by the presence of a low-affinity binding site and in vivo hydrolysis of NPA. In Arabidopsis, colocalization of NPA amidase and aminopeptidase (AP) activities, inhibition of auxin transport by artificial β-naphthylamide substrates, and saturable displacement of NPA by the AP inhibitor bestatin suggest that PM APs may be involved in both low-affinity NPA binding and hydrolysis. We report the purification and molecular cloning of NPA-binding PM APs and associated proteins from Arabidopsis. This is the first report of PM APs in plants. PM proteins were purified by gel permeation, anion exchange, and NPA affinity chromatography monitored for tyrosine-AP activity. Lower affinity fractions contained two orthologs of mammalian APs involved in signal transduction and cell surface-extracellular matrix interactions. AtAPM1 and ATAPP1 have substrate specificities and inhibitor sensitivities similar to their mammalian orthologs, and have temporal and spatial expression patterns consistent with previous in planta histochemical data. Copurifying proteins suggest that the APs interact with secreted cell surface and cell wall proline-rich proteins. AtAPM1 and AtAPP1 are encoded by single genes. In vitro translation products of ATAPM1 and AtAPP1 have enzymatic activities similar to those of native proteins.

Purification and Immunological Identification of Metallothioneins 1 and 2 from Arabidopsis thaliana

Gene families encoding two types of metallothioneins (MTs), MT1 and MT2, have been identified in Arabidopsis thaliana, and their respective mRNAs have been shown to be regulated by copper in a tissue-specific manner (J. Zhou and P.B. Goldsbrough [1994] Plant Cell 6: 875–884; J. Zhou and P.B. Goldsbrough [1995] Mol Gen Genet 248: 318–328; A.S. Murphy and L. Taiz [1995] Plant Physiol 109:1–10). However, to date the protein products have not been identified. To purify MT proteins from Arabidopsis, we isolated low-molecular-mass, copper-binding, thiol-rich proteins using selective precipitation followed by size-exclusion, copper-affinity, and thiol-affinity chromatographies. Polyclonal antibodies raised against Arabidopsis MT-glutathione-S-transferase fusion proteins cross-reacted with the 4.5- and 8-kD bands in immunoblots of low-molecular-mass, copper-binding proteins purified from seedling, mature leaf, and mature root tissues. The identity of the proteins was subsequently confirmed by amino acid sequencing. MT1 expression was constitutive in roots and inducible by copper in mature leaves; the reverse pattern was observed for MT2. MT2 expression was also concentrated in the growing tip of the root. The accumulation of the MT1- and MT2-encoded proteins thus parallels the regulation of their respective mRNAs with regard to tissue specificity and induction by copper. In addition, a new type of MT, designated MT3, was derived from the database, detected by reverse transcription-polymerase chain reaction, and tentatively identified at the protein level by amino acid sequencing of a 7-kD cysteine-rich polypeptide.

A New Vertical Mesh Transfer Technique for Metal-Tolerance Studies in Arabidopsis (Ecotypic Variation and Copper-Sensitive Mutants)

A new vertical mesh transfer (VMT) technique has been developed to facilitate the rapid isolation of plant metal-tolerance mutants. The technique is quantitative, allowing comparisons of the growth responses of different strains or ecotypes. Using the VMT technique, we have characterized the dose responses of 10 ecotypes of Arabidopsis thaliana to Cu2+, Zn2+, Ni2+, Cr3+, Cd2+, and Al3+. Ecotypic variations in the highest concentration causing no inhibition and the lowest concentration causing complete inhibition for the six metals were observed. Two ecotypes, Ws and Enkheim, exhibited an inducible tolerance mechanism in response to copper. Pretreatment of Ws with the highest concentration causing no inhibition for copper resulted in a shifting of the lowest concentration causing complete inhibition to a higher value. Partial cross-induction and cross-tolerance between Cu2+ and Zn2+ were demonstrated. In addition, ethyl methanesulfonate-mutagenized Columbia seeds were screened for copper-sensitive (cus) mutants using the VMT procedure. Thus far, 59 putative cus mutants have survived retesting to the M4 or M5 generation. When grown on gellan gum supplemented with 30 [mu]M CuCl2, cus mutants develop marked toxicity symptoms. A copper dose-response curve of the cus1 mutant showed that the metal-sensitive phenotype is specific for the lower concentration range.

Evolution of structure and function of V-ATPases

Proton pumping ATPases/ATPsynthases are found in all groups of present-day organisms. The structure of V- and F-type ATPases/ATP synthases is very conserved throughout evolution. Sequence analysis shows that the V- and F-type ATPases evolved from the same enzyme already present in the last common ancestor of all known extant life forms. The catalytic and noncatalytic subunits found in the dissociable head groups of the V/F-type ATPases are paralogous subunits, i.e., these two types of subunits evolved from a common ancestral gene. The gene duplication giving rise to these two genes (i.e., encoding the catalytic and noncatalytic subunits) predates the time of the last common ancestor.Mapping of gene duplication events that occurred in the evolution of the proteolipid, the noncatalytic and the catalytic subunits, onto the tree of life leads to a prediction for the likely subunit structure of the encoded ATPases. A correlation between structure and function of V/F-ATPases has been established for present-day organisms. Implications resulting from this correlation for the bioenergetics operative in proto-eukaryotes and in the last common ancestor are presented. The similarities of the V/F-ATPase subunits to an ATPase-like protein that was implicated to play a role in flagellar assembly are evaluated.Different V-ATPase isoforms have been detected in some higher eukaryotes. These data are analyzed with respect to the possible function of the different isoforms (tissue specific, organelle specific) and with respect to the point in their evolution when these gene duplications giving rise to the isoforms had occurred, i.e., how far these isoforms are distributed.

Gene duplication as a means for altering H+/ATP ratios during the evolution of Fo F1 ATPases and synthases

In the evolution of the FoF1 family of proton‐translocating membrane complexes, two reversals in function appear to have occurred, first changing it from an ATPase to an ATP synthase and then back again to an ATPase. Here we suggest that with each change in function, the ratio of protons transported per ATP hydrolyzed or synthesized (H+/ATP) was altered in order for the complex to better adapt to its new role. We propose that this was accomplished by gene duplication with partial loss in the number of functional catalytic sites (to increase H+/ATP) or functional proton channels (to decrease H+/ATP). This method of changing the H+/ATP ratio preserved overall structural features of the complex essential to energy coupling.

Evolution of proton pumping ATPases: Rooting the tree of life.

Proton pumping ATPases are found in all groups of present day organisms. The F-ATPases of eubacteria, mitochondria and chloroplasts also function as ATP synthases, i.e., they catalyze the final step that transforms the energy available from reduction/oxidation reactions (e.g., in photosynthesis) into ATP, the usual energy currency of modern cells. The primary structure of these ATPases/ATP synthases was found to be much more conserved between different groups of bacteria than other parts of the photosynthetic machinery, e.g., reaction center proteins and redox carrier complexes.These F-ATPases and the vacuolar type ATPase, which is found on many of the endomembranes of eukaryotic cells, were shown to be homologous to each other; i.e., these two groups of ATPases evolved from the same enzyme present in the common ancestor. (The term eubacteria is used here to denote the phylogenetic group containing all bacteria except the archaebacteria.) Sequences obtained for the plasmamembrane ATPase of various archaebacteria revealed that this ATPase is much more similar to the eukaryotic than to the eubacterial counterpart. The eukaryotic cell of higher organisms evolved from a symbiosis between eubacteria (that evolved into mitochondria and chloroplasts) and a host organism. Using the vacuolar type ATPase as a molecular marker for the cytoplasmic component of the eukaryotic cell reveals that this host organism was a close relative of the archaebacteria.A unique feature of the evolution of the ATPases is the presence of a non-catalytic subunit that is paralogous to the catalytic subunit, i.e., the two types of subunits evolved from a common ancestral gene. Since the gene duplication that gave rise to these two types of subunits had already occurred in the last common ancestor of all living organisms, this non-catalytic subunit can be used to root the tree of life by means of an outgroup; that is, the location of the last common ancestor of the major domains of living organisms (archaebacteria, eubacteria and eukaryotes) can be located in the tree of life without assuming constant or equal rates of change in the different branches.A correlation between structure and function of ATPases has been established for present day organisms. Implications resulting from this correlation for biochemical pathways, especially photosynthesis, that were operative in the last common ancestor and preceding life forms are discussed.

Interactions between a blue-green reversible photoreceptor and a separate UV-B receptor in stomatal guard cells.

Stomatal opening exhibits two main peaks of activity in the visible range-a red peak, mediated by photosynthesis, and a blue peak, mediated by one or more blue light (BL) photoreceptors. In addition, a pronounced peak in the UV-B region has been characterized, as has a smaller UV-A peak. The BL-induced stomatal opening can be reversed by green light (GL). Here we report that UV-B-induced opening is also antagonized by GL. To determine whether UV-B is being absorbed by the BL photoreceptor or by a separate UV-B receptor, the UV-B responses of two different Arabidopsis mutants, npq1 and phot1/phot2, were tested. Both putative BL-photoreceptor mutants exhibited normal stomatal opening in response to UV-B, consistent with the existence of a separate UV-B photoreceptor. Moreover, GL failed to antagonize UV-B-induced stomatal opening in the phot1/phot2 double mutant and only partially antagonized UV-B opening in npq1. Thus, both phot1 and phot 2, as well as zeaxanthin, are required for the normal GL inhibition of UV-B. A model for a photoreceptor network that regulates stomatal opening is presented. Unlike the situation in guard cells, the UV-B bending response of Arabidopsis hypocotyls during phototropism appears to be mediated by phototropins.

Are biosynthetic reactions in plant cells thermodynamically coupled to glycolysis and the tonoplast proton motive force

The traditional model for anabolic processes, in which biosynthetic reactions are thought to be driven forward by the free energy of hydrolysis of pyrophosphate by soluble pyrophosphatases, is re-evaluated in the light of recent studies on pyrophosphate levels and pyrophosphate-dependent enzymes in plants. It is concluded that the evidence now favors the view that pyrophosphate is metabolized by two major energy-conserving reactions: a PPi-phosphofructokinase and a proton-pumping tonoplast pyrophosphatase. The implications for coupling between anabolic, catabolic and transport processes are discussed.