ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis.

Auxin transport is mediated at the cellular level by three independent mechanisms that are characterised by the PIN-formed (PIN), P-glycoprotein (ABCB/PGP) and AUX/LAX transport proteins. The PIN and ABCB transport proteins, best represented by PIN1 and ABCB19 (PGP19), have been shown to coordinately regulate auxin efflux. When PIN1 and ABCB19 coincide on the plasma membrane, their interaction enhances the rate and specificity of auxin efflux and the dynamic cycling of PIN1 is reduced. However, ABCB19 function is not regulated by the dynamic cellular trafficking mechanisms that regulate PIN1 in apical tissues, as localisation of ABCB19 on the plasma membrane was not inhibited by short-term treatments with latrunculin B, oryzalin, brefeldin A (BFA) or wortmannin--all of which have been shown to alter PIN1 and/or PIN2 plasma membrane localisation. When taken up by endocytosis, the styryl dye FM4-64 labels diffuse rather than punctuate intracellular bodies in abcb19 (pgp19), and some aggregations of PIN1 induced by short-term BFA treatment did not disperse after BFA washout in abcb19. Although the subcellular localisations of ABCB19 and PIN1 in the reciprocal mutant backgrounds were like those in wild type, PIN1 plasma membrane localisation in abcb19 roots was more easily perturbed by the detergent Triton X-100, but not other non-ionic detergents. ABCB19 is stably associated with sterol/sphingolipid-enriched membrane fractions containing BIG/TIR3 and partitions into Triton X-100 detergent-resistant membrane (DRM) fractions. In the wild type, PIN1 was also present in DRMs, but was less abundant in abcb19 DRMs. These observations suggested a rationale for the observed lack of auxin transport activity when PIN1 is expressed in a non-plant heterologous system. PIN1 was therefore expressed in Schizosaccharomyces pombe, which has plant-like sterol-enriched microdomains, and catalysed auxin transport in these cells. These data suggest that ABCB19 stabilises PIN1 localisation at the plasma membrane in discrete cellular subdomains where PIN1 and ABCB19 expression overlaps.

The ABC subfamily B auxin transporter AtABCB19 is involved in the inhibitory effects of N-1-naphthyphthalamic acid on the phototropic and gravitropic responses of Arabidopsis hypocotyls.

N-1-Naphthylphthalamic acid (NPA) causes the abnormal growth and development of plants by suppressing polar auxin transport. The mechanisms underlying this inhibition, however, have remained elusive. In Arabidopsis, we show that a defect in the ABC subfamily B auxin transporter AtABCB19 suppresses the inhibitory effects of NPA on hypocotyl phototropism and gravitropism, but not on hypocotyl elongation. Expression analysis using the auxin reporter gene DR5:GUS further suggests that NPA partially inhibits the asymmetric distribution of auxin in an AtABCB19-dependent manner. These data thus suggest that AtABCB19 plays an important role in the inhibitory effects of NPA on hypocotyl tropism induced by auxin.

Ancient trans-Acting siRNAs Confer Robustness and Sensitivity onto the Auxin Response.

Novel developmental programs often evolve via cooption of existing genetic networks. To understand this process, we explored cooption of the TAS3 tasiRNA pathway in the moss Physcomitrella patens. We find an ancestral function for this repeatedly redeployed pathway in the spatial regulation of a conserved set of Auxin Response Factors. In moss, this results in stochastic patterning of the filamentous protonemal tissue. Through modeling and experimentation, we demonstrate that tasiRNA regulation confers sensitivity and robustness onto the auxin response. Increased auxin sensitivity parallels increased developmental sensitivity to nitrogen, a key environmental signal. We propose that the properties lent to the auxin response network, along with the ability to stochastically modulate development in response to environmental cues, have contributed to repeated cooption of the tasiRNA-ARF module during evolution. The signaling properties of a genetic network, and not just its developmental output, are thus critical to understanding evolution of multicellular forms.

Digital gene expression profiling by 5'-end sequencing of cDNAs during reprogramming in the moss Physcomitrella patens.

Stem cells self-renew and repeatedly produce differentiated cells during development and growth. The differentiated cells can be converted into stem cells in some metazoans and land plants with appropriate treatments. After leaves of the moss Physcomitrella patens are excised, leaf cells reenter the cell cycle and commence tip growth, which is characteristic of stem cells called chloronema apical cells. To understand the underlying molecular mechanisms, a digital gene expression profiling method using mRNA 5'-end tags (5'-DGE) was established. The 5'-DGE method produced reproducible data with a dynamic range of four orders that correlated well with qRT-PCR measurements. After the excision of leaves, the expression levels of 11% of the transcripts changed significantly within 6 h. Genes involved in stress responses and proteolysis were induced and those involved in metabolism, including photosynthesis, were reduced. The later processes of reprogramming involved photosynthesis recovery and higher macromolecule biosynthesis, including of RNA and proteins. Auxin and cytokinin signaling pathways, which are activated during stem cell formation via callus in flowering plants, are also activated during reprogramming in P. patens, although no exogenous phytohormone is applied in the moss system, suggesting that an intrinsic phytohormone regulatory system may be used in the moss.

Phytochromes and cryptochromes regulate the differential growth of Arabidopsis hypocotyls in both a PGP19-dependent and a PGP19-independent manner.

Photoreceptors, phytochromes and cryptochromes regulate hypocotyl growth under specific conditions, by suppressing negative gravitropism, modulating phototropism and inhibiting elongation. Although these effects seem to be partially caused via the regulation of the phytohormone auxin, the molecular mechanisms underlying this process are still poorly understood. In our present study, we demonstrate that the flabby mutation enhances both phytochrome- and cryptochrome-inducible hypocotyl bending in Arabidopsis. The FLABBY gene encodes the ABC-type auxin transporter, PGP19, and its expression is suppressed by the activation of phytochromes and cryptochromes. Our current results therefore indicate that the phytochromes and cryptochromes have at least two effects upon the tropic responses of the hypocotyls in Arabidopsis: the enhancement of hypocotyl bending through the suppression of PGP19, and a PGP19-independent mechanism that induces hypocotyl bending. By the using an auxin polar transport assay and DR5:GUS expression analysis, we further find that the phytochromes inhibit basipetal auxin transport, and induce the asymmetric distribution of auxin in the hypocotyls. These data suggest that the control of auxin transport by phytochromes and cryptochromes is a critical regulatory component of hypocotyl growth in response to light.

Chloroplast ribosome release factor 1 (AtcpRF1) is essential for chloroplast development.

To study the functions of nuclear genes involved in chloroplast development, we systematically analyzed albino and pale green Arabidopsis thaliana mutants by use of the Activator/Dissociation (Ac/Ds) transposon tagging system. In this study, we focused on one of these albino mutants, designated apg3-1 (for a lbino or p ale g reen mutant 3). A gene encoding a ribosome release factor 1 (RF1) homologue was disrupted by the insertion of a Ds transposon into the APG3 gene; a T-DNA insertion into the same gene caused a similar phenotype (apg3-2). The APG3 gene (At3g62910) has 15 exons and encodes a protein (422-aa) with a transit peptide that functions in targeting the protein to chloroplasts. The amino acid sequence of APG3 showed 40.6% homology with an RF1 of Escherichia coli, and complementation analysis using the E. coli rf1 mutant revealed that APG3 functions as an RF1 in E. coli, although complementation was not successful in the RF2-deficient (rf2) mutants of E. coli. These results indicate that the APG3 protein is an orthologue of E. coli RF1, and is essential for chloroplast translation machinery; it was accordingly named AtcpRF1. Since the chloroplasts of apg3-1 plants contained few internal thylakoid membranes, and chloroplast proteins related to photosynthesis were not detected by immunoblot analysis, AtcpRF1 is thought to be essential for chloroplast development.

Interactions among PIN-FORMED and P-glycoprotein auxin transporters in Arabidopsis.

Directional transport of the phytohormone auxin is established primarily at the point of cellular efflux and is required for the establishment and maintenance of plant polarity. Studies in whole plants and heterologous systems indicate that PIN-FORMED (PIN) and P-glycoprotein (PGP) transport proteins mediate the cellular efflux of natural and synthetic auxins. However, aromatic anion transport resulting from PGP and PIN expression in nonplant systems was also found to lack the high level of substrate specificity seen in planta. Furthermore, previous reports that PGP19 stabilizes PIN1 on the plasma membrane suggested that PIN-PGP interactions might regulate polar auxin efflux. Here, we show that PGP1 and PGP19 colocalized with PIN1 in the shoot apex in Arabidopsis thaliana and with PIN1 and PIN2 in root tissues. Specific PGP-PIN interactions were seen in yeast two-hybrid and coimmunoprecipitation assays. PIN-PGP interactions appeared to enhance transport activity and, to a greater extent, substrate/inhibitor specificities when coexpressed in heterologous systems. By contrast, no interactions between PGPs and the AUXIN1 influx carrier were observed. Phenotypes of pin and pgp mutants suggest discrete functional roles in auxin transport, but pin pgp mutants exhibited phenotypes that are both additive and synergistic. These results suggest that PINs and PGPs characterize coordinated, independent auxin transport mechanisms but also function interactively in a tissue-specific manner.

The multiple-stress responsive plastid sigma factor, SIG5, directs activation of the psbD blue light-responsive promoter (BLRP) in Arabidopsis thaliana.

Transcription in higher plant plastids is performed by two types of RNA polymerases called NEP and PEP, and expression of photosynthesis genes in chloroplasts is largely dependent on PEP, a eubacteria-type multi-subunit enzyme. The transcription specificity of PEP is modulated by six nuclear-encoded sigma factors (SIG1 to SIG6) in Arabidopsis thaliana. Here, we show that one of the six sigma factors, SIG5, is induced under various stress conditions, such as high light, low temperature, high salt and high osmotic conditions. Interestingly, transcription from the psbD blue light-responsive promoter (psbD-BLRP) was activated by not only light but also various stresses, and the transcription and the transcriptional activation of psbD-BLRP were abolished in a sig5-2 mutant. This suggests that the PEP holoenzyme containing SIG5 transcribes the psbD-BLRP in response to multiple stresses. Since the seed germination under saline conditions and recovery from damage to the PSII induced by high light were delayed in the sig5-2 mutant, we postulate that SIG5 protects plants from stresses by enhancing repair of the PSII reaction center.

DNA microarray analysis of plastid gene expression in an Arabidopsis mutant deficient in a plastid transcription factor sigma, SIG2.

The plastid genome of higher plants contains more than one hundred genes for photosynthesis, gene expression, and other processes. Plastid transcription is done by two types of RNA polymerase, PEP and NEP. PEP is a eubacteria-type RNA polymerase that is essential for chloroplast development. In Arabidopsis thaliana, six sigma factors (SIG1-6) are encoded by the nuclear genome, and postulated to determine the transcription specificity of PEP. In this study, we constructed a DNA microarray for all of the plastid protein-coding genes, and analyzed the effects of the sig2 lesion on the global plastid gene expression. Of the 79 plastid protein genes, it was found that only the psaJ transcript was decreased in the mutant, whereas transcripts of 47 genes were rather increased. Since many of the up-regulated genes are under the control of NEP, it was suggested that the NEP activity was increased in the sig2-1 mutant.