PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism.

Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.

Root-Growth Behavior of the Arabidopsis Mutant rgr11 : Roles of Gravitropism and Circumnutation in the Waving/Coiling Phenomenon

In this study we investigated the kinetics of the gravitropic response of the Arabidopsis mutant rgr1 (reduced root gravitropism). Although the rate of curvature in rgr1, which is allelic to axr4, was smaller than in the wild type (ecotype Wassilewskija), curvature was initiated in the same region of the root, the distal elongation zone. The time lag for the response was unaffected in the mutant; however, the gravitropic response of rgr1 contained a feature not found in the wild type: when roots growing along the surface of an agar plate were gravistimulated, there was often an upward curvature that initiated in the central elongation zone. Because this response was dependent on the tactile environment of the root, it most likely resulted from the superposition of the waving/coiling phenomenon onto the gravitropic response. We found that the frequency of the waving pattern and circumnutation, a cyclic endogenous pattern of root growth, was the same in rgr1 and in the wild type, so the waving/coiling phenomenon is likely governed by circumnutation patterns. The amplitudes of these oscillations may then be selectively amplified by tactile stimulation to provide a directional preference to the slanting.

Light Promotion of Hypocotyl Gravitropism of a Starch-Deficient Tobacco Mutant Correlates with Plastid Enlargement and Sedimentation1

Dark-grown hypocotyls of a starch-deficient mutant (NS458) of tobacco (Nicotiana sylvestris) lack amyloplasts and plastid sedimentation, and have severely reduced gravitropism. However, gravitropism improved dramatically when NS458 seedlings were grown in the light. To determine the extent of this improvement and whether mutant hypocotyls contain sedimented amyloplasts, gravitropic sensitivity (induction time and intermittent stimulation) and plastid size and position in the endodermis were measured in seedlings grown for 8 d in the light. Light-grown NS458 hypocotyls were gravitropic but were less sensitive than the wild type (WT). Starch occupied 10% of the volume of NS458 plastids grown in both the light and the dark, whereas WT plastids were essentially filled with starch in both treatments. Light increased plastid size twice as much in the mutant as in the WT. Plastids in light-grown NS458 were sedimented, presumably because of their larger size and greater total starch content. The induction by light of plastid sedimentation in NS458 provides new evidence for the role of plastid mass and sedimentation in stem gravitropic sensing. Because the mutant is not as sensitive as the WT, NS458 plastids may not have sufficient mass to provide full gravitropic sensitivity.

Structural and Biophysical Characterization of Variants of the Mechanosensitive Channel of Large Conductance (MSCL)

The ability to sense mechanical force is vital to all organisms to interact with and respond to stimuli in their environment. Mechanosensation is critical to many physiological functions such as the senses of hearing and touch in animals, gravitropism in plants and osmoregulation in bacteria. Of these processes, the best understood at the molecular level involve bacterial mechanosensitive channels. Under hypo-osmotic stress, bacteria are able to alleviate turgor pressure through mechanosensitive channels that gate directly in response to tension in the membrane lipid bilayer. A key participant in this response is the mechanosensitive channel of large conductance (MscL), a non-selective channel with a high conductance of ~3 nS that gates at tensions close to the membrane lytic tension.

It has been appreciated since the original discovery by C. Kung that the small subunit size (~130 to 160 residues) and the high conductance necessitate that MscL forms a homo-oligomeric channel. Over the past 20 years of study, the proposed oligomeric state of MscL has ranged from monomer to hexamer. Oligomeric state has been shown to vary between MscL homologues and is influenced by lipid/detergent environment. In this thesis, we report the creation of a chimera library to systematically survey the correlation between MscL sequence and oligomeric state to identify the sequence determinants of oligomeric state. Our results demonstrate that although there is no combination of sequences uniquely associated with a given oligomeric state (or mixture of oligomeric states), there are significant correlations. In the quest to characterize the oligomeric state of MscL, an exciting discovery was made about the dynamic nature of the MscL complex. We found that in detergent solution, under mild heating conditions (37 °C – 60 °C), subunits of MscL can exchange between complexes, and the dynamics of this process are sensitive to the protein sequence.

Extensive efforts were made to produce high diffraction quality crystals of MscL for the determination of a high resolution X-ray crystal structure of a full length channel. The surface entropy reduction strategy was applied to the design of S. aureus MscL variants and while the strategy appears to have improved the crystallizability of S. aureus MscL, unfortunately the diffraction qualities of these crystals were not significantly improved. MscL chimeras were also screened for crystallization in various solubilization detergents, but also failed to yield high quality crystals.

MscL is a fascinating protein and continues to serve as a model system for the study of the structural and functional properties of mechanosensitive channels. Further characterization of the MscL chimera library will offer more insight into the characteristics of the channel. Of particular interest are the functional characterization of the chimeras and the exploration of the physiological relevance of intercomplex subunit exchange.

小麦TaRanBP基因功能的初步分析

小G蛋白（small GTPases）是真核生物中广泛存在的一类调节各种生命活动的信号分子。根据结构与功能的不同，小G蛋白家族成员可分成五个亚家族，分别为Ras，Rab，Rho，Arf和Ran。五类小G蛋白通过其活化态（GTP结合态）和非活化态（GDP结合态）的相互转换行使着各种功能。Ras GTPases在酵母和哺乳动物中调节细胞增殖过程; Rho GTPases调控肌动蛋白重组过程，并参与MAP 激酶的细胞信号转导过程等; Rab GTPases和Arf GTPases分别在膜转运过程中起着不同的重要作用;而Ran GTPases则在核孔位置调节着蛋白和RNA分子的运输过程。 小G蛋白附属蛋白调节着小G蛋白活化态与非活化态之间的转换，其中鸟核苷酸交换因子(guanine nucleotide exchange factors, GEFs)可以催化小G蛋白转换为GTP结合形式，即活化态;而GTPase 激活蛋白(GTPase-activating proteins, GAPs)和小G蛋白结合蛋白(small GTPases binding proteins)可以激活小G蛋白自身的水解活性，从而将其转变成非活化态形式。 相比其它小G蛋白，Ran GTPases及其附属蛋白在真核生物中的研究相对较少。已有的成果表明它们主要在核质运输过程中及对相应的信号转导途径起调节作用。而针对Ran GTPases及其附属蛋白在真核生物尤其是高等植物个体发育过程中的作用，目前报道还很少。 为了揭示Ran结合蛋白(Ran binding protein, RanBP)在植物发育过程中的作用，本文通过转基因手段对其功能进行 了研究。在此之前，本实验室已从小麦cDNA文库中成功克隆Ran结合蛋白基因：TaRanBP。该基因cDNA全长1035 bp，编码207个氨基酸。通过农杆菌介导叶圆片法，分别用正义、反义及TaRanBP与GFP融合蛋白等表达载体转化烟草，并成功获得转基因植株。亚细胞定位观察发现TaRanBP蛋白主要定位于细胞质内，尤其是在核膜附近富集。生理学和细胞学等方面的研究分析发现，TaRanBP基因在烟草个体发育过程中产生重要作用。过量表达TaRanBP基因的转基因植株在一定数量上表现出愈合的花冠筒上出现不同程度开裂，花冠筒上有附生舌状花瓣，及带有花瓣状颜 色的花萼等异常花表型。同时，转反义基因在一定程度上促进了转基因植株初生主根的生长（为对照烟草的2.3倍），而转正义基因烟草与对照烟草的初生主根长度差异不明显。用碘化丙锭(Propidium Iodide, PI)进行根部细胞染色。观察发现，不同的转基因烟草与对照烟草之间在根的各个不同形态区域的细胞大小差异不明显，推测根长的差异可能是由于整体细胞数目变化的原因导致。向重力性实验发现，转反义基因烟草幼苗较对照烟草的向重力性反应增加，而转正义基因的则表现为降低。激素吲哚乙酸(Indoleacetic Acid, IAA)的添加处理可以恢复转反义基因烟草的向重力性异常表型，而对转正义基因烟草几乎无影响。添加激动素(Kinetin, KT)的处理发现不同转基因烟草和对照烟草的向重力性均有减弱。观测后期，转正义基因的向重力敏感性较对照烟草得到恢复。测量不同转基因株系T1代幼苗鲜重，发现不同转基因烟草和对照烟草的幼苗鲜重动态变化在各个时间点有差异，且差异情况不尽相同。而不同转基因幼苗T1代幼苗可溶性蛋白含量较对照烟草有不同程度的下降。这种下降并没有影响转基因烟草的整体生长进程，开花期和结实情况与对照烟草相比也无明显变化。

Regulation of woody plant development by gibberellin catabolic and signaling genes

Gibberellin (GA) is a growth promoting hormone implicated in regulating a diversity of plant processes. This dissertation examines the role of GA metabolic and signaling genes in woody plant growth and development. Transgenic modifications, expression analysis, physiological/biochemical assays, biometric measurements and histological analysis were used to understand the regulatory roles these genes play in the model woody plant, Populus. Our results highlight the importance of GA regulatory genes in woody perennial growth, including: phenology, wood formation, phenotypic plasticity, and growth/survival under field conditions. We characterize two putative Populus orthologs of the SHORT INTERNODES (SHI) gene from Arabidopsis, a negative regulator of GA signaling. RNAi-mediated suppression of Populus SHI-like genes increased several growth-related traits, including extent of xylem proliferation, in a dose-dependent manner. Three Populus genes, sharing sequence homology to the positive regulator of GA signaling gene PHOTOPERIOD-RESPONSIVE 1 (PHOR1) from Solanum, are up-regulated in GA-deficient and insensitive plants suggesting a conserved role in GA signaling. We demonstrate that Populus PHOR1-like genes have overlapping and divergent function(s). Two PHOR1-like genes are highly expressed in roots, predominantly affect root growth (e.g., morphology, starch quantity and gravitropism), and induced by short-days (SD). The other PHOR1-like gene is ubiquitously expressed with a generalized function in root and shoot development. The effects of GA catabolic and signaling genes on important traits (e.g., adaptive and productivity traits) were studied in a multi-year field trial. Transgenics overexpressing GA 2-oxidase (GA2ox) and DELLA genes showed tremendous variation in growth, form, foliage, and phenology (i.e., vegetative and reproductive). Observed gradients in trait modifications were correlated to transgene expression levels, in a manner suggesting a dose-dependent relationship. We explore GA2ox and DELLA genes involvement in mediating growth responses to immediate short-term drought stress, and SD photoperiods, signaling prolonged periods of stress (e.g., winter bud dormancy). GA2ox and DELLA genes show substantial up-regulation in response to drought and SDs. Transgenics overexpressing homologs of these genes subjected to drought and SD photoperiods show hypersensitive growth restraint and increased stress resistances. These results suggest growth cessation (i.e., dormancy) in response to adverse conditions is mediated by GA regulatory genes.

Regulation of woody plant development by auxin and polar auxin transport

Auxin is a key regulator in plant growth and development. This dissertation examines the role of auxin and polar auxin transport in woody growth and development. Strategies of promoter reporter system, microarray expression analysis, transgenic modification, physiological assays, anatomical analysis, and histochemical/biochemical assays were employed to improve our understanding of auxin study in Populus. The results demonstrate various aspects of auxin regulation on shoot growth, root development, wood formation, and gravitropism in woody tissues. We describe the behavior of the DR5 reporter system for measuring auxin concentrations and response in stably transformed Populus trees. Our study shows that DR5 reporter system can be efficiently used in Populus to study auxin biology at a cellular resolution. We investigated the global gene expression in responding to auxin in Populus root. The results revealed groups of IBA up- and down- regulated genes involved in various biological processes including cell wall modification, root growth and lateral root formation, transporter activity and hormone crosstalk. We also verify two of the identified genes' function by transgenic modification in Populus, which encode auxin efflux carrier PtPIN9 and transcription factor PtERF72. We investigated the role of PtPIN9 in woody growth and development, especially in wood formation and gravitropic response in woody stem. We found that overexpressing PtPIN9 enhanced several growth parameters while suppression of PtPIN9 has inhibited tension wood formation. Our results show that PIN9 and other members from PIN family could be possible useful tools for increasing biomass productivity, wood quality, or in modifying plant form.

The Arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589–1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

Curvature Induced by Amyloplast Magnetophoresis in Protonemata of the Moss Ceratodon purpureus1

After gravistimulation of Ceratodon purpureus (Hedw.) Brid. protonemata in the dark, amyloplast sedimentation was followed by upward curvature in the wild-type (WT) and downward curvature in the wwr mutant (wrong way response). We used ponderomotive forces induced by high-gradient magnetic fields (HGMF) to simulate the effect of gravity and displace the presumptive statoliths. The field was applied by placing protonemata either between two permanent magnets at the edge of the gap, close to the edge of a magnetized ferromagnetic wedge, or close to a small (<1 mm) permanent magnet. Continuous application of an HGMF in all three configurations resulted in plastid displacement and induced curvature in tip cells of WT and wwr protonemata. WT cells curved toward the HGMF, and wwr cells curved away from the HGMF, comparable to gravitropism. Plastids isolated from protonemal cultures had densities ranging from 1.24 to 1.38 g cm−3. Plastid density was similar for both genotypes, but the mutant contained larger plastids than the WT. The size difference might explain the stronger response of the wwr protonemata to the HGMF. Our data support the plastid-based theory of gravitropic sensing and suggest that HGMF-induced ponderomotive forces can substitute for gravity.

NPH4, a Conditional Modulator of Auxin-Dependent Differential Growth Responses in Arabidopsis1

Although sessile in nature, plants are able to use a number of mechanisms to modify their morphology in response to changing environmental conditions. Differential growth is one such mechanism. Despite its importance in plant development, little is known about the molecular events regulating the establishment of differential growth. Here we report analyses of the nph4 (nonphototropic hypocotyl) mutants of Arabidopsis that suggest that the NPH4 protein plays a central role in the modulation of auxin-dependent differential growth. Results from physiological studies demonstrate that NPH4 activity is conditionally required for a number of differential growth responses, including phototropism, gravitropism, phytochrome-dependent hypocotyl curvature, apical hook maintenance, and abaxial/adaxial leaf-blade expansion. The nph4 mutants exhibited auxin resistance and severely impaired auxin-dependent gene expression, indicating that the defects associated with differential growth likely arise because of altered auxin responsiveness. Moreover, the auxin signaling events mediating phototropism are genetically correlated with the abundance of the NPH4 protein.

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis1

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m−2 s−1 of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.

Mapping the Functional Roles of Cap Cells in the Response of Arabidopsis Primary Roots to Gravity1

The cap is widely accepted to be the site of gravity sensing in roots because removal of the cap abolishes root curvature. Circumstantial evidence favors the columella cells as the gravisensory cells because amyloplasts (and often other cellular components) are polarized with respect to the gravity vector. However, there has been no functional confirmation of their role. To address this problem, we used laser ablation to remove defined cells in the cap of Arabidopsis primary roots and quantified the response of the roots to gravity using three parameters: time course of curvature, presentation time, and deviation from vertical growth. Ablation of the peripheral cap cells and tip cells did not alter root curvature. Ablation of the innermost columella cells caused the strongest inhibitory effect on root curvature without affecting growth rates. Many of these roots deviated significantly from vertical growth and had a presentation time 6-fold longer than the controls. Among the two inner columella stories, the central cells of story 2 contributed the most to root gravitropism. These cells also exhibited the largest amyloplast sedimentation velocities. Therefore, these results are consistent with the starch-statolith sedimentation hypothesis for gravity sensing.

Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes

The function of the extracytoplasmic AUXIN-BINDING-PROTEIN1 (ABP1) is largely enigmatic. We complemented a homozygous T-DNA insertion null mutant of ABP1 in Arabidopsis thaliana Wassilewskia with three mutated and one wild-type (wt) ABP1 cDNA, all tagged C-terminally with a strepII-FLAG tag upstream the KDEL signal. Based on in silico modelling, the abp1 mutants were predicted to have altered geometries of the auxin binding pocket and calculated auxin binding energies lower than the wt. Phenotypes linked to auxin transport were compromised in these three complemented abp1 mutants. Red light effects, such as elongation of hypocotyls in constant red (R) and far-red (FR) light, in white light supplemented by FR light simulating shade, and inhibition of gravitropism by R or FR, were all compromised in the complemented lines. Using auxin-or light-induced expression of marker genes, we showed that auxininduced expression was delayed already after 10 min, and light-induced expression within 60 min, even though TIR1/AFB or phyB are thought to act as receptors relevant for gene expression regulation. The expression of marker genes in seedlings responding to both auxin and shade showed that for both stimuli regulation of marker gene expression was altered after 10-20 min in the wild type and phyB mutant. The rapidity of expression responses provides a framework for the mechanics of functional interaction of ABP1 and phyB to trigger interwoven signalling pathways.

AUXIN-BINDING-PROTEIN1 (ABP1) in phytochrome-B-controlled responses

The auxin receptor ABP1 directly regulates plasma membrane activities including the number of PIN-formed (PIN) proteins and auxin efflux transport. Red light (R) mediated by phytochromes regulates the steady-state level of ABP1 and auxin-inducible growth capacity in etiolated tissues but, until now, there has been no genetic proof that ABP1 and phytochrome regulation of elongation share a common mechanism for organ elongation. In far red (FR)-enriched light, hypocotyl lengths were larger in the abp1-5 and abp1/ABP1 mutants, but not in tir1-1, a null mutant of the TRANSPORT-INHIBITOR-RESPONSE1 auxin receptor. The polar auxin transport inhibitor naphthylphthalamic acid (NPA) decreased elongation in the low R: FR light-enriched white light (WL) condition more strongly than in the high red: FR light-enriched condition WL suggesting that auxin transport is an important condition for FR-induced elongation. The addition of NPA to hypocotyls grown in R-and FR-enriched light inhibited hypocotyl gravitropism to a greater extent in both abp1 mutants and in phyB-9 and phyA-211 than the wild-type hypocotyl, arguing for decreased phytochrome action in conjunction with auxin transport in abp1 mutants. Transcription of FR-enriched light-induced genes, including several genes regulated by auxin and shade, was reduced 3-5-fold in abp1-5 compared with Col and was very low in abp1/ABP1. In the phyB-9 mutant the expression of these reporter genes was 5-15-fold lower than in Col. In tir1-1 and the phyA-211 mutants shade-induced gene expression was greatly attenuated. Thus, ABP1 directly or indirectly participates in auxin and light signalling.