Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings

Lateral root formation is profoundly affected by auxins. Here we present data which indicate that light influences the formation of indole-3-acetic acid (IAA) in germinating Arabidopsis seedlings. IAA transported from the developing leaves to the root system is detectable as a short-lived pulse in the roots and is required for the emergence of the lateral root primordia (LRP) during early seedling development. LRP emergence is inhibited by the removal of apical tissues prior to detection of the IAA pulse in the root, but this treatment has minimal effects on LRP initiation. Our results identify the first developing true leaves as the most likely source for the IAA required for the first emergence of the LRP, as removal of cotyledons has only a minor effect on LRP emergence in contrast to removal of the leaves. A basipetal IAA concentration gradient with high levels of IAA in the root tip appears to control LRP initiation, in contrast to their emergence. A significant increase in the ability of the root system to synthesize IAA is observed 10 days after germination, and this in turn is reflected in the reduced dependence of the lateral root emergence on aerial tissue-derived auxin at this stage. We propose a model for lateral root formation during early seedling development that can be divided into two phases: (i) an LRP initiation phase dependent on a root tip-localized IAA source, and (ii) an LRP emergence phase dependent on leaf-derived IAA up to 10 days after germination.     

A novel root gravitropism mutant of Arabidopsis thaliana exhibiting altered auxin physiology

A root gravitropism mutant was isolated from the DuPont Arabidopsis thaliana T-DNA insertional mutagenesis collection. This mutant has reduced root gravitropism, hence the name rgrl. Roots of rgrl are shorter than those of wild-type, and they have reduced lateral root formation. In addition, roots of rgrl coil clockwise on inclined agar plates, unlike wild-type roots which grow in a wavy pattern. The rgrl mutant has increased resistance, as measured by root elongation, to exogenously applied auxins (6-fold to indole-3-acetic acid, 3-fold to 2,4-dichlorophenoxyacetic acid, and 2-fold to napthyleneacetic acid). It is also resistant to polar auxin transport inhibitors (2-fold to triiodobenzoic acid and 3- to 5-fold lo napthyleneacetic acid). The rgrl mutant does not appear to be resistant to other plant hormone classes. When grown in the presence of 10^?2 M 2.4-dichlorophenoxyacetic acid, rgrl roots have fewer root hairs than wild type. All these rgrl phenotypes are Mendelian recessives. Complementation tests indicate that rgrl is not allelic to previously characterized agravitropic or auxin-resistant mutants. The rgrl locus was mapped using visible markers to 1.4 ± 0.6 map units from the CHI locus at 1–65.4. The rgrl mutation and the T-DNA cosegregate, suggesting that rgrl was caused by insertional gene inactivation.     

Adaptation to high salinity in poplar involves changes in xylem anatomy and auxin physiology

To investigate the physiological basis of salt adaptation in poplar, we compared the effect of salt stress on wood anatomy and auxin physiology of the salt-resistant Populus euphratica and salt-sensitive Populus x canescens. Both poplar species showed decreases in vessel lumina associated with increases in wall strength in response to salt, however, in P. euphratica at three-fold higher salt concentrations than in P. x canescens. The predicted hydraulic conductivity of the wood formed under salt stress decreased in P. x canescens, while in P. euphratica, no significant effects of salt on conductivity and transpiration were observed. The concentration of free indole-3-acetic acid (IAA) decreased under salt stress in the xylem of both poplar species, but to a larger extent in P. x canescens than in P. euphratica. Only salt-treated P. euphratica exhibited an increase in IAA-conjugates in the xylem. Genes homologous to the auxin-amidohydrolase ILL3 were isolated from the xylems of P. euphratica and P. x canescens. For functional analysis, the auxin-amidohydrolase from P. x canescens was overexpressed in Arabidopsis. Transgenic Arabidopsis plants were more resistant to salt stress than the wild-type plants. Increased sensitivity of the transgenic Arabidopsis to IAA-Leu showed that the encoded hydrolase used IAA-Leu as a substrate. These results suggest that poplar can use IAA-amidoconjugates in the stem as a source of auxin to balance the effects of salt stress on auxin physiology.     

Molecular cloning and characterization of Arabidopsis thaliana Golgi alpha-mannosidase II, a key enzyme in the formation of complex N-glycans in plants

N-glycosylation is one of the major post-translational modifications of proteins in eukaryotes; however, the processing reactions of oligomannosidic N-glycan precursors leading to hybrid-type and finally complex-type N-glycans are not fully understood in plants. To investigate the role of Golgi alpha-mannosidase II (GMII) in the formation of complex N-glycans in plants, we identified a putative GMII from Arabidopsis thaliana (AtGMII; EC 3.2.1.114) and characterized the enzyme at a molecular level. The putative AtGMII cDNA was cloned, and its deduced amino acid sequence revealed a typical type II membrane protein of 1173 amino acids. A soluble recombinant form of the enzyme produced in insect cells was capable of processing different physiologically relevant hybrid N-glycans. Furthermore, a detailed N-glycan analysis of two AtGMII knockout mutants revealed the predominant presence of unprocessed hybrid N-glycans. These results provide evidence that AtGMII plays a central role in the formation of complex N-glycans in plants. Furthermore, conclusive evidence was obtained that alternative routes in the conversion of hybrid N-glycans to complex N-glycans exist in plants. Transient expression of N-terminal AtGMII fragments fused to a GFP reporter molecule demonstrated that the transmembrane domain and 10 amino acids from the cytoplasmic tail are sufficient to retain a reporter molecule in the Golgi apparatus and that lumenal sequences are not involved in the retention mechanism. A GFP fusion construct containing only the transmembrane domain was predominantly retained in the ER, a result that indicates the presence of a motif promoting ER export within the last 10 amino acids of the cytoplasmic tail of AtGMII.     

In Arabidopsis, the spatial and dynamic organization of the endoplasmic reticulum and Golgi apparatus is influenced by the integrity of the C-terminal domain of RHD3, a non-essential GTPase

The mechanisms underlying the organization and dynamics of plant endomembranes are largely unknown. Arabidopsis RHD3, a distant member of the dynamin superfamily, has recently been implicated in plant ER morphology and Golgi movement through analyses of dominant-negative mutants of the putative GTPase domain in a heterologous system. Whether RHD3 is indispensable for ER architecture and what role regions other than the putative GTPase domain play in RHD3 function are unanswered questions. Here we characterized an EMS mutant, gom8, with disrupted Golgi movement and positioning and compromised ER shape and dynamics. gom8 mapped to a missense mutation in the RHD3 hairpin loop domain, causing accumulation of the mutant protein into large structures, a markedly different distribution compared with wild-type RHD3 over the ER network. Despite the GOM8 distribution, tubules fused in the peripheral ER of the gom8 mutant. These data imply that integrity of the hairpin region is important for the subcellular distribution of RHD3, and that reduced availability of RHD3 over the ER can cause ER morphology defects, but does not prevent peripheral fusion between tubules. This was confirmed by evidence that gom8 was phenocopied in an RHD3 null background. Furthermore, we established that the region encompassing the RHD3 hairpin domain and the C-terminal cytosolic domain is necessary for RHD3 function. We conclude that RHD3 is important in ER morphology, but is dispensable for peripheral ER tubulation in an endogenous context, and that its activity relies on the C-terminal region in addition to the GTPase domain.     

A novel subtilisin-like protease gene from Arabidopsis thaliana is expressed at sites of lateral root emergence.

Differential screening of a cDNA library for mRNA species that specifically accumulate during auxin-induced lateral root formation in Arabidopsis thaliana led to the isolation of the AIR3 cDNA clone. The corresponding single copy gene consists of 10 exons which encode a protein that possesses all the characteristics of subtilisin-like proteases. The promoter of the AIR3 gene was fused to the gusA (beta-glucuronidase) reporter gene and introduced into Arabidopsis. Expression was almost completely restricted to the outer layers of the parental root at sites of lateral root emergence and could be observed even before protrusion of the newly formed root tip. In the presence of external auxin, GUS activity was visible throughout the parts of the root that are competent for lateral root formation. By digesting structural proteins in the extracellular matrix of cells located above sites of lateral root formation, AIR3 might weaken cell-to-cell connections and thus facilitate lateral root emergence.     

Gibberellins inhibit adventitious rooting in hybrid aspen and Arabidopsis by affecting auxin transport

Knowledge of processes involved in adventitious rooting is important to improve both fundamental understanding of plant physiology and the propagation of numerous plants. Hybrid aspen (Populus tremula × tremuloïdes) plants overexpressing a key gibberellin (GA) biosynthesis gene (AtGA20ox1) grow rapidly but have poor rooting efficiency, which restricts their clonal propagation. Therefore, we investigated the molecular basis of adventitious rooting in Populus and the model plant Arabidopsis. The production of adventitious roots (ARs) in tree cuttings is initiated from the basal stem region, and involves the interplay of several endogenous and exogenous factors. The roles of several hormones in this process have been characterized, but the effects of GAs have not been fully investigated. Here, we show that a GA treatment negatively affects the numbers of ARs produced by wild‐type hybrid aspen cuttings. Furthermore, both hybrid aspen plants and intact Arabidopsis seedlings overexpressing AtGA20ox1, PttGID1.1 or PttGID1.3 genes (with a 35S promoter) produce few ARs, although ARs develop from the basal stem region of hybrid aspen and the hypocotyl of Arabidopsis. In Arabidopsis, auxin and strigolactones are known to affect AR formation. Our data show that the inhibitory effect of GA treatment on adventitious rooting is not mediated by perturbation of the auxin signalling pathway, or of the strigolactone biosynthetic and signalling pathways. Instead, GAs appear to act by perturbing polar auxin transport, in particular auxin efflux in hybrid aspen, and both efflux and influx in Arabidopsis.     

Elongation and gravitropic responses of Arabidopsis roots are regulated by brassinolide and IAA

Exogenously applied brassinolide (BL) increased both gravitropic curvature and length of primary roots of Arabidopsis at low concentration (10(-10) M), whereas at higher concentration, BL further increased gravitropic curvature while it inhibited primary root growth. BRI1-GFP plants possessing a high steady-state expression level of a brassinosteroid (BR) receptor kinase rendered the plant's responses to gravity and root growth more sensitive, while BR-insensitive mutants, bri1-301 and bak1, delayed root growth and reduced their response to the gravitropic stimulus. The stimulatory effect of BL on the root gravitropic curvature was also enhanced in auxin transport mutants, aux1-7 and pin2, relative to wild-type plants, and increasing concentration of auxin attenuated BL-induced root sensitivity to gravity. Interestingly, IAA treatment to the roots of bri1-301 and bak1 plants or of plants pretreated with a BL biosynthetic inhibitor, brassinazole, increased their sensitivity to gravity, while these treatments for the BL-hypersensitive transgenic plants, BRI1-GFP and 35S-BAK1, were less effective. Expression of a CYP79B2 gene, encoding an IAA biosynthetic enzyme, was suppressed in BL-hypersensitive plant types and enhanced in BL-insensitive or -deficient plants. In conclusion, our results indicate that BL interacts negatively with IAA in the regulation of plant gravitropic response and root growth, and its regulation is achieved partly by modulating biosynthetic pathways of the counterpart hormone.     

拟南芥矮小丛生突变体的分离与分子鉴定

顶端优势是指侧生分生组织的生长被主茎或主花序所抑制。最近的研究通过分离和鉴定顶端优势发生改变的突变体开始揭示顶端优势的分子机制。通过T-DNA标签法分离了拟南芥矮小丛生(bushy and dwarf l,budl)突变体。突变体植株的表型包括顶端优势丧失、株型矮小,表明budl突变体存在生长素代谢、运输或信号传导的缺陷。一个对生长素特异反应的启动子驱动的报告基因在budl中表达模式改变。生长素敏感性和运输能力的测定表明这两个过程在budl中均正常。以上结果显示budl表型是生长素代谢缺陷的结果。遗传分析表明BUDI为半显性突变且与一个T-DNA插入共分离,可通过iPCR方法分离。     

拟南芥抗旱转录因子CBF4基因区域的核苷酸多样性及其分子进化分析

以生长于不同气候条件下的17个拟南芥核心生态型为材料,分析了它们的抗旱转录因子CBF4基因区域的序列多态性。结果表明：拟南芥CBF4基因区域具有高密度的单核苷酸多态性(SNP)和插入缺失(Indel),多态性频率为每35．8bp一个SNP,每143bp一个Indel,基因非编码区的多态性是编码区的4倍;在编码区,SNP的频率为每96．4bp一个SNP,其中发现25av、203av和244av 3个生态型CBF4基因区域1034位(以Gen—Bank登录号AB015478序列第19696位的核苷酸为1)碱基变化：G←→T,引起第205位氨基酸变化：gly←→val。核苷酸多样性统计分析显示,该基因内部大范围内存在连锁不平衡(linkage disequilibrium,LD),5’端非编码区有一个重组。与拟南芥等的研究结果类似,选择压力对不同的区域作用不同。3’端非编码区核苷酸多样性程度最高,是平衡性选择的结果,编码区核苷酸变化符合中性突变假说,而5’端非编码区是自然选择作用的靶位点。     

Arabidopsis vacuolar H-ATPase subunit E isoform 1 is required for Golgi organization and vacuole function in embryogenesis

Vacuolar H(+)-ATPases play an important role in maintaining the pH of endomembrane compartments in eukaryotic cells. The functional relevance of this homeostasis for multicellular development has not been studied in plants. Here, we analyze the biological consequences resulting from the lack of subunit E isoform 1 (VHA-E1) encoded by the Arabidopsis TUFF gene. tuff mutant embryos are lethal, displaying variably enlarged cells with multiple nuclei, large vacuoles containing inclusions, abnormal organization of Golgi stacks, and cell wall defects. Rescue of embryo lethality by cell cycle-regulated expression of VHA-E1 results in abnormal seedlings with non-functional meristems and defective cell differentiation. VHA-E1 is the predominant isoform in embryogenesis whereas VHA-E3 is expressed mainly in the endosperm and surrounding maternal tissues during seed development, and VHA-E2 is pollen-specific. VHA-E1 protein accumulates at endomembrane compartments including vacuoles and endosomes, but appears absent from the plasma membrane. Our results suggest an essential role for VHA-E1 in maintaining a functional secretory system during somatic development but not in the haploid gametophytes.     

Differential IAA dose response relations of the axr1 and axr2 mutants of Arabidopsis

In comparison to wild type Arabidopsis thaliana, the auxin resistant mutants axr1 and axr2 exhibit reduced inhibition of root elongation in response to auxins. Several auxin-regulated physiological processes are also altered in the mutant plants. When wild-type, axr1 and axr2 seedlings were grown in darkness on media containing indoleacetic acid (IAA), promotion of root growth was observed at low concentrations of IAA (10^?11 to 10^?7M) in 5-day-old axr2 seedlings, but not in axr1 or wild-type seedlings. In axr1 there was little or no measurable root growth response over the same concentration range. In wild type, root growth was inhibited at concentrations greater than 10^?10M and no detectable root growth response was observed at lower concentrations. In addition, production of lateral roots in response to IAA increased in axr2 seedlings and decreased in axr1 seedlings relative to wild type. Promotion of root elongation and initiation of lateral roots in axr2 seedlings in response to auxin indicate that axr2 seedlings are able to perceive and respond to IAA.     

铵减弱拟南芥主根向地性及其相关作用途径

向地性是决定植物根系空间构型的主要因素之一,对植物锚定和水分养分吸收至关重要。除了重力,根系向地性还受土壤环境因子影响。本文采用琼脂培养方法,研究了铵对拟南芥主根向地性反应的影响及相关作用途径。结果表明：短期内,不同浓度（NH4）2SO4均显著抑制主根向地性弯曲,但随着时间的延长,根尖向地性角度逐渐变小。而等（NH4）2SO4浓度的NaCl对主根向地性抑制效应较小,不同浓度的甘露醇不阻碍主根向地性弯曲。纽织化学染色结果显示铵处理12h以内,Col-0根尖没有淀粉体的快速降解过程,并且铵对淀粉体缺失突变体pgm—1主根向地性的影响同Col-0相似。铵处理部分恢复生长素转运载体突变体auxl-22和eir1-1主根向地性缺失。这些结果表明,铵对拟南芥主根向地性的影响独立于根尖淀粉体参与的重力感应途径。     

The Arabidopsis concentration-dependent influx/efflux transporter ABCB4 regulates cellular auxin levels in the root epidermis

Arabidopsis ATP-binding cassette B4 (ABCB4) is a root-localised auxin efflux transporter with reported auxin uptake activity in low auxin concentrations. Results reported here demonstrate that ABCB4 is a substrate-activated regulator of cellular auxin levels. The contribution of ABCB4 to shootward auxin movement at the root apex increases with auxin concentration, but in root hair elongation assays ABCB4-mediated uptake is evident at low concentrations as well. Uptake kinetics of ABCB4 heterologously expressed in Schizosaccharomyces pombe differed from the saturation kinetics of AUX1 as uptake converted to efflux at threshold indole-3-acetic acid (IAA) concentrations. The concentration dependence of ABCB4 appears to be a direct effect on transporter activity, as ABCB4 expression and ABCB4 plasma membrane (PM) localisation at the root apex are relatively insensitive to changes in auxin concentration. However, PM localization of ABCB4 decreases with 1-naphthylphthalamic acid (NPA) treatment. Unlike other plant ABCBs studied to date, and consistent with decreased detergent solubility, ABCB4(pro) :ABCB4-GFP is partially internalised in all cell types by 0.05% DMSO, but not 0.1% ethanol. In trichoblasts, ABCB4(pro) :ABCB4-GFP PM signals are reduced by >200 nm IAA and 2,4-dichlorophenoxyacetic acid (2,4-D). In heterologous systems and in planta, ABCB4 transports benzoic acid with weak affinity, but not the oxidative catabolism products 2-oxindole-3-acetic-acid and 2-oxindole-3-acetyl-β-D-glucose. ABCB4 mediates uptake, but not efflux, of the synthetic auxin 2,4-D in cells lacking AUX1 activity. Results presented here suggest that 2,4-D is a non-competitive inhibitor of IAA transport by ABCB4 and indicate that ABCB4 is a target of 2,4-D herbicidal activity.     

Fertilization-dependent auxin response in ovules triggers fruit development through the modulation of gibberellin metabolism in Arabidopsis

Fruit development is usually triggered by ovule fertilization, and it requires coordination between seed development and the growth and differentiation of the ovary to host the seeds. Hormones are known to synchronize these two processes, but the role of each hormone, and the mechanism by which they interact, are still unknown. Here we show that auxin and gibberellins (GAs) act in a hierarchical scheme. The synthetic reporter construct DR5:GFP showed that fertilization triggered an increase in auxin response in the ovules, which could be mimicked by blocking polar auxin transport. As the application of GAs did not affect auxin response, the most likely sequence of events after fertilization involves auxin-mediated activation of GA synthesis. We have confirmed this, and have shown that GA biosynthesis upon fertilization is localized specifically in the fertilized ovules. Furthermore, auxin treatment caused changes in the expression of GA biosynthetic genes similar to those triggered by fertilization, and also restricted to the ovules. Finally, GA signaling was activated in ovules and valves, as shown by the rapid downregulation of the fusion protein RGA-GFP after pollination and auxin treatment. Taken together, this evidence suggests a model in which fertilization would trigger an auxin-mediated promotion of GA synthesis specifically in the ovule. The GAs synthesized in the ovules would be then transported to the valves to promote GA signaling and thus coordinate growth of the silique.