Arabidopsis ASA1 Is Important for Jasmonate-Mediated Regulation of Auxin Biosynthesis and Transport during Lateral Root Formation

Plant roots show an impressive degree of plasticity in adapting their branching patterns to ever-changing growth conditions. An important mechanism underlying this adaptation ability is the interaction between hormonal and developmental signals. Here, we analyze the interaction of jasmonate with auxin to regulate lateral root (LR) formation through characterization of an Arabidopsis thaliana mutant, jasmonate-induced defective lateral root1 (jdl1/asa1-1). We demonstrate that, whereas exogenous jasmonate promotes LR formation in wild-type plants, it represses LR formation in jdl1/asa1-1. JDL1 encodes the auxin biosynthetic gene ANTHRANILATE SYNTHASE α1 (ASA1), which is required for jasmonate-induced auxin biosynthesis. Jasmonate elevates local auxin accumulation in the basal meristem of wild-type roots but reduces local auxin accumulation in the basal meristem of mutant roots, suggesting that, in addition to activating ASA1-dependent auxin biosynthesis, jasmonate also affects auxin transport. Indeed, jasmonate modifies the expression of auxin transport genes in an ASA1-dependent manner. We further provide evidence showing that the action mechanism of jasmonate to regulate LR formation through ASA1 differs from that of ethylene. Our results highlight the importance of ASA1 in jasmonate-induced auxin biosynthesis and reveal a role for jasmonate in the attenuation of auxin transport in the root and the fine-tuning of local auxin distribution in the root basal meristem.     

Characterisation of root amino acid exudation in white clover (Trifolium repens L.)

Lateral roots are crucial for the plasticity of root responses to environmental conditions in soil. The bacterivorous microfauna has been shown to increase root branching and to foster auxin producing soil bacteria. However, information on modifications of plant internal auxin content by soil bacteria and bacterivores is missing. Therefore, the effects of a rhizosphere bacterial community and a common soil amoeba (Acanthamoeba castellanii) on root branching and on auxin (indole-3-acetic acid) metabolism in Lepidium sativum and Arabidopsis thaliana were investigated. In a first experimental series, bacteria increased conjugated auxin concentrations in L. sativum shoots, but did not alter free bioactive auxin content nor root branching. In contrast, in presence of soil bacteria plus amoebae free auxin concentrations in shoots and root branching increased, demonstrating that effects of bacteria on auxin metabolism in plants were strongly modified by the bacterivorous amoebae. In a second experiment, A. thaliana reporter plants for auxin (DR5) and cytokinin (ARR5) responded similarly with increased root branching in the presence of amoebae. Surprisingly, in reporter plants cytokinin but not auxin responses were detectable, accompanied by higher soil nitrate concentrations in the presence of amoebae. Likely, increased nitrate concentrations in the rhizosphere led to an accumulation of cytokinin and interactions with free auxin in plants and finally to increased root growth in the presence of amoebae. Altogether, the results show that mutual control mechanisms exist between plant hormone metabolism and microbial signalling, and that effects on hormonal concentrations of plants by free-living bacteria are strongly influenced by bacterial grazers like amoebae.     

Auxin quantification based on histochemical staining of GUS under the control of auxin-responsive promoter

To visualize phytohormone localization in plant tissues, transgenic plants comprising the GUS reporter gene are often used. However, until now only qualitative assessment of the hormone presence was available. In this work, we suggested the method for IAA quantification in transgenic DR5::GUS Arabidopsis thaliana L. plants by the analysis of digital images. An empirical quadratic dependence was established between the IAA concentration in medium and the level of GUS-dependent staining. Using this method, we demonstrated that, after A. thaliana root gravistimulation for 90 min, auxin lateral redistribution occurred. It resulted in the increase in the IAA concentration in the lower root part (in the elongation zone and apical meristem) by 200% on the average.     

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme

Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole-3-acetonitrile to the plant growth hormone, indole-3-acetic acid in vitro. To probe the capacity of this enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV-35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg^?1 protein with indole3-acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1-[¹³C]-indole-3-acetonitrile to 1-[¹³C]-indole-3-acetic acid in vivo as determined by HPLC/ GC-MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole-3-acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole-3-acetonitrile, a strong auxin-overproducing phenotype developed resulting in increased lateral root formation (at 10 µM indole-3-acetonitrile) or stunted shoot growth, excessive lateral root initiation, inhibition of root out-growth and callus formation at the root/shoot interface (at 100 µM indole-3-acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole-3-acetonitrile to indole-3-acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high-auxin phenotype upon transgenic plants. Thus, the A. thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.     

MdARF8: An Auxin Response Factor Involved in Jasmonate Signaling Pathway in Malus domestica

Auxin response factor (ARF) is a key component of auxin signal. The study of MdARF8 gene in apple shows that it is involved in the process of jasmonate regulating plant growth and development. Methyl jasmonate (MeJA) treatment inhibited the growth process of apple calli, and ARF8 played a negative regulatory role in this pathway. The results of ectopic expression in Arabidopsis showed that MdARF8 could reduce the sensitivity of Arabidopsis to MeJA and alleviate the phenotype of promoting leaf senescence and inhibiting taproot elongation. Further results showed that the dysplastic phenotype of transgenic Arabidopsis root hair could be partially recovered by MeJA treatment. This study provided valuable clues for functional characterization of ARF8 and signal crosstalk between jasmonate and auxin in apple.

     

Aeromonas punctata PNS-1: a promising candidate to change the root morphogenesis of Arabidopsis thaliana in MS and sand system

A model system of sand, comprising Arabidopsis plants inoculated with Aeromonas punctata PNS-1 strain, was used to evaluate the bacterial effect in modulation of plant root structure at second-order lateral root level. In MS media, the root morphogenesis was changed only at first-order lateral root level when inoculated with PNS-1 strain. Inoculation with PNS-1 strain was subjected to significant (P < 0.01) increase in primary root length and lateral root density in both MS and sand system. However, this strain modulated the root structure in the sand environment in a complex manner that may be helpful for incitation of the plant–microbe interaction close to natural environment. In order to determine whether this change in root morphology was due to bacterial auxin, Arabidopsis transgenic line (DR5:GUS) was used to reveal the change in homeostasis of endogenous auxin. In PNS-1 inoculated transgenic seedlings of Arabidopsis plant (DR5:GUS), endogenous auxin in primary root apices and lateral roots was enhanced. For confirmation, PNS-1 was evaluated for auxin production in vitro, showed an increase in auxin production after supplementation of l-tryptophan. The presence of ACC deaminase activity in PNS-1 showed its possible involvement in primary root elongation. In the present study Aeromonas punctata PNS-1 is the potential candidate for triggering the change in root morphogenesis of Arabidopsis thaliana with the involvement of auxin and ACC deaminase production.     

The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass

Switchgrass (Panicum virgatum L.; family Poaceae) is a warm-season C4 perennial grass. Tillering plays an important role in determining the morphology of aboveground parts and the final biomass yield of switchgrass. Auxin distribution in plants can affect a variety of important growth and developmental processes, including the regulation of shoot and root branching, plant resistance and biological yield. Auxin transport and gradients in plants are mediated by influx and efflux carriers. PvPIN1, a switchgrass PIN1-like gene that is involved in regulating polar transport, is a putative auxin efflux carrier. Neighbor-joining analysis using sequences deposited in NCBI databases showed that the PvPIN1gene belongs to the PIN1 family and is evolutionarily closer to the Oryza sativa japonica group. Tiller emergence and development was significantly promoted in plants subjected toPvPIN1 RNA interference (RNAi), which yielded a phenotype similar to that of wild-type plants treated with the auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid). A transgenic approach that inducedPvPIN1 gene overexpression or suppression altered tiller number and the shoot/root ratio. These data suggest that PvPIN1plays an important role in auxin-dependent adventitious root emergence and tillering.     

Quantitative analysis of IAA in DR5::GUS transgenic arabidopsis plants

In the study of auxin transport, transgenic constructs, including DR5::GUS, are widely used for visualization of phytohormone localization. Previously we proposed a method for quantitative evaluation of the IAA content by histochemical staining for glucuronidase activity. In this work, this method was complemented by quantitative data on the content of IAA in plants obtained by gas chromatography-mass spectrometry (GC/MS), which allowed more accurate characterization of the lateral IAA gradient arising at the Arabidopsis thaliana (L.) Heynh (ecotype Columbia 0) root gravistimulation. Applied method of IAA analysis, combining GC/MS and histochemistry, can be used for quantitatification of the other plant hormone distribution in transgenic plants with the GUS reporter.     

A Unique Phenotype in Heterozygotes of the Auxin-Insensitive Mutant of Tomato, diageotropica

Tomato (Lycopersicon esculentum Mill.) plants heterozygous for the diageotropica (dgt) mutation exhibit a unique phenotype, termed `mottled.' Unlike dgt, mottled individuals grow upright, exhibit normal root branching, and produce normal levels of ethylene in response to applied auxin. Leaves of mottled plants are deformed and reduced in size and are characterized by a mottled appearance on their surfaces with small dark-green islands clustered along the leaf veins. The lack of phenotypic overlap between dgt and mottled may represent interallelic interaction at a locus which influences auxin sensitivity or action in the tomato.     

Regulation of root morphogenesis in arbuscular mycorrhizae: what role do fungal exudates,phosphate, sugars and hormones play in lateral root formation?

Background

Arbuscular mycorrhizae (AMs) form a widespread root–fungus symbiosis that improves plant phosphate (Pi) acquisition and modifies the physiology and development of host plants. Increased branching is recognized as a general feature of AM roots, and has been interpreted as a means of increasing suitable sites for colonization. Fungal exudates, which are involved in the dialogue between AM fungi and their host during the pre-colonization phase, play a well-documented role in lateral root (LR) formation. In addition, the increased Pi content of AM plants, in relation to Pi-starved controls, as well as changes in the delivery of carbohydrates to the roots and modulation of phytohormone concentration, transport and sensitivity, are probably involved in increasing root system branching.

Scope

This review discusses the possible causes of increased branching in AM plants. The differential root responses to Pi, sugars and hormones of potential AM host species are also highlighted and discussed in comparison with those of the non-host Arabidopsis thaliana.

Conclusions

Fungal exudates are probably the main compounds regulating AM root morphogenesis during the first colonization steps, while a complex network of interactions governs root development in established AMs. Colonization and high Pi act synergistically to increase root branching, and sugar transport towards the arbusculated cells may contribute to LR formation. In addition, AM colonization and high Pi generally increase auxin and cytokinin and decrease ethylene and strigolactone levels. With the exception of cytokinins, which seem to regulate mainly the root:shoot biomass ratio, these hormones play a leading role in governing root morphogenesis, with strigolactones and ethylene blocking LR formation in the non-colonized, Pi-starved plants, and auxin inducing them in colonized plants, or in plants grown under high Pi conditions.     

Composite Cucurbita pepo plants with transgenic roots as a tool to study root development

Background and Aims

In most plant species, initiation of lateral root primordia occurs above the elongation zone. However, in cucurbits and some other species, lateral root primordia initiation and development takes place in the apical meristem of the parental root. Composite transgenic plants obtained by Agrobacterium rhizogenes-mediated transformation are known as a suitable model to study root development. The aim of the present study was to establish this transformation technique for squash.

Methods

The auxin-responsive promoter DR5 was cloned into the binary vectors pKGW-RR-MGW and pMDC162-GFP. Incorporation of 5-ethynyl-2′-deoxyuridine (EdU) was used to evaluate the presence of DNA-synthesizing cells in the hypocotyl of squash seedlings to find out whether they were suitable for infection. Two A. rhizogenes strains, R1000 and MSU440, were used. Roots containing the respective constructs were selected based on DsRED1 or green fluorescent protein (GFP) fluorescence, and DR5::Egfp-gusA or DR5::gusA insertion, respectively, was verified by PCR. Distribution of the response to auxin was visualized by GFP fluorescence or β-glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively.

Key Results

Based on the distribution of EdU-labelled cells, it was determined that 6-day-old squash seedlings were suited for inoculation by A. rhizogenes since their root pericycle and the adjacent layers contain enough proliferating cells. Agrobacterium rhizogenes R1000 proved to be the most virulent strain on squash seedlings. Squash roots containing the respective constructs did not exhibit the hairy root phenotype and were morphologically and structurally similar to wild-type roots.

Conclusions

The auxin response pattern in the root apex of squash resembled that in arabidopsis roots. Composite squash plants obtained by A. rhizogenes-mediated transformation are a good tool for the investigation of root apical meristem development and root branching.     

Expression of the Full‐length Coat Protein Gene of Tomato leaf curl Taiwan virus is Not Necessary for Recovery Phenotype in Transgenic Tomato

Transgenic tomato plants expressing full‐length (CPV1) and truncated coat protein (CP) gene (CPV2) of Tomato leaf curl Taiwan virus (ToLCTWV) were generated by Agrobacterium‐mediated transformation. Transgene integration and expression was confirmed by PCR and Southern blotting and Northern analysis, respectively. Resistance was evaluated both in plants of T0 and T1 progenies using viruliferous whiteflies under two different inoculum pressures (10–15 and 40–50 whiteflies/plant). Upon inoculation with ToLCTWV using viruliferous whiteflies, various levels of phenotypic reaction were observed. No complete resistance was observed in any of the plants tested. The reaction of the transgenic tomato lines carrying full‐length and truncated CP gene to ToLCTWV phenotype was (i) susceptible as non‐transgenic control, (ii) delayed symptom expression, (iii) complete susceptible (from delayed symptom expression phenotype) and (iv) recovered phenotype (either plants from symptom expression as non‐transgenic plants or delayed symptom expression phenotype). Dot blot quantification of the ToLCTWV using the replicase gene as a probe revealed that the recovered phenotypes accumulated a low level of ToLCTWV, and virus concentration was gradually reduced from 10 to 14 weeks postinoculation. The possible mechanisms of CP‐mediated resistance are discussed.     

Inhibition of primary roots and stimulation of lateral root development in Arabidopsis thaliana by the rhizobacterium Serratia marcescens 90–166 is through both auxin-dependent and -independent signaling pathways

The rhizobacterium Serratia marcescens strain 90–166 was previously reported to promote plant growth and induce resistance in Arabidopsis thaliana. In this study, the influence of strain 90-166 on root development was studied in vitro. We observed inhibition of primary root elongation, enhanced lateral root emergence, and early emergence of second order lateral roots after inoculation with strain 90–166 at a certain distance from the root. Using the DR5::GUS transgenic A. thaliana plant and an auxin transport inhibitor, N-1-naphthylphthalamic acid, the altered root development was still elicited by strain 90–166, indicating that this was not a result of changes in plant auxin levels. Intriguingly, indole-3-acetic acid, a major auxin chemical, was only identified just above the detection limit in liquid culture of strain 90–166 using liquid chromatography-mass spectrometry. Focusing on bacterial determinants of the root alterations, we found that primary root elongation was inhibited in seedlings treated with cell supernatant (secreted compounds), while lateral root formation was induced in seedlings treated with lysate supernatant (intracellular compounds). Further study revealed that the alteration of root development elicited by strain 90–166 involved the jasmonate, ethylene, and salicylic acid signaling pathways. Collectively, our results suggest that strain 90–166 can contribute to plant root development via multiple signaling pathways.     

The role of AUX1 gene and auxin content to the branching phenotype of Kenaf (Hibiscus cannabinus L.)

The objectives of this research were to identify auxin gene, AUX1, and to determine the plant auxin content and their role in conferring branching on Kenaf. PCR analysis using AUX1 primer capable to amplify the DNA of non branching (KR11) and branching kenaf mutant, resulting in 800 bp PCR product. The sequence of the PCR product showed high degree of homology with the sequence of AUX1 gene of other plants in the NCBI GenBank database, confirming kenaf possession of the gene AUX1. However, some variation on the DNA sequence was found between branching and non branching phenotype indicated allele differences of the same gene which were responsible for the variation in the type of branching. Identification of auxin content in the roots, apical shoot, and axillary branches using spectrophotometry method showed that the branching plant has higher auxin content in the apical shoot compared to the content in the branches. This indicate that AUX1 controls the formation of branches by controlling either the content of auxin in the apical shoot and branches, or the ratio of auxin content in the shoot and branches.     

Geminivirus C4 protein alters Arabidopsis development

The C4 protein of beet curly top virus [BCTV-B (US:Log:76)] induces hyperplasia in infected phloem tissue and tumorigenic growths in transgenic plants. The protein offers an excellent model for studying cell cycle control, cell differentiation, and plant development. To investigate the role of the C4 protein in plant development, transgenic Arabidopsis thaliana plants were generated in which the C4 transgene was expressed under the control of an inducible promoter. A detailed analysis of the developmental changes that occur in cotyledons and hypocotyls of seedlings expressing the C4 transgene showed extensive cell division in all tissues types examined, radically altered tissue layer organization, and the absence of a clearly defined vascular system. Induced seedlings failed to develop true leaves, lateral roots, and shoot and root apical meristems, as well as vascular tissue. Specialized epidermis structures, such as stomata and root hairs, were either absent or developmentally impaired in seedlings that expressed C4 protein. Exogenous application of brassinosteroid and abscisic acid weakly rescued the C4-induced phenotype, while induced seedlings were hypersensitive to gibberellic acid and kinetin. These results indicate that ectopic expression of the BCTV C4 protein in A. thaliana drastically alters plant development, possibly through the disruption of multiple hormonal pathways.     

Agrobacterium T‐DNA‐encoded protein Atu6002 interferes with the host auxin response

Several genes in the Agrobacterium tumefaciens transferred (T)‐DNA encode proteins that are involved in developmental alterations, leading to the formation of tumours in infected plants. We investigated the role of the protein encoded by the Atu6002 gene, the function of which is completely unknown. Atu6002 expression occurs in Agrobacterium‐induced tumours, and is also activated on activation of plant cell division by growth hormones. Within the expressing plant cells, the Atu6002 protein is targeted to the plasma membrane. Interestingly, constitutive ectopic expression of Atu6002 in transgenic tobacco plants leads to a severe developmental phenotype characterized by stunted growth, shorter internodes, lanceolate leaves, increased branching and modified flower morphology. These Atu6002‐expressing plants also display impaired response to auxin. However, auxin cellular uptake and polar transport are not significantly inhibited in these plants, suggesting that Atu6002 interferes with auxin perception or signalling pathways.