Free IAA Levels Research Articles

Alternate bearing in 'Hass' avocado: fruit load-induced changes in bud auxin homeostasis are associated with flowering repression.

In 'Hass' avocado (Persea americana), fruit presence reduces next season flowering. Recent fruit tree studies proposed that heavy fruit load (HFL) generates an auxin (indole-3-acetic acid, IAA) signal in the buds that represses flowering. However, the nature of this signal remains unknown. Here, we investigated the effect of avocado HFL on bud IAA accumulation and flowering transition. We found that IAA-aspartate and IAA-glutamate conjugate levels were significantly higher in buds from fully loaded ('on') than low-loaded ('off') trees, hinting that free IAA levels were higher in the former. Expression analysis showed that coinciding with flowering reduction, HFL induced the floral repressor PaTFL1, and suggested that accumulation of IAA in buds as imposed by HFL was associated with its conjugation to aspartate and glutamate and resulted both from de novo IAA synthesis and from reduced IAA export. Accordingly, experiments involving radiolabelled [14C]IAA demonstrated that HFL reduced shoot basipetal IAA transport. Finally, we confirmed the negative effects of IAA on flowering, showing that IAA and polar auxin transport blocker (2,3,5-triiodobenzoic acid) treatments delayed 'off' trees' inflorescence development, reducing their inflorescence axis and inducing PaTFL1 expression. Together, our data indicate that avocado HFL generates IAA signalling in buds that induces PaTFL1, leading to repression of inflorescence development.

The GH3 Protein from the Clubroot Pathogen Plasmodiophora brassicae Causes Hormone-Related Phenotypes in Arabidopsis thaliana

In the genome of the obligate biotrophic protist Plasmodiophora brassicae, which causes clubroot disease in the Brassicaceae family, a single gene was identified with homology to the group of GH3 proteins synthesizing amino acid conjugates with carboxylic acids. In comparison to plant members the P. brassicae GH3 protein seems to be very promiscuous with respect to the substrates converted. In vitro assays showed conversion of three different auxins (indole-3-acetic, IAA; indole-propionic, IPA; and indole-3-butyric acids, IBA), jasmonic acid (JA) and also 12-oxo-phytodienoic acid (OPDA). Auxin and Methyl-JA responsive elements were found in the promoter sequence of PbGH3. For further analysis Arabidopsis thaliana was transformed with PbGH3. Seedlings with confirmed constitutive PbGH3 protein production were grown to adult plants that were shown to produce fewer inflorescences per plant. Growth on 10 µM IAA or JA levels also altered the phenotype of the seedlings, but not so much between wild types and transgenic lines. The hormone profiles in seedlings and adult plants were examined for IAA, JA, OPDA, and respective conjugates. Transgenic seedlings displayed changes that could be partially attributed to the overexpression of a GH3 gene like lower levels of free IAA and the JA precursor cis-OPDA under control conditions, and higher levels of some auxin conjugates accumulated after IAA treatment compared to the corresponding wild types. However, the hormone patterns in adult plants were more complex and varied based on different tissue types analyzed. After inoculation with P. brassicae the transgenic plants did not show any changes in infection rate and disease index, but had higher shoot and lower root weight in infected plants compared to controls. Based on the above findings we discuss a role for PbGH3 during pathogenesis.

Physcomitrium patens Mutants in Auxin Conjugating GH3 Proteins Show Salt Stress Tolerance but Auxin Homeostasis Is Not Involved in Regulation of Oxidative Stress Factors.

Salt stress is among the most challenging abiotic stress situations that a plant can experience. High salt levels do not only occur in areas with obvious salty water, but also during drought periods where salt accumulates in the soil. The moss Physcomitrium patens became a model for studying abiotic stress in non-vascular plants. Here, we show that high salt concentrations can be tolerated in vitro, and that auxin homeostasis is connected to the performance of P. patens under these stress conditions. The auxin levels can be regulated by conjugating IAA to amino acids by two members of the family of GH3 protein auxin amino acid-synthetases that are present in P. patens. Double GH3 gene knock-out mutants were more tolerant to high salt concentrations. Furthermore, free IAA levels were differentially altered during the time points investigated. Since, among the mutant lines, an increase in IAA on at least one NaCl concentration tested was observed, we treated wild type (WT) plants concomitantly with NaCl and IAA. This experiment showed that the salt tolerance to 100 mM NaCl together with 1 and 10 µM IAA was enhanced during the earlier time points. This is an additional indication that the high IAA levels in the double GH3-KO lines could be responsible for survival in high salt conditions. While the high salt concentrations induced several selected stress metabolites including phenols, flavonoids, and enzymes such as peroxidase and superoxide dismutase, the GH3-KO genotype did not generally participate in this upregulation. While we showed that the GH3 double KO mutants were more tolerant of high (250 mM) NaCl concentrations, the altered auxin homeostasis was not directly involved in the upregulation of stress metabolites.

Difference in Profiles of the Gut-Derived Tryptophan Metabolite Indole Acetic Acid between Transplanted and Non-Transplanted Patients with Chronic Kidney Disease.

Background: Uremic toxins have emerged as potential mediators of morbidity and mortality in patients with chronic kidney disease (CKD). Indole-3-acetic acid (IAA, a tryptophan-derived uremic toxin) might be a useful biomarker in patients with CKD. The objectives of the present study were to (i) describe IAA concentrations in a cohort of non-transplanted patients with CKD and a cohort of transplanted patients with CKD, and (ii) investigate the possible relationship between IAA levels and adverse outcomes in the two cohorts. Methods: Levels of free and total IAA were assayed in the two prospective CKD cohorts (140 non-transplanted patients and 311 transplanted patients). Cox multivariate analyses were used to evaluate the association between IAA levels and outcomes (mortality, cardiovascular events, and graft loss). Results: In the non-transplanted CKD cohort, free and total IAA increased progressively with the CKD stage. In the transplanted CKD cohort, free and total IAA levels were elevated at the time of transplantation but had fallen substantially at one-month post-transplantation. Indole acetic acid concentrations were lower in transplanted patients than non-dialysis non-transplanted patients matched for estimated glomerular filtration rate (eGFR), age, and sex. After adjustment for multiple confounders, the free IAA level predicted overall mortality and cardiovascular events in the non-transplanted CKD cohort (hazard ratio [95% confidence interval]: 2.5 [1.2–5.1] and 2.5 [1.3–4.8], respectively). In the transplanted CKD cohort, however, no associations were found between free or total IAA on one hand, and mortality, CV event, or graft survival on the other. Conclusion: We demonstrated that levels of IAA increase with the CKD stage, and fall substantially, even normalizing, after kidney transplantation. Free IAA appears to be a valuable outcome-associated biomarker in non-transplanted patients, but—at least in our study setting—not in transplanted patients.

Downregulation of the auxin transporter gene SlPIN8 results in pollen abortion in tomato

SlPIN8 is expressed specifically within tomato pollen, and that it is involved in tomato pollen development and intracellular auxin homeostasis. The auxin (IAA) transport protein PIN-FORMED (PIN) plays key roles in various aspects of plant development. The biological role of the auxin transporter SlPIN8 in tomato development remains unclear. Here, we examined the expression pattern of the SlPIN8 gene in vegetative and reproductive organs of tomato. RNA interference (RNAi) transgenic lines specifically silenced for the SlPIN8 gene were generated to identify the role of SlPIN8 in pollen development. We found that SlPIN8 mRNA is expressed specifically within tomato pollen. In the anthers, the highest mRNA expression and β-glucuronidase (GUS) activity of promoter-SlPIN8-GUS was detected during late stages of anther development, when pollen maturation occurred. The downregulation of SlPIN8 did not drastically affect the vegetative growth of tomato. However, in SlPIN8-RNAi transgenic plants, approximately 80% of the pollen grains were identified to be abnormal and lack viability; they were shriveled and flattened. Furthermore, the downregulation of SlPIN8 affected the gene expression of some anther development-specific proteins. SlPIN8-RNAi transgenic plants induced seedless fruits because of defective pollen function rather than defective female gametophyte function. In addition, SlPIN8 was found to localize to the endoplasmic reticulum, consistent with the changes in the auxin levels of SlPIN8-RNAi lines, whereas the level of free IAA was increased in SlPIN8-overexpressing protoplasts, indicating that SlPIN8 is involved in intracellular auxin homeostasis.

Transgenic poplar expressing Arabidopsis YUCCA6 exhibits auxin-overproduction phenotypes and increased tolerance to abiotic stress

YUCCA6, a member of the YUCCA family of flavin monooxygenase-like proteins, is involved in the tryptophan-dependent IAA biosynthesis pathway and responses to environmental cues in Arabidopsis. However, little is known about the role of the YUCCA pathway in auxin biosynthesis in poplar. Here, we generated transgenic poplar (Populus alba × P. glandulosa) expressing the Arabidopsis YUCCA6 gene under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SY plants). Three SY lines (SY7, SY12 and SY20) were selected based on the levels of AtYUCCA6 transcript. SY plants displayed auxin-overproduction morphological phenotypes, such as rapid shoot growth and retarded main root development with increased root hair formation. In addition, SY plants had higher levels of free IAA and early auxin-response gene transcripts. SY plants exhibited tolerance to drought stress, which was associated with reduced levels of reactive oxygen species. Furthermore, SY plants showed delayed hormone- and dark-induced senescence in detached leaves due to higher photosystem II efficiency and less membrane permeability. These results suggest that the conserved IAA biosynthesis pathway mediated by YUCCA family members exists in poplar.

Tiller formation in rice is altered by overexpression of OsIAGLU gene encoding an IAA-conjugating enzyme or exogenous treatment of free IAA

Optimization of plant architecture is important for cultivation and yield of cereal crops in the field. Tillering is an essential factor used to determine the overall architecture of cereal crops. It has long been recognized that the development of branching patterns is controlled by the level and distribution of auxin within a plant. To better understand the relationship between auxin levels and tillering in rice, we examined rice plants with increased or decreased levels of free IAA. To decrease IAA levels, we selected the rice IAA-glucose synthase gene (OsIAGLU) from the rice genome database based on high sequence homology with IAA-glucose synthase from maize (ZmIAGLU), which is known to generate IAAglucose conjugate from free IAA. The OsIAGLU gene driven by the Cauliflower Mosaic Virus 35S promoter was transformed into a rice cultivar to generate transgenic rice plants constitutively over-expressing this gene. The number of tillers and panicles significantly increased in the transgenic lines compared to the wild-type plants, while plant height and panicle length decreased. These results indicate that decreased levels of free IAA likely enhance tiller formation in rice. To increase levels of free IAA, we treated rice plants with three different concentrations of exogenous IAA (1 μM, 10 μM and 100 μM) twice a week by spraying. Exogenous IAA treatment at concentrations of 10 μM and 100 μM significantly reduced tiller number in three different rice cultivars. These results indicate that exogenously applied IAA inhibits shoot branching in rice. Overall, auxin tightly controls tiller formation in rice in a negative way.

Impaired Auxin Biosynthesis in the defective endosperm18 Mutant Is Due to Mutational Loss of Expression in the ZmYuc1 Gene Encoding Endosperm-Specific YUCCA1 Protein in Maize

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.

The endoplasmic reticulum localized PIN8 is a pollen‐specific auxin carrier involved in intracellular auxin homeostasis

The plant hormone auxin is a mobile signal which affects nuclear transcription by regulating the stability of auxin/indole-3-acetic acid (IAA) repressor proteins. Auxin is transported polarly from cell to cell by auxin efflux proteins of the PIN family, but it is not as yet clear how auxin levels are regulated within cells and how access of auxin to the nucleus may be controlled. The Arabidopsis genome contains eight PINs, encoding proteins with a similar membrane topology. While five of the PINs are typically targeted polarly to the plasma membranes, the smallest members of the family, PIN5 and PIN8, seem to be located not at the plasma membrane but in endomembranes. Here we demonstrate by electron microscopy analysis that PIN8, which is specifically expressed in pollen, resides in the endoplasmic reticulum and that it remains internally localized during pollen tube growth. Transgenic Arabidopsis and tobacco plants were generated overexpressing or ectopically expressing functional PIN8, and its role in control of auxin homeostasis was studied. PIN8 ectopic expression resulted in strong auxin-related phenotypes. The severity of phenotypes depended on PIN8 protein levels, suggesting a rate-limiting activity for PIN8. The observed phenotypes correlated with elevated levels of free IAA and ester-conjugated IAA. Activation of the auxin-regulated synthetic DR5 promoter and of auxin response genes was strongly repressed in seedlings overexpressing PIN8 when exposed to 1-naphthalene acetic acid. Thus, our data show a functional role for endoplasmic reticulum-localized PIN8 and suggest a mechanism whereby PIN8 controls auxin thresholds and access of auxin to the nucleus, thereby regulating auxin-dependent transcriptional activity.

PIF4–Mediated Activation of YUCCA8 Expression Integrates Temperature into the Auxin Pathway in Regulating Arabidopsis Hypocotyl Growth

Higher plants adapt their growth to high temperature by a dramatic change in plant architecture. It has been shown that the transcriptional regulator phytochrome-interacting factor 4 (PIF4) and the phytohormone auxin are involved in the regulation of high temperature–induced hypocotyl elongation in Arabidopsis. Here we report that PIF4 regulates high temperature–induced hypocotyl elongation through direct activation of the auxin biosynthetic gene YUCCA8 (YUC8). We show that high temperature co-upregulates the transcript abundance of PIF4 and YUC8. PIF4–dependency of high temperature–mediated induction of YUC8 expression as well as auxin biosynthesis, together with the finding that overexpression of PIF4 leads to increased expression of YUC8 and elevated free IAA levels in planta, suggests a possibility that PIF4 directly activates YUC8 expression. Indeed, gel shift and chromatin immunoprecipitation experiments demonstrate that PIF4 associates with the G-box–containing promoter region of YUC8. Transient expression assay in Nicotiana benthamiana leaves support that PIF4 directly activates YUC8 expression in vivo. Significantly, we show that the yuc8 mutation can largely suppress the long-hypocotyl phenotype of PIF4–overexpression plants and also can reduce high temperature–induced hypocotyl elongation. Genetic analyses reveal that the shy2-2 mutation, which harbors a stabilized mutant form of the IAA3 protein and therefore is defective in high temperature–induced hypocotyl elongation, largely suppresses the long-hypocotyl phenotype of PIF4–overexpression plants. Taken together, our results illuminate a molecular framework by which the PIF4 transcriptional regulator integrates its action into the auxin pathway through activating the expression of specific auxin biosynthetic gene. These studies advance our understanding on the molecular mechanism underlying high temperature–induced adaptation in plant architecture.

Distribution and change patterns of free IAA, ABP 1 and PM H +-ATPase during ovary and ovule development of Nicotiana tabacum L.

Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H +-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H +-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H +-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H +-ATPase may play roles in the ovary and ovule initiation, formation and differentiation.

YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana.

The Arabidopsis thaliana YUCCA family of flavin monooxygenase proteins catalyses a rate-limiting step in de novo auxin biosynthesis. A YUCCA6 activation mutant, yuc6-1D, has been shown to contain an elevated free IAA level and to display typical high-auxin phenotypes. It is reported here that Arabidopsis plants over-expressing YUCCA6, such as the yuc6-1D activation mutant and 35S:YUC6 transgenic plants, displayed dramatic longevity. In addition, plants over-expressing YUCCA6 exhibited classical, delayed dark-induced and hormone-induced senescence in assays using detached rosette leaves. However, plants over-expressing an allele of YUCCA6, that carries mutations in the NADPH cofactor binding site, exhibited neither delayed leaf senescence phenotypes nor phenotypes typical of auxin overproduction. When the level of free IAA was reduced in yuc6-1D by conjugation to lysine, yuc6-1D leaves senesced at a rate similar to the wild-type leaves. Dark-induced senescence in detached leaves was accompanied by a decrease in their free IAA content, by the reduced expression of auxin biosynthesis enzymes such as YUCCA1 and YUCCA6 that increase cellular free IAA levels, and by the increased expression of auxin-conjugating enzymes encoded by the GH3 genes that reduce the cellular free auxin levels. Reduced transcript abundances of SAG12, NAC1, and NAC6 during senescence in yuc6-1D compared with the wild type suggested that auxin delays senescence by directly or indirectly regulating the expression of senescence-associated genes.

Micropropagation of Wild Service Tree (Sorbus torminalis [L.] Crantz): The Regulative Role of Different Aromatic Cytokinins During Organogenesis

The influences of three different aromatic cytokinin derivatives [6-benzylaminopurine, meta-topolin, and 6-(3-methoxybenzylamino)purine-9-s-D-ribofuranoside (MeOBAPR)] on in vitro multiplication and rhizogenesis of the wild service tree (Sorbus torminalis [L.] Crantz) were compared. The highest micropropagation rate (24 new shoots per explant after 3 months of cultivation) was achieved on media containing BAP. On the other hand, the best rooting microcuttings were those multiplied on a medium containing MeoBAPR. To compare these results with the levels of endogenous cytokinins in multiplied explants, a newly developed UPLC-ESI(+)-MS/MS method was used to determine levels of 50 cytokinin metabolites in explants cultivated 12 weeks on media supplemented by BAP and of the two other aromatic cytokinin analogs used. Several significant differences among the levels of endogenous cytokinins, extracted from the explants, were found. The concentration of BAP9G, an important metabolite suspected to be responsible for inhibition of rooting and acclimatization problems of newly formed plantlets, was found to be the highest in microcuttings grown on media supplemented with BAP. This agrees well with the results of our rooting experiments; the lowest percentages of rooted plantlets 6 weeks after transferring shoots on rooting medium were present on explants multiplied on BAP. In contrast, BAP was still the most effective for the induction of bud formation on primary explants. Levels of the most active endogenous isoprenoid cytokinins, tZ, tZR, and iPR, as well as O-glucosides were also suppressed in explants grown on BAP compared with those of explants treated with other cytokinin derivatives. This may be the result of a very high BAP uptake into the explants grown on this cytokinin. On the other hand, endogenous concentrations of cis-zeatin derivatives as well as dihydrozeatin derivatives were not affected. Differences in the production of another plant hormone, ethylene, that plays an important role in controlling organogenesis in tissue culture, were also observed among S. torminalis plantlets grown in vitro on media containing different cytokinins tested. The highest ethylene levels were detected in the vessels containing media supplemented with mT. They were two to four times higher compared with the production by the S. torminalis explants cultivated on other media used. Finally, the levels of free IAA were also determined in the explants. S. torminalis plantlets grown on media containing BAP contained the lowest level of auxin, which is again in good agreement with their loss of rooting capacity. The results found in this study about optimal plant hormone concentrations may be used to improve in vitro rooting efficiency of the wild service tree and possibly also of other plant species.

Interaction of jasmonic acid and blue light in the regulation of arabidopsis morphogenesis

The effects of blue light (BL) and jasmonic acid (JA) on morphogenesis of Arabidopsis thaliana (L.) Heynh seedlings of genotypes Col and Ler and their mutants, namely, axr1-3 and jar1-1 mutants resistant to IAA and JA, respectively, and a CRY1 photoreceptor-deficient mutant hy4 were studied. Both 1 μM JA and BL exposure retarded hypocotyl growth of Ler, Col, and jar1-1 seedlings, whereas JA had no effect on hypocotyl growth of axr1-3, but the suppression of hypocotyl growth of this mutant by BL was even more noticeable than that of Ler, Col, and jar1-1. JA and BL applied simultaneously inhibited hypocotyl growth of axr1-3 and especially of Ler, Col, and jar1-1 more than either of factors applied separately. The hy4 mutant did not respond to BL, whereas JA stimulated its hypocotyl growth. JA did not change the cotyledon size of Col, axr1-3, and jar1-1 and reduced the cotyledon size of Ler and hy4. BL enhanced the cotyledon growth of all wild-type and mutant plants used in the study. The cotyledon sizes of all plants except Ler were also increased when JA and BL were applied together. Some of the growth responses correlated with the endogenous IAA and ABA contents. Thus, for example, the hypocotyl and cotyledon growth retardation of Ler seedlings in the presence of JA correlated with a reduced level of free IAA and a considerable increase in the free ABA level in plants grown both in darkness and in BL. Under other growth conditions, no correlation between the endogenous IAA and ABA levels and A. thaliana seedling growth was noted. The interaction between the signal transduction pathways triggered by BL and JA at the early stages of arabidopsis morphogenesis is discussed on the basis of Col, Ler, axr1-3, and jar1-1 hypocotyl growth responses.

Free IAA in stigmas and styles during pollen germination and pollen tube growth of Nicotiana tabacum

Although many studies have emphasized the importance of auxin in plant growth and development, the thorough understanding of its effect on pollen-pistil interactions is largely unknown. In this study, we investigated the role of free IAA in pollen-pistil interactions during pollen germination and tube growth in Nicotiana tabacum L. through using histo and subcellular immunolocalization with auxin monoclonal antibodies, quantification by HPLC and ELISA together with GUS staining in DR5::GUS-transformed plants. The results showed that free IAA in unpollinated styles was higher in the apical part and basal part than in the middle part, and it was more abundant in the transmitting tissue (TT). At the stage of pollen germination, IAA reached its highest content in the stigma and was mainly distributed in TT. After the pollen tubes entered the styles, the signal increased in the part where pollen tubes would enter and then rapidly declined in the part where pollen tubes had penetrated. Subcellular localization confirmed the presence of IAA in TT cells of stigmas and styles. Accordingly, a schematic diagram summarizes the changing pattern of free IAA level during flowering, pollination and pollen tube growth. Furthermore, we presented evidence that low concentration of exogenous IAA could, to a certain extent, facilitate in vitro pollen tube growth. These results suggest that IAA may be directly or indirectly involved in the pollen-pistil interactions. Additionally, some improvements of the IAA immunolocalization technique were made.

옥수수 (Zea mays) 뿌리의 알데히드 산화효소와 생장에 미치는 텅스텐산 나트륨의 영향

We tested the effect of sodium tungstate, which disturbs the molybdenum cofactor formation, on the activities of aldehyde oxidase(AO) and the growth of maize(Zea mays) primary roots. As reported in other plants, sodium tungstate inhibited AO also in the maize root concentration-dependently. The inhibitory effect of sodium tungstate was observed only when the inhibitor was applied to the living plants. Application of tungstate to the extracted protein did not show any effect. Western analysis revealed slightly decreased level of AO protein in the presence of tungstate, indicating a positive feedback of gene regulation by the product. We also tested the effects of tungstate on the root growth. The elongation of primary root and the development of lateral roots, which are sensitive to the absolute level of auxin, were decreased in the presence of sodium tungstate. However, the gravitropic curvature of the primary root, which is dependent on the relative amount of auxin at both sides, was unaffected. These data suggested the decrease of auxin biosynthesis by the application of tungstate. However, the level of free IAA was unaffected by tungstate application. We discuss the possible explanations for the observed results.

Involvement of HLS1 in Sugar and Auxin Signaling in Arabidopsis Leaves

Sugar regulates a variety of genes and controls plant growth and development similarly to phytohormones. As part of a screen for Arabidopsis mutants with defects in sugar-responsive gene expression, we identified a loss-of-function mutation in the HOOKLESS1 (HLS1) gene. HLS1 was originally identified to regulate apical hook formation of dark-grown seedlings (Lehman et al., 1996, Cell 85: 183-194). In hls1, sugar-induced gene expression in excised leaf petioles was more sensitive to exogenous sucrose than that in the wild type. Exogenous IAA partially repressed sugar-induced gene expression and concomitantly activated some auxin response genes such as AUR3 encoding GH3-like protein. The repression and the induction of gene expression by auxin were attenuated and enhanced, respectively, by the hls1 mutation. These results suggest that HLS1 plays a negative role in sugar and auxin signaling. Because AUR3 GH3-like protein conjugates free IAA to amino acids (Staswick et al., 2002, Plant Cell 14: 1405-1415; Staswick et al., 2005, Plant Cell 17: 616-627), enhanced expression of GH3-like genes would result in a decrease in the free IAA level. Indeed, hls1 leaves accumulated a reduced level of free IAA, suggesting that HLS1 may be involved in negative feedback regulation of IAA homeostasis through the control of GH3-like genes. We discuss the possible mechanisms by which HLS1 is involved in auxin signaling for sugar- and auxin-responsive gene expression and in IAA homeostasis.

Changes in endogenous auxins during winter dormancy in tea (Camellia sinensis L.) O. Kuntze

Changes in endogenous auxin (IAA) were determined in tea shoots with the onset of winter dormancy (30 October) and subsequent to dormancy release (15 April). Very low levels of free IAA were detected in dormant shoots when they were still in deep dormancy. The levels increased after 30 January and reached highest value after dormancy release. Conjugated IAA levels increased with onset of dormancy reaching its maximum value when free IAA levels were lowest. With increase in free IAA levels, the conjugated IAA levels decreased in shoots prior to dormancy release suggesting of metabolic interconversion of IAA during these periods. The possible roles of free and conjugated IAA in dormancy and following its release are discussed in relation to winter dormancy in tea shoots.

Modification in indole-3-acetic acid metabolism, growth and development of strawberry through transformation with maize IAA-glucose synthase gene (iaglu)

Strawberry (Fragaria × ananassa Duch.) was transformed with maize IAA-glucose synthase gene (iaglu) via Agrobacterium tumefaciens using binary vector system. Incorporation of the transgene was confirmed by PCR and Southern blot analysis and its transcription by RT-PCR. Transformation resulted in a significant increase of ester-conjugated IAA level in tissue of all tranegenic plants while free IAA level was significantly lower in two trangenic clones tested and in other two it was comparable to that in wild-type plants. The level of amide-conjugated hormone was not affected by transformation with iaglu. The change in endogenous IAA metabolism affected the growth and development of transgenic strawberry plants but the effect was not fully correlated with changes in endogenous IAA level. In general, the transgenic clones were dwarfish — their leaf laminas were smaller, leaf petioles shorter and the crown diameter smaller in comparison to wild-type ones, although in one clone the difference was significant only for leaf lamina area. Shoots of all transgenic clones formed more roots in vitro than the wild-type plants and in two clones the roots were longer than in the control.

Disruption and overexpression of auxin response factor 8 gene of Arabidopsis affect hypocotyl elongation and root growth habit, indicating its possible involvement in auxin homeostasis in light condition.

Auxin response factor (ARF) family genes play a central role in controlling sensitivity to the plant hormone auxin. We characterized the function of ARF8 in Arabidopsis by investigating a T-DNA insertion line (arf8-1) and overexpression lines (ARF8 OX) of ARF8. arf8-1 showed a long-hypocotyl phenotype in either white, blue, red or far-red light conditions, in contrast to ARF8 OX that displayed short hypocotyls in the light. Stronger and weaker apical dominance, and promotion and inhibition of lateral root formation were observed in arf8-1 and ARF8 OX respectively. Sensitivity to auxin was unaltered in arf8-1 hypocotyls with respect to growth inhibition caused by exogenously applied auxin and growth promotion induced by higher temperatures. ARF8 expression was observed constitutively in shoot and root apexes, and was induced in the light condition in hypocotyls. Free IAA contents were approximately 30% reduced in light-grown hypocotyls of ARF8 OX, but were similar between those of arf8-1 and wild type. Expression of the three GH3 genes was reduced in arf8-1 and increased in ARF8 OX, indicating that they are targets of ARF8 transcriptional control. Because the three GH3 proteins may be involved in the conjugation of IAA as suggested by Staswick et al. (2002), and because two of the three GH3 genes are auxin inducible, ARF8 may control the free IAA level in a negative feedback fashion by regulating GH3 gene expression. ARF family genes seem to control both auxin sensitivity and homeostasis in Arabidopsis.