Changes In Auxin Levels Research Articles

A lipid synthase maintains metabolic flux for jasmonate synthesis to regulate root growth and phosphate homeostasis.

Plants require phosphate (Pi) for proper growth and development but often face scarcity of this vital nutrient in the soil. Pi-starvation triggers membrane lipid remodeling to utilize the membrane phospholipid-bound Pi in plants. In this process, phospholipids are replaced by non-Pi-containing galactolipids (MGDG, DGDG) and sulfolipids. The galactolipids ratio (MGDG:DGDG) is suggested to influence jasmonic acid (JA) biosynthesis. However, how the MGDG:DGDG ratio, JA levels, and root growth are coordinated under Pi deficiency in rice (Oryza sativa) remains unknown. Here, we characterized DGDG synthase 1 (OsDGD1) for its role in regulating root development by maintaining metabolic flux for JA biosynthesis. We showed that OsDGD1 is responsive under low Pi and is under the direct control of Phosphate Starvation Response 2 (OsPHR2), the master regulator of low Pi adaptations. Further, OsDGD1 knockout (KO) lines showed marked phenotypic differences compared to the wild type (WT), including a significant reduction in root length and biomass, leading to reduced Pi uptake. Further, lipidome analyses revealed reduced DGDG levels in the KO line, leading to reduced membrane remodeling, thus affecting P utilization efficiency. We also observed an increase in the MGDG: DGDG ratio in KO lines, which enhanced the endogenous JA levels and signaling. This imbalance of JA in KO plants led to changes in auxin levels, causing drastic root growth inhibition. These findings indicate the critical role of OsDGD1 in maintaining optimum levels of JA during Pi deficiency for conducive root growth. Besides acting as signaling molecules and structural components, our study widens the role of lipids as metabolic flux controllers for phytohormone biosynthesis.

Molecular and cellular insights into auxin-regulated primary root growth: a comparative study of Arabidopsis and rice.

Auxin regulation of primary root growth in Arabidopsis and rice was compared by analyzing root growth in response to changes in auxin levels. A bell-shaped root-growth curve was identified in both Arabidopsis and rice in response to change in auxin levels. In Arabidopsis, cell division was the main regulator of root growth in response to auxin; in rice, auxin promoted root growth by regulating cell division and cell length. The expression levels of PLETHORA (PLT) genes in response to change in auxin level followed a bell-shaped curve and closely correlated with cell division in Arabidopsis but not in rice, implying that PLT gene expression plays key role to control root growth in Arabidopsis. The level of auxin in Arabidopsis was optimal for primary root elongation, while in rice it was higher than optimal. These differences may explain the species-dependent development of root systems.

Clade-D auxin response factors regulate auxin signaling and development in the moss Physcomitrium patens.

Auxin response factors (ARFs) are a family of transcription factors that are responsible for regulating gene expression in response to changes in auxin level. The analysis of ARF sequence and activity indicates that there are 2 major groups: activators and repressors. One clade of ARFs, clade-D, is sister to clade-A activating ARFs, but are unique in that they lack a DNA-binding domain. Clade-D ARFs are present in lycophytes and bryophytes but absent in other plant lineages. The transcriptional activity of clade-D ARFs, as well as how they regulate gene expression, is not well understood. Here, we report that clade-D ARFs are transcriptional activators in the model bryophyte Physcomitrium patens and have a major role in the development of this species. Δarfddub protonemata exhibit a delay in filament branching, as well as a delay in the chloronema to caulonema transition. Additionally, leafy gametophore development in Δarfddub lines lags behind wild type. We present evidence that ARFd1 interacts with activating ARFs via their PB1 domains, but not with repressing ARFs. Based on these results, we propose a model in which clade-D ARFs enhance gene expression by interacting with DNA bound clade-A ARFs. Further, we show that ARFd1 must form oligomers for full activity.

Dynamics of Endogenous Auxin and Its Role in Somatic Embryogenesis Induction and Progression in Cork Oak

Somatic embryogenesis (SE) is a feasible in vitro regeneration system with biotechnological applications in breeding programs, although, in many forest species, SE is highly inefficient, mainly due to their recalcitrance. On the other hand, SE represents a valuable model system for studies on cell reprogramming, totipotency acquisition, and embryogenic development. The molecular mechanisms that govern the transition of plant somatic cells to embryogenic cells are largely unknown. There is increasing evidence that auxins mediate this transition and play a key role in somatic embryo development, although data on woody species are very limited. In this study, we analyzed the dynamics and possible role of endogenous auxin during SE in cork oak (Quercus suber L.). The auxin content was low in somatic cells before cell reprogramming, while it increased after induction of embryogenesis, as revealed by immunofluorescence assays. Cellular accumulation of endogenous auxin was also detected at the later stages of somatic embryo development. These changes in auxin levels correlated with the expression patterns of the auxin biosynthesis (QsTAR2) and signaling (QsARF5) genes, which were upregulated after SE induction. Treatments with the inhibitor of auxin biosynthesis, kynurenine, reduced the proliferation of proembryogenic masses and impaired further embryo development. QsTAR2 and QsARF5 were downregulated after kynurenine treatment. Our findings indicate a key role of endogenous auxin biosynthesis and signaling in SE induction and multiplication, as well as somatic embryo development of cork oak.

Auxin homeostasis in maize (Zea mays) is regulated via 1-O-indole-3-acetyl-myo-inositol synthesis at early stages of seedling development and under abiotic stress

Main conclusionIndole-3-acetyl-myo-inositol biosynthesis is regulated during maize seedling development and in response to drought and cold stress. The main purpose of this pathway is maintenance of auxin homeostasis.Indole-3-acetic acid (IAA) conjugation to myo-inositol is a part of a mechanism controlling free auxin level in maize. In this work, we investigated changes in the indole-3-acetyl-myo-inositol (IAInos) biosynthesis pathway in 3-d- and 6-d-old maize seedlings and germinating seeds as well as in seedlings subjected to drought and cold stress to evaluate a role of this pathway in maize development and stress response. In germinating seeds, activity of the enzymes involved in IAInos biosynthesis remains unchanged between 3-d- and 6-d-old material but increases in coleoptiles and radicles of the seedlings. Under cold stress, in germinating seeds and in coleoptiles, activity of the enzymes decreases and increases, respectively; however, it does not entail changes in auxin level. In drought-exposed germinating maize seeds, totally diminished activities of IAInos synthesis pathway enzymes resulted in almost twofold increase of free IAA content. Similar increase of auxin level was observed in radicles of drought-subjected seedlings together with lack of catalytic activity of the first enzyme of the pathway. Exogenous IAInos has no effect on the level of non-enzymatic antioxidant, ascorbate. It has also either no effect on the protein carbonylation and lipid peroxidation, or it affects it in a similar way as exogenously applied IAA and myo-inositol, which are products of IAInos hydrolysis. Thus, IAInos biosynthesis pathway acts in maize development and stress responses by regulation of free IAA concentration, as IAInos itself does not appear to have a distinct role in these processes.

Role of auxin in the symbiotic relationship between Piriformospora indica and rice plants

Piriformospora indica (P. indica) is a beneficial endophytic fungus colonized with roots of a wide range of host plant species; however, the mechanisms of P. indica symbiosis with different plants could vary and need to be further addressed. Our study showed that root architecture and growth behaviors were changed in P. indica-colonized rice plants. The P. indica-induced primary root shorting and coiling phenomena were consistent with the auxin-responsive phenotype, suggesting that the auxin levels of P. indica-colonized roots were modulated. To investigate the changes in auxin levels, auxin-responsive gene expressions and promoter activities were used as indicators to monitor auxin levels in root tissues. The results showed that the auxin levels of primary roots were increased in P. indica-inoculated seedlings. Furthermore, an auxin inhibitor (p-chlorophenoxyisobutyric acid, PCIB) was applied to block auxin action to confirm whether auxin is a critical factor in regulating P. indica-induced root growth. The P. indica-induced root architecture modification was significantly repressed by PCIB which was added to the rice culture solutions at the initiation of P. indica inoculation. A proposed hypothesis was that the PCIB treatment might result in lower colonization and further reduce the influence of P. indica colonization on root architecture. The effect of PCIB on colonization was observed by counting the percent root colonization. The data showed that the colonization levels of P. indica in PCIB-treated seedlings were significantly decreased. Taken together, our data indicated that auxin levels would be increased in P. indica-colonized rice roots. Moreover, it was demonstrated that auxin has dual roles in the symbiotic relationship between rice and P. indica, which is a signal that triggers P. indica-induced root growth and a positive factor involved in the colonization mechanism.

Indole pyruvate decarboxylase gene regulates the auxin synthesis pathway in rice by interacting with the indole-3-acetic acid-amido synthetase gene, promoting root hair development under cadmium stress.

This research focused on cadmium (Cd), which negatively affects plant growth and auxin hemostasis. In plants, many processes are indirectly controlled through the expression of certain genes due to the secretion of bacterial auxin, as indole-3-acetic acid (IAA) acts as a reciprocal signaling molecule in plant-microbe interaction. The aim of current studies was to investigate responsible genes in rice for plant-microbe interaction and lateral root development due to the involvement of several metabolic pathways. Studies revealed that GH3-2 interacts with endogenous IAA in a homeostasis manner without directly providing IAA. In rice, indole-3-pyruvate decarboxylase (IPDC) transgenic lines showed a 40% increase in lateral roots. Auxin levels and YUCCA (auxin biosynthesis gene) expression were monitored in osaux1 mutant lines inoculated with Bacillus cereus exposed to Cd. The results showed an increase in root hairs (RHs) and lateral root density, changes in auxin levels, and expression of the YUCCA gene. B. cereus normalizes the oxidative stress caused by Cd due to the accumulation of and H2O2 in osaux1 mutant lines. Furthermore, the inoculation of B. cereus increases DR5:GUS expression, indicating that bacterial species have a positive role in auxin regulation. Thus, the current study suggests that B. cereus and IPDC transgenic lines increase the RH development in rice by interacting with IAA synthetase genes in the host plant, alleviating Cd toxicity and enhancing plant defense mechanisms.

AUXIN RESPONSE FACTOR 18-HISTONE DEACETYLASE 6 module regulates floral organ identity in rose (Rosa hybrida).

The phytohormone auxin plays a pivotal role in floral meristem initiation and gynoecium development, but whether and how auxin controls floral organ identity remain largely unknown. Here, we found that auxin levels influence organ specification, and changes in auxin levels influence homeotic transformation between petals and stamens in rose (Rosa hybrida). The PIN-FORMED-LIKES (PILS) gene RhPILS1 governs auxin levels in floral buds during floral organogenesis. RhAUXIN RESPONSE FACTOR 18 (RhARF18), whose expression decreases with increasing auxin content, encodes a transcriptional repressor of the C-class gene RhAGAMOUS (RhAG), and controls stamen-petal organ specification in an auxin-dependent manner. Moreover, RhARF18 physically interacts with the histone deacetylase (HDA) RhHDA6. Silencing of RhHDA6 increases H3K9/K14 acetylation levels at the site adjacent to the RhARF18-binding site in the RhAG promoter and reduces petal number, indicating that RhARF18 might recruit RhHDA6 to the RhAG promoter to reinforce the repression of RhAG transcription. We propose a model for how auxin homeostasis controls floral organ identity via regulating transcription of RhAG.

A fish scale-like magnetic nanomaterial as a highly efficient sorbent for monitoring the changes in auxin levels under cadmium stress.

Sorbents with high surface utilization and good dispersibility are of great importance for the extraction performance of magnetic solid-phase extraction (MSPE). In this study, a fish scale-like magnetic nanomaterial (Co@Co3O4/OCN) was synthesized, which can be used as a highly efficient MSPE sorbent due to its strong magnetism, special morphology, doping of N element, numerous micro-mesopore cavities and organic functional groups (hydroxyl and carboxyl). Furthermore, a Co@Co3O4/OCN-based MSPE method for monitoring the changes in the levels of three auxins (indole-3-acetic acid, indole-3-propionic acid and 3-indole butyric acid) was successfully established. Wide linear ranges (1.0-1000.0 pg mL-1) with good correlation coefficients (R > 0.9992), low limits of detection (LODs, 0.2-4.0 pg mL-1) and satisfactory repeatability (RSD ≤5.9%, n = 3) were obtained. Using the developed method, various growth parts and different growth periods of plants under Cd stress were monitored. The results showed that auxins in various parts of plants showed differential response under Cd stress, and there was a threshold for the changes in auxin levels against Cd stress. This indicates that the developed fish scale-like Co@Co3O4/OCN nanomaterial has a good application prospect for enriching small molecular targets containing hydroxyl and carboxyl groups.

Fungal-Induced Formation of Auxin Maxima in Arabidopsis thaliana Roots

Phytohormones are crucial molecules for plant development and the interaction with microbes. This study focused on the phytohormone auxin (indole-3-acetic acid, IAA) and its role in the interaction of Arabidopsis thaliana (L.) Heynh. roots with two beneficial (Piriformospora indica and Mortierella hyalina) and two pathogenic (Alternaria brassicicola and Verticillium dahliae) root-colonizing fungi. Arabidopsis plants expressing the dual reporter construct DR5::EGFP-DR5v2::tdTomato allow visualization of auxin maxima during early stages of the plant–fungus interaction. Fluorescence microscopy was used to monitor changes in auxin levels and distribution patterns. We hereby demonstrate that only the beneficial P. indica activates the IAA reporter system. M. hyalina- but not P. indica-colonized roots accumulate jasmonates which might prevent the activation of the IAA reporter system. Additionally, both the necrotrophic fungus A. brassicicola and the biotrophic fungus V. dahliae completely inhibit the fluorescence emission from the IAA reporter system within 3–6 h. The results indicate that the reporter system responds to IAA accumulation in symbiotic roots, but the activation process might be controlled by a crosstalk with other phytohormones, such as jasmonates.

Aux/IAA Gene Family in Plants: Molecular Structure, Regulation, and Function.

Auxin plays a crucial role in the diverse cellular and developmental responses of plants across their lifespan. Plants can quickly sense and respond to changes in auxin levels, and these responses involve several major classes of auxin-responsive genes, including the Auxin/Indole-3-Acetic Acid (Aux/IAA) family, the auxin response factor (ARF) family, small auxin upregulated RNA (SAUR), and the auxin-responsive Gretchen Hagen3 (GH3) family. Aux/IAA proteins are short-lived nuclear proteins comprising several highly conserved domains that are encoded by the auxin early response gene family. These proteins have specific domains that interact with ARFs and inhibit the transcription of genes activated by ARFs. Molecular studies have revealed that Aux/IAA family members can form diverse dimers with ARFs to regulate genes in various ways. Functional analyses of Aux/IAA family members have indicated that they have various roles in plant development, such as root development, shoot growth, and fruit ripening. In this review, recently discovered details regarding the molecular characteristics, regulation, and protein–protein interactions of the Aux/IAA proteins are discussed. These details provide new insights into the molecular basis of the Aux/IAA protein functions in plant developmental processes.

Early Arabidopsis root hair growth stimulation by pathogenic strains of Pseudomonas syringae.

Selected beneficial Pseudomonas spp. strains have the ability to influence root architecture in Arabidopsis thaliana by inhibiting primary root elongation and promoting lateral root and root hair formation. A crucial role for auxin in this long-term (1week), long-distance plant-microbe interaction has been demonstrated. Arabidopsis seedlings were cultivated in vitro on vertical plates and inoculated with pathogenic strains Pseudomonas syringae pv. maculicola (Psm) and P. syringae pv. tomato DC3000 (Pst), as well as Agrobacterium tumefaciens (Atu) and Escherichia coli (Eco). Root hair lengths were measured after 24 and 48h of direct exposure to each bacterial strain. Several Arabidopsis mutants with impaired responses to pathogens, impaired ethylene perception and defects in the exocyst vesicle tethering complex that is involved in secretion were also analysed. Arabidopsis seedling roots infected with Psm or Pst responded similarly to when infected with plant growth-promoting rhizobacteria; root hair growth was stimulated and primary root growth was inhibited. Other plant- and soil-adapted bacteria induced similar root hair responses. The most compromised root hair growth stimulation response was found for the knockout mutants exo70A1 and ein2. The single immune pathways dependent on salicylic acid, jasmonic acid and PAD4 are not directly involved in root hair growth stimulation; however, in the mutual cross-talk with ethylene, they indirectly modify the extent of the stimulation of root hair growth. The Flg22 peptide does not initiate root hair stimulation as intact bacteria do, but pretreatment with Flg22 prior to Psm inoculation abolished root hair growth stimulation in an FLS2 receptor kinase-dependent manner. These early response phenomena are not associated with changes in auxin levels, as monitored with the pDR5::GUS auxin reporter. Early stimulation of root hair growth is an effect of an unidentified component of living plant pathogenic bacteria. The root hair growth response is triggered in the range of hours after bacterial contact with roots and can be modulated by FLS2 signalling. Bacterial stimulation of root hair growth requires functional ethylene signalling and an efficient exocyst-dependent secretory machinery.

Alteration in Auxin Homeostasis and Signaling by Overexpression Of PINOID Kinase Causes Leaf Growth Defects in Arabidopsis thaliana.

In plants many developmental processes are regulated by auxin and its directional transport. PINOID (PID) kinase helps to regulate this transport by influencing polar recruitment of PIN efflux proteins on the cellular membranes. We investigated how altered auxin levels affect leaf growth in Arabidopsis thaliana. Arabidopsis mutants and transgenic plants with altered PID expression levels were used to study the effect on auxin distribution and leaf development. Single knockouts showed small pleiotropic growth defects. Contrastingly, several leaf phenotypes related to changes in auxin concentrations and transcriptional activity were observed in PID overexpression (PIDOE) lines. Unlike in the knockout lines, the leaves of PIDOE lines showed an elevation in total indole-3-acetic acid (IAA). Accordingly, enhanced DR5-visualized auxin responses were detected, especially along the leaf margins. Kinematic analysis revealed that ectopic expression of PID negatively affects cell proliferation and expansion rates, yielding reduced cell numbers and small-sized cells in the PIDOE leaves. We used PIDOE lines as a tool to study auxin dose effects on leaf development and demonstrate that auxin, above a certain threshold, has a negative affect on leaf growth. RNA sequencing further showed how subtle PIDOE-related changes in auxin levels lead to transcriptional reprogramming of cellular processes.

Auxin and chloroplast movements.

Auxin is involved in a wide spectrum of physiological processes in plants, including responses controlled by the blue light photoreceptors phototropins: phototropic bending and stomatal movement. However, the role of auxin in phototropin-mediated chloroplast movements has never been studied. To address this question we searched for potential interactions between auxin and the chloroplast movement signaling pathway using different experimental approaches and two model plants, Arabidopsis thaliana and Nicotiana tabacum. We observed that the disturbance of auxin homeostasis by shoot decapitation caused a decrease in chloroplast movement parameters, which could be rescued by exogenous auxin application. In several cases, the impairment of polar auxin transport, by chemical inhibitors or in auxin carrier mutants, had a similar negative effect on chloroplast movements. This inhibition was not correlated with changes in auxin levels. Chloroplast relocations were also affected by the antiauxin p-chlorophenoxyisobutyric acid and mutations in genes encoding some of the elements of the SCF(TIR1)-Aux/IAA auxin receptor complex. The observed changes in chloroplast movement parameters are not prominent, which points to a modulatory role of auxin in this process. Taken together, the obtained results suggest that auxin acts indirectly to regulate chloroplast movements, presumably by regulating gene expression via the SCF(TIR1)-Aux/IAA-ARF pathway. Auxin does not seem to be involved in controlling the expression of phototropins.

CONSTANS-LIKE 7 (COL7) Is Involved in Phytochrome B (phyB)-Mediated Light-Quality Regulation of Auxin Homeostasis

Arabidopsis phytochrome B (phyB) is the major photoreceptor that senses the ratio of red to far-red light (R:FR) to regulate the shade-avoidance response (SAR). It has been hypothesized that altered homeostasis of phytohormones such as auxin and strigolactone is at least partially responsible for SAR, but the mechanism underlying phyB regulation of the hormonal change is not fully understood. Previously we reported that CONSTANS-LIKE 7 (COL7) enhances branching number under high R:FR but not under low R:FR, implying that COL7 may be involved in the phyB-mediated SAR. In this study, we provide evidence that COL7 reduces auxin levels in a high R:FR-dependent manner. We found that the phyB mutation suppresses the COL7-induced branching proliferation. Moreover, COL7 promotes mRNA expression of SUPERROOT 2 (SUR2), which encodes a suppressor of auxin biosynthesis, in high R:FR but not in low R:FR. Consistently with these results, deficiency of phyB suppresses the elevated transcription of SUR2 in COL7 overexpression plants grown in high R:FR. Taking these results together with data suggesting that photo-excited phyB is required for stabilization of the COL7 protein, we argue that COL7 is a critical factor linking light perception to changes in auxin level in Arabidopsis.

Changes in auxin level in the course of growth of a sunflower crown-gall suspension culture

The auxin level in the cell mass and culture medium was determined by means of the <em>Avena</em> straight caleoptile test in various periods of the suspension culture cycle of the sunflower crown-gall tumour. The investigations were performed in the course of the zero passage (PO) and first one (Pl), differing in their time of duration of maximum growth and its intensity. In both passages the intra- and extra-cellular auxin levels reach values of the same order. At the beginning of the maximal growth phase the activity corresponding to IAA in the cells prevails over that of the other auxin-like compounds. This disproportion diminishes with further development of the culture, and with the beginning of the stationary phase the cellular IAA level is lower than that of the remaining auxin-like compounds. The short phase of maximal growth (PO) occurs with an auxin level decreasing in the cell mass and increasing in the medium, and towards the end of the cycle these levels become equal. During the long phase of maximal growth (Pl) the total amount of auxins in the cells increases and is 2-3 times higher than in the medium, whereas IAA in the cells remains at a constant level. These results suggest that the participation of IAA in the intracellular pool of auxin-like substances is decisive for the mitotic activity of the cells and maintenance of growth in the culture.

Auxin‐mediated nitrate signalling by NRT1.1 participates in the adaptive response of Arabidopsis root architecture to the spatial heterogeneity of nitrate availability

To optimize their nitrogen nutrition, plants are able to direct root growth in nitrate-rich patches. This depends in Arabidopsis on the NRT1.1 nitrate transporter/sensor. NRT1.1 was shown to display on homogenous medium, an auxin transport activity that lowers auxin accumulation in lateral roots and inhibits their growth at low nitrate. Using a split-root system, we explored the hypothesis that preferential lateral root growth in the nitrate-rich side involves the NRT1.1-dependent repression of lateral root growth in the low nitrate side. Data show that NRT1.1 acts locally to modulate both auxin levels and meristematic activity in response to the low nitrate concentration directly experienced by lateral roots leading to a repression of their growth. A stimulatory role of NRT1.1 in the high nitrate side, which does not rely on changes in auxin levels, is also observed. Altogether, our data suggest that NRT1.1 allows preferential root colonization of nitrate-rich patches by both preventing root growth in response to low nitrate, through modulation of auxin traffic, and stimulating root growth in response to high nitrate, through a yet uncharacterized mechanism. In addition, transcriptional regulation of NRT1.1 affects both mechanisms allowing plants to modulate the effect of nitrate on root branching.

ABA-deficiency results in reduced plant and fruit size in tomato

Abscisic acid (ABA) deficient mutants, such as notabilis and flacca, have helped elucidating the role of ABA during plant development and stress responses in tomato (Solanum lycopersicum L.). However, these mutants have only moderately decreased ABA levels. Here we report on plant and fruit development in the more strongly ABA-deficient notabilis/flacca (not/flc) double mutant. We observed that plant growth, leaf-surface area, drought-induced wilting and ABA-related gene expression in the different genotypes were strongly correlated with the ABA levels and thus most strongly affected in the not/flc double mutants. These mutants also had reduced fruit size that was caused by an overall smaller cell size. Lower ABA levels in fruits did not correlate with changes in auxin levels, but were accompanied by higher ethylene evolution rates. This suggests that in a wild-type background ABA stimulates cell enlargement during tomato fruit growth via a negative effect on ethylene synthesis.

Changes in auxin distribution patterns during lateral root development in rice

Root differentiation in plants is mediated by the hormone auxin. As auxin is implicated in coordinating the development and organization of tissues in lateral roots, we investigated the inferred cellular distribution of auxin in various cell types in root prior to and during lateral root formation in rice ( Oryza sativa cv. Murasaki R86) using the transgenic plants carrying the auxin responsive soybean GH3 promoter fused to gusA. Modulation in the expression of the Pr GH3:gusA fusion was assessed by histological GUS staining in rice tissues following the treatment of transgenic rice seedlings with or without auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) or N-naphthaleneacetic acid (NAA), and the auxin transport inhibitors N-(1-naphthyl)phthalamic acid (NPA), kaempferol, quercetin, apigenin or Nod factors under axenic conditions. In this work we demonstrate that soybean GH3 promoter is active in rice, and the GH3-gusA reporter system can be utilized as a molecular marker to monitor the changes in auxin levels during organogenesis of this monocot plant. Our studies revealed distinct patterns of GH3 activity prior to and during various developmental phases of lateral root differentiation in rice. The GH3 expression patterns during lateral root development in rice showed considerable similarities with those occurring during comparable stages of lateral root development in dicots, implying that the requirement of auxin for the morphogenesis of lateral roots is similar in evolutionarily distinct monocots and dicots.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus × H. tuberosus

The clone EMB-2 of the interspecific hybrid Helianthus annuus x H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography-mass spectrometry-selected ion monitoring method and an immuno-cytochemical approach. Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.