Exogenous Auxin Research Articles

The ARF7 and ARF19 Transcription Factors Positively Regulate PHOSPHATE STARVATION RESPONSE1 in Arabidopsis Roots.

PHOSPHATE STARVATION RESPONSE1 (PHR1) is a key regulatory component of the response to phosphate (Pi) starvation. However, the regulation of PHR1 in this response remains poorly understood. Here, we report that PHR1 is a target of the transcription factors AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 and is positively regulated by auxin signaling in Arabidopsis (Arabidopsis thaliana) roots. PHR1 expression was induced by exogenous auxin and suppressed by auxin transport inhibitors in Arabidopsis roots. In the PHR1 promoter, three auxin-response elements, which are bound directly by ARF7 and ARF19, were shown to be essential for PHR1 expression. The arf7, arf19, and arf7 arf19 mutants showed down-regulated expression of PHR1 and downstream Pi starvation-induced genes in roots; they also exhibited defective Pi uptake in roots and overaccumulation of anthocyanin in shoots. The induction of lateral root formation in response to low Pi and to exogenous auxin was decreased in the phr1 mutant, whereas the expression of LATERAL ORGAN BOUNDARIES-DOMAIN16 (LBD16) and LBD29 was not changed significantly. PHR1 acted independently of LBD16 and LBD29 in the regulation of lateral root formation in response to low Pi. Under low-Pi conditions, lateral root impairment in the arf7 arf19 mutant was partially rescued by constitutive expression of PHR1, demonstrating that reduced PHR1 expression contributed to the arf7 arf19 phenotype. In addition to PHR1, other genes encoding MYB-CC members also were targets of ARF7 and ARF19. Our work thus reveals a mechanism coordinating auxin signaling and the PHR1 regulon in Arabidopsis responses to Pi deficiency.

Transcriptional regulation of auxin metabolic-enzyme genes during tomato fruit development

Abstract Auxin plays crucial roles in many stages of fruit development. To understand the mechanism of regulation exerted by auxin during fruit development, we analyzed the variations in the levels of indole-3-acetic acid (IAA) and its precursors, as well as the variations in the expression of the genes involved in IAA metabolism in developing tomato (Solanum lycopersicum ‘Ailsa Craig’) fruits. The concentrations of IAA and its precursor indole-3-pyruvic acid (IPyA) increased in the ovary three days after anthesis. The transcript levels of the auxin biosynthetic-enzyme genes SlTAR2, ToFZY1, ToFZY2, and ToFZY5 also increased during this stage. These results suggest that the de novo IAA biosynthesis in the pollinated ovaries is controlled through the IPyA pathway, and is involved in fruit set and early fruit growth. Contrarily, the transcripts of the auxin inactivating-enzyme genes SlGH3-9 and SlGH3-15 were accumulated in the unpollinated ovaries and decreased after pollination. This suggests that the auxin inactivating-enzymes might be involved in the control of IAA levels in the unpollinated ovaries. Further, we found that application of synthetic auxin 2,4-dichlorophenoxyacetic acid on the unpollinated ovaries increased the transcript levels of SlTAR2, ToFZY1, ToFZY2, and ToFZY5 and decreased those of SlGH3-9 and SlGH3-15. These results suggest that elevated auxin signaling via pollination or exogenous auxin treatment might promote further auxin accumulation in the ovaries.

A role for ALF4 during gall and giant cell development in the biotic interaction between Arabidopsis and Meloidogyne spp.

Root-knot nematodes (RKNs; Meloidogyne spp.) are a major pest for the agriculture worldwide. RKNs induce specialized feeding cells (giant cells, GCs) inside galls which are de novo formed pseudo-organs in the roots that share similarities with other developmental processes as lateral root (LR) and callus formation or grafting involving new vascular development or pericycle proliferation. Hence, it is pertinent to study the molecular mechanisms directing the plant-nematode interaction. In this respect, ALF4 is a key gene during LR formation, vascular vessels reconnection in grafting, hormone-induced callus formation or de novo root organogenesis from leaf explants. Our results show that ALF4 is also induced in galls at early infection stages in an auxin-independent way. Furthermore, ALF4 activity is necessary for the formation of proper galls and GCs, as the mutant alf4-1 presents aberrant galls and GCs with severe structural abnormalities leading to a dramatic reduction in the nematode egg production. However, a low-reproduction rate is maintained, that might be explained by the local auxin maximum build by the nematodes in galls, partially rescuing alf4-1 phenotype. This would be similar to the partial rescue described for LR formation with exogenous auxins and also agrees with the LR emergence from alf4-1 galls but not from uninfected roots. In addition, ALF4 is also induced in syncytia formed by cyst nematodes. All these data support a pivotal role for ALF4 during de novo organogenesis processes induced by endoparasitic nematodes, in addition to its role in LR formation, callus development or vessel reconnection during grafting.

Growth, Metabolite Profile, Oxidative Status, and Phytohormone Levels in the Green Alga Acutodesmus obliquus Exposed to Exogenous Auxins and Cytokinins

The effects of auxins and cytokinins at the range of concentrations 0.0001–100 µM on Acutodesmus obliquus (Chlorophyceae) cultures were studied. Microalga exhibited sensitivity to cytokinins in the following order: 0.01 µM tZ > 0.1 µM Kin > 1 µM DPU, whereas the hierarchy of auxin activity was: 0.01 µM IAA > 0.1 µM IBA > 0.1 µM PAA. Cytokinins possessed higher stimulating properties on the cell number, whereas auxins increased the size of cells. Differences in the metabolite profiles of the cultures treated with phytohormones were observed. Auxins and cytokinins had a positive effect on the photosynthetic apparatus enhancing the level of chlorophylls, carotenes, and xanthophylls. In comparison with auxins, cytokinins more effectively delayed oxidative damage by increasing the level of non-enzymatic antioxidants (ascorbate, glutathione) and the activity of enzymes scavenging reactive oxidative species (catalase, glutathione reductase, ascorbate peroxidase). On the other hand, auxins stimulated superoxide dismutase activity and provoked hydrogen peroxide generation, which may be involved in cell enlargement. All phytohormones reduced the content of abscisic acid and controlled the level of endogenous auxin and cytokinins suggesting complex interactions. Different dynamics of A. obliquus responses to auxins and cytokinins clearly demonstrated their diverse roles in algal growth and metabolism.

Lateral root initiation and formation within the parental root meristem of Cucurbita pepo: is auxin a key player?

In some plant families, including Cucurbitaceae, initiation and development of lateral roots (LRs) occur in the parental root apical meristem. The objective of this study was to identify the general mechanisms underlying LR initiation (LRI). Therefore, the first cellular events leading to LRI as well as the role of auxin in this process were studied in the Cucurbita pepo root apical meristem. Transgenic hairy roots harbouring the auxin-responsive promoter DR5 fused to different reporter genes were used for visualizing of cellular auxin response maxima (ARMs) via confocal laser scanning microscopy and 3-D imaging. The effects of exogenous auxin and auxin transport inhibitors on root branching were analysed. The earliest LRI event involved a group of symmetric anticlinal divisions in pericycle cell files at a distance of 250-350 µm from the initial cells. The visualization of the ARMs enabled the precise detection of cells involved in determining the site of LR primordium formation. A local ARM appeared in sister cells of the pericycle and endodermis files before the first division. Cortical cells contributed to LR development after the anticlinal divisions in the pericycle via the formation of an ARM. Exogenous auxins did not increase the total number of LRs and did not affect the LRI index. Although exogenous auxin transport inhibitors acted in different ways, they all reduced the number of LRs formed. Literature data, as well as results obtained in this study, suggest that the formation of a local ARM before the first anticlinal formative divisions is the common mechanism underlying LRI in flowering plants. We propose that the mechanisms of the regulation of root branching are independent of the position of the LRI site relative to the parental root tip.

Class C ARFs evolved before the origin of land plants and antagonize differentiation and developmental transitions in Marchantia polymorpha

A plethora of developmental and physiological processes in land plants is influenced by auxin, to a large extent via alterations in gene expression by AUXIN RESPONSE FACTORs (ARFs). The canonical auxin transcriptional response system is a land plant innovation, however, charophycean algae possess orthologues of at least some classes of ARF and AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, suggesting that elements of the canonical land plant system existed in an ancestral alga. We reconstructed the phylogenetic relationships between streptophyte ARF and AUX/IAA genes and functionally characterized the solitary class C ARF, MpARF3, in Marchantia polymorpha. Phylogenetic analyses indicate that multiple ARF classes, including class C ARFs, existed in an ancestral alga. Loss- and gain-of-function MpARF3 alleles result in pleiotropic effects in the gametophyte, with MpARF3 inhibiting differentiation and developmental transitions in multiple stages of the life cycle. Although loss-of-function Mparf3 and Mpmir160 alleles respond to exogenous auxin treatments, strong miR-resistant MpARF3 alleles are auxin-insensitive, suggesting that class C ARFs act in a context-dependent fashion. We conclude that two modules independently evolved to regulate a pre-existing ARF transcriptional network. Whereas the auxin-TIR1-AUX/IAA pathway evolved to repress class A/B ARF activity, miR160 evolved to repress class C ARFs in a dynamic fashion.

Auxin protects spikelet fertility and grain yield under drought and heat stresses in rice

Rice is highly sensitive to drought and high temperature (HT) stresses at reproductive phase. Since auxin is a major regulator of reproductive development, we analysed the role of indole-3-acetic acid (IAA) in spikelet fertility of rice under drought and HT stresses in 4 different experiments with 23 diverse rice genotypes. The results showed that both of these stresses reduced the pollen viability, spikelet fertility, and yield components. Exogenous IAA (10−5 M) sprayed on plants during drought or heat stresses mitigated the adverse stress effects on pollen viability, spikelet fertility and yield components across experiments in most genotypes. Exogenous IAA application reduced the membrane lipid peroxidation and ROS accumulation in spikelets under both stresses. Drought and HT stresses significantly reduced the mean endogenous IAA content of rice panicles. Endogenous IAA levels significantly and positively correlated with pollen viability and yield component traits, and negatively correlated with lipid peroxidation of spikelets. Expression of most of the YUCCA genes and their transcriptional regulators SPL, NGA and TFL were downregulated in the spikelets of rice genotypes under stress. Further, auxin co-receptor genes TIR1/AFB2-AUX/IAA, ARF target gene HAF TF, 3 out of 4 PIN genes, and 5 ARFs analysed in this study were significantly downregulated by drought/heat stresses in rice spikelets. These results showed that drought and HT stress-mediated decline in endogenous IAA level and impairment of IAA signalling are the major causes of spikelet sterility in rice. Exogenous auxin and genetic manipulation of auxin synthesis and signalling will be useful to mitigate spikelet sterility and stabilize the grain yield of rice under drought and heat stresses.

Effects of GhWUS from upland cotton (Gossypium hirsutum L.) on somatic embryogenesis and shoot regeneration

The WUSCHEL (WUS) gene encodes a plant-specific homeodomain-containing transcriptional regulator, which plays important roles during embryogenesis, as well as in the formation of shoot and flower meristems. Here, we isolated two homologues of Arabidopsis thaliana WUS (AtWUS), GhWUS1a_At and GhWUS1b_At, from upland cotton (Gossypium hirsutum). Domain analysis suggested that the two putative GhWUS proteins contained a highly conserved DNA-binding HOX domain and a WUS-box. Expression profile analysis showed that GhWUSs were predominantly expressed during the embryoid stage. Ectopic expression of GhWUSs in Arabidopsis could induce somatic embryo and shoot formation from seedling root tips. Furthermore, in the absence of exogenous hormone, overexpression of GhWUSs in Arabidopsis could promote shoot regeneration from excised roots, and in the presence of exogenous auxin, excised roots expressing GhWUS could be induced to produce somatic embryo. In addition, expression of the chimeric GhWUS repressor in cotton callus inhibited embryogenic callus formation. Our results show that GhWUS is an important regulator of somatic embryogenesis and shoot regeneration.

Aeroponics for adventitious rhizogenesis in evergreen haloxeric tree Tamarix aphylla (L.) Karst.: influence of exogenous auxins and cutting type.

Tamarix aphylla (L.) Karst., a drought resistant halophyte tree, is an agroforestry species which can be used for reclamation of waterlogged saline and marginal lands. Due to very low seed viability and unsuitable conditions for seed germination, the tree is becoming rare in Indian Thar desert. Present study concerns the evaluation of aeroponics technique for vegetative propagation of T. aphylla. Effect of various exogenous auxins (indole-3-acetic acid, indole-3-butyric acid, naphthalene acetic acid) at different concentrations (0.0, 1.0, 2.0, 3.0, 5.0, 10.0mgl-1) was examined for induction of adventitious rooting and other morphological features. Among all three auxins tested individually, maximum rooting response (79%) was observed with IBA 2.0mgl-1. However, stem cuttings treated with a combination of auxins (2.0mgl-1 IBA and 1.0mgl-1 IAA) for 15min resulted in 87% of rooting response. Among three types of stem cuttings (apical shoot, newly sprouted cuttings, mature stem cuttings), maximum rooting (~90%) was observed on mature stem cuttings. Number of roots and root length were significantly higher in aeroponically rooted stem cuttings as compared to stem cuttings rooted in soil conditions. Successfully rooted and sprouted plants were transferred to polybags with 95% survival rate. This is the first report on aeroponic culture of Tamarix aphylla which can be utilized in agroforestry practices, marginal land reclamation and physiological studies.

Smoke tree (Cotinus coggygria Scop.) propagation and biotechnology: A mini-review

Cotinus coggygria Scop. (Anacardiaceae), commonly referred to as ‘smoke tree’ or ‘smoke bush’, is an attractive ornamental tree or large bush that has medicinal properties and multiple bioactivities. Seed germination can be high after treating seeds with H2SO4, but to avoid growing male trees that will not form attractive inflorescences, the rooting of cuttings from female trees is a preferred method of vegetative propagation. One method to intensify the rooting of stem cuttings is by shading mother plants and simultaneously applying an exogenous auxin or biostimulator. This method can increase the quality of rooted cuttings. In addition, shading causes anatomical changes in the stem structure and leads to the formation of adventitious root primordia. New EU regulations have forced member states to change or withdraw existing authorization for products used in plant protection that contain active substances like auxin (indole-3-butyric acid, IBA). Consequently, there is an active search for alternative measures to support the process of rooting woody plants. Examples of such preparations are biostimulators, including AlgaminoPlant, HumiPlant and Route®, which can increase the rooting of cuttings, affect processes associated with oxidative stress, and increase the intensity of gas exchange and the content of organic compounds. The most effective in vitro micropropagation protocol for C. coggygria involves the use of Murashige and Skoog basal solid or liquid media enriched with one of two cytokinins, 6N-benzyladenine or meta-metoxytopolins. The use of IBA has shown most success, both ex vitro and in vitro. All other aspects of Cotinus species biotechnology still need to be developed.

Efficient extraction of indole from wash oil by quaternary ammonium salts via forming deep eutectic solvents

Indole plays a significant role in the production of products such as spices, medicines, and exogenous auxins. Deep eutectic solvents (DESs) formed by indole and quaternary ammonium salts (QASs, including tetraethylammonium chloride-TEAC, tetrapropylammonium chloride-TPAC, triethylmethyl-ammonium chloride-TEMAC) were found and proposed to efficiently extract indole from model wash oil. The effects of time, temperature, mole ratio of QAS to indole and initial indole concentration on the extraction of indole were studied. The results indicate that these QASs can extract indole from wash oil with efficiencies up to 96.7% (TPAC). The extraction process can be finished within 5 min. It is also found that the ultimate concentrations are almost identical for one kind of QAS although their initial concentrations are different, and the indole concentration in model wash oil can be reduced to a very low level of 1.3 g/dm3 (TPAC). The maximum distribution coefficients of indole are 40.8 for TEAC, 91.9 for TPAC, and 29.5 for TEMAC at the studied conditions, and the extraction ability follows the order of TPAC > TEAC > TEMAC. Moreover, QASs in DESs can be reused without obvious reduction in extraction efficiency. The extraction mechanism was studied by FT-IR, and the results indicate that there is a hydrogen bond between QAS and indole. Finally, a comparison was made between this method and other methods to verify and demonstrate its desirable properties of efficiency and environmentally friendliness.

Genome-wide analysis of the auxin/indoleacetic acid (Aux/IAA) gene family in allotetraploid rapeseed (Brassica napus L.)

BackgroundAuxin/Indoleacetic acid (Aux/IAA) genes participate in the auxin signaling pathway and play key roles in plant growth and development. Although the Aux/IAA gene family has been identified in many plants, within allotetraploid Brassica napus little is known.ResultsIn this study, a total of 119 Aux/IAA genes were found in the genome of B. napus. They were distributed non-randomly across all 19 chromosomes and other non-anchored random scaffolds, with a symmetric distribution in the A and C subgenomes. Evolutionary and comparative analysis revealed that 111 (94.1%) B. napus Aux/IAA genes were multiplied due to ancestral Brassica genome triplication and recent allotetraploidy from B. rapa and B. oleracea. Phylogenetic analysis indicated seven subgroups containing 29 orthologous gene sets and two Brassica-specific gene sets. Structures of genes and proteins varied across different genes but were conserved among homologous genes in B. napus. Furthermore, analysis of transcriptional profiles revealed that the expression patterns of Aux/IAA genes in B. napus were tissue dependent. Auxin-responsive elements tend to be distributed in the proximal region of promoters, and are significantly associated with early exogenous auxin up-regulation.ConclusionsMembers of the Aux/IAA gene family were identified and analyzed comprehensively in the allotetraploid B. napus genome. This analysis provides a deeper understanding of diversification of the Aux/IAA gene family and will facilitate further dissection of Aux/IAA gene function in B. napus.

Genome-wide analysis and transcriptomic profiling of the auxin biosynthesis, transport and signaling family genes in moso bamboo (Phyllostachys heterocycla)

BackgroundAuxin is essential for plant growth and development. Although substantial progress has been made in understanding auxin pathways in model plants such as Arabidopsis and rice, little is known in moso bamboo which is famous for its fast growth resulting from the rapid cell elongation and division.ResultsHere we showed that exogenous auxin has strong effects on crown and primary roots. Genes involved in auxin action, including 13 YUCCA (YUC) genes involved in auxin synthesis, 14 PIN-FORMED/PIN-like (PIN/PILS) and 7 AUXIN1/LIKE-AUX1 (AUX1/LAX) members involved in auxin transport, 10 auxin receptors (AFB) involved in auxin perception, 43 auxin/indole-3-aceticacid (AUX/IAA) genes, and 41 auxin response factors (ARF) involved in auxin signaling were identified through genome-wide analysis. Phylogenetic analysis of these genes from Arabidopsis, Oryza sativa and bamboo revealed that auxin biosynthesis, transport, and signaling pathways are conserved in these species. A comprehensive study of auxin-responsive genes using RNA sequencing technology was performed, and the results also supported that moso bamboo shared a conserved regulatory mechanism for the expression of auxin pathway genes; meanwhile it harbors its own specific properties.ConclusionsIn summary, we generated an overview of the auxin pathway in bamboo, which provides information for uncovering the precise roles of auxin pathway in this important species in the future.

A sensitive synthetic reporter for visualizing cytokinin signaling output in rice

BackgroundCytokinins play many essential roles in plant growth and development, mainly through signal transduction pathways. Although the cytokinin signaling pathway in rice has been clarified, no synthetic reporter for cytokinin signaling output has been reported for rice. The sensitive synthetic reporter two-component signaling sensor (TCSn) is used in the model plant Arabidopsis; however, whether the reporter reflects the cytokinin signaling output pattern in rice remains unclear.ResultsEarly-cytokinin-responsive type-A OsRR-binding element (A/G)GAT(C/T) was more clustered in the 15 type-A OsRRs than in the 13 control genes. Quantitative polymerase chain reaction analysis showed that the relative expression of seven type-A OsRRs in roots and shoots was significantly induced by exogenous cytokinin application, and that of seven OsRRs, mainly in roots, was inhibited by exogenous auxin application. We constructed a transgenic rice plant harboring a beta-glucuronidase (GUS) driven by the synthetic promoter TCSn. TCSn::GUS was expressed in the meristem of germinated rice seed and rice seedlings. Furthermore, TCSn::GUS expression in rice seedlings was induced specifically by exogenous cytokinin application and decreased by exogenous auxin application. Moreover, no obvious reduction in GUS levels was observed after three generations of selfing of transgenic plants, indicating that TCSn::GUS is not subject to transgene silencing.ConclusionsWe report here a robust and sensitive synthetic sensor for monitoring the transcriptional output of the cytokinin signaling network in rice.

Events Associated with Early Age-Related Decline in Adventitious Rooting Competence of Eucalyptus globulus Labill.

The development of adventitious roots is affected by several factors, including the age of the cutting donor plant, which negatively affects rooting capacity. Eucalyptus globulus quickly loses rooting capacity of cuttings as the donor plant ages, although the molecular and biochemical mechanisms behind this process are still unclear. To better understand the bases of rooting competence loss in E. globulus, the time required for a significant decline in rhizogenic ability without exogenous auxin was determined in microcuttings derived from donor plants of different ages after sowing. Tip cuttings of donor plants were severed before and after loss of rooting competence of microcuttings to test the hypothesis that auxin and carbohydrate homeostasis regulate rooting competence decline. There were no significant changes in concentration of carbohydrates, flavonoids, or proteins before and after the loss of rooting capacity. Peroxidase (EC 1.11.1.7) total activity increased with loss of rooting competence. Auxin concentration showed the opposite pattern. In good agreement, TAA1, a key gene in auxin biosynthesis, had lower expression after loss of rooting capacity. The same applied to the auxin receptor gene TIR1, suggesting reduced auxin sensitivity. On the other hand, genes associated with auxin response repression (TPL, IAA12) or with the action of cytokinins, the rhizogenesis inhibitor-related ARR1, showed higher expression in plants with lower rooting competence. Taken together, data suggest that age negatively affects E. globulus rooting by a combination of factors. Decreased endogenous auxin concentration, possibly caused by less biosynthesis, lower auxin sensitivity, higher expression of genes inhibiting auxin action, as well as of genes related to the action of cytokinins, appear to play roles in this process.

Exogenous auxin represses soybean seed germination through decreasing the gibberellin/abscisic acid (GA/ABA) ratio

Auxin is an important phytohormone which mediates diverse development processes in plants. Published research has demonstrated that auxin induces seed dormancy. However, the precise mechanisms underlying the effect of auxin on seed germination need further investigation, especially the relationship between auxins and both abscisic acid (ABA) and gibberellins (GAs), the latter two phytohormones being the key regulators of seed germination. Here we report that exogenous auxin treatment represses soybean seed germination by enhancing ABA biosynthesis, while impairing GA biogenesis, and finally decreasing GA1/ABA and GA4/ABA ratios. Microscope observation showed that auxin treatment delayed rupture of the soybean seed coat and radicle protrusion. qPCR assay revealed that transcription of the genes involved in ABA biosynthetic pathway was up-regulated by application of auxin, while expression of genes involved in GA biosynthetic pathway was down-regulated. Accordingly, further phytohormone quantification shows that auxin significantly increased ABA content, whereas the active GA1 and GA4 levels were decreased, resulting insignificant decreases in the ratiosGA1/ABA and GA4/ABA.Consistent with this, ABA biosynthesis inhibitor fluridone reversed the delayed-germination phenotype associated with auxin treatment, while paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Altogether, exogenous auxin represses soybean seed germination by mediating ABA and GA biosynthesis.

ERECTA Regulates Cell Elongation by Activating Auxin Biosynthesis in Arabidopsis thaliana.

The ERECTA family genes, ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERECTA-LIKE2 (ERL2), encode leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. Knocking out these three genes can cause severe phenotypes, which indicates that they play significant roles in plant growth and development. However, the molecular mechanism within remains unclear. Here we show that the short hypocotyl phenotypes of er erl1 erl2 mutants are mainly due to the defects of cell elongation rather than the cell division. In contrast, in the ERECTA overexpression transgenic plants, the hypocotyl length is increased with elongated cells. Moreover, we show that the er erl1 erl2 triple mutant contains a low level of auxin, and the expression levels of the key auxin biosynthesis genes are significantly reduced. Consistent with this observation, increasing exogenous or endogenous auxin levels could partially rescue the cell elongation defects of the er erl1 erl2 triple mutant. Therefore, our results provide a molecular basis for auxin mediated ERECTA control of the hypocotyl length in Arabidopsis thaliana.

Sensory role of actin in auxin-dependent responses of tobacco BY-2

Polar auxin transport depends on the polar localization of auxin-efflux carriers. The cycling of these carriers between cell interior and plasma membrane depends on actin. The dynamic of actin not only affects auxin transport, but also changes the auxin-responsiveness. To study the potential link between auxin responsiveness and actin dynamics, we investigated developmental responses of the non-transformed BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cell line and the transgenic BY-2 strain GF11 (stably transformed BY-2 cells with a GFP-fimbrin actin-binding domain 2 construct). The developmental process was divided into three distinct stages: cell cycling, cell elongation and file disintegration. Several phenotypes were measured to monitor the cellular responses to different concentrations of exogenous natural auxin (Indole-3-acetic acid, IAA). We found that auxin stimulated and prolonged the mitotic activity, and delayed the exit from the proliferation phase. However, both responses were suppressed in the GF11 line. At the stationary phase of the cultivation cycle, auxin strongly accelerated the cell file disintegration. Interestingly, it was not suppressed but progressed to a more complete disintegration in the GF11 line. During the cultivation cycle, we also followed the organization of actin in the GF11 line and did not detect any significant difference in actin organization from untreated control or exogenous IAA treatment. Therefore, our findings indicate that the specific differences observed in the GF11 line must be linked with a function of actin that is not structural. It means that there is a sensory role of actin for auxin signaling.

Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity

BackgroundIndole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control adventitious root (AR) formation in planta. Adventitious roots are also crucial for propagation via cuttings. However, IBA role(s) is/are still far to be elucidated. In Arabidopsis thaliana stem cuttings, 10 μM IBA is more AR-inductive than 10 μM IAA, and, in thin cell layers (TCLs), IBA induces ARs when combined with 0.1 μM kinetin (Kin). It is unknown whether arabidopsis TCLs produce ARs under IBA alone (10 μM) or IAA alone (10 μM), and whether they contain endogenous IAA/IBA at culture onset, possibly interfering with the exogenous IBA/IAA input. Moreover, it is unknown whether an IBA-to-IAA conversion is active in TCLs, and positively affects AR formation, possibly through the activity of the nitric oxide (NO) deriving from the conversion process.ResultsRevealed undetectable levels of both auxins at culture onset, showing that arabidopsis TCLs were optimal for investigating AR-formation under the total control of exogenous auxins. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants was analyzed under different treatments. It was shown that ARs are better induced by IBA than IAA and IBA + Kin. IBA induced IAA-efflux (PIN1) and IAA-influx (AUX1/LAX3) genes, IAA-influx carriers activities, and expression of ANTHRANILATE SYNTHASE -alpha1 (ASA1), a gene involved in IAA-biosynthesis. ASA1 and ANTHRANILATE SYNTHASE -beta1 (ASB1), the other subunit of the same enzyme, positively affected AR-formation in the presence of exogenous IBA, because the AR-response in the TCLs of their mutant wei2wei7 was highly reduced. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA-conversion, was also strongly reduced. Nitric oxide, an IAA downstream signal and a by-product of IBA-to-IAA conversion, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter explants.ConclusionsAltogether, results showed that IBA induced AR-formation by conversion into IAA involving NO activity, and by a positive action on IAA-transport and ASA1/ASB1-mediated IAA-biosynthesis. Results are important for applications aimed to overcome rooting recalcitrance in species of economic value, but mainly for helping to understand IBA involvement in the natural process of adventitious rooting.

Exogenous Auxin Elicits Changes in the Arabidopsis thaliana Root Proteome in a Time-Dependent Manner.

Auxin is involved in many aspects of root development and physiology, including the formation of lateral roots. Improving our understanding of how the auxin response is mediated at the protein level over time can aid in developing a more complete molecular framework of the process. This study evaluates the effects of exogenous auxin treatment on the Arabidopsis root proteome after exposure of young seedlings to auxin for 8, 12, and 24 h, a timeframe permitting the initiation and full maturation of individual lateral roots. Root protein extracts were processed to peptides, fractionated using off-line strong-cation exchange, and analyzed using ultra-performance liquid chromatography and data independent acquisition-based mass spectrometry. Protein abundances were then tabulated using label-free techniques and evaluated for significant changes. Approximately 2000 proteins were identified during the time course experiment, with the number of differences between the treated and control roots increasing over the 24 h time period, with more proteins found at higher abundance with exposure to auxin than at reduced abundance. Although the proteins identified and changing in levels at each time point represented similar biological processes, each time point represented a distinct snapshot of the response. Auxin coordinately regulates many physiological events in roots and does so by influencing the accumulation and loss of distinct proteins in a time-dependent manner. Data are available via ProteomeXchange with the identifier PXD001400.