High Auxin Levels Research Articles

AUXIN RESPONSE FACTOR 2 mediates repression of strawberry receptacle ripening via auxin-ABA interplay.

Cultivated strawberry (Fragaria × ananassa) is a popular, economically important fruit. The ripening of the receptacle (pseudocarp), the main edible part, depends on endogenously produced abscisic acid (ABA) and is suppressed by the high level of auxin produced from achenes (true fruit) during early development. However, the mechanism whereby auxin regulates receptacle ripening through inhibiting ABA biosynthesis remains unclear. Here, we identified AUXIN RESPONSE FACTOR 2 (FaARF2), which showed decreased expression with reduced auxin content in the receptacle, leading to increased ABA levels and accelerated ripening. Dual-luciferase, yeast one-hybrid, and electrophoretic mobility shift assays demonstrated that FaARF2 could bind to the AuxRE element in the promoter of 9-CIS-EPOXYCAROT-ENOID DIOXYGENASE 1 (FaNCED1), a key ABA biosynthetic gene, to suppress its transcriptional activity. Transiently overexpressing FaARF2 in the receptacles decreased FaNCED1 expression and ABA levels, resulting in inhibition of receptacle ripening and of development of quality attributes, such as pigmentation, aroma, and sweetness. This inhibition caused by overexpressing FaARF2 was partially recovered by the injection of exogenous ABA; conversely, transient silencing of FaARF2 using RNA interference produced the opposite results. The negative targeting of FaNCED1 by FaARF2 is a key link between auxin-ABA interactions and regulation of strawberry ripening.

Shade-induced ROS/NO reinforce COP1-mediated diffuse cell growth

Canopy shade enhances the activity of PHYTOCHROME INTERACTING FACTORs (PIFs) to boost auxin synthesis in the cotyledons. Auxin, together with local PIFs and their positive regulator CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), promotes hypocotyl growth to facilitate access to light. Whether shade alters the cellular redox status thereby affecting growth responses, remains unexplored. Here, we show that, under shade, high auxin levels increased reactive oxygen species and nitric oxide accumulation in the hypocotyl of Arabidopsis. This nitroxidative environment favored the promotion of hypocotyl growth by COP1 under shade. We demonstrate that COP1 is S-nitrosylated, particularly under shade. Impairing this redox regulation enhanced COP1 degradation by the proteasome and diminished the capacity of COP1 to interact with target proteins and to promote hypocotyl growth. Disabling this regulation also generated transversal asymmetries in hypocotyl growth, indicating poor coordination among different cells, which resulted in random hypocotyl bending and predictably low ability to compete with neighbors. These findings highlight the significance of redox signaling in the control of diffuse growth during shade avoidance.

Genome-wide gene network uncover temporal and spatial changes of genes in auxin homeostasis during fruit development in strawberry (F. × ananassa)

BackgroundThe plant hormone auxin plays a crucial role in regulating important functions in strawberry fruit development. Although a few studies have described the complex auxin biosynthetic and signaling pathway in wild diploid strawberry (Fragaria vesca), the molecular mechanisms underlying auxin biosynthesis and crosstalk in octoploid strawberry fruit development are not fully characterized. To address this knowledge gap, comprehensive transcriptomic analyses were conducted at different stages of fruit development and compared between the achene and receptacle to identify developmentally regulated auxin biosynthetic genes and transcription factors during the fruit ripening process. Similar to wild diploid strawberry, octoploid strawberry accumulates high levels of auxin in achene compared to receptacle.ResultsGenes involved in auxin biosynthesis and conjugation, such as Tryptophan Aminotransferase of Arabidopsis (TAAs), YUCCA (YUCs), and Gretchen Hagen 3 (GH3s), were found to be primarily expressed in the achene, with low expression in the receptacle. Interestingly, several genes involved in auxin transport and signaling like Pin-Formed (PINs), Auxin/Indole-3-Acetic Acid Proteins (Aux/IAAs), Transport Inhibitor Response 1 / Auxin-Signaling F-Box (TIR/AFBs) and Auxin Response Factor (ARFs) were more abundantly expressed in the receptacle. Moreover, by examining DEGs and their transcriptional profiles across all six developmental stages, we identified key auxin-related genes co-clustered with transcription factors from the NAM-ATAF1,2-CUC2/ WRKYGQK motif (NAC/WYKY), Heat Shock Transcription Factor and Heat Shock Proteins (HSF/HSP), APETALA2/Ethylene Responsive Factor (AP2/ERF) and MYB transcription factor groups.ConclusionsThese results elucidate the complex regulatory network of auxin biosynthesis and its intricate crosstalk within the achene and receptacle, enriching our understanding of fruit development in octoploid strawberries.

Ectopic assembly of an auxin efflux control machinery shifts developmental trajectories.

Polar auxin transport in the Arabidopsis (Arabidopsis thaliana) root tip maintains high auxin levels around the stem cell niche that gradually decrease in dividing cells but increase again once they transition toward differentiation. Protophloem differentiates earlier than other proximal tissues and employs a unique auxin "canalization" machinery that is thought to balance auxin efflux with retention. It consists of a proposed activator of PIN-FORMED (PIN) auxin efflux carriers, the cAMP-, cGMP- and Calcium-dependent (AGC) kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX); its inhibitor, BREVIS RADIX (BRX); and PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K) enzymes, which promote polar PAX and BRX localization. Because of a dynamic PAX-BRX-PIP5K interplay, the net cellular output of this machinery remains unclear. In this study, we deciphered the dosage-sensitive regulatory interactions among PAX, BRX, and PIP5K by their ectopic expression in developing xylem vessels. The data suggest that the dominant collective output of the PAX-BRX-PIP5K module is a localized reduction in PIN abundance. This requires PAX-stimulated clathrin-mediated PIN endocytosis upon site-specific phosphorylation, which distinguishes PAX from other AGC kinases. An ectopic assembly of the PAX-BRX-PIP5K module is sufficient to cause cellular auxin retention and affects root growth vigor by accelerating the trajectory of xylem vessel development. Our data thus provide direct evidence that local manipulation of auxin efflux alters the timing of cellular differentiation in the root.

The ratio of auxin to cytokinin controls leaf development and meristem initiation in Physcomitrium patens.

Crosstalk between auxin and cytokinin contributes to widespread developmental processes, including root and shoot meristem maintenance, phyllotaxy, and vascular patterning. However, our understanding of crosstalk between these hormones is limited primarily to angiosperms. The moss Physcomitrium patens (formerly Physcomitrella patens) is a powerful system for studying plant hormone function. Auxin and cytokinin play similar roles in regulating moss gametophore (shoot) architecture, to those in flowering plant shoots. However, auxin-cytokinin crosstalk is poorly understood in moss. Here we find that the ratio of auxin to cytokinin is an important determinant of development in P. patens, especially during leaf development and branch stem cell initiation. Addition of high levels of auxin to P. patens gametophores blocks leaf outgrowth. However, simultaneous addition of high levels of both auxin and cytokinin partially restores leaf outgrowth, suggesting that the ratio of these hormones is the predominant factor. Likewise, during branch initiation and outgrowth, chemical inhibition of auxin synthesis phenocopies cytokinin application. Finally, cytokinin-insensitive mutants resemble plants with altered auxin signaling and are hypersensitive to auxin. In summary, our results suggest that the ratio between auxin and cytokinin signaling is the basis for developmental decisions in the moss gametophore.

Differences in the Growth of Seedlings and the Selection of Fast-Growing Species in the Gleditsia Genus

The Gleditsia genus has various uses, including those for medicinal, edible, chemical, timber, and ornamental purposes, and the genus is widely distributed in China. However, there is still a lack of understanding about the phenotypic and growth differences seen among species within the Gleditsia genus. In this study, we compared and analyzed the various species of Gleditsia seedlings in terms of their genotypes, chromosome numbers, physiological growth, photosynthesis, hormone content, and gene expression. The results showed that the genome size of the Gleditsia genus ranges from 686.08 M to 1034.24 M and that all Gleditsia species are diploid. Among the species studied, G. fera can be divided into fast-growing genotype, exhibited several advantages in terms of leaf type and photosynthetic capacity, high levels of GA3, and fast stem growth, making it a species with the potential for promotion and application. G. delavayi exhibited high levels of auxin and cytokinin and strong photosynthetic capacity, with rapid growth in terms of plant height. G. microphylla had the lowest levels of IAA, IBA, and NAA in the apical, and showed slow growth in terms of plant height. Weighted correlation network analysis (WGCNA) identified the hub genes associated with traits. This study lays a material and theoretical foundation for the development of new resources for Gleditsia breeding and rootstock selection and provides a basis for the mechanism of rootstock–scion interaction.

Disruption of the amino acid transporter CsAAP2 inhibits auxin-mediated root development in cucumber.

Amino acid transporters are the principal mediators of organic nitrogen distribution within plants and are essential for plant growth and development. Despite this importance, relatively few amino acid transporter genes have been explored and elucidated in cucumber (Cucumis sativus). Here, a total of 86 amino acid transporter genes were identified in the cucumber genome. We further identified Amino Acid Permease (AAP) subfamily members that exhibited distinct expression patterns in different tissues. We found that the CsAAP2 as a candidate gene encoding a functional amino acid transporter is highly expressed in cucumber root vascular cells. CsAAP2 knockout lines exhibited arrested development of root meristem, which then caused the delayed initiation of lateral root and the inhibition of root elongation. What is more, the shoot growth of aap2 mutants was strongly retarded due to defects in cucumber root development. Moreover, aap2 mutants exhibited higher concentrations of amino acids and lignin in roots. We found that the mutant roots had a stronger ability to acidize medium. Furthermore, in the aap2 mutants, polar auxin transport was disrupted in the root tip, leading to high auxin levels in roots. Interestingly, slightly alkaline media rescued their severely reduced root growth by stimulating auxin pathway.

High auxin stimulates callus through SDG8-mediated histone H3K36 methylation in Arabidopsis.

Callus induction, which results in fate transition in plant cells, is considered as the first and key step for plant regeneration. This process can be stimulated in different tissues by a callus-inducing medium (CIM), which contains a high concentration of phytohormone auxin. Although a few key regulators for callus induction have been identified, the multiple aspects of the regulatory mechanism driven by high levels of auxin still need further investigation. Here, we find that high auxin induces callus through a H3K36 histone methylation-dependent mechanism, which requires the methyltransferase SET DOMAIN GROUP 8 (SDG8). During callus induction, the increased auxin accumulates SDG8 expression through a TIR1/AFBs-based transcriptional regulation. SDG8 then deposits H3K36me3 modifications on the loci of callus-related genes, including a master regulator WOX5 and the cell proliferation-related genes, such as CYCB1.1. This epigenetic regulation in turn is required for the transcriptional activation of these genes during callus formation. These findings suggest that the massive transcriptional reprogramming for cell fate transition by auxin during callus formation requires epigenetic modifications including SDG8-mediated histone H3K36 methylation. Our results provide insight into the coordination between auxin signaling and epigenetic regulation during fundamental processes in plant development.

The thiol-reductase activity of YUCCA6 enhances nickel heavy metal stress tolerance in Arabidopsis.

Anthropogenic activities cause the leaching of heavy metals into groundwater and their accumulation in soil. Excess levels of heavy metals cause toxicity in plants, inducing the production of reactive oxygen species (ROS) and possible death caused by the resulting oxidative stress. Heavy metal stresses repress auxin biosynthesis and transport, inhibiting plant growth. Here, we investigated whether nickel (Ni) heavy metal toxicity is reduced by exogenous auxin application and whether Ni stress tolerance in Arabidopsis thaliana is mediated by the bifunctional enzyme YUCCA6 (YUC6), which functions as an auxin biosynthetic enzyme and a thiol-reductase (TR). We found that an application of up to 1 µM exogenous indole-3-acetic acid (IAA) reduces Ni stress toxicity. yuc6-1D, a dominant mutant of YUC6 with high auxin levels, was more tolerant of Ni stress than wild-type (WT) plants, despite absorbing significantly more Ni. Treatments of WT plants with YUCASIN, a specific inhibitor of YUC-mediated auxin biosynthesis, increased Ni toxicity; however yuc6-1D was not affected by YUCASIN and remained tolerant of Ni stress. This suggests that rather than the elevated IAA levels in yuc6-1D, the TR activity of YUC6 might be critical for Ni stress tolerance. The loss of TR activity in YUC6 caused by the point-mutation of Cys85 abolished the YUC6-mediated Ni stress tolerance. We also found that the Ni stress–induced ROS accumulation was inhibited in yuc6-1D plants, which consequently also showed reduced oxidative damage. An enzymatic assay and transcriptional analysis revealed that the peroxidase activity and transcription of PEROXIREDOXIN Q were enhanced by Ni stress to a greater level in yuc6-1D than in the WT. These findings imply that despite the need to maintain endogenous IAA levels for basal Ni stress tolerance, the TR activity of YUC6, not the elevated IAA levels, plays the predominant role inNi stress tolerance by lowering Ni-induced oxidative stress.

Morphological and Physiological Indicators for Screening Cell Lines with High Potential for Somatic Embryo Maturation at an Early Stage of Somatic Embryogenesis in Pinus Koraiensis.

Many cell lines in the embryogenic callus cannot produce somatic embryos (SEs) even if they meet the optimal SE maturation culture conditions during conifer somatic embryogenesis. This phenomenon hinders the progress of the industrial-scale reproduction of conifers. Therefore, there is an urgent need to obtain morphological and physiological markers to screen embryogenic calli in response to SE maturation conditions. To detect cell lines with high somatic embryogenesis potential during the proliferation process, we counted the number of pro-embryos and early SEs (ESEs) in different cell lines and storage substances, endogenous hormones, and polyamine contents. The results showed that the yield of P. koraiensis SEs was heavily dependent on genotype (p = 0.001). There were high levels of PE III (pro-embryo III) number, ESE number, and soluble protein content, in the response cell lines (R cell lines), which were 1.6-, 3-, and 1.1-fold those of the obstructive cell lines (B cell lines), respectively. The B cell line had high levels of starch, auxin (IAA), Put, Spd, and putrescine: spermine (Put: Spm) compared to the R cell line. In addition, the numbers of PE III, ESEs, and soluble protein content were significantly positively correlated with SE yield. In contrast, the contents of starch, abscisic acid (ABA), Put, Spm, and Spd were significantly negatively correlated with SE yield. To ensure the accuracy of the results, we used nine cell lines to test the results. The PE III and ESE numbers and the Spm and Spd contents were positively correlated with SE yield, while the levels of starch, ABA, IAA, Put: Spd, and Put: Spm were negatively correlated with SE yield. Thus, we recommend using high PE III and ESEs as morphological indicators and low levels of starch, IAA, ABA, and Put: Spm as physiological markers to screen cell lines with a high somatic embryogenesis potential. In addition, we also found that the relationship between Spd, Spm, and SE yield was opposite in the two experimental results. Therefore, we speculate that the differences in Spd and Spm content are mainly affected by genotype. In conclusion, this study obtained the morphological and physiological markers of some high-somatic embryogenic cell lines by comparing the differences between nine somatic embryogenic cell lines. Our results can guide the improvement of conifer somatic embryogenesis technology and can provide a theoretical basis for accelerating the application of biotechnology in large-scale artificial breeding.

Auxin receptors as integrators of developmental and hormonal signals during reproductive development in pea.

Auxins regulate many aspects of plant growth and development. In pea, three of the five TIR1/AFB members (PsTIR1a, PsTIR1b, and PsAFB2) have been implicated in auxin-related responses during fruit/seed development; however, the roles of PsAFB4 and PsAFB6 in these processes are unknown. Using yeast two-hybrid assays, we found that all five pea TIR1/AFB receptor proteins interacted with the pea AUX/IAAs PsIAA6 and/or PsIAA7 in an auxin-dependent manner, a requirement for functional auxin receptors. All five auxin receptors are expressed in young ovaries (pericarps) and rapidly developing seeds, with overlapping and unique developmental and hormone-regulated gene expression patterns. Pericarp PsAFB6 expression was suppressed by seeds and increased in response to deseeding, and exogenous hormone treatments suggest that seed-derived auxin and deseeding-induced ethylene are involved in these responses, respectively. Ethylene-induced elevation of pericarp PsAFB6 expression was associated with 4-Cl-IAA-specific reduction in ethylene responsiveness. In developing seeds, expression of PsTAR2 and PsYUC10 auxin biosynthesis genes was associated with high auxin levels in seed coat and cotyledon tissues, and PsAFB2 dominated the seed tissue transcript pool. Overall, auxin receptors had overlapping and unique developmental and hormone-regulated gene expression patterns during fruit/seed development, suggesting mediation of diverse responses to auxin, with PsAFB6 linking auxin and ethylene signaling.

The interplay of auxin and brassinosteroid signaling tunes root growth under low and different nitrogen forms.

The coordinated signaling activity of auxin and brassinosteroids (BRs) is critical for optimal plant growth and development. Nutrient-derived signals regulate root growth by modulating the levels and spatial distribution of growth hormones to optimize nutrient uptake and assimilation. However, the effect of the interaction of these two hormones and their signaling on root plasticity during low and differential availability of nitrogen (N) forms (NH4+/NO3-) remains elusive. We demonstrate that root elongation under low N (LN) is an outcome of the interdependent activity of auxin and BR signaling pathways in Arabidopsis (Arabidopsis thaliana). LN promotes root elongation by increasing BR-induced auxin transport activity in the roots. Increased nuclear auxin signaling and its transport efficiency have a distinct impact on root elongation under LN conditions. High auxin levels reversibly inhibit BR signaling via BRI1 KINASE INHIBITOR1. Using the tissue-specific approach, we show that BR signaling from root vasculature (stele) tissues is sufficient to promote cell elongation and, hence, root growth under LN condition. Further, we show that N form-defined root growth attenuation or enhancement depends on the fine balance of BR and auxin signaling activity. NH4+ as a sole N source represses BR signaling and response, which in turn inhibits auxin response and transport, whereas NO3- promotes root elongation in a BR signaling-dependent manner. In this study, we demonstrate the interplay of auxin and BR-derived signals, which are critical for root growth in a heterogeneous N environment and appear essential for root N foraging response and adaptation.

Specification of leaf dorsiventrality via a prepatterned binary readout of a uniform auxin input.

Developmental boundaries play an important role in coordinating the growth and patterning of lateral organs. In plants, specification of dorsiventrality is critical to leaf morphogenesis. Despite its central importance, the mechanism by which leaf primordia acquire adaxial versus abaxial cell fates to establish dorsiventrality remains a topic of much debate. Here, by combining time-lapse confocal imaging, cell lineage tracing and molecular genetic analyses, we demonstrate that a stable boundary between adaxial and abaxial cell fates is specified several plastochrons before primordium emergence when high auxin levels accumulate on a meristem prepattern formed by the AS2 and KAN1 transcription factors. This occurrence triggers a transient induction of ARF3 and an auxin transcriptional response in AS2-marked progenitors that distinguishes adaxial from abaxial identity. As the primordium emerges, dynamic shifts in auxin distribution and auxin-related gene expression gradually resolve this initial polarity into the stable regulatory network known to maintain adaxial-abaxial polarity within the developing organ. Our data show that spatial information from an AS2-KAN1 meristem prepattern governs the conversion of a uniform auxin input into an ARF-dependent binary auxin response output to specify adaxial-abaxial polarity. Auxin thus serves as a single morphogenic signal that orchestrates distinct, spatially separated responses to coordinate the positioning and emergence of a new organ with its patterning.

The epigenetic regulator ULTRAPETALA1 suppresses de novo root regeneration from Arabidopsis leaf explants

ABSTRACT Plants have the potency to regenerate adventitious roots from aerial organs after detachment. In Arabidopsis thaliana, de novo root regeneration (DNRR) from leaf explants is triggered by wounding signaling that rapidly induces the expression of the ETHYLENE RESPONSE FACTOR (ERF) transcription factors ERF109 and ABR1 (ERF111). In turn, the ERFs promote the expression of ASA1, an essential enzyme of auxin biosynthesis, which contributes to rooting by providing high levels of auxin near the wounding side of the leaf. Here, we show that the loss of the epigenetic regulator ULTRAPETALA1 (ULT1), which interacts with Polycomb and Trithorax Group proteins, accelerates and reinforces adventitious root formation. Expression of ERF109 and ASA1 was increased in ult1 mutants, whereas ABR1 was not significantly changed. Cultivation of explants on media with exogenous auxin equates adventitious root formation in wild-type with ult1 mutants, suggesting that ULT1 negatively regulates DNRR by suppressing auxin biosynthesis. Based on these findings, we propose that ULT1 is involved in a novel mechanism that prevents overproliferation of adventitious roots during DNRR.

Biphasic control of cell expansion by auxin coordinates etiolated seedling development.

Seedling emergence is critical for food security. It requires rapid hypocotyl elongation and apical hook formation, both of which are mediated by regulated cell expansion. How these events are coordinated in etiolated seedlings is unclear. Here, we show that biphasic control of cell expansion by the phytohormone auxin underlies this process. Shortly after germination, high auxin levels restrain elongation. This provides a temporal window for apical hook formation, involving a gravity-induced auxin maximum on the eventual concave side of the hook. This auxin maximum induces PP2C.D1 expression, leading to asymmetrical H+-ATPase activity across the hypocotyl that contributes to the differential cell elongation underlying hook development. Subsequently, auxin concentrations decline acropetally and switch from restraining to promoting elongation, thereby driving hypocotyl elongation. Our findings demonstrate how differential auxin concentrations throughout the hypocotyl coordinate etiolated development, leading to successful soil emergence.

N-glucosyltransferase GbNGT1 from ginkgo complements the auxin metabolic pathway

As auxins are among the most important phytohormones, the regulation of auxin homeostasis is complex. Generally, auxin conjugates, especially IAA glucosides, are predominant at high auxin levels. Previous research on terminal glucosylation focused mainly on the O-position, while IAA-N-glucoside and IAA-Asp-N-glucoside have been neglected since their discovery in 2001. In our study, IAA-Asp-N-glucoside was found to be specifically abundant (as high as 4.13 mg/g) in the seeds of 58 ginkgo cultivars. Furthermore, a novel N-glucosyltransferase, termed GbNGT1, was identified via differential transcriptome analysis and in vitro enzymatic testing. It was found that GbNGT1 could catalyze IAA-Asp and IAA to form their corresponding N-glucosides. The enzyme was demonstrated to possess a specific catalytic capacity toward the N-position of the IAA-amino acid or IAA from 52 substrates. Docking and site-directed mutagenesis of this enzyme confirmed that the E15G mutant could almost completely abolish its N-glucosylation ability toward IAA-Asp and IAA in vitro and in vivo. The IAA modification of GbNGT1 and GbGH3.5 was verified by transient expression assay in Nicotiana benthamiana. The effect of GbNGT1 on IAA distribution promotes root growth in Arabidopsis thaliana.

Characterization of embryo and protocorm development of Paphiopedilum spicerianum

Paphiopedilum spicerianum (P. spicerianum) is a rare orchid species with high ornamental value. Asymbiotic germination is the most efficient propagation method for conservation and commercial purposes because clonal propagation is very difficult and the separation of native species of Paphiopedilum through aseptic seeding is uncommon owing to their conservatism. However, a high protocorm developmental arresting rate during the asymbiotic germination is the major obstacle for seedling establishment. The fundamental understanding of embryo and protocorm developmental mechanisms will guide the development of an effective propagation method. The morphological and physiological characterization of the key developmental process of embryos and protocorms shows that the mature seeds of P. spicerianum consist of a spherical embryo without an endosperm. Seed coats become heavily lignified once the embryo is mature. Embryo cell size is relatively uniform, and significant structure polarity and cell size gradients occur at the early protocorm stage. The high level of auxin and cytokinin accumulation at the early stage of embryo development and protocorm stage may help to facilitate cell division. The transcriptome profiles of protocorms at three different developmental stages were compared to explore the regulatory mechanism of protocorm development. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that differentially expressed genes were implicated in secondary metabolite metabolism, plant hormone signal transduction and photosynthesis. The temporal expression patterns of candidate genes related to embryo and shoot development were analyzed to reveal their roles in protocorm development: in the early stage of protocorm development, embryonic development related genes such as SERKs and BBM1 were active, while in the late stage of protocorm, shoot apical meristem related genes such as WOX8, CLAVATA2, CUC2, and SCR were active.

Systematic analysis of specific and nonspecific auxin effects on endocytosis and trafficking.

The phytohormone auxin and its directional transport through tissues are intensively studied. However, a mechanistic understanding of auxin-mediated feedback on endocytosis and polar distribution of PIN auxin transporters remains limited due to contradictory observations and interpretations. Here, we used state-of-the-art methods to reexamine the auxin effects on PIN endocytic trafficking. We used high auxin concentrations or longer treatments versus lower concentrations and shorter treatments of natural indole-3-acetic acid (IAA) and synthetic naphthalene acetic acid (NAA) auxins to distinguish between specific and nonspecific effects. Longer treatments of both auxins interfere with Brefeldin A-mediated intracellular PIN2 accumulation and also with general aggregation of endomembrane compartments. NAA treatment decreased the internalization of the endocytic tracer dye, FM4-64; however, NAA treatment also affected the number, distribution, and compartment identity of the early endosome/trans-Golgi network, rendering the FM4-64 endocytic assays at high NAA concentrations unreliable. To circumvent these nonspecific effects of NAA and IAA affecting the endomembrane system, we opted for alternative approaches visualizing the endocytic events directly at the plasma membrane (PM). Using total internal reflection fluorescence microscopy, we saw no significant effects of IAA or NAA treatments on the incidence and dynamics of clathrin foci, implying that these treatments do not affect the overall endocytosis rate. However, both NAA and IAA at low concentrations rapidly and specifically promoted endocytosis of photo-converted PIN2 from the PM. These analyses identify a specific effect of NAA and IAA on PIN2 endocytosis, thus, contributing to its polarity maintenance and furthermore illustrate that high auxin levels have nonspecific effects on trafficking and endomembrane compartments.

Alternating temperatures increase germination and emergence in relation to endogenous hormones and enzyme activities in aubergine seeds

High seed quality is important for obtaining high quality seedlings for commercial vegetable production. The rate of seed germination affects seedling quality since seeds which tend to germinate more slowly generally result in low-quality seedlings, especially in aubergine seeds. Alternating temperatures play a crucial role in determining the rate of seed germination in many plant species. This study investigated the effect of alternating temperatures on aubergine seed germination, emergence rates, and physiological changes regulating dormancy. Seeds from four open-pollinated aubergine cultivars were incubated for 14 days under the following temperature regimes: 35/20°C-24/24h, 35/20°C-16/8h, 35/20°C-8/16h, 30/20°C-24/24h, 30/20°C-16/8h, 30/20°C-8/16h, or at constant 25°C. The highest mean germination was 92%, with a corresponding highest mean emergence of 88%, which was found with the 35/20°C-16/8h treatment. All alternating temperature treatments displayed faster germination and emergence compared to the control (25°C), as well as high catalase, superoxide dismutase and ascorbate peroxidase antioxidant enzyme activities, and high auxin and salicylic acid levels. In contrast, lipase enzyme activity, abscisic acid and jasmonic acid content decreased. In conclusion, these findings showed that alternating temperatures improved aubergine seed germination rate, and seedling emergence, and also demonstrated positive correlations with enzymatic and hormonal fluctuations within the seeds.

Nitrate reductase is a key enzyme responsible for nitrogen-regulated auxin accumulation in Arabidopsis roots

Nitrate reductase (NR) is one of the key enzymes for plant nitrogen assimilation and root architecture remodeling. However, crosstalk between NR-mediated signaling and auxin-mediated root development in nitrogen-status responses has not been investigated in details before. In this study, root phenotype and auxin distribution in nia1/nia2 (nitrate reductase) double mutant and chl1-5 (nitrate transporter NRT1.1) mutant under different nitrogen availabilities were compared. The nia1/nia2 mutant showed very low expression levels of auxin biosynthetic/signaling genes and was insensitive to nitrogen changes. While the chl1-5 mutant showed a high NR activity with a high level of auxin in the meristematic zone and a weaker response to nitrogen changes, when compared with the wild-type plants. We firstly found that NR activity was roughly positive-correlated with the root auxin level, and there is a crosstalk between nitrate signaling and auxin signaling. The putative signaling pathways downstream of NR have been discussed.