Atmospheric nitrogen dioxide suppresses the activity of phytochrome interacting factor 4 to suppress hypocotyl elongation.

MAIN CONCLUSION: Ambient concentrations of atmospheric nitrogen dioxide (NO2) inhibit the binding of PIF4 to promoter regions of auxin pathway genes to suppress hypocotyl elongation in Arabidopsis. Ambient concentrations (10-50 ppb) of atmospheric nitrogen dioxide (NO2) positively regulate plant growth to the extent that organ size and shoot biomass can nearly double in various species, including Arabidopsis thaliana (Arabidopsis). However, the precise molecular mechanism underlying NO2-mediated processes in plants, and the involvement of specific molecules in these processes, remain unknown. We measured hypocotyl elongation and the transcript levels of PIF4, encoding a bHLH transcription factor, and its target genes in wild-type (WT) and various pif mutants grown in the presence or absence of 50 ppb NO2. Chromatin immunoprecipitation assays were performed to quantify binding of PIF4 to the promoter regions of its target genes. NO2 suppressed hypocotyl elongation in WT plants, but not in the pifq or pif4 mutants. NO2 suppressed the expression of target genes of PIF4, but did not affect the transcript level of the PIF4 gene itself or the level of PIF4 protein. NO2 inhibited the binding of PIF4 to the promoter regions of two of its target genes, SAUR46 and SAUR67. In conclusion, NO2 inhibits the binding of PIF4 to the promoter regions of genes involved in the auxin pathway to suppress hypocotyl elongation in Arabidopsis. Consequently, PIF4 emerges as a pivotal participant in this regulatory process. This study has further clarified the intricate regulatory mechanisms governing plant responses to environmental pollutants, thereby advancing our understanding of how plants adapt to changing atmospheric conditions.

FERONIA and microtubules independently contribute to mechanical integrity in the Arabidopsis shoot.

To survive, cells must constantly resist mechanical stress. In plants, this involves the reinforcement of cell walls, notably through microtubule-dependent cellulose deposition. How wall sensing might contribute to this response is unknown. Here, we tested whether the microtubule response to stress acts downstream of known wall sensors. Using a multistep screen with 11 mutant lines, we identify FERONIA (FER) as the primary candidate for the cell's response to stress in the shoot. However, this does not imply that FER acts upstream of the microtubule response to stress. In fact, when performing mechanical perturbations, we instead show that the expected microtubule response to stress does not require FER. We reveal that the feronia phenotype can be partially rescued by reducing tensile stress levels. Conversely, in the absence of both microtubules and FER, cells appear to swell and burst. Altogether, this shows that the microtubule response to stress acts as an independent pathway to resist stress, in parallel to FER. We propose that both pathways are required to maintain the mechanical integrity of plant cells.

Sugar inhibits brassinosteroid signaling by enhancing BIN2 phosphorylation of BZR1.

Sugar, light, and hormones are major signals regulating plant growth and development, however, the interactions among these signals are not fully understood at the molecular level. Recent studies showed that sugar promotes hypocotyl elongation by activating the brassinosteroid (BR) signaling pathway after shifting Arabidopsis seedlings from light to extended darkness. Here, we show that sugar inhibits BR signaling in Arabidopsis seedlings grown under light. BR induction of hypocotyl elongation in seedlings grown under light is inhibited by increasing concentration of sucrose. The sugar inhibition of BR response is correlated with decreased effect of BR on the dephosphorylation of BZR1, the master transcription factor of the BR signaling pathway. This sugar effect is independent of the sugar sensors Hexokinase 1 (HXK1) and Target of Rapamycin (TOR), but requires the GSK3-like kinase Brassinosteroid-Insensitive 2 (BIN2), which is stabilized by sugar. Our study uncovers an inhibitory effect of sugar on BR signaling in plants grown under light, in contrast to its promotive effect in the dark. Such light-dependent sugar-BR crosstalk apparently contributes to optimal growth responses to photosynthate availability according to light-dark conditions.

The epidermis coordinates auxin-induced stem growth in response to shade.

Growth of a complex multicellular organism requires coordinated changes in diverse cell types. These cellular changes generate organs of the correct size, shape, and functionality. In plants, the growth hormone auxin induces stem elongation in response to shade; however, which cell types of the stem perceive the auxin signal and contribute to organ growth is poorly understood. Here, we blocked the transcriptional response to auxin within specific tissues to show that auxin signaling is required in many cell types for correct hypocotyl growth in shade, with a key role for the epidermis. Combining genetic manipulations in Arabidopsis thaliana with transcriptional profiling of the hypocotyl epidermis from Brassica rapa, we show that auxin acts in the epidermis in part by inducing activity of the locally acting, growth-promoting brassinosteroid pathway. Our findings clarify cell-specific auxin function in the hypocotyl and highlight the complexity of cell type interactions within a growing organ.

Synthesis, Biological Activity, and Molecular-Docking Studies of New Brassinosteroid Analogs.

Much work has been dedicated to the quest to determine the structure-activity relationship in synthetic brassinosteroid (BR) analogs. Recently, it has been reported that analogs with phenyl or benzoate groups in the alkyl chain present activities comparable to those shown by natural BRs, depending on the nature of the substituent in the aromatic ring. However, as it is well known that the activity depends on the structure of the whole molecule, in this work, we have synthesized a series of compounds with the same substituted benzoate in the alkyl chain and a hydroxyl group at C3. The main goal was to compare the activities with analogs with -OH at C2 and C3. Additionally, a molecular-docking study and molecular dynamics simulations were performed to establish a correlation between the experimental and theoretical results. The synthesis of eight new BR analogs was described. All the analogs were fully characterized by spectroscopical methods. The bioactivity of these analogs was assessed using the rice lamina inclination test (RLIT) and the inhibition of the root and hypocotyl elongation of Arabidopsis thaliana. The results of the RLIT indicate that at the lowest tested concentration (1 × 10-8 M), in the BR analogs in which the aromatic ring was substituted at the para position with methoxy, the I and CN substituents were more active than brassinolide (50-72%) and 2-3 times more active than those analogs in which the substituent group was F, Cl or Br atoms. However, at the highest concentrations, brassinolide was the most active compound, and the structure-activity relationship changed. On the other hand, the results of the A. thaliana root sensitivity assay show that brassinolide and the analogs with I and CN as substituents on the benzoyl group were the most active compounds. These results are in line with those obtained via the RLIT. A comparison of these results with those obtained for similar analogs that had a hydroxyl group at C2 indicates the importance of considering the whole structure. The molecular-docking results indicate that all the analogs adopted a brassinolide-like orientation, while the stabilizing effect of the benzoate group on the interactions with the receptor complex provided energy binding values ranging between -10.17 and -13.17 kcal mol-1, where the analog with a nitrile group was the compound that achieved better contact with the amino acids present in the active site.

Physiological Parameters and Transcriptomic Levels Reveal the Response Mechanism of Maize to Deep Sowing and the Mechanism of Exogenous MeJA to Alleviate Deep Sowing Stress.

Deep sowing, as a method to mitigate drought and preserve soil moisture and seedlings, can effectively mitigate the adverse effects of drought stress on seedling growth. The elongation of the hypocotyl plays an important role in the emergence of maize seeds from deep-sowing stress. This study was designed to explore the function of exogenous methyl jasmonate (MeJA) in the growth of the maize mesocotyl and to examine its regulatory network. The results showed that the addition of a 1.5 µ mol L-1 MeJA treatment significantly increased the mesocotyl length (MES), mesocotyl and coleoptile length (MESCOL), and seedling length (SDL) of maize seedlings. Transcriptome analysis showed that exogenous MeJA can alleviate maize deep-sowing stress, and the differentially expressed genes (DEGs) mainly include ornithine decarboxylase, terpene synthase 7, ethylene responsive transcription factor 11, and so on. In addition, candidate genes that may regulate the length of maize hypocotyls were screened by Weighted Gene Co-expression Network Analysis (WGCNA). These genes may be involved in the growth of maize hypocotyls through transcriptional regulation, histones, ubiquitin protease, protein binding, and chlorophyll biosynthesis and play an important role in maize deep-sowing tolerance. Our research findings may provide a theoretical basis for determining the tolerance of maize to deep-sowing stress and the mechanism of exogenous hormone regulation of deep-sowing stress.

Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-Glucan Synthase That Functions during Plant Cell Wall Synthesis.

In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the ß-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual ß-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent ß-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6.

Salicylic Acid Suppresses Apical Hook Formation via NPR1-Mediated Repression of EIN3 and EIL1 in Arabidopsis.

Salicylic acid (SA) and ethylene (ET) are important phytohormones that regulate numerous plant growth, development, and stress response processes. Previous studies have suggested functional interplay of SA and ET in defense responses, but precisely how these two hormones coregulate plant growth and development processes remains unclear. Our present work reveals antagonism between SA and ET in apical hook formation, which ensures successful soil emergence of etiolated dicotyledonous seedlings. Exogenous SA inhibited ET-induced expression of HOOKLESS1 (HLS1) in Arabidopsis (Arabidopsis thaliana) in a manner dependent on ETHYLENE INSENSITIVE3 (EIN3) and EIN3-LIKE1 (EIL1), the core transcription factors in the ET signaling pathway. SA-activated NONEXPRESSER OF PR GENES1 (NPR1) physically interacted with EIN3 and interfered with the binding of EIN3 to target gene promoters, including the HLS1 promoter. Transcriptomic analysis revealed that NPR1 and EIN3/EIL1 coordinately regulated subsets of genes that mediate plant growth and stress responses, suggesting that the interaction between NPR1 and EIN3/EIL1 is an important mechanism for integrating the SA and ET signaling pathways in multiple physiological processes. Taken together, our findings illuminate the molecular mechanism underlying SA regulation of apical hook formation as well as the antagonism between SA and ET in early seedling establishment and possibly other physiological processes.

Strigolactone and Karrikin Signaling Pathways Elicit Ubiquitination and Proteolysis of SMXL2 to Regulate Hypocotyl Elongation in Arabidopsis.

Strigolactones (SLs) and karrikins (KARs) are related butenolide signaling molecules that control plant development. In Arabidopsis (Arabidopsis thaliana), they are recognized separately by two closely related receptors but use the same F-box protein MORE AXILLARY GROWTH2 (MAX2) for signal transduction, targeting different members of the SMAX1-LIKE (SMXL) family of transcriptional repressors for degradation. Both signals inhibit hypocotyl elongation in seedlings, raising the question of whether signaling is convergent or parallel. Here, we show that synthetic SL analog GR244DO enhanced the interaction between the SL receptor DWARF14 (D14) and SMXL2, while the KAR surrogate GR24 ent-5DS induced association of the KAR receptor KARRIKIN INSENSITIVE2 (KAI2) with SMAX1 and SMXL2. Both signals trigger polyubiquitination and degradation of SMXL2, with GR244DO dependent on D14 and GR24 ent-5DS dependent mainly on KAI2. SMXL2 is critical for hypocotyl responses to GR244DO and functions redundantly with SMAX1 in hypocotyl response to GR24 ent-5DS Furthermore, GR244DO induced response of D14-LIKE2 and KAR-UP F-BOX1 through SMXL2, whereas GR24 ent-5DS induced expression of these genes via both SMAX1 and SMXL2. These findings demonstrate that both SLs and KARs could trigger polyubiquitination and degradation of SMXL2, thus uncovering an unexpected but important convergent pathway in SL- and KAR-regulated gene expression and hypocotyl elongation.

IPyA glucosylation mediates light and temperature signaling to regulate auxin-dependent hypocotyl elongation in Arabidopsis.

Auxin is a class of plant hormone that plays a crucial role in the life cycle of plants, particularly in the growth response of plants to ever-changing environments. Since the auxin responses are concentration-dependent and higher auxin concentrations might often be inhibitory, the optimal endogenous auxin level must be closely controlled. However, the underlying mechanism governing auxin homeostasis remains largely unknown. In this study, a UDP-glycosyltransferase (UGT76F1) was identified from Arabidopsis thaliana, which participates in the regulation of auxin homeostasis by glucosylation of indole-3-pyruvic acid (IPyA), a major precursor of the auxin indole-3-acetic acid (IAA) biosynthesis, in the formation of IPyA glucose conjugates (IPyA-Glc). In addition, UGT76F1 was found to mediate hypocotyl growth by modulating active auxin levels in a light- and temperature-dependent manner. Moreover, the transcription of UGT76F1 was demonstrated to be directly and negatively regulated by PIF4, which is a key integrator of both light and temperature signaling pathways. This study sheds a light on the trade-off between IAA biosynthesis and IPyA-Glc formation in controlling auxin levels and reveals a regulatory mechanism for plant growth adaptation to environmental changes through glucosylation of IPyA.

The chemical compound 'Heatin' stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases.

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.

Strigolactone mimic 2-nitrodebranone is highly active in Arabidopsis growth and development.

Strigolactones play crucial roles in regulating plant architecture and development, as endogenous hormones, and orchestrating symbiotic interactions with fungi and parasitic plants, as components of root exudates. rac-GR24 is currently the most widely used strigolactone analog and serves as a reference compound in investigating the action of strigolactones. In this study, we evaluated a suite of debranones and found that 2-nitrodebranone (2NOD) exhibited higher biological activity than rac-GR24 in various aspects of plant growth and development in Arabidopsis, including hypocotyl elongation inhibition, root hair promotion and senescence acceleration. The enhanced activity of 2NOD in promoting AtD14-SMXL7 and AtD14-MAX2 interactions indicates that the molecular structure of 2NOD is a better match for the ligand perception site pocket of D14. Moreover, 2NOD showed lower activity than rac-GR24 in promoting Orobanche cumana seed germination, suggesting its higher ability to control plant architecture than parasitic interactions. In combination with the improved stability of 2NOD, these results demonstrate that 2NOD is a strigolactone analog that can specifically mimic the activity of strigolactones and that 2NOD exhibits strong potential as a tool for studying the strigolactone signaling pathway in plants.

Karrikins control seedling photomorphogenesis and anthocyanin biosynthesis through a HY5-BBX transcriptional module.

The butenolide molecule, karrikin (KAR), emerging in smoke of burned plant material, enhances light responses such as germination, inhibition of hypocotyl elongation, and anthocyanin accumulation in Arabidopsis. The KAR signaling pathway consists of KARRIKIN INSENSITIVE 2 (KAI2) and MORE AXILLARY GROWTH 2 (MAX2), which, upon activation, act in an SCF E3 ubiquitin ligase complex to target the downstream signaling components SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE 2 (SMXL2) for degradation. How degradation of SMAX1 and SMXL2 is translated into growth responses remains unknown. Although light clearly influences the activity of KAR, the molecular connection between the two pathways is still poorly understood. Here, we demonstrate that the KAR signaling pathway promotes the activity of a transcriptional module consisting of ELONGATED HYPOCOTYL 5 (HY5), B-BOX DOMAIN PROTEIN 20 (BBX20), and BBX21. The bbx20 bbx21 mutant is largely insensitive to treatment with KAR2 , similar to a hy5 mutant, with regards to inhibition of hypocotyl elongation and anthocyanin accumulation. Detailed analysis of higher order mutants in combination with RNA-sequencing analysis revealed that anthocyanin accumulation downstream of SMAX1 and SMXL2 is fully dependent on the HY5-BBX module. However, the promotion of hypocotyl elongation by SMAX1 and SMXL2 is, in contrast to KAR2 treatment, only partially dependent on BBX20, BBX21, and HY5. Taken together, these results suggest that light- and KAR-dependent signaling intersect at the HY5-BBX transcriptional module.

An essential role for abscisic acid in the regulation of xylem fibre differentiation.

Division of the cambial cells and their subsequent differentiation into xylem and phloem drives radial expansion of the hypocotyl. Following the transition to reproductive growth, a phase change occurs in the Arabidopsis hypocotyl. During this second phase, the relative rate of xylem production is dramatically increased compared with that of phloem, and xylem fibres that contain thick secondary cell walls also form. Using two different genetic backgrounds and different environmental conditions, we identified a set of core transcriptional changes that is associated with the switch to the second phase of growth in the hypocotyl. Abscisic acid (ABA) signalling pathways are significantly over-represented in this set of core genes. Reverse genetic analysis demonstrated that mutants that are defective in ABA-biosynthesis enzymes exhibited significantly delayed fibre production without affecting the xylem:phloem ratio, and that these effects can be reversed by the application of ABA. The altered morphology is also reflected at the transcript level, with a reduced expression of marker genes that are associated with fibre formation in aba1 mutants. Taken together, the data reveal an essential role for ABA in the regulation of fibre formation.

Exogenous Auxin Induces Transverse Microtubule Arrays Through TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX Receptors.

Auxin plays a central role in controlling plant cell growth and morphogenesis. Application of auxin to light-grown seedlings elicits both axial growth and transverse patterning of the cortical microtubule cytoskeleton in hypocotyl cells. Microtubules respond to exogenous auxin within 5 min, although repatterning of the array does not initiate until 30 min after application and is complete by 2 h. To examine the requirements for auxin-induced microtubule array patterning, we used an Arabidopsis (Arabidopsis thaliana) double auxin f-box (afb) receptor mutant, afb4-8 afb5-5, that responds to conventional auxin (indole-3-acetic acid) but has a strongly diminished response to the auxin analog, picloram. We show that 5 µm picloram induces immediate changes to microtubule density and later transverse microtubule patterning in wild-type plants, but does not cause microtubule array reorganization in the afb4-8 afb5-5 mutant. Additionally, a dominant mutant (axr2-1) for the auxin coreceptor AUXIN RESPONSIVE2 (AXR2) was strongly suppressed for auxin-induced microtubule array reorganization, providing additional evidence that auxin functions through a transcriptional pathway for transverse patterning. We observed that brassinosteroid application mimicked the auxin response, showing both early and late microtubule array effects, and induced transverse patterning in the axr2-1 mutant. Application of auxin to the brassinosteroid synthesis mutant, diminuto1, induced transverse array patterning but did not produce significant axial growth. Thus, exogenous auxin induces transverse microtubule patterning through the TRANSPORT INHIBITOR 1/AUXIN F-BOX (TIR1/AFB) transcriptional pathway and can act independently of brassinosteroids.

Retrograde Induction of phyB Orchestrates Ethylene-Auxin Hierarchy to Regulate Growth.

Exquisitely regulated plastid-to-nucleus communication by retrograde signaling pathways is essential for fine-tuning of responses to the prevailing environmental conditions. The plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) has emerged as a stress signal transduced into a diverse ensemble of response outputs. Here, we demonstrate enhanced phytochrome B protein abundance in red light-grown MEcPP-accumulating ceh1 mutant Arabidopsis (Arabidopsis thaliana) plants relative to wild-type seedlings. We further establish MEcPP-mediated coordination of phytochrome B with auxin and ethylene signaling pathways and uncover differential hypocotyl growth of red light-grown seedlings in response to these phytohormones. Genetic and pharmacological interference with ethylene and auxin pathways outlines the hierarchy of responses, placing ethylene epistatic to the auxin signaling pathway. Collectively, our findings establish a key role of a plastidial retrograde metabolite in orchestrating the transduction of a repertoire of signaling cascades. This work positions plastids at the zenith of relaying information coordinating external signals and internal regulatory circuitry to secure organismal integrity.

Light-induced stabilization of ACS contributes to hypocotyl elongation during the dark-to-light transition in Arabidopsis seedlings.

Hypocotyl growth during seedling emergence is a crucial developmental transition influenced by light and phytohormones such as ethylene. Ethylene and light antagonistically control hypocotyl growth in either continuous light or darkness. However, how ethylene and light regulate hypocotyl growth, including seedling emergence, during the dark-to-light transition remains elusive. Here, we show that ethylene and light cooperatively stimulate a transient increase in hypocotyl growth during the dark-to-light transition via the light-mediated stabilization of 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs), the rate-limiting enzymes in ethylene biosynthesis. We found that, in contrast to the known inhibitory role of light in hypocotyl growth, light treatment transiently increases hypocotyl growth in wild-type etiolated seedlings. Moreover, ACC, the direct precursor of ethylene, accentuates the effects of light on hypocotyl elongation during the dark-to-light transition. We determined that light leads to the transient elongation of hypocotyls by stabilizing the ACS5 protein during the dark-to-light transition. Furthermore, biochemical analysis of an ACS5 mutant protein bearing an alteration in the C-terminus indicated that light stabilizes ACS5 by inhibiting the degradation mechanism that acts through the C-terminus of ACS5. Our study reveals that plants regulate hypocotyl elongation during seedling establishment by coordinating light-induced ethylene biosynthesis at the post-translational level. Moreover, the stimulatory role of light on hypocotyl growth during the dark-to-light transition provides additional insights into the known inhibitory role of light in hypocotyl development.

A highly efficient organogenesis protocol based on zeatin riboside for in vitro regeneration of eggplant.

BACKGROUND: Efficient organogenesis induction in eggplant (Solanum melongena L.) is required for multiple in vitro culture applications. In this work, we aimed at developing a universal protocol for efficient in vitro regeneration of eggplant mainly based on the use of zeatin riboside (ZR). We evaluated the effect of seven combinations of ZR with indoleacetic acid (IAA) for organogenic regeneration in five genetically diverse S. melongena and one S. insanum L. accessions using two photoperiod conditions. In addition, the effect of six different concentrations of indolebutyric acid (IBA) in order to promote rooting was assessed to facilitate subsequent acclimatization of plants. The ploidy level of regenerated plants was studied. RESULTS: In a first experiment with accessions MEL1 and MEL3, significant (p < 0.05) differences were observed for the four factors evaluated for organogenesis from cotyledon, hypocotyl and leaf explants, with the best results obtained (9 and 11 shoots for MEL1 and MEL3, respectively) using cotyledon tissue, 16 h light / 8 h dark photoperiod conditions, and medium E6 (2 mg/L of ZR and 0 mg/L of IAA). The best combination of conditions was tested in the other four accessions and confirmed its high regeneration efficiency per explant when using both cotyledon and hypocotyl tissues. The best rooting media was R2 (1 mg/L IBA). The analysis of ploidy level revealed that between 25 and 50% of the regenerated plantlets were tetraploid. CONCLUSIONS: An efficient protocol for organogenesis of both cultivated and wild accessions of eggplant, based on the use of ZR, is proposed. The universal protocol developed may be useful for fostering in vitro culture applications in eggplant requiring regeneration of plants and, in addition, allows developing tetraploid plants without the need of antimitotic chemicals.

Photoexcited CRY1 and phyB interact directly with ARF6 and ARF8 to regulate their DNA-binding activity and auxin-induced hypocotyl elongation in Arabidopsis.

Arabidopsis CRY1 and phyB are the primary blue and red light photoreceptors mediating blue and red light inhibition of hypocotyl elongation, respectively. Auxin is a pivotal phytohormone involved in promoting hypocotyl elongation. CRY1 and phyB interact with and stabilize auxin/indole acetic acid proteins (Aux/IAAs) to inhibit auxin signaling. The present study investigated whether photoreceptors might interact directly with Auxin Response Factors (ARFs) to regulate auxin signaling. Protein-protein interaction studies demonstrated that CRY1 and phyB interact physically with ARF6 and ARF8 through their N-terminal domains in a blue and red light-dependent manner, respectively. Moreover, the N-terminal DNA-binding domain of ARF6 and ARF8 is involved in mediating their interactions with CRY1. Genetic studies showed that ARF6 and ARF8 act partially downstream from CRY1 and PHYB to regulate hypocotyl elongation under blue and red light, respectively. Chromatin immunoprecipitation-PCR assays demonstrated that CRY1 and phyB mediate blue and red light repression of the DNA-binding activity of ARF6 and ARF6-target gene expression, respectively. Altogether, the results herein suggest that the direct repression of auxin-responsive gene expression mediated by the interactions of CRY1 and phyB with ARFs constitutes a new layer of the regulatory mechanisms by which light inhibits auxin-induced hypocotyl elongation.

Pinstatic Acid Promotes Auxin Transport by Inhibiting PIN Internalization.

Polar auxin transport plays a pivotal role in plant growth and development. PIN-FORMED (PIN) auxin efflux carriers regulate directional auxin movement by establishing local auxin maxima, minima, and gradients that drive multiple developmental processes and responses to environmental signals. Auxin has been proposed to modulate its own transport by regulating subcellular PIN trafficking via processes such as clathrin-mediated PIN endocytosis and constitutive recycling. Here, we further investigated the mechanisms by which auxin affects PIN trafficking by screening auxin analogs and identified pinstatic acid (PISA) as a positive modulator of polar auxin transport in Arabidopsis (Arabidopsis thaliana). PISA had an auxin-like effect on hypocotyl elongation and adventitious root formation via positive regulation of auxin transport. PISA did not activate SCFTIR1/AFB signaling and yet induced PIN accumulation at the cell surface by inhibiting PIN internalization from the plasma membrane. This work demonstrates PISA to be a promising chemical tool to dissect the regulatory mechanisms behind subcellular PIN trafficking and auxin transport.