Regulation Of Auxin Homeostasis Research Articles

Significance of NatB-mediated N-terminal acetylation of auxin biosynthetic enzymes in maintaining auxin homeostasis in Arabidopsis thaliana

The auxin IAA (Indole-3-acetic acid) plays key roles in regulating plant growth and development, which depends on an intricate homeostasis that is determined by the balance between its biosynthesis, metabolism and transport. YUC flavin monooxygenases catalyze the rate-limiting step of auxin biosynthesis via IPyA (indole pyruvic acid) and are critical targets in regulating auxin homeostasis. Despite of numerous reports on the transcriptional regulation of YUC genes, little is known about those at the post-translational protein level. Here, we show that loss of function of CKRC3/TCU2, the auxiliary subunit (Naa25) of Arabidopsis NatB, and/or of its catalytic subunit (Naa20), NBC, led to auxin-deficiency in plants. Experimental evidences show that CKRC3/TCU2 can interact with NBC to form a NatB complex, catalyzing the N-terminal acetylation (NTA) of YUC proteins for their intracellular stability to maintain normal auxin homeostasis in plants. Hence, our findings provide significantly new insight into the link between protein NTA and auxin biosynthesis in plants.

Chemical inhibition of the auxin inactivation pathway uncovers the roles of metabolic turnover in auxin homeostasis

The phytohormone auxin, indole-3-acetic acid (IAA), plays a prominent role in plant development. Auxin homeostasis is coordinately regulated by auxin synthesis, transport, and inactivation; however, the physiological contribution of auxin inactivation to auxin homeostasis has not been determined. The GH3 IAA-amino acid conjugating enzymes play a central role in auxin inactivation. Chemical inhibition of GH3 proteins in planta is challenging because the inhibition of these enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we report the characterization of a potent GH3 inhibitor, kakeimide, that selectively targets IAA-conjugating GH3 proteins. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min) and indicates that both auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.

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.

Arabidopsis NHX5 and NHX6 regulate PIN6-mediated auxin homeostasis and growth

NHX5 and NHX6, endosomal Na+,K+/H+ antiporters in Arabidopsis thaliana, play a vital role in growth and development. Our previous study has shown that NHX5 and NHX6 function as H+ leak to regulate auxin-mediated growth in Arabidopsis. In this report, we investigated the function of NHX5 and NHX6 in controlling PIN6-mediated auxin homeostasis and growth in Arabidopsis. Phenotypic analyses found that NHX5 and NHX6 were critical for the function of PIN6, an auxin transporter. We further showed that PIN6 depended on NHX5 and NHX6 in regulating auxin homeostasis. NHX5 and NHX6 were colocalized with PIN6, but they did not interact physically. The conserved acidic residues that are vital for the activity of NHX5 and NHX6 were critical for PIN6 function. Together, NHX5 and NHX6 may regulate PIN6 function by their transport activity.

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.

ZmEHD1 Is Required for Kernel Development and Vegetative Growth through Regulating Auxin Homeostasis.

The roles of C-terminal Eps15 homology domain (EHD) proteins in clathrin-mediated endocytosis in plants are poorly understood. Here, we isolated a maize (Zea mays) mutant, designated ehd1, which showed defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that ehd1 encodes an EHD protein. Internalization of the endocytic tracer FM4-64 was substantially reduced in the ehd1 mutant and ZmEHD1 knockout mutants. We further demonstrated that ZmEHD1 and the ZmAP2 σ subunit physically interact at the plasma membrane. Auxin distribution and ZmPIN1a-YFP localization were altered in the ehd1 mutant. Kernel indole-3-acetic acid levels were substantially lower in the ehd1 mutant than in wild-type maize. Exogenous application of 1-naphthaleneacetic acid, but not GA3 or 2-naphthaleneacetic acid, rescued the seed germination and seedling emergency phenotypic defects of ehd1 mutants. Taken together, these results indicate that ZmEHD1 regulates auxin homeostasis by mediating clathrin-mediated endocytosis through its interaction with the ZmAP2 σ subunit, which is crucial for kernel development and vegetative growth of maize.

Mediator subunit OsMED14_1 plays an important role in rice development.

Mediator, a multisubunit co-activator complex, regulates transcription in eukaryotes and is involved in diverse processes in Arabidopsis through its different subunits. Here, we have explored developmental aspects of one of the rice Mediator subunit gene OsMED14_1. We analyzed its expression pattern through RNA in situ hybridization and pOsMED14_1:GUS transgenics that showed its expression in roots, leaves, anthers and seeds prominently at younger stages, indicating possible involvement of this subunit in multiple aspects of rice development. To understand the developmental roles of OsMED14_1 in rice, we generated and studied RNAi-based knockdown rice plants that showed multiple effects including less height, narrower leaves and culms with reduced vasculature, lesser lateral root branching, defective microspore development, reduced panicle branching and seed set, and smaller seeds. Histological analyses showed that slender organs were caused by reduction in both cell number and cell size in OsMED14_1 knockdown plants. Flow cytometric analyses and expression analyses of cell cycle-related genes revealed that defective cell-cycle progression led to these defects. Expression analyses of auxin-related genes and indole-3-acetic acid (IAA) immunolocalization study indicated altered auxin level in these knockdown plants. Reduction of lateral root branching in knockdown plants was corrected by exogenous IAA supplement. OsMED14_1 physically interacts with transcription factors YABBY5, TAPETUM DEGENERATION RETARDATION (TDR) and MADS29, possibly regulating auxin homeostasis and ultimately leading to lateral organ/leaf, microspore and seed development.

A multidrug and toxic compound extrusion (MATE) transporter modulates auxin levels in root to regulate root development and promotes aluminium tolerance.

MATE (multidrug and toxic compound extrusion) transporters play multiple roles in plants including detoxification, secondary metabolite transport, aluminium (Al) tolerance, and disease resistance. Here we identify and characterize the role of the Arabidopsis MATE transporter DETOXIFICATION30. AtDTX30 regulates auxin homeostasis in Arabidopsis roots to modulate root development and Al-tolerance. DTX30 is primarily expressed in roots and localizes to the plasma membrane of root epidermal cells including root hairs. dtx30 mutants exhibit reduced elongation of the primary root, root hairs, and lateral roots. The mutant seedlings accumulate more auxin in their root tips indicating role of DTX30 in maintaining auxin homeostasis in the root. Al induces DTX30 expression and promotes its localization to the distal transition zone. dtx30 seedlings accumulate more Al in their roots but are hyposensitive to Al-mediated rhizotoxicity perhaps due to saturation in root growth inhibition. Increase in expression of ethylene and auxin biosynthesis genes in presence of Al is absent in dtx30. The mutants exude less citrate under Al conditions, which might be due to misregulation of AtSTOP1 and the citrate transporter AtMATE. In conclusion, DTX30 modulates auxin levels in root to regulate root development and in the presence of Al indirectly modulates citrate exudation to promote Al tolerance.

CSN5A Subunit of COP9 Signalosome Temporally Buffers Response to Heat in Arabidopsis.

The COP9 (constitutive photomorphogenesis 9) signalosome (CSN) is an evolutionarily conserved protein complex which regulates various growth and developmental processes. However, the role of CSN during environmental stress is largely unknown. Using Arabidopsis as model organism, we used CSN hypomorphic mutants to study the role of the CSN in plant responses to environmental stress and found that heat stress specifically enhanced the growth of csn5a-1 but not the growth of other hypomorphic photomorphogenesis mutants tested. Following heat stress, csn5a-1 exhibits an increase in cell size, ploidy, photosynthetic activity, and number of lateral roots and an upregulation of genes connected to the auxin response. Immunoblot analysis revealed an increase in deneddylation of CUL1 but not CUL3 following heat stress in csn5a-1, implicating improved CUL1 activity as a basis for the improved growth of csn5a-1 following heat stress. Studies using DR5::N7-VENUS and DII-VENUS reporter constructs confirm that the heat-induced growth is due to an increase in auxin signaling. Our results indicate that CSN5A has a specific role in deneddylation of CUL1 and that CSN5A is required for the recovery of AUX/IAA repressor levels following recurrent heat stress to regulate auxin homeostasis in Arabidopsis.

Genome-wide comprehensive analysis of transcriptomes and small RNAs offers insights into the molecular mechanism of alkaline stress tolerance in a citrus rootstock

Alkaline stress has serious-negative effects on citrus production. Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka) (Cj) is a rootstock that is tolerant to alkaline stress and iron deficiency. Trifoliate orange (Poncirus trifoliata (L.) Raf.) (Pt), the most widely used rootstock in China, is sensitive to alkaline stress. To investigate the molecular mechanism underlying the tolerance of Cj to alkaline stress, next-generation sequencing was employed to profile the root transcriptomes and small RNAs of Cj and Pt seedlings that were cultured in nutrient solutions along a three pH gradient. This two-level regulation data set provides a system-level view of molecular events with a precise resolution. The data suggest that the auxin pathway may play a central role in the inhibitory effect of alkaline stress on root growth and that the regulation of auxin homeostasis under alkaline stress is important for the adaptation of citrus to alkaline stress. Moreover, the jasmonate (JA) pathway exhibits the opposite response to alkaline stress in Cj and Pt and may contribute to the differences in the alkaline stress tolerance and iron acquisition between Cj and Pt. The dataset provides a wealth of genomic resources and new clues to further study the mechanisms underlying alkaline stress resistance in Cj.

Auxin and Gibberellins Are Required for the Receptor-Like Kinase ERECTA Regulated Hypocotyl Elongation in Shade Avoidance in Arabidopsis.

Plants use shade avoidance strategy to escape the canopy shade when grown under natural conditions. Previous studies showed that the Arabidopsis receptor-like kinase ERECTA (ER) is involved in shade avoidance syndrome. However, the mechanisms of ER in modulating SAR by promoting hypocotyl elongation are unknown yet. Here, we report that ER regulated hypocotyl elongation in shade avoidance requires auxin and gibberellins (GAs). The T-DNA insertional ER mutant er-3 shows a less hypocotyl length than that in Col-0 wild type. Promoter::GUS staining analysis shows that ER and its paralogous genes ERECTA-LIKE1 (ERL1) and ERECTA-LIKE2 (ERL2) are differentially expressed in the seedlings, of which only ER is most obviously upregulated in the hypocotyl by shade treatment. Exogenous feeding assay by using media-application with vertical-grown of Arabidopsis seedlings showed that the hypocotyl length of er-3 is partially promoted by indol-3-acetic acid (IAA), while it is relatively insensitive of er-3 to various concentrations of IAA than that of Col-0. Hypocotyl elongation of er-3 is promoted similar to that of Col-0 by high temperature in the white light condition, but the elongation was not significantly affected by the treatment of the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA). Exogenous GA3 increased the hypocotyl elongation of both er-3 and the wild type in the shade condition, and the GA3 biosynthesis inhibitor paclobutrazol (PAC) severely inhibits the hypocotyl elongation of Col-0 and er-3. Further analysis showed that auxin biosynthesis inhibitors yucasin and L-kynurenine remarkably inhibited the hypocotyl elongation of er-3 while yucasin shows a more severe inhibition to er-3 than Col-0. Relative expression of genes regulating auxin homeostasis and signaling, and GA homeostasis is less in er-3 than that in Col-0. Furthermore, genetic evidences show that ER regulated hypocotyl elongation is dependent of PHYTOCHROME B (PHYB). Overall, we propose that ER regulated shade avoidance by promoting hypocotyl elongation is PHYB-dependent and requires auxin and GAs.

Cytokinin-Auxin Crosstalk in the Gynoecial Primordium Ensures Correct Domain Patterning.

The Arabidopsis (Arabidopsis thaliana) gynoecium consists of two congenitally fused carpels made up of two lateral valve domains and two medial domains, which retain meristematic properties and later fuse to produce the female reproductive structures vital for fertilization. Polar auxin transport (PAT) is important for setting up distinct apical auxin signaling domains in the early floral meristem remnants allowing for lateral domain identity and outgrowth. Crosstalk between auxin and cytokinin plays an important role in the development of other meristematic tissues, but hormone interaction studies to date have focused on more accessible later-stage gynoecia and the spatiotemporal interactions pivotal for patterning of early gynoecium primordia remain unknown. Focusing on the earliest stages, we propose a cytokinin-auxin feedback model during early gynoecium patterning and hormone homeostasis. Our results suggest that cytokinin positively regulates auxin signaling in the incipient gynoecial primordium and strengthen the concept that cytokinin regulates auxin homeostasis during gynoecium development. Specifically, medial cytokinin promotes auxin biosynthesis components [YUCCA1/4 (YUC1/4)] in, and PINFORMED7 (PIN7)-mediated auxin efflux from, the medial domain. The resulting laterally focused auxin signaling triggers ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN6 (AHP6), which then represses cytokinin signaling in a PAT-dependent feedback. Cytokinin also down-regulates PIN3, promoting auxin accumulation in the apex. The yuc1, yuc4, and ahp6 mutants are hypersensitive to exogenous cytokinin and 1-napthylphthalamic acid (NPA), highlighting their role in mediolateral gynoecium patterning. In summary, these mechanisms self-regulate cytokinin and auxin signaling domains, ensuring correct domain specification and gynoecium development.

Abscisic Acid Regulates Auxin Homeostasis in Rice Root Tips to Promote Root Hair Elongation.

Abscisic acid (ABA) plays an essential role in root hair elongation in plants, but the regulatory mechanism remains to be elucidated. In this study, we found that exogenous ABA can promote rice root hair elongation. Transgenic rice overexpressing SAPK10 (Stress/ABA-activated protein kinase 10) had longer root hairs; rice plants overexpressing OsABIL2 (OsABI-Like 2) had attenuated ABA signaling and shorter root hairs, suggesting that the effect of ABA on root hair elongation depends on the conserved PYR/PP2C/SnRK2 ABA signaling module. Treatment of the DR5-GUS and OsPIN-GUS lines with ABA and an auxin efflux inhibitor showed that ABA-induced root hair elongation depends on polar auxin transport. To examine the transcriptional response to ABA, we divided rice root tips into three regions: short root hair, long root hair and root tip zones; and conducted RNA-seq analysis with or without ABA treatment. Examination of genes involved in auxin transport, biosynthesis and metabolism indicated that ABA promotes auxin biosynthesis and polar auxin transport in the root tip, which may lead to auxin accumulation in the long root hair zone. Our findings shed light on how ABA regulates root hair elongation through crosstalk with auxin biosynthesis and transport to orchestrate plant development.

DAO1 catalyzes temporal and tissue-specific oxidative inactivation of auxin in Arabidopsis thaliana

Tight homeostatic regulation of the phytohormone auxin [indole-3-acetic acid (IAA)] is essential to plant growth. Auxin biosynthetic pathways and the processes that inactivate auxin by conjugation to amino acids and sugars have been thoroughly characterized. However, the enzyme that catalyzes oxidation of IAA to its primary catabolite 2-oxindole-3-acetic acid (oxIAA) remains uncharacterized. Here, we show that DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1) catalyzes formation of oxIAA in vitro and in vivo and that this mechanism regulates auxin homeostasis and plant growth. Null dao1-1 mutants contain 95% less oxIAA compared with wild type, and complementation of dao1 restores wild-type oxIAA levels, indicating that DAO1 is the primary IAA oxidase in seedlings. Furthermore, dao1 loss of function plants have altered morphology, including larger cotyledons, increased lateral root density, delayed sepal opening, elongated pistils, and reduced fertility in the primary inflorescence stem. These phenotypes are tightly correlated with DAO1 spatiotemporal expression patterns as shown by DAO1pro:β-glucuronidase (GUS) activity and DAO1pro:YFP-DAO1 signals, and transformation with DAO1pro:YFP-DAO1 complemented the mutant phenotypes. The dominant dao1-2D mutant has increased oxIAA levels and decreased stature with shorter leaves and inflorescence stems, thus supporting DAO1 IAA oxidase function in vivo. A second isoform, DAO2, is very weakly expressed in seedling root apices. Together, these data confirm that IAA oxidation by DAO1 is the principal auxin catabolic process in Arabidopsis and that localized IAA oxidation plays a role in plant morphogenesis.

Physiological and Biochemical Responses of Merwilla plumbea Cultured In Vitro with Different Cytokinins After 1 Year of Growth Under Ex Vitro Conditions

Since their discovery in the 1950s, cytokinins (CKs) have become integral, almost indispensable, components of plant growth media in in vitro propagation. However, despite the extensive use of exogenous CKs in plant tissue culture systems, there is a paucity of knowledge on the longevity of their physiological effect post in vitro. The objective of this study was to evaluate the long-term physiological effect of CKs (including a novel meta-topolin derivative) applied during in vitro propagation of Merwilla plumbea after 1 year of ex vitro growth. Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was used for the quantification of endogenous auxins and phenolic compounds in the 1-year-old M.plumbea plants. Compared to the control, exogenous CKs had a significant carry-over effect on ex vitro growth of M.plumbea plants after 1 year growing under ex vitro conditions. M. plumbea plants treated with the novel CK 6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine (mTTHP, 1.0 µM) had significantly higher leaf area per plant, total leaf fresh weight and bulb diameter. Varying concentrations of free indole-3-acetic acid (IAA), oxindole-3-acetic acid (OxIAA) and indole-3-acetyl-l-aspartic acid (IAA-Asp) were detected in leaves of plants originally treated with exogenous CKs, whereas these compounds were absent in leaves of plants without exogenous CKs. Likewise, a CK-type and concentration-dependent response was observed for hydroxybenzoic acids, hydroxycinnamic acids, flavonoids and antioxidant activity of the extracts. The current study showed the influence of type and concentration of exogenous CKs used in vitro on production of phytochemical compounds during ex vitro growth. Taken together, the study provides significant insights on the residual effect of exogenous CKs on the regulation of auxin homeostasis and phenolic acid and/or flavonoid metabolism in 1-year-old M.plumbea plants growing under ex vitro conditions.

Auxin-mediated expression of a GH3 gene in relation to ontogenic state in Chestnut

The CsGH3 - 1 gene was isolated from chestnut microshoots. Expression analysis during induction of adventitious roots indicates an alteration in shoot auxin homeostasis during maturation that negatively affects adventitious rooting. A new auxin inducible gene isolated from chestnut microshoots was found to encode a protein belonging to group II of the Gretchen Hagen 3 (GH3) family. The gene was, therefore, named CsGH3-1. Predicted protein sequence analysis revealed the presence of conserved domains involved in the conjugation of amino acids to indole-3-acetic acid (IAA). Modelling of the protein and molecular docking of IAA, indole-3-butyric-acid (IBA), 1-naphthaleneacetic acid (NAA), and benzothiazole-2-oxyacetic acid (BTOA) into the active site of CsGH3-1 indicated a high and similar binding affinity for the four substrates. Expression analysis by qPCR indicated that CsGH3-1 is regulated by wounding, darkness, and auxin in chestnut microshoots, in an ontogenetic-dependent manner. Under IBA treatment, upregulation of CsGH3-1 was higher in mature than in juvenile shoots and was negatively correlated with the ability of microshoots to form roots. High levels of auxin-induced expression of CsGH3-1 were detected in mature shoots 24 h after the IBA treatment, whereas transcript levels decreased in rooting-competent shoots with cell-type-specific expression. CsGH3-1 transcripts were specifically localized in cells involved in the initiation of adventitious roots only in rooting-competent shoots at the time when cells switch their fate to root initial cells. Thus, these data show a correlation between the specific localization of transcripts and rooting competence, and they also suggest a role for CsGH3-1 in regulating auxin homeostasis during the onset of adventitious root formation.

Hydrolases of the ILR1-like family of Arabidopsis thaliana modulate auxin response by regulating auxin homeostasis in the endoplasmic reticulum.

Amide-linked conjugates of indole-3-acetic acid (IAA) have been identified in most plant species. They function in storage, inactivation or inhibition of the growth regulator auxin. We investigated how the major known endogenous amide-linked IAA conjugates with auxin-like activity act in auxin signaling and what role ILR1-like proteins play in this process in Arabidopsis. We used a genetically encoded auxin sensor to show that IAA-Leu, IAA-Ala and IAA-Phe act through the TIR1-dependent signaling pathway. Furthermore, by using the sensor as a free IAA reporter, we followed conjugate hydrolysis mediated by ILR1, ILL2 and IAR3 in plant cells and correlated the activity of the hydrolases with a modulation of auxin response. The conjugate preferences that we observed are in agreement with available in vitro data for ILR1. Moreover, we identified IAA-Leu as an additional substrate for IAR3 and showed that ILL2 has a more moderate kinetic performance than observed in vitro. Finally, we proved that IAR3, ILL2 and ILR1 reside in the endoplasmic reticulum, indicating that in this compartment the hydrolases regulate the rates of amido-IAA hydrolysis which results in activation of auxin signaling.

Plant Hormone Homeostasis, Signaling, and Function during Adventitious Root Formation in Cuttings.

Adventitious root (AR) formation in cuttings is a multiphase developmental process, resulting from wounding at the cutting site and isolation from the resource and signal network of the whole plant. Though, promotive effects of auxins are widely used for clonal plant propagation, the regulation and function of plant hormones and their intricate signaling networks during AR formation in cuttings are poorly understood. In this focused review, we discuss our recent publications on the involvement of polar auxin transport (PAT) and transcriptional regulation of auxin and ethylene action during AR formation in petunia cuttings in a broad context. Integrating new findings on cuttings of other plant species and general models on plant hormone networks, a model on the regulation and function of auxin, ethylene, and jasmonate in AR formation of cuttings is presented. PAT and cutting off from the basipetal auxin drain are considered as initial principles generating early accumulation of IAA in the rooting zone. This is expected to trigger a self-regulatory process of auxin canalization and maximization to responding target cells, there inducing the program of AR formation. Regulation of auxin homeostasis via auxin influx and efflux carriers, GH3 proteins and peroxidases, of flavonoid metabolism, and of auxin signaling via AUX/IAA proteins, TOPLESS, ARFs, and SAUR-like proteins are postulated as key processes determining the different phases of AR formation. NO and H2O2 mediate auxin signaling via the cGMP and MAPK cascades. Transcription factors of the GRAS-, AP2/ERF-, and WOX-families link auxin signaling to cell fate specification. Cyclin-mediated governing of the cell cycle, modifications of sugar metabolism and microtubule and cell wall remodeling are considered as important implementation processes of auxin function. Induced by the initial wounding and other abiotic stress factors, up-regulation of ethylene biosynthesis, and signaling via ERFs and early accumulation of jasmonic acid stimulate AR formation, while both pathways are linked to auxin. Future research on the function of candidate genes should consider their tissue-specific role and regulation by environmental factors. Furthermore, the whole cutting should be regarded as a system of physiological units with diverse functions specifically responding to the environment and determining the rooting response.

The WRKY Transcription Factor WRKY71/EXB1 Controls Shoot Branching by Transcriptionally Regulating RAX Genes in Arabidopsis.

Plant shoot branching is pivotal for developmental plasticity and crop yield. The formation of branch meristems is regulated by several key transcription factors including REGULATOR OF AXILLARY MERISTEMS1 (RAX1), RAX2, and RAX3. However, the regulatory network of shoot branching is still largely unknown. Here, we report the identification of EXCESSIVE BRANCHES1 (EXB1), which affects axillary meristem (AM) initiation and bud activity. Overexpression of EXB1 in the gain-of-function mutant exb1-D leads to severe bushy and dwarf phenotypes, which result from excessive AM initiation and elevated bud activities. EXB1 encodes the WRKY transcription factor WRKY71, which has demonstrated transactivation activities. Disruption of WRKY71/EXB1 by chimeric repressor silencing technology leads to fewer branches, indicating that EXB1 plays important roles in the control of shoot branching. We demonstrate that EXB1 controls AM initiation by positively regulating the transcription of RAX1, RAX2, and RAX3. Disruption of the RAX genes partially rescues the branching phenotype caused by EXB1 overexpression. We further show that EXB1 also regulates auxin homeostasis in control of shoot branching. Our data demonstrate that EXB1 plays pivotal roles in shoot branching by regulating both transcription of RAX genes and auxin pathways.

LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis.

Somatic embryogenesis (SE) is an efficient tool for the propagation of plant species and also, a useful model for studying the regulatory networks in embryo development. However, the regulatory networks underlying the transition from nonembryogenic callus to somatic embryos during SE remain poorly understood. Here, we describe an upland cotton (Gossypium hirsutum) CASEIN KINASE I gene, GhCKI, which is a unique key regulatory factor that strongly affects SE. Overexpressing GhCKI halted the formation of embryoids and plant regeneration because of a block in the transition from nonembryogenic callus to somatic embryos. In contrast, defective GhCKI in plants facilitated SE. To better understand the mechanism by which GhCKI regulates SE, the regulatory network was analyzed. A direct upstream negative regulator protein, cotton LEAFY COTYLEDON1, was identified to be targeted to a cis-element, CTTTTC, in the promoter of GhCKI. Moreover, GhCKI interacted with and phosphorylated cotton CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF transcription factor15 by coordinately regulating the expression of cotton PHYTOCHROME INTERACTING FACTOR4, finally disrupting auxin homeostasis, which led to increased cell proliferation and aborted somatic embryo formation in GhCKI-overexpressing somatic cells. Our results show a complex process of SE that is negatively regulated by GhCKI through a complex regulatory network.