WAV E3 ubiquitin ligases mediate degradation of IAA32/34 in the TMK1-mediated auxin signaling pathway during apical hook development.

Auxin regulates plant growth and development through downstream signaling pathways, including the best-known SCFTIR1/AFB-Aux/IAA-ARF pathway and several other less characterized "noncanonical" pathways. Recently, one SCFTIR1/AFB-independent noncanonical pathway, mediated by Transmembrane Kinase 1 (TMK1), was discovered through the analyses of its functions in Arabidopsis apical hook development. Asymmetric accumulation of auxin on the concave side of the apical hook triggers DAR1-catalyzed release of the C-terminal of TMK1, which migrates into the nucleus, where it phosphorylates and stabilizes IAA32/34 to inhibit cell elongation, which is essential for full apical hook formation. However, the molecular factors mediating IAA32/34 degradation have not been identified. Here, we show that proteins in the CYTOKININ INDUCED ROOT WAVING 1 (CKRW1)/WAVY GROWTH 3 (WAV3) subfamily act as E3 ubiquitin ligases to target IAA32/34 for ubiquitination and degradation, which is inhibited by TMK1c-mediated phosphorylation. This antagonistic interaction between TMK1c and CKRW1/WAV3 subfamily E3 ubiquitin ligases regulates IAA32/34 levels to control differential cell elongation along opposite sides of the apical hook.

The Roles of GRETCHEN HAGEN3 (GH3)-Dependent Auxin Conjugation in the Regulation of Plant Development and Stress Adaptation.

The precise control of free auxin (indole-3-acetic acid, IAA) gradient, which is orchestrated by biosynthesis, conjugation, degradation, hydrolyzation, and transport, is critical for all aspects of plant growth and development. Of these, the GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetase family, pivotal in conjugating IAA with amino acids, has garnered significant interest. Recent advances in understanding GH3-dependent IAA conjugation have positioned GH3 functional elucidation as a hot topic of research. This review aims to consolidate and discuss recent findings on (i) the enzymatic mechanisms driving GH3 activity, (ii) the influence of chemical inhibitor on GH3 function, and (iii) the transcriptional regulation of GH3 and its impact on plant development and stress response. Additionally, we explore the distinct biological functions attributed to IAA-amino acid conjugates.

Precise Regulation of the TAA1/TAR-YUCCA Auxin Biosynthesis Pathway in Plants.

The indole-3-pyruvic acid (IPA) pathway is the main auxin biosynthesis pathway in the plant kingdom. Local control of auxin biosynthesis through this pathway regulates plant growth and development and the responses to biotic and abiotic stresses. During the past decades, genetic, physiological, biochemical, and molecular studies have greatly advanced our understanding of tryptophan-dependent auxin biosynthesis. The IPA pathway includes two steps: Trp is converted to IPA by TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS/TRYPTOPHAN AMINOTRANSFERASE RELATED PROTEINs (TAA1/TARs), and then IPA is converted to IAA by the flavin monooxygenases (YUCCAs). The IPA pathway is regulated at multiple levels, including transcriptional and post-transcriptional regulation, protein modification, and feedback regulation, resulting in changes in gene transcription, enzyme activity and protein localization. Ongoing research indicates that tissue-specific DNA methylation and miRNA-directed regulation of transcription factors may also play key roles in the precise regulation of IPA-dependent auxin biosynthesis in plants. This review will mainly summarize the regulatory mechanisms of the IPA pathway and address the many unresolved questions regarding this auxin biosynthesis pathway in plants.

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.

The roles of epigenetic modifications in the regulation of auxin biosynthesis.

Auxin is one of the most important plant growth regulators of plant morphogenesis and response to environmental stimuli. Although the biosynthesis pathway of auxin has been elucidated, the mechanisms regulating auxin biosynthesis remain poorly understood. The transcription of auxin biosynthetic genes is precisely regulated by complex signaling pathways. When the genes are expressed, epigenetic modifications guide mRNA synthesis and therefore determine protein production. Recent studies have shown that different epigenetic factors affect the transcription of auxin biosynthetic genes. In this review, we focus our attention on the molecular mechanisms through which epigenetic modifications regulate auxin biosynthesis.

WRKY46 promotes ammonium tolerance in Arabidopsis by repressing NUDX9 and indole-3-acetic acid-conjugating genes and by inhibiting ammonium efflux in the root elongation zone.

Ammonium (NH4+ ) is toxic to root growth in most plants, even at moderate concentrations. Transcriptional regulation is one of the most important mechanisms in the response of plants to NH4+ toxicity, but the nature of the involvement of transcription factors (TFs) in this regulation remains unclear. Here, RNA-seq analysis was performed on Arabidopsis roots to screen for ammonium-responsive TFs. WRKY46, the member of the WRKY transcription factor family most responsive to NH4+ , was selected. We defined the role of WRKY46 using mutation and overexpression assays, and characterized the regulation of NUDX9 and indole-3-acetic acid (IAA)-conjugating genes by WRKY46 via yeast one-hybrid and electrophoretic mobility shift assays and chromatin immunoprecipitation-quantitative real-time polymerase chain reaction (ChIP-qPCR). Knockout of WRKY46 increased, while overexpression of WRKY46 decreased, NH4+ -suppression of the primary root. WRKY46 is shown to directly bind to the promoters of the NUDX9 and IAA-conjugating genes (GH3.1, GH3.6, UGT75D1, UGT84B2) and to inhibit their transcription, thus positively regulating free IAA content and stabilizing protein N-glycosylation, leading to an inhibition of NH4+ efflux in the root elongation zone (EZ). We identify TF involvement in the regulation of NH4+ efflux in the EZ, and show that WRKY46 inhibits NH4+ efflux by negative regulation of NUDX9 and IAA-conjugating genes.

High ammonium inhibits root growth in Arabidopsis thaliana by promoting auxin conjugation rather than inhibiting auxin biosynthesis.

Ammonium (NH4+) inhibits primary root (PR) growth in most plant species when present even at moderate concentrations. Previous studies have shown that transport of indole-3-acetic acid (IAA) is critical to maintaining root elongation under high-NH4+ stress. However, the precise regulation of IAA homeostasis under high-NH4+ stress (HAS) remains unclear. In this study, qRT-PCR, RNA-seq, free IAA and IAA conjugate and PR elongation measurements were conducted in genetic mutants to investigate the role of IAA biosynthesis and conjugation under HAS. Our data clearly show that HAS decreases free IAA in roots by increasing IAA inactivation but does not decrease IAA biosynthesis, and that the IAA-conjugating genes GH3.1, GH3.2, GH3.3, GH3.4, and GH3.6 function as the key genes in regulating high-NH4+ sensitivity in the roots. Furthermore, the analysis of promoter::GUS staining in situ and genetic mutants reveals that HAS promotes IAA conjugation in the elongation zone (EZ), which may be responsible for the PR inhibition observed under HAS. This study provides potential new insight into the role of auxin in the improvement of tolerance to NH4+.

Function of histone H2B monoubiquitination in transcriptional regulation of auxin biosynthesis in Arabidopsis.

The auxin IAA is a vital plant hormone in controlling growth and development, but our knowledge about its complicated biosynthetic pathways and molecular regulation are still limited and fragmentary. cytokinin induced root waving 2 (ckrw2) was isolated as one of the auxin-deficient mutants in a large-scale forward genetic screen aiming to find more genes functioning in auxin homeostasis and/or its regulation. Here we show that CKRW2 is identical to Histone Monoubiquitination 1 (HUB1), a gene encoding an E3 ligase required for histone H2B monoubiquitination (H2Bub1) in Arabidopsis. In addition to pleiotropic defects in growth and development, loss of CKRW2/HUB1 function also led to typical auxin-deficient phenotypes in roots, which was associated with significantly lower expression levels of several functional auxin synthetic genes, namely TRP2/TSB1, WEI7/ASB1, YUC7 and AMI1. Corresponding defects in H2Bub1 were detected in the coding regions of these genes by chromatin immunoprecipitation (ChIP) analysis, indicating the involvement of H2Bub1 in regulating auxin biosynthesis. Importantly, application of exogenous cytokinin (CK) could stimulate CKRW2/HUB1 expression, providing an epigenetic avenue for CK to regulate the auxin homeostasis. Our results reveal a previously unknown mechanism for regulating auxin biosynthesis via HUB1/2-mediated H2Bub1 at the chromatin level.

Transcriptome analysis of rice (Oryza sativa L.) in response to ammonium resupply reveals the involvement of phytohormone signaling and the transcription factor OsJAZ9 in reprogramming of nitrogen uptake and metabolism.

NH4+ is not only the primary nitrogen for rice, a well-known NH4+ specialist, but is also the chief limiting factor for its production. Limiting NH4+ triggers a series of physiological and biochemical responses that help rice optimise its nitrogen acquisition. However, the dynamic nature and spatial distribution of the adjustments at the whole plant level during this response are still unknown. Here, nitrogen-starved rice seedlings were treated with 0.1 mM (NH4)2SO4 for 4 or 12 h, and then the shoots and roots were harvested for RNA-Seq analysis. We identified 138 and 815 differentially expressed genes (DEGs) in shoots, and 597 and 1074 in roots following 4 and 12 h treatment, respectively. Up-regulated DEGs mainly participated in phenylpropanoid, sugar, and amino acid metabolism, which was confirmed by chemical content analysis. The transcription factor OsJAZ9 was the most pronouncedly induced component under low NH4+ in roots, and a significant increase in root growth, NH4+ absorption, amino acid, and sugar metabolism in response to resupplied NH4+ following nitrogen starvation was identified in JAZ9ox (OsJAZ9-overexpressed) and coi1 (OsCOI1-RNAi). Our data provide comprehensive insight into the whole-plant transcriptomic response in terms of metabolic processes and signaling transduction to a low-NH4+ signal, and identify the transcription factor OsJAZ9 and its involvement in the regulation of carbon/nitrogen metabolism as central to the response to low NH4+.

Endogenous ABA alleviates rice ammonium toxicity by reducing ROS and free ammonium via regulation of the SAPK9-bZIP20 pathway.

Ammonium (NH4+) is one of the principal nitrogen (N) sources in soils, but is typically toxic already at intermediate concentrations. The phytohormone abscisic acid (ABA) plays a pivotal role in responses to environmental stresses. However, the role of ABA under high-NH4+ stress in rice (Oryza sativa L.) is only marginally understood. Here, we report that elevated NH4+ can significantly accelerate tissue ABA accumulation. Mutants with high (Osaba8ox) and low levels of ABA (Osphs3-1) exhibit elevated tolerance or sensitivity to high-NH4+ stress, respectively. Furthermore, ABA can decrease NH4+-induced oxidative damage and tissue NH4+ accumulation by enhancing antioxidant and glutamine synthetase (GS)/glutamate synthetasae (GOGAT) enzyme activities. Using RNA sequencing and quantitative real-time PCR approaches, we ascertain that two genes, OsSAPK9 and OsbZIP20, are induced both by high NH4+ and by ABA. Our data indicate that OsSAPK9 interacts with OsbZIP20, and can phosphorylate OsbZIP20 and activate its function. When OsSAPK9 or OsbZIP20 are knocked out in rice, ABA-mediated antioxidant and GS/GOGAT activity enhancement under high-NH4+ stress disappear, and the two mutants are more sensitive to high-NH4+ stress compared with their wild types. Taken together, our results suggest that ABA plays a positive role in regulating the OsSAPK9-OsbZIP20 pathway in rice to increase tolerance to high-NH4+ stress.

Functional roles of Arabidopsis CKRC2/YUCCA8 gene and the involvement of PIF4 in the regulation of auxin biosynthesis by cytokinin.

Auxin and cytokinin (CK) are both important hormones involved in many aspects of plant growth and development. However, the details of auxin biosynthesis and the interaction between auxin and CK are still unclear. Isolation and characterization of an auxin deficient mutant cytokinin induced root curling 2 (ckrc2) in this work reveal that CKRC2 encodes a previously identified member of YUCCA (YUC) flavin monooxygenase-like proteins (YUC8). Our results show that, like other YUCs, CKRC2/YUC8 is a rate-limiting enzyme for catalyzing the conversion of indole-3-pyruvic acid (IPyA) to indole-3-acetic acid (IAA), acting downstream of CKRC1/TAA1 in the IPyA pathway. Here we show that the transcription of both CKRC1/TAA and CKRC2/YUC8 can be induced by CK and that the phytochrome-interacting factor 4 (PIF4) is required for this upregulation. Transcription of PIF4 itself is induced by CK via the AHKs-ARR1/12 signalling pathway. These results indicate that PIF4 plays an essential role in mediating the regulatory effect of CK on the transcriptions of CKRC1 and CKRC2 genes in the IPyA pathway of auxin biosynthesis.

Forward genetic screen for auxin-deficient mutants by cytokinin.

Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

Frequent problems and their resolutions by using thermal asymmetric interlaced PCR (TAIL-PCR) to clone genes in Arabidopsis T-DNA tagged mutants.

T-DNA insertional mutagenesis is a powerful tool in Arabidopsis functional genomics research. Previous studies have developed thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) as an efficient strategy in isolation of DNA sequences adjacent to known sequences in T-DNA tagged mutants. However, a number of problems are encountered when attempts are made to clone flanking sequences in T-DNA tagged mutants. Therefore, it is necessary to improve the efficiency of cloning mutagenesis. Here, we present the most frequent problems and provide an improved method to increase TAIL-PCR efficiency. Even then, it is not always possible to successfully obtain flanking sequences; in such cases, we recommend using high-throughput sequencing to determine the mutations.

Involvement of secondary messengers and small organic molecules in auxin perception and signaling.

Auxin is a major phytohormone involved in most aspects of plant growth and development. Generally, auxin is perceived by three distinct receptors: TRANSPORT INHIBITOR RESISTANT1-Auxin/INDOLE ACETIC ACID, S-Phase Kinase-Associated Protein 2A and AUXIN-BINDING PROTEIN1. The auxin perception is regulated by a variety of secondary messenger molecules, including nitric oxide, reactive oxygen species, calcium, cyclic GMP, cyclic AMP, inositol triphosphate, diacylglycerol and by physiological pH. In addition, some small organic molecules, including inositol hexakisphosphate, yokonolide B, p-chlorophenoxyisobutyric acid, toyocamycin and terfestatin A, are involved in auxin signaling. In this review, we summarize and discuss the recent progress in understanding the functions of these secondary messengers and small organic molecules, which are now thoroughly demonstrated to be pervasive and important in auxin perception and signal transduction.