【目的】谷氨酰胺合成酶（glutamine synthetase, GS）是氮代谢“GS-GOGAT循环”中的关键酶，研究GS在大豆中的家族成员以及对外界胁迫的响应情况。【方法】利用生物信息学方法，在大豆中全面鉴定GS基因，明确大豆GmGSs基因的位置与结构、蛋白的理化性质以及组织表达模式等，并对非生物胁迫的应答响应进行研究。【结果】从大豆中共鉴定出8个GS基因，位于8条染色体上，对应编码的氨基酸序列长度为356-432 aa。在10个保守基序中，8个GmGS都包含9个保守基序，GmGS7和GmGS8比其他成员多一个motif10保守基序。启动子顺式作用元件分析表明，GmGSs的启动子中包含丰富的光响应元件、激素响应元件和逆境胁迫响应元件。转录组数据分析表明GmGSs基因在所有组织中均有表达，其中GmGS3和GmGS4在各组织中表达量较高。荧光定量qPCR结果表明：在不同浓度的氯化铵处理后，大豆GS家族中GmGS4、GmGS5和GmGS7对低浓度铵盐处理后期响应最显著。且高盐胁迫处理后，GmGS5在根、茎、叶组织中表达量下降；GmGS7在根、茎组织中表达量上升。【结论】GS在大豆中共有8个成员，其中GmGS7在氯化铵处理和盐胁迫中均参与响应。

The phytohormone auxin is essential for plant development and contributes to nearly every aspect of the plant life cycle. The spatio-temporal distribution of auxin depends on a complex interplay between auxin metabolism and cell-to-cell auxin transport. Auxin metabolism and transport are both crucial for plant development; however, it largely remains to be seen how these processes are integrated to ensure defined cellular auxin levels or even gradients within tissues or organs. In this review, we provide a glance at very diverse topics of auxin biology, such as biosynthesis, conjugation, oxidation, and transport of auxin. This broad, but certainly superficial, overview highlights the mutual importance of auxin metabolism and transport. Moreover, it allows pinpointing how auxin metabolism and transport get integrated to jointly regulate cellular auxin homeostasis. Even though these processes have been so far only separately studied, we assume that the phytohormonal crosstalk integrates and coordinates auxin metabolism and transport. Besides the integrative power of the global hormone signaling, we additionally introduce the hypothetical concept considering auxin transport components as gatekeepers for auxin responses.

The engineering of human tissues to cure diseases is an interdisciplinary and a very attractive field of research both in academia and the biotechnology industrial sector. Three-dimensional (3D) biomaterial scaffolds can play a critical role in the development of new tissue morphogenesis via interacting with human cells. Although simple polymeric biomaterials can provide mechanical and physical properties required for tissue development, insufficient biomimetic property and lack of interactions with human progenitor cells remain problematic for the promotion of functional tissue formation. Therefore, the developments of advanced functional biomaterials that respond to stimulus could be the next choice to generate smart 3D biomimetic scaffolds, actively interacting with human stem cells and progenitors along with structural integrity to form functional tissue within a short period. To date, smart biomaterials are designed to interact with biological systems for a wide range of biomedical applications, from the delivery of bioactive molecules and cell adhesion mediators to cellular functioning for the engineering of functional tissues to treat diseases.

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical-basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical-basal position or the identity of the induced structures.

To investigate possible roles of polar auxin transport in vein patterning, cotyledon and leaf vein patterns were compared for plants grown in medium containing polar auxin transport inhibitors (N-1-naphthylphthalamic acid, 9-hydroxyfluorene-9-carboxylic acid, and 2,3,5-triiodobenzoic acid) and in medium containing a less well-characterized inhibitor of auxin-mediated processes, 2-(p-chlorophynoxy)-2-methylpropionic acid. Cotyledon vein pattern was not affected by any inhibitor treatments, although vein morphology was altered. In contrast, leaf vein pattern was affected by inhibitor treatments. Growth in polar auxin transport inhibitors resulted in leaves that lacked vascular continuity through the petiole and had broad, loosely organized midveins, an increased number of secondary veins, and a dense band of misshapen tracheary elements adjacent to the leaf margin. Analysis of leaf vein pattern developmental time courses suggested that the primary vein did not develop in polar auxin transport inhibitor-grown plants, and that the broad midvein observed in these seedlings resulted from the coalescence of proximal regions of secondary veins. Possible models for leaf vein patterning that could account for these observations are discussed.

The distribution and biosynthesis of indole‐3‐acetic acid (IAA) was investigated during early plant development in Arabidopsis. The youngest leaves analysed, less than 0.5 mm in length, contained 250 pg mg−1 of IAA and also exhibited the highest relative capacity to synthesize this hormone. A decrease of nearly one hundred‐fold in IAA content occurred as the young leaves expanded to their full size, and this was accompanied by a clear shift in both pool size and IAA synthesis capacity. The correlation between high IAA content and intense cell division was further verified in tobacco leaves, where a detailed analysis revealed that dividing mesophyll tissue contained ten‐fold higher IAA levels than tissue growing solely by elongation. We demonstrated that all parts of the young Arabidopsis plant can potentially contribute to the auxin needed for growth and development, as not only young leaves, but also all other parts of the plant such as cotyledons, expanding leaves and root tissues have the capacity to synthesize IAA de novo. We also observed that naphthylphthalamic acid (NPA) treatment induced tissue‐dependent feedback inhibition of IAA biosynthesis in expanding leaves and cotyledons, but intriguingly not in young leaves or in the root system. This observation supports the hypothesis that there is a sophisticated tissue‐specific regulatory mechanism for auxin biosynthesis. Finally, a strict requirement for maintaining the pool sizes of IAA was revealed as reductions in leaf expansion followed both decreases and increases in the IAA levels in developing leaves. This indicates that leaves are not only important sources for IAA synthesis, but that normal leaf expansion depends on rigorous control of IAA homeostasis.

AUXIN BINDING PROTEIN1 (ABP1) has long been characterized as a potentially important mediator of auxin action in plants. Analysis of the functional requirement for ABP1 during development was hampered because of embryo lethality of the null mutant in Arabidopsis thaliana. Here, we used conditional repression of ABP1 to investigate its function during vegetative shoot development. Using an inducible cellular immunization approach and an inducible antisense construct, we showed that decreased ABP1 activity leads to a severe retardation of leaf growth involving an alteration in cell division frequency, an altered pattern of endocycle induction, a decrease in cell expansion, and a change in expression of early auxin responsive genes. In addition, local repression of ABP1 activity in the shoot apical meristem revealed an additional role for ABP1 in cell plate formation and cell shape. Moreover, cells at the site of presumptive leaf initiation were more sensitive to ABP1 repression than other regions of the meristem. This spatial context-dependent response of the meristem to ABP1 inactivation and the other data presented here are consistent with a model in which ABP1 acts as a coordinator of cell division and expansion, with local auxin levels influencing ABP1 effectiveness.

The phytohormone auxin is critical for plant growth and orchestrates many developmental processes.This review considers the complex array of mechanisms plants use to control auxin levels, the movement of auxin through the plant, the emerging view of auxin-signalling mechanisms, and several interactions between auxin and other phytohormones. Though many natural and synthetic compounds exhibit auxin-like activity in bioassays, indole-3-acetic acid (IAA) is recognized as the key auxin in most plants. IAA is synthesized both from tryptophan (Trp) using Trp-dependent pathways and from an indolic Trp precursor via Trp-independent pathways; none of these pathways is fully elucidated. Plants can also obtain IAA by beta-oxidation of indole-3-butyric acid (IBA), a second endogenous auxin, or by hydrolysing IAA conjugates, in which IAA is linked to amino acids, sugars or peptides. To permanently inactivate IAA, plants can employ conjugation and direct oxidation. Consistent with its definition as a hormone, IAA can be transported the length of the plant from the shoot to the root; this transport is necessary for normal development, and more localized transport is needed for tropic responses. Auxin signalling is mediated, at least in large part, by an SCFTIR1 E3 ubiquitin ligase complex that accelerates Aux/IAA repressor degradation in response to IAA, thereby altering gene expression. Two classes of auxin-induced genes encode negatively acting products (the Aux/IAA transcriptional repressors and GH3 family of IAA conjugating enzymes), suggesting that timely termination of the auxin signal is crucial. Auxin interaction with other hormone signals adds further challenges to understanding auxin response.Nearly six decades after the structural elucidation of IAA, many aspects of auxin metabolism, transport and signalling are well established; however, more than a few fundamental questions and innumerable details remain unresolved.

Substantial evidence indicates that amino acid conjugates of indole-3-acetic acid (IAA) function in auxin homeostasis, yet the plant enzymes involved in their biosynthesis have not been identified. We tested whether several Arabidopsis thaliana enzymes that are related to the auxin-induced soybean (Glycine max) GH3 gene product synthesize IAA–amino acid conjugates. In vitro reactions with six recombinant GH3 enzymes produced IAA conjugates with several amino acids, based on thin layer chromatography. The identity of the Ala, Asp, Phe, and Trp conjugates was verified by gas chromatography–mass spectrometry. Insertional mutations in GH3.1, GH3.2, GH3.5, and GH3.17 resulted in modestly increased sensitivity to IAA in seedling root. Overexpression of GH3.6 in the activation-tagged mutant dfl1-D did not significantly alter IAA level but resulted in 3.2- and 4.5-fold more IAA-Asp than in wild-type seedlings and mature leaves, respectively. In addition to IAA, dfl1-D was less sensitive to indole-3-butyric acid and naphthaleneacetic acid, consistent with the fact that GH3.6 was active on each of these auxins. By contrast, GH3.6 and the other five enzymes tested were inactive on halogenated auxins, and dfl1-D was not resistant to these. This evidence establishes that several GH3 genes encode IAA-amido synthetases, which help to maintain auxin homeostasis by conjugating excess IAA to amino acids.

Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid-specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how a highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.

Plant architecture is determined by genetic and developmental programs as well as by environmental factors. Sessile plants have evolved a subtle adaptive mechanism that allows them to alter their growth and development during periods of stress. Phytohormones play a central role in this process; however, the molecules responsible for integrating growth- and stress-related signals are unknown. Here, we report a gain-of-function rice (Oryza sativa) mutant, tld1-D, characterized by (and named for) an increased number of tillers, enlarged leaf angles, and dwarfism. TLD1 is a rice GH3.13 gene that encodes indole-3-acetic acid (IAA)-amido synthetase, which is suppressed in aboveground tissues under normal conditions but which is dramatically induced by drought stress. The activation of TLD1 reduced the IAA maxima at the lamina joint, shoot base, and nodes, resulting in subsequent alterations in plant architecture and tissue patterning but enhancing drought tolerance. Accordingly, the decreased level of free IAA in tld1-D due to the conjugation of IAA with amino acids greatly facilitated the accumulation of late-embryogenesis abundant mRNA compared with the wild type. The direct regulation of such drought-inducible genes by changes in the concentration of IAA provides a model for changes in plant architecture via the process of drought adaptation, which occurs frequently in nature.

Plant responses to abiotic stresses are coordinated by arrays of growth and developmental processes. Indole-3-acetic acid (IAA) and abscisic acid (ABA) play critical roles in developmental programmes and environmental responses, respectively, through complex signalling and metabolism networks. However, crosstalk between the two phytohormones in the stress responses remains largely unknown. Here, it is reported that a GH3 family gene, OsGH3-2, encoding an enzyme catalysing IAA conjugation to amino acids, is involved in the modulation of ABA level and stress tolerance. Expression of OsGH3-2 was induced by drought but was suppressed by cold. Overexpression of OsGH3-2 in rice caused significant morphological aberrations related to IAA deficiency, such as dwarfism, smaller leaves, and fewer crown roots and root hairs. The overexpressing line showed significantly reduced carotene, ABA, and free IAA levels, greater stomata aperture, and faster water loss, and was hypersensitive to drought stress. However, the overexpressing line showed increased cold tolerance, which was due to the combined effects of reduced free IAA content, alleviated oxidative damage, and decreased membrane penetrability. Furthermore, expression levels of some ABA synthesis- and stress-related genes were significantly changed in the overexpression line. It was conclude that OsGH3-2 modulates both endogenous free IAA and ABA homeostasis and differentially affects drought and cold tolerance in rice.

GH3 genes are main components of the hormonal mechanism regulating growth and development and, hence, are deeply involved in a broad range of physiological processes. They are implicated in hormonal homeostasis through the conjugation to amino acids of the free form of essential plant growth regulators such as indoleacetic and jasmonic acids. In this work, we showed that OsGH3.1 overexpression in rice caused dwarfism and significantly reduced both free auxin content and cell elongation. Functional classification of the transcriptomic profiling revealed that most genes involved in auxin biosynthesis and auxin signaling inhibition were induced and repressed, respectively. Many genes related to cell organization and biogenesis were also significantly downregulated. The survey also showed that, although the response to abiotic stresses was not clearly stimulated, OsGH3.1 overexpression did activate a significant number of defense-related genes. In successive bioassays, it was demonstrated that the resistance of rice plants to pathogen infection, evaluated with two different Magnaporthe grisea strains, was higher in the transformants overexpressing OsGH3.1. Taken together, these results indicate that OsGH3.1 overexpression reduces auxin content, inhibits cell growth and cell wall loosening, and enhances resistance to a fungal pathogen. Our results provide evidence that auxin homeostasis can regulate the activation of the defense response in rice.

To study the GH3 gene family of Arabidopsis, we investigated a flanking sequence database of Arabidopsis activation-tagged lines. We found a dwarf mutant, named yadokari 1-D (ydk1-D), that had a T-DNA insertion proximal to a GH3 gene. ydk1-D is dominant and has a short hypocotyl not only in light but also in darkness. Moreover, ydk1-D has a short primary root, a reduced lateral root number, and reduced apical dominance. A GH3 gene, named YDK1, was upregulated in ydk1-D, and YDK1 transgenic plants showed the ydk1-D phenotype. YDK1 gene expression was induced by exogenously applied auxin and regulated by auxin-response factor (ARF)7. In addition, YDK1 gene expression was downregulated by blue and far-red (FR) lights. Strong promoter activity of YDK1 was observed in roots and flowers. These results suggest that YDK1 may function as a negative component in auxin signaling by regulating auxin activity.

The hormone auxin is a key regulator of plant growth and development, and great progress has been made understanding auxin transport and signaling. Here, we show that auxin metabolism and homeostasis are also regulated in a complex manner. The principal auxin degradation pathways in Arabidopsis include oxidation by Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1/2 (AtDAO1/2) and conjugation by Gretchen Hagen3s (GH3s). Metabolic profiling of dao1-1 root tissues revealed a 50% decrease in the oxidation product 2-oxoindole-3-acetic acid (oxIAA) and increases in the conjugated forms indole-3-acetic acid aspartic acid (IAA-Asp) and indole-3-acetic acid glutamic acid (IAA-Glu) of 438- and 240-fold, respectively, whereas auxin remains close to the WT. By fitting parameter values to a mathematical model of these metabolic pathways, we show that, in addition to reduced oxidation, both auxin biosynthesis and conjugation are increased in dao1-1 Transcripts of AtDAO1 and GH3 genes increase in response to auxin over different timescales and concentration ranges. Including this regulation of AtDAO1 and GH3 in an extended model reveals that auxin oxidation is more important for auxin homoeostasis at lower hormone concentrations, whereas auxin conjugation is most significant at high auxin levels. Finally, embedding our homeostasis model in a multicellular simulation to assess the spatial effect of the dao1-1 mutant shows that auxin increases in outer root tissues in agreement with the dao1-1 mutant root hair phenotype. We conclude that auxin homeostasis is dependent on AtDAO1, acting in concert with GH3, to maintain auxin at optimal levels for plant growth and development.

Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.© 2020 The Authors New Phytologist © 2020 New Phytologist Trust.

A general gas chromatography/mass spectrometry (MS)-based screen was performed to identify catabolites and conjugates of indole-3-acetic acid (IAA) during vegetative growth of Arabidopsis. This experiment revealed the existence of two new conjugates:N-(indole-3-acetyl)-alfa-alanine (IA-Ala) andN-(indole-3-acetyl)-alfa-leucine (IA-Leu). A method for quantitative analysis of IAA metabolites in plant extracts by liquid chromatography-electrospray tandem MS has been developed. The accuracy and precision of the new method are better than 10% for standards close to the detection limit, and are between 6% and 16% for the entire protocol applied to plant extracts. The low detection limits, 0.02 to 0.1 pmol for the different metabolites, made it possible to use as little as 50 to 100 mg of tissue for quantitative analysis. The analysis was performed on different tissues of an Arabidopsis plant at two stages of development, using heavy labeled internal standards of the catabolite 2-oxoindole-3-acetic acid as well as IAA conjugated to amino acids: aspartate, glutamate, Ala, and Leu. Expanding leaves and roots that generally contain high amounts of the free hormone also contained the highest levels of IA-aspartate, IA-glutamate, and 2-oxoindole-3-acetic acid, supporting their role as irreversible catabolic products. The levels of IA-Leu and IA-Ala did not follow the general distribution of IAA. Interestingly, the level of IA-Leu was highest in roots and IA-Ala in the aerial tissues.