| 1. |
- Brunoni, Federica, et al.
(författare)
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A bacterial assay for rapid screening of IAA catabolic enzymes
- 2019
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Ingår i: Plant Methods. - : BioMed Central (BMC). - 1746-4811. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-L-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays.Results: In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein.Conclusions: This new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.
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| 2. |
- Brunoni, Federica, et al.
(författare)
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Control of root meristem establishment in conifers
- 2019
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Ingår i: Physiologia Plantarum. - : John Wiley & Sons. - 0031-9317 .- 1399-3054. ; 165:1, s. 81-89
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Tidskriftsartikel (refereegranskat)abstract
- The evolution of terrestrial plant life was made possible by the establishment of a root system, which enabled plants to migrate from aquatic to terrestrial habitats. During evolution, root organization has gradually progressed from a very simple to a highly hierarchical architecture. Roots are initiated during embryogenesis and branch afterward through lateral root formation. Additionally, adventitious roots can be formed post-embryonically from aerial organs. Induction of adventitious roots (ARs) forms the basis of the vegetative propagation via cuttings in horticulture, agriculture and forestry. This method, together with somatic embryogenesis, is routinely used to clonally multiply conifers. In addition to being utilized as propagation techniques, adventitious rooting and somatic embryogenesis have emerged as versatile models to study cellular and molecular mechanisms of embryo formation and organogenesis of coniferous species. Both formation of the embryonic root and the AR primordia require the establishment of auxin gradients within cells that coordinate the developmental response. These processes also share key elements of the genetic regulatory networks that, e.g. are triggering cell fate. This minireview gives an overview of the molecular control mechanisms associated with root development in conifers, from initiation in the embryo to post-embryonic formation in cuttings.
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| 3. |
- Lakehal, Abdellah, et al.
(författare)
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A DAO1-Mediated Circuit Controls Auxin and Jasmonate Crosstalk Robustness during Adventitious Root Initiation in Arabidopsis
- 2019
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 20:18
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Tidskriftsartikel (refereegranskat)abstract
- Adventitious rooting is a post-embryonic developmental program governed by a multitude of endogenous and environmental cues. Auxin, along with other phytohormones, integrates and translates these cues into precise molecular signatures to provide a coherent developmental output. Auxin signaling guides every step of adventitious root (AR) development from the early event of cell reprogramming and identity transitions until emergence. We have previously shown that auxin signaling controls the early events of AR initiation (ARI) by modulating the homeostasis of the negative regulator jasmonate (JA). Although considerable knowledge has been acquired about the role of auxin and JA in ARI, the genetic components acting downstream of JA signaling and the mechanistic basis controlling the interaction between these two hormones are not well understood. Here we provide evidence that COI1-dependent JA signaling controls the expression of DAO1 and its closely related paralog DAO2. In addition, we show that the dao1-1 loss of function mutant produces more ARs than the wild type, probably due to its deficiency in accumulating JA and its bioactive metabolite JA-Ile. Together, our data indicate that DAO1 controls a sensitive feedback circuit that stabilizes the auxin and JA crosstalk during ARI.
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| 4. |
- Lakehal, Abdellah, et al.
(författare)
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A Molecular Framework for the Control of Adventitious Rooting by TIR1/AFB2-Aux/IAA-Dependent Auxin Signaling in Arabidopsis
- 2019
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Ingår i: Molecular Plant. - : Elsevier. - 1674-2052 .- 1752-9867. ; 12:11, s. 1499-1514
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Tidskriftsartikel (refereegranskat)abstract
- In Arabidopsis thaliana, canonical auxin-dependent gene regulation is mediated by 23 transcription factors from the AUXIN RESPONSE FACTOR (ARF) family that interact with auxin/indole acetic acid repressors (Aux/IAAs), which themselves form co-receptor complexes with one of six TRANSPORT INHIBITOR1/AUXIN-SIGNALLING F-BOX (TIR1/AFB) proteins. Different combinations of co-receptors drive specific sensing outputs, allowing auxin to control a myriad of processes. ARF6 and ARF8 are positive regulators of adventitious root initiation upstream of jasmonate, but the exact auxin co-receptor complexes controlling the transcriptional activity of these proteins has remained unknown. Here, using loss-of-function mutants we show that three Aux/IAA genes, IAA6, IAA9, and IAA17, act additively in the control of adventitious root (AR) initiation. These three IAA proteins interact with ARF6 and/or ARF8 and likely repress their activity in AR development. We show that TIR1 and AFB2 are positive regulators of AR formation and TIR1 plays a dual role in the control of jasmonic acid (JA) biosynthesis and conjugation, as several JA biosynthesis genes are up-regulated in the tir1-1 mutant. These results lead us to propose that in the presence of auxin, TIR1 and AFB2 form specific sensing complexes with IAA6, IAA9, and/or IAA17 to modulate JA homeostasis and control AR initiation.
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| 5. |
- Lakehal, Abdellah, 1984-
(författare)
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A molecular network mediating adventitious root initiation in Arabidopsis thaliana
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- To adapt to the ever-changing rhizosphere conditions, land plants evolved a sophisticated root system. The genetic determinants of the root system establishment have been the targets of natural selection, resulting in a very complex but robust molecular networks and circuits. These networks provide the plant with precise cell-fate and developmental decisions. The plant root system consists of primary root, lateral roots and often adventitious roots (ARs). ARs derive from the aboveground organs in response to either intrinsic developmental cues or in response to the environmental ones. AR formation is a pre-requisite step for vegetative propagation, which is widely used to multiply elite genotypes in forestry and agriculture. The main focus of this study is to unravel the molecular networks controlling AR initiation (ARI) using the intact-etiolated Arabidopsis hypocotyl as a model system. Previous data from our laboratory showed that ARI in Arabidopsis is controlled by a crosstalk between the positive regulator auxin (IAA) and the negative regulator jasmonate (JA). First, combining genetic, biochemical and hormonomics approaches, we identified the auxin coreceptor complexes involved in ARI. We found that IAA is perceived by two F-box proteins (TRANSPORT INHIBITOR1/AUXIN-SIGNALLING F-BOX (TIR1) and its closest homolog AFB2 as well as three Auxin/Inodole-3-acetic acid (Aux/IAA) repressors (IAA6, IAA9 and IAA17). These coreceptor proteins possibly act in combinatorial manner to fine-tune the auxin signaling machinery during ARI. In addition, in a genetic screen, we also revealed that the COP9 SIGNALOSOME SUBUNIT 4 (CSN4) protein plays a central role in ARI by modulating the function of the auxin perception machinery. Next, in silico search for genes acting downstream of JA involved in ARI, we retrieved the recently characterized DIOXYGENASE FOR AUXIN OXIDATION (DAO1) and DAO2 genes. The DAOs encode for enzymes that catalyze the conversion of free IAA into 2-oxindole-3-acetic acid (oxIAA), a rate-limiting step in auxin degradation. We found that the DAO1 gene mediates a molecular circuit to stabilize the interaction between IAA and JA. Combining genetics, genome-wide transcriptome profiling, hormononics and cell biological approaches, we found that MYC2-mediated JA signaling controls the expression of the ETHYLENE RESPONSE FACTOR 115 (ERF115) gene, which is a repressor of ARI. Our genetic data revealed that ERF115-mediated ARI inhibition requires cytokinins (CKs). CKs have long been established as inhibitors of ARI. Altogether, ARI seems to be controlled by a complex molecular network guided by three hormonal pathways (IAA, JA and CK), in which JA-induced ERF115 plays a role of "molecular switch".
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| 6. |
- Le Hir, Rozenn, et al.
(författare)
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AtbHLH68 transcription factor contributes to the regulation of ABA homeostasis and drought stress tolerance in Arabidopsis thaliana
- 2017
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Ingår i: Physiologia Plantarum. - : Wiley-Blackwell. - 0031-9317 .- 1399-3054. ; 160:3, s. 312-327
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Tidskriftsartikel (refereegranskat)abstract
- Basic helix-loop-helix (bHLH) transcription factors are involved in a wide range of developmental processes and in response to biotic and abiotic stresses. They represent one of the biggest families of transcription factors but only few of them have been functionally characterized. Here we report the characterization of AtbHLH68 and show that, although the knock out mutant did not have an obvious development phenotype, it was slightly more sensitive to drought stress than the Col-0, and AtbHLH68 overexpressing lines displayed defects in lateral root (LR) formation and a significant increased tolerance to drought stress, likely related to an enhanced sensitivity to abscisic acid (ABA) and/or increased ABA content. AtbHLH68 was expressed in the vascular system of Arabidopsis and its expression was modulated by exogenously applied ABA in an organ-specific manner. We showed that the expression of genes involved in ABA metabolism [AtAAO3 (AtALDEHYDE OXIDASE 3) and AtCYP707A3 (AtABSCISIC ACID 8HYDROXYLASE 3)], in ABA-related response to drought-stress (AtMYC2, AtbHLH122 and AtRD29A) or during LRs development (AtMYC2 and AtABI3) was de-regulated in the overexpressing lines. We propose that AtbHLH68 has a function in the regulation of LR elongation, and in the response to drought stress, likely through an ABA-dependent pathway by regulating directly or indirectly components of ABA signaling and/or metabolism.
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| 7. |
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| 8. |
- Pacurar, Daniel Ioan, et al.
(författare)
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The Arabidopsis Cop9 signalosome subunit 4 (CNS4) is involved in adventitious root formation
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The COP9 signalosome (CSN) is an evolutionary conserved multiprotein complex that regulates many aspects of plant development by controlling the activity of CULLIN-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate and target for proteasomal degradation a vast number of specific substrate proteins involved in many developmental and physiological processes, including light and hormone signaling and cell division. As a consequence of CSN pleiotropic function, complete loss of CSN activity results in seedling lethality. Therefore, a detailed analysis of CSN physiological functions in adult Arabidopsis plants has been hampered by the early seedling lethality of csn null mutants. Here we report the identification and characterization of a viable allele of the Arabidopsis COP9 signalosome subunit 4 (CSN4). The allele, designated csn4-2035, suppresses the adventitious root (AR) phenotype of the Arabidopsis superroot2-1 mutant, potentially by altering its auxin signaling. Furthermore, we show that although the csn4-2035 mutation affects primary and lateral root (LR) formation in the 2035 suppressor mutant, CSN4 and other subunits of the COP9 complex seem to differentially control AR and LR development.
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| 9. |
- Rahneshan, Zahra, et al.
(författare)
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Unravelling salt stress responses in two pistachio (Pistacia vera L.) genotypes
- 2018
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Ingår i: Acta Physiologiae Plantarum. - : Springer Berlin/Heidelberg. - 0137-5881 .- 1861-1664. ; 40:9
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Tidskriftsartikel (refereegranskat)abstract
- Pistachio tree (Pistacia vera L.) is among the tree species that are most tolerant to salinity stress. In the present investigation, we analyzed the behavior of two pistachio genotypes (Badami-e-Zarand (BZ) and Badami-e-Sefid (BS)) under different NaCl concentrations to reveal the mechanisms involved in salinity tolerance. A greater decline in several growth-related traits and biomass as well as relative water content was observed in BS seedlings than in BZ seedlings. Proline content, which is an indicator of stress, increased in both genotypes. Salinity induced oxidative stress in both genotypes, but the levels were higher for the BS genotype. The negative impact of salinity on photosynthetic process in BS was represented by a remarkable decrease in total chlorophyll and carotenoids, while the better performance of the BZ genotype under high salinity was accompanied by an increase in the activities of ascorbate peroxidase, catalase and guaiacol peroxidase. A significant increase in the superoxide dismutase activity in the leaves of BZ was observed under moderate salinity treatment. In both genotypes, Na+ content in leaf and root tissues increased progressively after salinity treatment. However, the leaves of BZ contained less Na+ and retained a lower Na+/K+ ratio. Moreover, under salinity treatment, BZ seedlings had a greater amount of NHX1 transcripts, which suggests that excess Na+ may be sequestered into root vacuoles to avoid deleterious effects of these toxic ions.
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