Chemical induction of hypocotyl rooting reveals extensive conservation of auxin signalling controlling lateral and adventitious root formation.

Upon exposure to light, etiolated Arabidopsis seedlings form adventitious roots (AR) along the hypocotyl. While processes underlying lateral root formation are studied intensively, comparatively little is known about the molecular processes involved in the initiation of hypocotyl AR. AR and LR formation were studied using a small molecule named Hypocotyl Specific Adventitious Root INducer (HYSPARIN) that strongly induces AR but not LR formation. HYSPARIN does not trigger rapid DR5-reporter activation, DII-Venus degradation or Ca2+ signalling. Transcriptome analysis, auxin signalling reporter lines and mutants show that HYSPARIN AR induction involves nuclear TIR1/AFB and plasma membrane TMK auxin signalling, as well as multiple downstream LR development genes (SHY2/IAA3, PUCHI, MAKR4 and GATA23). Comparison of the AR and LR induction transcriptome identified SAURs, AGC kinases and OFP transcription factors as specifically upregulated by HYSPARIN. Members of the SAUR19 subfamily, OFP4 and AGC2 suppress HYS-induced AR formation. While SAUR19 and OFP subfamily members also mildly modulate LR formation, AGC2 regulates only AR induction. Analysis of HYSPARIN-induced AR formation uncovers an evolutionary conservation of auxin signalling controlling LR and AR induction in Arabidopsis seedlings and identifies SAUR19, OFP4 and AGC2 kinase as novel regulators of AR formation.

Species-specific transcriptional reprogramming during adventitious root initiation.

Adventitious roots or shoot-borne roots transdifferentiate from cells close to vascular tissues after cell reprogramming, which is associated with increased transcriptional activity. Recently, Garg et al. provided a genome-wide landscape of transcriptional signatures during the early stages of adventitious root initiation in rice and showed that conserved transcription factors acquire species-specific function.

Natural variation in the long-distance transport of nutrients and photoassimilates in response to N availability.

Phloem and xylem tissues are necessary for the allocation of nutrients and photoassimilates. However, how the long-distance transport of carbon (C) and nitrogen (N) is coordinated with the central metabolism is largely unknown. To better understand how the genetic and environmental factors influence C and N transport, we analysed the metabolite profiles of phloem exudates and xylem saps of five Arabidopsis thaliana accessions grown in low or non-limiting N supply. We observed that xylem saps were composed of 46 or 56% carbohydrates, 27 or 45% amino acids, and 5 or 13% organic acids in low or non-limiting N supply, respectively. In contrast, phloem exudates were composed of 76 or 86% carbohydrates, 7 or 18% amino acids, and 5 or 6% organic acids. Variation in N supply impacted amino acid, organic acid and sugar contents. When comparing low N and non-limiting N, the most striking differences were variations of glutamine, aspartate, and succinate abundance in the xylem saps and citrate and fumarate abundance in phloem exudates. In addition, we observed a substantial variation of metabolite content between genotypes, particularly under high N. The content of several organic acids, such as malate, citrate, fumarate, and succinate was affected by the genotype alone or by the interaction between genotype and N supply. This study confirmed that the response of the transport of nutrients in the phloem and the xylem to N availability is associated with the regulation of the central metabolism and could be an adaptive trait.

Molecular basis of differential adventitious rooting competence in poplar genotypes.

Recalcitrant adventitious root (AR) development is a major hurdle in propagating commercially important woody plants. Although significant progress has been made to identify genes involved in subsequent steps of AR development, the molecular basis of differences in apparent recalcitrance to form AR between easy-to-root and difficult-to-root genotypes remains unknown. To address this, we generated cambium tissue-specific transcriptomic data from stem cuttings of hybrid aspen, T89 (difficult-to-root) and hybrid poplar OP42 (easy-to-root), and used transgenic approaches to verify the role of several transcription factors in the control of adventitious rooting. Increased peroxidase activity was positively correlated with better rooting. We found differentially expressed genes encoding reactive oxygen species scavenging proteins to be enriched in OP42 compared with T89. A greater number of differentially expressed transcription factors in cambium cells of OP42 compared with T89 was revealed by a more intense transcriptional reprograming in the former. PtMYC2, a potential negative regulator, was less expressed in OP42 compared with T89. Using transgenic approaches, we demonstrated that PttARF17.1 and PttMYC2.1 negatively regulate adventitious rooting. Our results provide insights into the molecular basis of genotypic differences in AR and implicate differential expression of the master regulator MYC2 as a critical player in this process.

A vacuolar hexose transport is required for xylem development in the inflorescence stem.

In Angiosperms, the development of the vascular system is controlled by a complex network of transcription factors. However, how nutrient availability in the vascular cells affects their development remains to be addressed. At the cellular level, cytosolic sugar availability is regulated mainly by sugar exchanges at the tonoplast through active and/or facilitated transport. In Arabidopsis (Arabidopsis thaliana), among the genes encoding tonoplastic transporters, SUGAR WILL EVENTUALLY BE EXPORTED TRANSPORTER 16 (SWEET16) and SWEET17 expression has been previously detected in the vascular system. Here, using a reverse genetics approach, we propose that sugar exchanges at the tonoplast, regulated by SWEET16, are important for xylem cell division as revealed in particular by the decreased number of xylem cells in the swt16 mutant and the accumulation of SWEET16 at the procambium-xylem boundary. In addition, we demonstrate that transport of hexoses mediated by SWEET16 and/or SWEET17 is required to sustain the formation of the xylem secondary cell wall. This result is in line with a defect in the xylem cell wall composition as measured by Fourier-transformed infrared spectroscopy in the swt16swt17 double mutant and by upregulation of several genes involved in secondary cell wall synthesis. Our work therefore supports a model in which xylem development partially depends on the exchange of hexoses at the tonoplast of xylem-forming cells.

Jasmonate inhibits adventitious root initiation through repression of CKX1 and activation of RAP2.6L transcription factor in Arabidopsis.

Adventitious rooting is a de novo organogenesis process that enables plants to propagate clonally and cope with environmental stresses. Adventitious root initiation (ARI) is controlled by interconnected transcriptional and hormonal networks, but there is little knowledge of the genetic and molecular programs orchestrating these networks. Thus, we have applied genome-wide transcriptome profiling to elucidate the transcriptional reprogramming events preceding ARI. These reprogramming events are associated with the down-regulation of cytokinin (CK) signaling and response genes, which could be triggers for ARI. Interestingly, we found that CK free base (iP, tZ, cZ, and DHZ) content declined during ARI, due to down-regulation of de novo CK biosynthesis and up-regulation of CK inactivation pathways. We also found that MYC2-dependent jasmonate (JA) signaling inhibits ARI by down-regulating the expression of the CYTOKININ OXIDASE/DEHYDROGENASE1 (CKX1) gene. We also demonstrated that JA and CK synergistically activate expression of the transcription factor RELATED to APETALA2.6 LIKE (RAP2.6L), and constitutive expression of this transcription factor strongly inhibits ARI. Collectively, our findings reveal that previously unknown genetic interactions between JA and CK play key roles in ARI.

Adventitious Rooting in Populus Species: Update and Perspectives.

Populus spp. are among the most economically important species worldwide. These trees are used not only for wood and fiber production, but also in the rehabilitation of degraded lands. Since they are clonally propagated, the ability of stem cuttings to form adventitious roots is a critical point for plant establishment and survival in the field, and consequently for the forest industry. Adventitious rooting in different Populus clones has been an agronomic trait targeted in breeding programs for many years, and many factors have been identified that affect this quantitative trait. A huge variation in the rooting capacity has been observed among the species in the Populus genus, and the responses to some of the factors affecting this trait have been shown to be genotype-dependent. This review analyses similarities and differences between results obtained from studies examining the role of internal and external factors affecting rooting of Populus species cuttings. Since rooting is the most important requirement for stand establishment in clonally propagated species, understanding the physiological and genetic mechanisms that promote this trait is essential for successful commercial deployment.

Red Light Controls Adventitious Root Regeneration by Modulating Hormone Homeostasis in Picea abies Seedlings.

Vegetative propagation relies on the capacity of plants to regenerate de novo adventitious roots (ARs), a quantitative trait controlled by the interaction of endogenous factors, such as hormones and environmental cues among which light plays a central role. However, the physiological and molecular components mediating light cues during AR initiation (ARI) remain largely elusive. Here, we explored the role of red light (RL) on ARI in de-rooted Norway spruce seedlings. We combined investigation of hormone metabolism and gene expression analysis to identify potential signaling pathways. We also performed extensive anatomical characterization to investigate ARI at the cellular level. We showed that in contrast to white light, red light promoted ARI likely by reducing jasmonate (JA) and JA-isoleucine biosynthesis and repressing the accumulation of isopentyl-adenine-type cytokinins. We demonstrated that exogenously applied JA and/or CK inhibit ARI in a dose-dependent manner and found that they possibly act in the same pathway. The negative effect of JA on ARI was confirmed at the histological level. We showed that JA represses the early events of ARI. In conclusion, RL promotes ARI by repressing the accumulation of the wound-induced phytohormones JA and CK.

ETHYLENE RESPONSE FACTOR 115 integrates jasmonate and cytokinin signaling machineries to repress adventitious rooting in Arabidopsis.

Adventitious root initiation (ARI) is a de novo organogenesis program and a key adaptive trait in plants. Several hormones regulate ARI but the underlying genetic architecture that integrates the hormonal crosstalk governing this process remains largely elusive. In this study, we use genetics, genome editing, transcriptomics, hormone profiling and cell biological approaches to demonstrate a crucial role played by the APETALA2/ETHYLENE RESPONSE FACTOR 115 transcription factor. We demonstrate that ERF115 functions as a repressor of ARI by activating the cytokinin (CK) signaling machinery. We also demonstrate that ERF115 is transcriptionally activated by jasmonate (JA), an oxylipin-derived phytohormone, which represses ARI in NINJA-dependent and independent manners. Our data indicate that NINJA-dependent JA signaling in pericycle cells blocks early events of ARI. Altogether, our results reveal a previously unreported molecular network involving cooperative crosstalk between JA and CK machineries that represses ARI.

Conifers exhibit a characteristic inactivation of auxin to maintain tissue homeostasis.

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.

Multiple Roles of Jasmonates in Shaping Rhizotaxis: Emerging Integrators.

The root system and its architecture enormously contribute to plant survival and adaptation to the environment. Depending on the intrinsic genetic information and the surrounding rhizosphere, plants develop a highly plastic root system, which is a critical determinant for survival. Plant root system, which includes primary root (PR), lateral roots (LR) and adventitious roots (AR), is shaped by tightly controlled developmental programs. Phytohormones are the main signaling components that orchestrate and coordinate the genetic information and the external stimuli to shape the root system patterning or rhizotaxis. Besides their role in plant stress responses and defense against herbivory and pathogen attacks, jasmonic acid and its derivatives, including the receptor-active conjugate jasmonoyl-L-isoleucine (JA-Ile), emerge as potential regulators of rhizotaxis. In this chapter, we summarize and discuss the recent progress achieved during the recent years to understand the JA-mediated genetic and molecular networks guiding PR, LR, and AR initiation. We highlight the role of JAs as critical integrators in shaping rhizotaxis.

A bacterial assay for rapid screening of IAA catabolic enzymes.

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.

A Molecular Framework for the Control of Adventitious Rooting by TIR1/AFB2-Aux/IAA-Dependent Auxin Signaling in Arabidopsis.

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.

A DAO1-Mediated Circuit Controls Auxin and Jasmonate Crosstalk Robustness during Adventitious Root Initiation in Arabidopsis.

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.

Lateral Transport of Organic and Inorganic Solutes.

Organic (e.g., sugars and amino acids) and inorganic (e.g., K⁺, Na⁺, PO42-, and SO42-) solutes are transported long-distance throughout plants. Lateral movement of these compounds between the xylem and the phloem, and vice versa, has also been reported in several plant species since the 1930s, and is believed to be important in the overall resource allocation. Studies of Arabidopsis thaliana have provided us with a better knowledge of the anatomical framework in which the lateral transport takes place, and have highlighted the role of specialized vascular and perivascular cells as an interface for solute exchanges. Important breakthroughs have also been made, mainly in Arabidopsis, in identifying some of the proteins involved in the cell-to-cell translocation of solutes, most notably a range of plasma membrane transporters that act in different cell types. Finally, in the future, state-of-art imaging techniques should help to better characterize the lateral transport of these compounds on a cellular level. This review brings the lateral transport of sugars and inorganic solutes back into focus and highlights its importance in terms of our overall understanding of plant resource allocation.

Control of root meristem establishment in conifers.

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.

Control of adventitious root formation: insights into synergistic and antagonistic hormonal interactions.

Plants have evolved sophisticated root systems that help them to cope with harsh environmental conditions. They are typically composed of a primary root and lateral roots (LRs), but may also include adventitious roots (ARs). Unlike LRs, ARs may be initiated not only from pericycle cells, but from various cell types and tissues depending on the species. Phytohormones, together with many other internal and external stimuli, coordinate and guide every step of AR formation from the first event of cell reprogramming until emergence and outgrowth. In this review, we summarize recent advances in the molecular mechanisms controlling AR formation and highlight the main hormonal cross talk involved in its regulation under different conditions and in different model systems.

Erratum: The Arabidopsis Cop9 signalosome subunit 4 (CSN4) is involved in adventitious root formation.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.