Summary
The gaseous plant hormone ethylene is produced by a fairly simple two‐step biosynthesis route. Despite this pathway’s simplicity, recent molecular and genetic studies have revealed that the ...regulation of ethylene biosynthesis is far more complex and occurs at different layers. Ethylene production is intimately linked with the homeostasis of its general precursor S‐adenosyl‐l‐methionine (SAM), which experiences transcriptional and posttranslational control of its synthesising enzymes (SAM synthetase), as well as the metabolic flux through the adjacent Yang cycle. Ethylene biosynthesis continues from SAM by two dedicated enzymes: 1‐aminocyclopropane‐1‐carboxylic (ACC) synthase (ACS) and ACC oxidase (ACO). Although the transcriptional dynamics of ACS and ACO have been well documented, the first transcription factors that control ACS and ACO expression have only recently been discovered. Both ACS and ACO display a type‐specific posttranslational regulation that controls protein stability and activity. The nonproteinogenic amino acid ACC also shows a tight level of control through conjugation and translocation. Different players in ACC conjugation and transport have been identified over the years, however their molecular regulation and biological significance is unclear, yet relevant, as ACC can also signal independently of ethylene. In this review, we bring together historical reports and the latest findings on the complex regulation of the ethylene biosynthesis pathway in plants.
SUMMARY
The phytohormone cytokinin plays a significant role in nearly all aspects of plant growth and development. Cytokinin signaling has primarily been studied in the dicot model Arabidopsis, with ...relatively little work done in monocots, which include rice (Oryza sativa) and other cereals of agronomic importance. The cytokinin signaling pathway is a phosphorelay comprised of the histidine kinase receptors, the authentic histidine phosphotransfer proteins (AHPs) and type‐B response regulators (RRs). Two negative regulators of cytokinin signaling have been identified: the type‐A RRs, which are cytokinin primary response genes, and the pseudo histidine phosphotransfer proteins (PHPs), which lack the His residue required for phosphorelay. Here, we describe the role of the rice PHP genes. Phylogenic analysis indicates that the PHPs are generally first found in the genomes of gymnosperms and that they arose independently in monocots and dicots. Consistent with this, the three rice PHPs fail to complement an Arabidopsis php mutant (aphp1/ahp6). Disruption of the three rice PHPs results in a molecular phenotype consistent with these elements acting as negative regulators of cytokinin signaling, including the induction of a number of type‐A RR and cytokinin oxidase genes. The triple php mutant affects multiple aspects of rice growth and development, including shoot morphology, panicle architecture, and seed fill. In contrast to Arabidopsis, disruption of the rice PHPs does not affect root vascular patterning, suggesting that while many aspects of key signaling networks are conserved between monocots and dicots, the roles of at least some cytokinin signaling elements are distinct.
Significance Statement
We demonstrate that the function of the rice pseudo histidine phosphotransfer proteins has diverged from their Arabidopsis counterparts, despite data indicating that these rice proteins are also negative regulators of cytokinin signaling.
EIL2 is an ethylene-dependent transcription factor that evolved exclusively in the mustard family and operates downstream from EIN3/EIL1. EIL2is involved in fine-tuning hypocotyl elongation, lateral ...root formation, and flowering time in Arabidopsis thaliana.
Abstract
Ethylene signaling directs a pleiotropy of developmental processes in plants. In Arabidopsis, ethylene signaling converges at the master transcription factor Ethylene Insensitive 3 (EIN3), which has five homologs, EIN3-like 1–5 (EIL1–EIL5). EIL1 is most fully characterized and operates similarly to EIN3, while EIL3–5 are not involved in ethylene signaling. EIL2 remains less investigated. Our phylogenetic analysis revealed that EIL2 homologs have only been retrieved in the Brassicaceae family, suggesting that EIL2 diverged to have specific functions in the mustard family. By characterizing eil2 mutants, we found that EIL2 is involved in regulating ethylene-specific developmental processes in Arabidopsis thaliana, albeit in a more subtle way compared with EIN3/EIL1. EIL2 steers ethylene-triggered hypocotyl elongation in light-grown seedlings and is involved in lateral root formation. Furthermore, EIL2 takes part in regulating flowering time as eil2 mutants flower on average 1 d earlier and have fewer leaves. A pEIL2:EIL2:GFP translational reporter line revealed that EIL2 protein abundance is restricted to the stele of young developing roots. EIL2 expression, and not EIL2 protein stability, is regulated by ethylene in an EIN3/EIL1-dependent way. Despite EIL2 taking part in several developmental processes, the precise upstream and downstream regulation of this ethylene- and Brassicaceae-specific transcription factor remains to be elucidated.
•Cell division and cell fate specification determine vascular pattern.•A group of transcription factors specify the size of the vascular cylinder.•Auxin, cytokinin and class III HD-ZIP protein levels ...determine cell fate.•Recent research is uncovering how these processes interact to control patterning.
The Arabidopsis root has provided an excellent model for understanding patterning processes and cell fate specification. Vascular patterning represents an especially interesting process, as new positional information must be generated to transform an approximately radially symmetric root pole into a bisymmetric structure with a single xylem axis. This process requires both growth of the embryonic tissue alongside the subsequent patterning. Recently researchers have identified a series of transcription factors that modulate cell divisions to control vascular tissues growth. Spatial regulation in the signalling of two hormones, auxin and cytokinin, combine with other transcription factors to pattern the xylem axis. We are now witnessing the discovery of increasingly complex interactions between these hormones that can be interpreted through the use of mathematical models.
Plant adaptation, growth and development rely on the integration of many environmental and endogenous signals that collectively determine the overall plant phenotypic plasticity. Plant signaling ...molecules, also known as phytohormones, are fundamental to this process. These molecules act at low concentrations and regulate multiple aspects of plant fitness and development via complex signaling networks. By its nature, phytohormone research lies at the interface between chemistry and biology. Classically, the scientific community has always used synthetic phytohormones and analogs to study hormone functions and responses. However, recent advances in synthetic and combinational chemistry, have allowed a new field, plant chemical biology, to emerge and this has provided a powerful tool with which to study phytohormone function. Plant chemical biology is helping to address some of the most enduring questions in phytohormone research such as: Are there still undiscovered plant hormones? How can we identify novel signaling molecules? How can plants activate specific hormone responses in a tissue-specific manner? How can we modulate hormone responses in one developmental context without inducing detrimental effects on other processes? The chemical genomics approaches rely on the identification of small molecules modulating different biological processes and have recently identified active forms of plant hormones and molecules regulating many aspects of hormone synthesis, transport and response. We envision that the field of chemical genomics will continue to provide novel molecules able to elucidate specific aspects of hormone-mediated mechanisms. In addition, compounds blocking specific responses could uncover how complex biological responses are regulated. As we gain information about such compounds we can design small alterations to the chemical structure to further alter specificity, enhance affinity or modulate the activity of these compounds.
Plants produce the volatile hormone ethylene to regulate many developmental processes and to deal with (a)biotic stressors. In seed plants, ethylene is synthesized from ...1-aminocyclopropane-1-carboxylic acid (ACC) by the dedicated enzyme ACC oxidase (ACO). Ethylene biosynthesis is tightly regulated at the level of ACC through ACC synthesis, conjugation and transport. ACC is a non-proteinogenic amino acid, which also has signaling roles independent from ethylene. In this work, we investigated the biological function of an uncharacterized ACC dipeptide. The custom-synthesized di-ACC molecule can be taken up by Arabidopsis in a similar way as ACC, in part
via
Lysine Histidine Transporters (e.g., LHT1). Using Nano-Particle Assisted Laser Desoprtion/Ionization (Nano-PALDI) mass-spectrometry imaging, we revealed that externally fed di-ACC predominantly localizes to the vasculature tissue, despite it not being detectable in control hypocotyl segments. Once taken up, the ACC dimer can evoke a triple response phenotype in dark-grown seedlings, reminiscent of ethylene responses induced by ACC itself, albeit less efficiently compared to ACC. Di-ACC does not act
via
ACC-signaling, but operates
via
the known ethylene signaling pathway.
In vitro
ACO activity and molecular docking showed that di-ACC can be used as an alternative substrate by ACO to form ethylene. The promiscuous nature of ACO for the ACC dimer also explains the higher ethylene production rates observed
in planta
, although this reaction occurred less efficiently compared to ACC. Overall, the ACC dipeptide seems to be transported and converted into ethylene in a similar way as ACC, and is able to augment ethylene production levels and induce subsequent ethylene responses in Arabidopsis.
Studying the developmental genetics of plant organs requires following gene expression in specific tissues. To facilitate this, we have developed dual expression anatomy lines, which incorporate a ...red plasma membrane marker alongside a fluorescent reporter for a gene of interest in the same vector. Here, we adapted the GreenGate cloning vectors to create two destination vectors showing strong marking of cell membranes in either the whole root or specifically in the lateral roots. This system can also be used in both embryos and whole seedlings. As proof of concept, we follow both gene expression and anatomy in Arabidopsis (Arabidopsis thaliana) during lateral root organogenesis for a period of over 24 h. Coupled with the development of a flow cell and perfusion system, we follow changes in activity of the DII auxin sensor following application of auxin.
Pattern formation is typically controlled through the interaction between molecular signals within a given tissue. During early embryonic development, roots of the model plant
have a radially ...symmetric pattern, but a heterogeneous input of the hormone auxin from the two cotyledons forces the vascular cylinder to develop a diarch pattern with two xylem poles. Molecular analyses and mathematical approaches have uncovered the regulatory circuit that propagates this initial auxin signal into a stable cellular pattern. The diarch pattern seen in
is relatively uncommon among flowering plants, with most species having between three and eight xylem poles. Here, we have used multiscale mathematical modelling to demonstrate that this regulatory module does not require a heterogeneous auxin input to specify the vascular pattern. Instead, the pattern can emerge dynamically, with its final form dependent upon spatial constraints and growth. The predictions of our simulations compare to experimental observations of xylem pole number across a range of species, as well as in transgenic systems in
in which we manipulate the size of the vascular cylinder. By considering the spatial constraints, our model is able to explain much of the diversity seen in different flowering plant species.
The vascular anatomy of rice roots is an important factor in drought tolerance, but little is known about the mechanisms governing its patterning. The Arabidopsis thaliana root vascular tissues are ...patterned by a mutually inhibitory feedback loop between auxin and cytokinin signalling (Mähönen et al. 2006). Cytokinin signalling in the procambium promotes accumulation of auxin in neighbouring cells which consequently differentiate as xylem. In these cells, high auxin levels promote expression of the pseudo-histidine phosphotransfer protein (PHP) AHP6, an inhibitor of cytokinin signalling (Bishopp et al. 2011). Whereas the root vasculature of Arabidopsis consists of a single xylem axis with phloem poles on either side, that of rice roots show a central metaxylem vessel surrounded by multiple xylem poles, between which are phloem. I hypothesised that the basic interaction between auxin and cytokinin in vascular patterning is conserved between Arabidopsis and rice, but is adapted to enable development of these different tissue patterns. Chemical treatments revealed that auxin and cytokinin are central regulators of root vascular patterning in rice, as is the case in Arabidopsis. In an effort to test whether this conservation extended to individual components of the molecular circuitry, I examined the role of the three OsPHPs, homologues of AHP6. These genes are not auxin inducible and are not able to rescue the Arabidopsis AHP6 mutant, suggesting they do not function in rice root vascular patterning. However, screening known inhibitors of cytokinin signalling for in auxin inducibility in the root tip revealed a subset of the type-A RRs (OsRR1, OsRR6 and OsRR7) are auxin inducible, and were able to rescue the Arabidopsis ahp6 mutant, suggesting these genes may have been recruited to function in the hormonal crosstalk regulating root vascular patterning. Phylogenetic analyses revealed a single amino acid substitution in a conserved region of the PHPs which differs between the dicots and the monocots. To directly determine activity of the OsPHPs, and the relevance of these substitutions, in-vitro phosphotransfer assays were performed. Preliminary results suggest these substitutions do not affect protein function. To characterise candidate genes, transcriptional reporters and mutant plants were generated. Reporters show expression of most candidate genes in the root, with some localised to root protoxylem. Mutants were generated using CRISPR and homozygous and heterozygous mutants were identified. Together, results described here give insight into how regulatory networks may be adapted to bring about differing responses.
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