Publications by authors named "Julian I Schroeder"

172 Publications

Raf-like kinases and receptor-like (pseudo)kinase GHR1 are required for stomatal vapor pressure difference response.

Proc Natl Acad Sci U S A 2021 Nov;118(47)

Cell and Developmental Biology Section, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093;

Stomatal pores close rapidly in response to low-air-humidity-induced leaf-to-air vapor pressure difference (VPD) increases, thereby reducing excessive water loss. The hydroactive signal-transduction mechanisms mediating high VPD-induced stomatal closure remain largely unknown. The kinetics of stomatal high-VPD responses were investigated by using time-resolved gas-exchange analyses of higher-order mutants in guard-cell signal-transduction branches. We show that the slow-type anion channel SLAC1 plays a relatively more substantial role than the rapid-type anion channel ALMT12/QUAC1 in stomatal VPD signaling. VPD-induced stomatal closure is not affected in / double mutants that completely disrupt stomatal CO signaling, indicating that VPD signaling is independent of the early CO signal-transduction pathway. Calcium imaging shows that osmotic stress causes cytoplasmic Ca transients in guard cells. Nevertheless, //// Ca-permeable channel quintuple, /-channel double, /-channel double, -channel single, /-channel double, /-channel double, kinase quintuple, //// quintuple, and / double mutants showed wild-type-like stomatal VPD responses. A B3-family Raf-like mitogen-activated protein (MAP)-kinase kinase kinase, M3Kδ5/RAF6, activates the OST1/SnRK2.6 kinase in plant cells. Interestingly, B3 Raf-kinase and /// (///) quadruple mutants, but not a 14-gene mutant including osmotic stress-linked B4-family Raf-kinases, exhibited slowed high-VPD responses, suggesting that B3-family Raf-kinases play an important role in stomatal VPD signaling. Moreover, high VPD-induced stomatal closure was impaired in receptor-like pseudokinase GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) mutant alleles. Notably, the classical transient "wrong-way" VPD response was absent in mutant alleles. These findings reveal genes and signaling mechanisms in the elusive high VPD-induced stomatal closing response pathway.
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http://dx.doi.org/10.1073/pnas.2107280118DOI Listing
November 2021

Boolink: a graphical interface for open access Boolean network simulations and use in guard cell CO2 signaling.

Plant Physiol 2021 Jul 24. Epub 2021 Jul 24.

Physics Department, University of California, San Diego, La Jolla, California 92093, USA.

Signaling networks are at the heart of almost all biological processes. Most of these networks contain large number of components, and often either the connections between these components are not known or the rate equations that govern the dynamics of soluble signaling components are not quantified. This uncertainty in network topology and parameters can make it challenging to formulate detailed mathematical models. Boolean networks, in which all components are either on or off, have emerged as viable alternatives to detailed mathematical models that contain rate constants and other parameters. Therefore, open-source platforms of Boolean models for community use are desirable. Here, we present Boolink, a freely available graphical user interface that allows users to easily construct and analyze existing Boolean networks. Boolink can be applied to any Boolean network. We demonstrate its application using a previously published network for abscisic acid (ABA)-driven stomatal closure in Arabidopsis spp. (Arabidopsis thaliana). We also show how Boolink can be used to generate testable predictions by extending the network to include CO2 regulation of stomatal movements. Predictions of the model were experimentally tested, and the model was iteratively modified based on experiments showing that ABA effectively closes Arabidopsis stomata at near-zero CO2 concentrations (1.5-ppm CO2). Thus, Boolink enables public generation and the use of existing Boolean models, including the prior developed ABA signaling model with added CO2 signaling components.
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http://dx.doi.org/10.1093/plphys/kiab344DOI Listing
July 2021

Jasmonic acid and salicylic acid play minor roles in stomatal regulation by CO , abscisic acid, darkness, vapor pressure deficit and ozone.

Plant J 2021 Oct 14;108(1):134-150. Epub 2021 Aug 14.

Plant Signal Research Group, Institute of Technology, University of Tartu, Nooruse 1, Tartu, 50411, Estonia.

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas-exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO , light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2 and npr1 alleles. Although the stomata in the mutants studied clearly responded to ABA, CO , light and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in the coi1-16 mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl-JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine-induced stomatal opening was initiated slowly after 1.5-2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole-plant stomatal response to environmental cues in Arabidopsis and Solanum lycopersicum (tomato).
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http://dx.doi.org/10.1111/tpj.15430DOI Listing
October 2021

Molecular mechanisms of stomatal closure in response to rising vapour pressure deficit.

New Phytol 2021 10 29;232(2):468-475. Epub 2021 Jul 29.

Institute of Technology, University of Tartu, Tartu, 50411, Estonia.

Vapour pressure deficit (VPD), the difference between the saturation and actual air vapour pressures, indicates the level of atmospheric drought and evaporative pressure on plants. VPD increases during climate change due to changes in air temperature and relative humidity. Rising VPD induces stomatal closure to counteract the VPD-mediated evaporative water loss from plants. There are important gaps in our understanding of the molecular VPD-sensing and signalling mechanisms in stomatal guard cells. Here, we discuss recent advances, research directions and open questions with respect to the three components that participate in VPD-induced stomatal closure in Arabidopsis, including: (1) abscisic acid (ABA)-dependent and (2) ABA-independent regulation of the protein kinase OPEN STOMATA 1 (OST1), and (3) the passive hydraulic stomatal response. In the ABA-dependent component, two models are proposed: ABA may be rapidly synthesised or its basal levels may be involved in the stomatal VPD response. Further studies on stomatal VPD signalling should clarify: (1) whether OST1 activation above basal activity is needed for VPD responses, (2) which components are involved in ABA-independent regulation of OST1, (3) the role of other potential OST1 targets in VPD signalling, and (4) to which extent OST1 contributes to stomatal VPD sensitivity in other plant species.
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http://dx.doi.org/10.1111/nph.17592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8455429PMC
October 2021

The SLIM1 transcription factor is required for arsenic resistance in Arabidopsis thaliana.

FEBS Lett 2021 06 19;595(12):1696-1707. Epub 2021 May 19.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California, La Jolla, CA, USA.

The transcriptional regulators of arsenic-induced gene expression remain largely unknown. Sulfur assimilation is tightly linked with arsenic detoxification. Here, we report that mutant alleles in the SLIM1 transcription factor are substantially more sensitive to arsenic than cadmium. Arsenic treatment caused high levels of oxidative stress in the slim1 mutants, and slim1 alleles were impaired in both thiol accumulation and sulfate accumulation. We further found enhanced arsenic accumulation in roots of slim1 mutants. Transcriptome analyses indicate an important role for SLIM1 in arsenic-induced tolerance mechanisms. The present study identifies the SLIM1 transcription factor as an essential component in arsenic tolerance and arsenic-induced gene expression. Our results suggest that the severe arsenic sensitivity of the slim1 mutants is caused by altered redox status.
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http://dx.doi.org/10.1002/1873-3468.14096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238853PMC
June 2021

An amiRNA screen uncovers redundant CBF and ERF34/35 transcription factors that differentially regulate arsenite and cadmium responses.

Plant Cell Environ 2021 05 25;44(5):1692-1706. Epub 2021 Feb 25.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, California, USA.

Arsenic stress causes rapid transcriptional responses in plants. However, transcriptional regulators of arsenic-induced gene expression in plants remain less well known. To date, forward genetic screens have proven limited for dissecting arsenic response mechanisms. We hypothesized that this may be due to the extensive genetic redundancy present in plant genomes. To overcome this limitation, we pursued a forward genetic screen for arsenite tolerance using a randomized library of plants expressing >2,000 artificial microRNAs (amiRNAs). This library was designed to knock-down diverse combinations of homologous gene family members within sub-clades of transcription factor and transporter gene families. We identified six transformant lines showing an altered response to arsenite in root growth assays. Further characterization of an amiRNA line targeting closely homologous CBF and ERF transcription factors show that the CBF1,2 and 3 transcription factors negatively regulate arsenite sensitivity. Furthermore, the ERF34 and ERF35 transcription factors are required for cadmium resistance. Generation of CRISPR lines, higher-order T-DNA mutants and gene expression analyses, further support our findings. These ERF transcription factors differentially regulate arsenite sensitivity and cadmium tolerance.
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http://dx.doi.org/10.1111/pce.14023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068611PMC
May 2021

Identification and characterization of SaeIF1 from the eukaryotic translation factor SUI1 family in cadmium hyperaccumulator Sedum alfredii.

Planta 2021 Jan 3;253(1):12. Epub 2021 Jan 3.

School of Life Sciences, and Hubei Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan, 430079, Hubei, People's Republic of China.

Main Conclusion: Cadmium-sensitive yeast screening resulted in the isolation of protein translation factor SaeIF1 from the hyperaccumulator Sedum alfredii which has both general and special regulatory roles in controlling cadmium accumulation. The hyperaccumulator of Sedum alfredii has the extraordinary ability to hyperaccumulate cadmium (Cd) in shoots. To investigate its underlying molecular mechanisms of Cd hyperaccumulation, a cDNA library was generated from leaf tissues of S. alfredii. SaeIF1, belonging to the eukaryotic protein translation factor SUI1 family, was identified by screening Cd-sensitive yeast transformants with this library. The full-length cDNA of SaeIF1 has 582 bp and encodes a predicted protein with 120 amino acids. Transient expression assays showed subcellular localization of SaeIF1 in the cytoplasm. SaeIF1 was constitutively and highly expressed in roots and shoots of the hyperaccumulator of S. alfredii, while its transcript levels showed over 100-fold higher expression in the hyperaccumulator of S. alfredii relative to the tissues of a nonhyperaccumulating ecotype of S. alfredii. However, the overexpression of SaeIF1 in yeast cells increased Cd accumulation, but conferred more Cd sensitivity. Transgenic Arabidopsis thaliana expressing SaeIF1 accumulated more Cd in roots and shoots without changes in the ratio of Cd content in shoots and roots, but were more sensitive to Cd stress than wild type. Both special and general roles of SaeIF1 in Cd uptake, transportation, and detoxification are discussed, and might be responsible for the hyperaccumulation characteristics of S. alfredii.
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http://dx.doi.org/10.1007/s00425-020-03539-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847809PMC
January 2021

A role for calcium-dependent protein kinases in differential CO - and ABA-controlled stomatal closing and low CO -induced stomatal opening in Arabidopsis.

New Phytol 2021 03 9;229(5):2765-2779. Epub 2020 Dec 9.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, La Jolla, CA, 92093-0116, USA.

Low concentrations of CO cause stomatal opening, whereas [CO ] elevation leads to stomatal closure. Classical studies have suggested a role for Ca and protein phosphorylation in CO -induced stomatal closing. Calcium-dependent protein kinases (CPKs) and calcineurin-B-like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca into specific phosphorylation events. However, Ca -binding proteins that function in the stomatal CO response remain unknown. Time-resolved stomatal conductance measurements using intact plants, and guard cell patch-clamp experiments were performed. We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO , light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO -induced stomatal closure and, unexpectedly, also in low CO -induced stomatal opening. Furthermore, K -uptake-channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light-mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO -regulated stomatal movement kinetics were not clearly affected in plasma membrane-targeted cbl1/4/5/8/9 quintuple mutant plants. Our findings describe combinatorial cpk mutants that function in CO control of stomatal movements and support the results of classical studies showing a role for Ca in this response.
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http://dx.doi.org/10.1111/nph.17079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902375PMC
March 2021

Signaling mechanisms in abscisic acid-mediated stomatal closure.

Plant J 2021 01 9;105(2):307-321. Epub 2020 Dec 9.

Cell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA, 92093-0116, USA.

The plant hormone abscisic acid (ABA) plays a central role in the regulation of stomatal movements under water-deficit conditions. The identification of ABA receptors and the ABA signaling core consisting of PYR/PYL/RCAR ABA receptors, PP2C protein phosphatases and SnRK2 protein kinases has led to studies that have greatly advanced our knowledge of the molecular mechanisms mediating ABA-induced stomatal closure in the past decade. This review focuses on recent progress in illuminating the regulatory mechanisms of ABA signal transduction, and the physiological importance of basal ABA signaling in stomatal regulation by CO and, as hypothesized here, vapor-pressure deficit. Furthermore, advances in understanding the interactions of ABA and other stomatal signaling pathways are reviewed here. We also review recent studies investigating the use of ABA signaling mechanisms for the manipulation of stomatal conductance and the enhancement of drought tolerance and water-use efficiency of plants.
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http://dx.doi.org/10.1111/tpj.15067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902384PMC
January 2021

Dynamic regulation of Pep-induced immunity through post-translational control of defence transcript splicing.

Nat Plants 2020 08 20;6(8):1008-1019. Epub 2020 Jul 20.

Section of Cell and Developmental Biology, UC San Diego, San Diego, CA, USA.

The survival of all living organisms requires the ability to detect attacks and swiftly counter them with protective immune responses. Despite considerable mechanistic advances, the interconnectivity of signalling modules often remains unclear. A newly characterized protein, IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR), negatively regulates immune responses in both maize and Arabidopsis, with disrupted function resulting in enhanced disease resistance. IRR associates with and promotes canonical splicing of transcripts encoding defence signalling proteins, including the key negative regulator of pattern-recognition receptor signalling complexes, CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28). On immune activation by Plant Elicitor Peptides (Peps), IRR is dephosphorylated, disrupting interaction with CPK28 transcripts and resulting in the accumulation of an alternative splice variant encoding a truncated CPK28 protein with impaired kinase activity and diminished function as a negative regulator. We demonstrate a new mechanism linking Pep-induced post-translational modification of IRR with post-transcriptionally mediated attenuation of CPK28 function to dynamically amplify Pep signalling and immune output.
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http://dx.doi.org/10.1038/s41477-020-0724-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7482133PMC
August 2020

FRET kinase sensor development reveals SnRK2/OST1 activation by ABA but not by MeJA and high CO during stomatal closure.

Elife 2020 05 28;9. Epub 2020 May 28.

Cell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, San Diego, United States.

Sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s) are critical for plant abiotic stress responses, including abscisic acid (ABA) signaling. Here, we develop a genetically encoded reporter for SnRK2 kinase activity. This sensor, named SNACS, shows an increase in the ratio of yellow to cyan fluorescence emission by OST1/SnRK2.6-mediated phosphorylation of a defined serine residue in SNACS. ABA rapidly increases FRET efficiency in leaf cells and guard cells. Interestingly, protein kinase inhibition decreases FRET efficiency in guard cells, providing direct experimental evidence that basal SnRK2 activity prevails in guard cells. Moreover, in contrast to ABA, the stomatal closing stimuli, elevated CO and MeJA, did not increase SNACS FRET ratios. These findings and gas exchange analyses of quintuple/sextuple ABA receptor mutants show that stomatal CO signaling requires basal ABA and SnRK2 signaling, but not SnRK2 activation. A recent model that CO signaling is mediated by PYL4/PYL5 ABA-receptors could not be supported here in two independent labs. We report a potent approach for real-time live-cell investigations of stress signaling.
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http://dx.doi.org/10.7554/eLife.56351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289597PMC
May 2020

Monitoring and mitigation of toxic heavy metals and arsenic accumulation in food crops: A case study of an urban community garden.

Plant Direct 2020 Jan 14;4(1):e00198. Epub 2020 Jan 14.

Division of Biological Sciences, Cell and Developmental Biology Section University of California, San Diego La Jolla CA USA.

Urban community gardens have increased in prevalence as a means to generate fresh fruits and vegetables, including in areas lacking access to healthy food options. However, urban soils may have high levels of toxic heavy metals, including lead and cadmium and the metalloid arsenic, which can lead to severe health risks. In this study, fruit and vegetable samples grown at an urban community garden in southeastern San Diego, the Ocean View Growing Grounds, were sampled repeatedly over a four-year time period in order to measure potential contamination of toxic heavy metals and metalloids and to develop solutions for this problem. Metal nutrient, heavy metal, and metalloid concentrations were monitored in the leaf and fruit tissues of fruit trees over the sampling period. Several of the fruit trees showed uptake of lead in the leaf samples, with Black Mission fig measuring 0.843-1.531 mg/kg dry weight and Mexican Lime measuring 1.103-1.522 mg/kg dry weight over the sampling period. Vegetables that were grown directly in the ground at this community garden and surrounding areas showed arsenic, 0.80 + 0.073 mg/kg dry weight for Swiss chard, and lead, 0.84 ± 0.404 mg/kg dry weight for strawberries, in their edible tissues. The subsequent introduction of raised beds with uncontaminated soil is described, which eliminated any detectable heavy metal or metalloid contamination in these crops during the monitoring period. Recommendations for facilitating the monitoring of edible tissues and for reducing risk are discussed, including introduction of raised beds and collaborations with local universities and research groups.
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http://dx.doi.org/10.1002/pld3.198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957986PMC
January 2020

MAP3Kinase-dependent SnRK2-kinase activation is required for abscisic acid signal transduction and rapid osmotic stress response.

Nat Commun 2020 01 2;11(1):12. Epub 2020 Jan 2.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, La Jolla, CA, 92093, USA.

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction.
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http://dx.doi.org/10.1038/s41467-019-13875-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940395PMC
January 2020

Genetic strategies for improving crop yields.

Nature 2019 11 6;575(7781):109-118. Epub 2019 Nov 6.

Cell and Developmental Biology Section, Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA.

The current trajectory for crop yields is insufficient to nourish the world's population by 2050. Greater and more consistent crop production must be achieved against a backdrop of climatic stress that limits yields, owing to shifts in pests and pathogens, precipitation, heat-waves and other weather extremes. Here we consider the potential of plant sciences to address post-Green Revolution challenges in agriculture and explore emerging strategies for enhancing sustainable crop production and resilience in a changing climate. Accelerated crop improvement must leverage naturally evolved traits and transformative engineering driven by mechanistic understanding, to yield the resilient production systems that are needed to ensure future harvests.
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http://dx.doi.org/10.1038/s41586-019-1679-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7024682PMC
November 2019

Toward a better understanding of signaling networks in plants: yeast has the power!

EMBO J 2019 09 6;38(17):e102478. Epub 2019 Aug 6.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, La Jolla, CA, USA.

In response to abiotic stresses, plants produce the hormone abscisic acid (ABA). The ABA signaling pathway is highly complex and relies on a large number of gene copies encoding homologous signaling components, theoretically enabling numerous permutations. In this issue, Ruschhaupt et al (2019) used yeast as a reconstitution system to examine the functionality, plasticity, and efficiency of this complex and highly multiplexed core signaling pathway.
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http://dx.doi.org/10.15252/embj.2019102478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717886PMC
September 2019

Cryo-EM structure of OSCA1.2 from elucidates the mechanical basis of potential membrane hyperosmolality gating.

Proc Natl Acad Sci U S A 2019 07 21;116(28):14309-14318. Epub 2019 Jun 21.

Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, CO 80309;

Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation.
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http://dx.doi.org/10.1073/pnas.1900774116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628804PMC
July 2019

Intact leaf gas exchange provides a robust method for measuring the kinetics of stomatal conductance responses to abscisic acid and other small molecules in and grasses.

Plant Methods 2019 17;15:38. Epub 2019 Apr 17.

1Cell and Developmental Biology Section, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0116 USA.

Background: Guard cells perceive external and internal stimuli and regulate stomatal conductance in plants. With the use of gas exchange analyzers, time-resolved stomatal conductance responses to light intensity, [CO] concentration and relative humidity changes can be measured. This is more difficult to achieve when measuring stomatal responses to small soluble molecules such as the plant hormone abscisic acid (ABA) or the bacterial peptide flagellin 22 (flg22), in particular when investigating mutants with response phenotypes.

Results: A method to evaluate the dynamic effects of small molecules on stomatal conductance in a time-resolved fashion using gas exchange analyzers is presented here. ABA-induced stomatal closure was investigated by adding ABA to the transpiration stream of intact leaves placed in a microcentrifuge tube containing water. Strong ABA responses were resolved in time- and in a dose-dependent manner in wild-type leaves, whereas the same response was not observed in leaves of the ABA-insensitive mutant - (-). Moreover, when leaves of the Plasma membrane Intrinsic Protein (PIP) aquaporin quadruple mutant were tested, robust wild-type-like responses to ABA were observed. When the bacterial peptide flg22 was added to the transpiration stream of intact wild-type leaves, a strong flg22-induced stomatal closure effect was observed. Finally, the proposed technique was further developed and optimized for evaluation of stomatal conductance responses to small molecules in leaves of grasses using the reference plant .

Conclusions: Due to the variable size of stomata in and the limited dynamic response of stomata in isolated epidermal strips, evaluation of the effect of small molecules on stomatal physiology has been challenging and has led in some cases to inconsistent results. Moreover, potential signals from the mesophyll are missing when using epidermal peels to evaluate stomatal aperture responses. Here we propose a less invasive technique which allows for time-resolved measurements of stomatal conductance responses to small molecules optimized for both and leaves.
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http://dx.doi.org/10.1186/s13007-019-0423-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472101PMC
April 2019

Calcium signals are necessary to establish auxin transporter polarity in a plant stem cell niche.

Nat Commun 2019 02 13;10(1):726. Epub 2019 Feb 13.

Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA, 91125, USA.

In plants mechanical signals pattern morphogenesis through the polar transport of the hormone auxin and through regulation of interphase microtubule (MT) orientation. To date, the mechanisms by which such signals induce changes in cell polarity remain unknown. Through a combination of time-lapse imaging, and chemical and mechanical perturbations, we show that mechanical stimulation of the SAM causes transient changes in cytoplasmic calcium ion concentration (Ca) and that transient Ca response is required for downstream changes in PIN-FORMED 1 (PIN1) polarity. We also find that dynamic changes in Ca occur during development of the SAM and this Ca response is required for changes in PIN1 polarity, though not sufficient. In contrast, we find that Ca is not necessary for the response of MTs to mechanical perturbations revealing that Ca specifically acts downstream of mechanics to regulate PIN1 polarity response.
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http://dx.doi.org/10.1038/s41467-019-08575-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374474PMC
February 2019

Chemical genetic identification of a lectin receptor kinase that transduces immune responses and interferes with abscisic acid signaling.

Plant J 2019 05 7;98(3):492-510. Epub 2019 Mar 7.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA, 92093-0116, USA.

Insight into how plants simultaneously cope with multiple stresses, for example, when challenged with biotic stress from pathogen infection and abiotic stress from drought, is important both for understanding evolutionary trade-offs and optimizing crop responses to these stresses. Mechanisms by which initial plant immune signaling antagonizes abscisic acid (ABA) signal transduction require further investigation. Using a chemical genetics approach, the small molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) has previously been identified due to its ability to suppress ABA signaling via plant immune signaling components. Here, we have used forward chemical genetics screening to identify DFPM-insensitive loci by monitoring the activity of ABA-inducible pRAB18::GFP in the presence of DFPM and ABA. The ability of DFPM to attenuate ABA signaling was reduced in rda mutants (resistant to DFPM inhibition of ABA signaling). One of the mutants, rda2, was mapped and is defective in a gene encoding a lectin receptor kinase. RDA2 functions in DFPM-mediated inhibition of ABA-mediated reporter expression. RDA2 is required for DFPM-mediated activation of immune signaling, including phosphorylation of mitogen-activated protein kinase (MAPK) 3 (MPK3) and MPK6, and induction of immunity marker genes. Our study identifies a previously uncharacterized receptor kinase gene that is important for DFPM-mediated immune signaling and inhibition of ABA signaling. We demonstrate that the lectin receptor kinase RDA2 is essential for perceiving the DFPM signal and activating MAPKs, and that MKK4 and MKK5 are required for DFPM interference with ABA signal transduction.
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http://dx.doi.org/10.1111/tpj.14232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488365PMC
May 2019

Insights into the Molecular Mechanisms of CO-Mediated Regulation of Stomatal Movements.

Curr Biol 2018 12;28(23):R1356-R1363

Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, La Jolla, CA 92093, USA. Electronic address:

Plants must continually balance the influx of CO for photosynthesis against the loss of water vapor through stomatal pores in their leaves. This balance can be achieved by controlling the aperture of the stomatal pores in response to several environmental stimuli. Elevation in atmospheric [CO] induces stomatal closure and further impacts leaf temperatures, plant growth and water-use efficiency, and global crop productivity. Here, we review recent advances in understanding CO-perception mechanisms and CO-mediated signal transduction in the regulation of stomatal movements, and we explore how these mechanisms are integrated with other signaling pathways in guard cells.
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http://dx.doi.org/10.1016/j.cub.2018.10.015DOI Listing
December 2018

A seed resource for screening functionally redundant genes and isolation of new mutants impaired in CO2 and ABA responses.

J Exp Bot 2019 01;70(2):641-651

Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, La Jolla, CA, USA.

The identification of homologous genes with functional overlap in forward genetic screens is severely limited. Here, we report the generation of over 14000 artificial microRNA (amiRNA)-expressing plants that enable screens of the functionally redundant gene space in Arabidopsis. A protocol was developed for isolating robust and reproducible amiRNA mutants. Examples of validation approaches and essential controls are presented for two new amiRNA mutants that exhibit genetically redundant phenotypes and circumvent double mutant lethality. In a forward genetic screen for abscisic acid (ABA)-mediated inhibition of seed germination, amiRNAs that target combinations of known redundant ABA receptor and SnRK2 kinase genes were rapidly isolated, providing a strong proof of principle for this approach. A new ABA-insensitive amiRNA line that targets three avirulence-induced gene 2(-like) genes was isolated . A thermal imaging screen for plants with impaired stomatal opening in response to low CO2 exposure led to the isolation of a new amiRNA targeting two essential proteasomal subunits, PAB1 and PAB2. The seed library of 11000 T2 amiRNA lines (with 3000 lines in progress) generated here provides a new platform for forward genetic screens and has been made available to the Arabidopsis Biological Resource Center (ABRC). Optimized procedures for amiRNA screening and controls are described.
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http://dx.doi.org/10.1093/jxb/ery363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322574PMC
January 2019

A transportome-scale amiRNA-based screen identifies redundant roles of Arabidopsis ABCB6 and ABCB20 in auxin transport.

Nat Commun 2018 10 11;9(1):4204. Epub 2018 Oct 11.

School of Plant Sciences and Food Security, Tel Aviv University, Tel Aviv, 69978, Israel.

Transport of signaling molecules is of major importance for regulating plant growth, development, and responses to the environment. A prime example is the spatial-distribution of auxin, which is regulated via transporters to govern developmental patterning. A critical limitation in our ability to identify transporters by forward genetic screens is their potential functional redundancy. Here, we overcome part of this functional redundancy via a transportome, multi-targeted forward-genetic screen using artificial-microRNAs (amiRNAs). We generate a library of 3000 plant lines expressing 1777 amiRNAs, designed to target closely homologous genes within subclades of transporter families and identify, genotype and quantitatively phenotype, 80 lines showing reproducible shoot growth phenotypes. Within this population, we discover and characterize a strong redundant role for the unstudied ABCB6 and ABCB20 genes in auxin transport and response. The unique multi-targeted lines generated in this study could serve as a genetic resource that is expected to reveal additional transporters.
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http://dx.doi.org/10.1038/s41467-018-06410-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182007PMC
October 2018

Identification of SLAC1 anion channel residues required for CO/bicarbonate sensing and regulation of stomatal movements.

Proc Natl Acad Sci U S A 2018 10 9;115(44):11129-11137. Epub 2018 Oct 9.

Cell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0116;

Increases in CO concentration in plant leaves due to respiration in the dark and the continuing atmospheric [CO] rise cause closing of stomatal pores, thus affecting plant-water relations globally. However, the underlying CO/bicarbonate (CO/HCO ) sensing mechanisms remain unknown. [CO] elevation in leaves triggers stomatal closure by anion efflux mediated via the SLAC1 anion channel localized in the plasma membrane of guard cells. Previous reconstitution analysis has suggested that intracellular bicarbonate ions might directly up-regulate SLAC1 channel activity. However, whether such a CO/HCO regulation of SLAC1 is relevant for CO control of stomatal movements in planta remains unknown. Here, we computationally probe for candidate bicarbonate-interacting sites within the SLAC1 anion channel via long-timescale Gaussian accelerated molecular dynamics (GaMD) simulations. Mutations of two putative bicarbonate-interacting residues, R256 and R321, impaired the enhancement of the SLAC1 anion channel activity by CO/HCO in oocytes. Mutations of the neighboring charged amino acid K255 and residue R432 and the predicted gate residue F450 did not affect HCO regulation of SLAC1. Notably, gas-exchange experiments with -transformed plants expressing mutated SLAC1 proteins revealed that the SLAC1 residue R256 is required for CO regulation of stomatal movements in planta, but not for abscisic acid (ABA)-induced stomatal closing. Patch clamp analyses of guard cells show that activation of S-type anion channels by CO/HCO , but not by ABA, was impaired, indicating the relevance of R256 for CO signal transduction. Together, these analyses suggest that the SLAC1 anion channel is one of the physiologically relevant CO/HCO sensors in guard cells.
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http://dx.doi.org/10.1073/pnas.1807624115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217375PMC
October 2018

Abscisic acid-independent stomatal CO signal transduction pathway and convergence of CO and ABA signaling downstream of OST1 kinase.

Proc Natl Acad Sci U S A 2018 10 3;115(42):E9971-E9980. Epub 2018 Oct 3.

Cell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093;

Stomatal pore apertures are narrowing globally due to the continuing rise in atmospheric [CO]. CO elevation and the plant hormone abscisic acid (ABA) both induce rapid stomatal closure. However, the underlying signal transduction mechanisms for CO/ABA interaction remain unclear. Two models have been considered: () CO elevation enhances ABA concentrations and/or early ABA signaling in guard cells to induce stomatal closure and () CO signaling merges with ABA at OST1/SnRK2.6 protein kinase activation. Here we use genetics, ABA-reporter imaging, stomatal conductance, patch clamp, and biochemical analyses to investigate these models. The strong ABA biosynthesis mutants and remain responsive to CO elevation. Rapid CO-triggered stomatal closure in PYR/RCAR ABA receptor quadruple and hextuple mutants is not disrupted but delayed. Time-resolved ABA concentration monitoring in guard cells using a FRET-based ABA-reporter, ABAleon2.15, and ABA reporter gene assays suggest that CO elevation does not trigger [ABA] increases in guard cells, in contrast to control ABA exposures. Moreover, CO activates guard cell S-type anion channels in and ABA receptor hextuple mutants. Unexpectedly, in-gel protein kinase assays show that unlike ABA, elevated CO does not activate OST1/SnRK2 kinases in guard cells. The present study points to a model in which rapid CO signal transduction leading to stomatal closure occurs via an ABA-independent pathway downstream of OST1/SnRK2.6. Basal ABA signaling and OST1/SnRK2 activity are required to facilitate the stomatal response to elevated CO These findings provide insights into the interaction between CO/ABA signal transduction in light of the continuing rise in atmospheric [CO].
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http://dx.doi.org/10.1073/pnas.1809204115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6196521PMC
October 2018

Mitogen-activated protein kinases MPK4 and MPK12 are key components mediating CO -induced stomatal movements.

Plant J 2018 12 23;96(5):1018-1035. Epub 2018 Oct 23.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, La Jolla, CA, 92093-0116, USA.

Respiration in leaves and the continued elevation in the atmospheric CO concentration cause CO -mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA) and CO are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevated CO , but not to ABA. Such mutants are invaluable in unraveling the molecular mechanisms of early CO signal transduction in guard cells. Recently, mutations in the mitogen-activated protein (MAP) kinase, MPK12, have been shown to partially impair CO -induced stomatal closure. Here, we show that mpk12 plants, in which MPK4 is stably silenced specifically in guard cells (mpk12 mpk4GC homozygous double-mutants), completely lack CO -induced stomatal responses and have impaired activation of guard cell S-type anion channels in response to elevated CO /bicarbonate. However, ABA-induced stomatal closure, S-type anion channel activation and ABA-induced marker gene expression remain intact in the mpk12 mpk4GC double-mutants. These findings suggest that MPK12 and MPK4 act very early in CO signaling, upstream of, or parallel to the convergence of CO and ABA signal transduction. The activities of MPK4 and MPK12 protein kinases were not directly modulated by CO /bicarbonate in vitro, suggesting that they are not direct CO /bicarbonate sensors. Further data indicate that MPK4 and MPK12 have distinguishable roles in Arabidopsis and that the previously suggested role of RHC1 in stomatal CO signaling is minor, whereas MPK4 and MPK12 act as key components of early stomatal CO signal transduction.
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http://dx.doi.org/10.1111/tpj.14087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261798PMC
December 2018

Eukaryotic lipid metabolic pathway is essential for functional chloroplasts and CO and light responses in guard cells.

Proc Natl Acad Sci U S A 2018 09 20;115(36):9038-9043. Epub 2018 Aug 20.

Department of Biology, Faculty of Science, Kyushu University, 819-0395 Fukuoka, Japan;

Stomatal guard cells develop unique chloroplasts in land plant species. However, the developmental mechanisms and function of chloroplasts in guard cells remain unclear. In seed plants, chloroplast membrane lipids are synthesized via two pathways: the prokaryotic and eukaryotic pathways. Here we report the central contribution of endoplasmic reticulum (ER)-derived chloroplast lipids, which are synthesized through the eukaryotic lipid metabolic pathway, in the development of functional guard cell chloroplasts. We gained insight into this pathway by isolating and examining an mutant, (), which had achlorophyllous stomatal guard cells and impaired stomatal responses to CO and light. The gene encodes a small glycine-rich protein, which is a putative regulatory component of the trigalactosyldiacylglycerol (TGD) protein complex that mediates ER-to-chloroplast lipid transport via the eukaryotic pathway. Lipidomic analysis revealed that in the wild type, the prokaryotic pathway is dysfunctional, specifically in guard cells, whereas in guard cells, the eukaryotic pathway is also abrogated. CO-induced stomatal closing and activation of guard cell S-type anion channels that drive stomatal closure were disrupted in guard cells. In conclusion, the eukaryotic lipid pathway plays an essential role in the development of a sensing/signaling machinery for CO and light in guard cell chloroplasts.
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http://dx.doi.org/10.1073/pnas.1810458115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130404PMC
September 2018

Starch biosynthesis by AGPase, but not starch degradation by BAM1/3 and SEX1, is rate-limiting for CO -regulated stomatal movements under short-day conditions.

FEBS Lett 2018 08 10;592(16):2739-2759. Epub 2018 Aug 10.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, La Jolla, CA, USA.

Starch in guard cells functions in osmoregulation during stomatal movements. Starch metabolism is controlled by the circadian clock. We investigated the role of starch metabolism in stomatal responses to CO under different photoperiodic conditions. Guard cell starch levels correlate with low/high [CO ] exposure. Starch biosynthesis-deficient AGPase (ADG1) mutants but, unexpectedly, not the starch degradation-deficient BAM1, BAM3, and SEX1 mutants alone, are rate-limiting for stomatal conductance responses to [CO ]-shifts. Interestingly, AGPase is rate-limiting solely under short- but not long-day conditions. These findings suggest a model of enhanced AGPase activity in guard cells under short days such that starch biosynthesis becomes rate-limiting for CO -induced stomatal closing.
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http://dx.doi.org/10.1002/1873-3468.13198DOI Listing
August 2018

Cytosolic malate and oxaloacetate activate S-type anion channels in Arabidopsis guard cells.

New Phytol 2018 10 4;220(1):178-186. Epub 2018 Jul 4.

Cell and Developmental Biology Section, Division of Biological Sciences, University of California at San Diego, La Jolla, CA, 92093-0116, USA.

Intracellular malate-starch interconversion plays an important role in stomatal movements. We investigated whether malate or oxaloacetate from the cytosolic membrane side regulate anion channels in the plasma membrane of Arabidopsis thaliana guard cells. Physiological concentrations of cytosolic malate have been reported in the range of 0.4-3 mM in leaf cells. Guard cell patch clamp and two-electrode oocyte voltage-clamp experiments were pursued. We show that a concentration of 1 mM cytosolic malate greatly activates S-type anion channels in Arabidopsis thaliana guard cells. Interestingly, 1 mM cytosolic oxaloacetate also activates S-type anion channels. Malate activation was abrogated at 10 mM malate and in SLAC1 anion channel mutant alleles. Interestingly, malate activation of S-type anion currents was disrupted at below resting cytosolic-free calcium concentrations ([Ca ] ), suggesting a key role for basal [Ca ] signaling. Cytosolic malate was not able to directly activate or enhance SLAC1-mediated anion currents in Xenopus oocytes, whereas in positive controls, cytosolic NaHCO enhanced SLAC1 activity, suggesting that malate may not directly modulate SLAC1. Cytosolic malate activation of S-type anion currents was impaired in ost1 and in cpk5/6/11/23 quadruple mutant guard cells. Together these findings show that these cytosolic organic anions function in guard cell 'plasma membrane' ion channel regulation.
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http://dx.doi.org/10.1111/nph.15292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115288PMC
October 2018

Control of seed dormancy and germination by DOG1-AHG1 PP2C phosphatase complex via binding to heme.

Nat Commun 2018 06 6;9(1):2132. Epub 2018 Jun 6.

Structural Biology Team, Advanced Analysis Center, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, 305-8602, Japan.

Abscisic acid (ABA) regulates abiotic stress and developmental responses including regulation of seed dormancy to prevent seeds from germinating under unfavorable environmental conditions. ABA HYPERSENSITIVE GERMINATION1 (AHG1) encoding a type 2C protein phosphatase (PP2C) is a central negative regulator of ABA response in germination; however, the molecular function and regulation of AHG1 remain elusive. Here we report that AHG1 interacts with DELAY OF GERMINATION1 (DOG1), which is a pivotal positive regulator in seed dormancy. DOG1 acts upstream of AHG1 and impairs the PP2C activity of AHG1 in vitro. Furthermore, DOG1 has the ability to bind heme. Binding of DOG1 to AHG1 and heme are independent processes, but both are essential for DOG1 function in vivo. Our study demonstrates that AHG1 and DOG1 constitute an important regulatory system for seed dormancy and germination by integrating multiple environmental signals, in parallel with the PYL/RCAR ABA receptor-mediated regulatory system.
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http://dx.doi.org/10.1038/s41467-018-04437-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989226PMC
June 2018

Abscisic acid-induced degradation of guanine nucleotide exchange factor requires calcium-dependent protein kinases.

Proc Natl Acad Sci U S A 2018 05 23;115(19):E4522-E4531. Epub 2018 Apr 23.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA 92093

Abscisic acid (ABA) plays essential roles in plant development and responses to environmental stress. ABA induces subcellular translocation and degradation of the guanine nucleotide exchange factor RopGEF1, thus facilitating ABA core signal transduction. However, the underlying mechanisms for ABA-triggered RopGEF1 trafficking/degradation remain unknown. Studies have revealed that RopGEFs associate with receptor-like kinases to convey developmental signals to small ROP GTPases. However, how the activities of RopGEFs are modulated is not well understood. Type 2C protein phosphatases stabilize the RopGEF1 protein, indicating that phosphorylation may trigger RopGEF1 trafficking and degradation. We have screened inhibitors followed by several protein kinase mutants and find that quadruple-mutant plants in the calcium-dependent protein kinases (CPKs) disrupt ABA-induced trafficking and degradation of RopGEF1. Moreover, partially impairs ABA inhibition of cotyledon emergence. Several CPKs interact with RopGEF1. CPK4 binds to and phosphorylates RopGEF1 and promotes the degradation of RopGEF1. CPK-mediated phosphorylation of RopGEF1 at specific N-terminal serine residues causes the degradation of RopGEF1 and mutation of these sites also compromises the RopGEF1 overexpression phenotype in root hair development in Our findings establish the physiological and molecular functions and relevance of CPKs in regulation of RopGEF1 and illuminate physiological roles of a CPK-GEF-ROP module in ABA signaling and plant development. We further discuss that CPK-dependent RopGEF degradation during abiotic stress could provide a mechanism for down-regulation of RopGEF-dependent growth responses.
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http://dx.doi.org/10.1073/pnas.1719659115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5948973PMC
May 2018
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