CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca(2+)- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling

The tropical Atlantic observing system

The tropical Atlantic is home to multiple coupled climate variations covering a wide range of timescales and impacting societally relevant phenomena such as continental rainfall, Atlantic hurricane activity, oceanic biological productivity, and atmospheric circulation in the equatorial Pacific. The tropical Atlantic also connects the southern and northern branches of the Atlantic meridional overturning circulation and receives freshwater input from some of the world’s largest rivers. To address these diverse, unique, and interconnected research challenges, a rich network of ocean observations has developed, building on the backbone of the Prediction and Research Moored Array in the Tropical Atlantic (PIRATA). This network has evolved naturally over time and out of necessity in order to address the most important outstanding scientific questions and to improve predictions of tropical Atlantic severe weather and global climate variability and change. The tropical Atlantic observing system is motivated by goals to understand and better predict phenomena such as tropical Atlantic interannual to decadal variability and climate change; multidecadal variability and its links to the meridional overturning circulation; air-sea fluxes of CO2 and their implications for the fate of anthropogenic CO2; the Amazon River plume and its interactions with biogeochemistry, vertical mixing, and hurricanes; the highly productive eastern boundary and equatorial upwelling systems; and oceanic oxygen minimum zones, their impacts on biogeochemical cycles and marine ecosystems, and their feedbacks to climate. Past success of the tropical Atlantic observing system is the result of an international commitment to sustained observations and scientific cooperation, a willingness to evolve with changing research and monitoring needs, and a desire to share data openly with the scientific community and operational centers. The observing system must continue to evolve in order to meet an expanding set of research priorities and operational challenges. This paper discusses the tropical Atlantic observing system, including emerging scientific questions that demand sustained ocean observations, the potential for further integration of the observing system, and the requirements for sustaining and enhancing the tropical Atlantic observing system

Proceedings of the 13th annual conference of INEBRIA

CITATION: Watson, R., et al. 2016. Proceedings of the 13th annual conference of INEBRIA. Addiction Science & Clinical Practice, 11:13, doi:10.1186/s13722-016-0062-9.The original publication is available at https://ascpjournal.biomedcentral.comENGLISH SUMMARY : Meeting abstracts.https://ascpjournal.biomedcentral.com/articles/10.1186/s13722-016-0062-9Publisher's versio

Guard Cell Expression of <i>CPK3</i> and <i>CPK6</i> CDPKs

<div><p>(A) Expression of <i>CPK3</i> and <i>CPK6</i> in guard cell (GC) and mesophyll cell (MC) protoplasts was examined by RT-PCR. Control amplifications of the guard cell-expressed <i>KAT1</i> gene and the mesophyll-expressed <i>CBP</i> marker genes [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040327#pbio-0040327-b054" target="_blank">54</a>] (Leonhardt et al., 2004) were used to test the purity of cell preparations (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040327#s2" target="_blank">Results</a>). <i>ACTIN2</i> was used for an internal loading control. To amplify each <i>CDPK</i>-specific band, RT-PCR was performed with primer sets as indicated by arrowheads in (B) for 36 cycles. Plants were sprayed with water (−ABA) or 100 μM ABA (+ABA) 4 h before isolation of protoplasts and RNA extraction.</p> <p>(B) Cartoon showing the T-DNA insertion positions in <i>cpk3</i> and <i>cpk6</i> T-DNA insertion alleles. PCR was performed with a left boarder primer of the T-DNA and a gene-specific primer, and the PCR products were sequenced to determine the T-DNA insertion positions. Arrowheads indicate primer locations for RT-PCR in (A) and (C). ATG and TGA indicate start and stop codons. White boxes indicate exons.</p> <p>(C) RT-PCR confirmed that <i>cpk3–1</i> and <i>cpk6–1</i> alleles were disruption mutants. PCRs (32 cycles) were performed with primer sets as indicated in (B) (black arrowheads) in the left three panels. Transcripts of wild-type (WT) and <i>cpk6–2</i> were examined with two sets of primers [white and black arrowheads in (B)] showing that <i>cpk6–2</i> lacks exon 1 and that the <i>cpk6–2</i> has 8% or less the mRNA level of wild-type based on densitometry analyses (<i>n</i> = 2). RNA was extracted from leaves of WT, homozygous <i>cpk3–1</i>, <i>cpk6–1,</i> and <i>cpk6–2</i> single mutants, and the <i>cpk3-1cpk6–1</i> double mutant.</p></div

ABA- and Ca²⁺-Induced Stomatal Closure Is Partially Impaired in <i>cpk3cpk6</i> Mutants

<div><p>(A) ABA-induced stomatal closing (white bars: wild-type; shaded bars: <i>cpk3-1cpk6–1</i> double mutant; black bars: <i>cpk3-2cpk6–2</i> double mutant). Average data from representative experiments are shown (wild-type: WT, <i>n</i> = 13 experiments, 260 total stomata; <i>cpk3-1cpk6–1</i> double mutant: <i>n</i> = 7 experiments, 140 stomata; <i>cpk3-2cpk6–2 n</i> = 4 experiments, <i>n</i> = 120 stomata).</p> <p>(B) External Ca²⁺-induced stomatal closing (white bars: wild-type, shaded bars: <i>cpk3-1cpk6–1</i> double mutant, black bars: <i>cpk3-2cpk6–2</i> double mutant). Average data from representative experiments are shown (wild-type: WT, <i>n</i> = 9 experiments including 4 blind experiments, 180 total stomata; <i>cpk3-1cpk6–1</i> double mutant, <i>n</i> = 9 experiments including 4 blind experiments, 180 total stomata; <i>cpk3-2cpk6–2</i>, <i>n</i> = 4 experiments, <i>n</i> = 80 stomata). Stomatal aperture widths are illustrated. Error bars represent SEM.</p></div