Calcium-Mediated Regulation of Proton-Coupled Sodium Transport - Final Report

The long-term goal of our experiments was to understand mechanisms that regulate energy coupling by ion currents in plants. Activities of living organisms require chemical, mechanical, osmotic or electrical work, the energy for which is supplied by metabolism. Adenosine triphosphate (ATP) has long been recognized as the universal energy currency, with metabolism supporting the synthesis of ATP and the hydrolysis of ATP being used for the subsequent work. However, ATP is not the only energy currency in living organisms. A second and very different energy currency links metabolism to work by the movement of ions passing from one side of a membrane to the other. These ion currents play a major role in energy capture and they support a range of physiological processes from the active transport of nutrients to the spatial control of growth and development. In Arabidopsis thaliana (Arabidopsis), the activity of a plasma membrane Na+/H+ exchanger, SALT OVERLY SENSITIVE1 (SOS1), is essential for regulation of sodium ion homeostasis during plant growth in saline conditions. Mutations in SOS1 result in severely reduced seedling growth in the presence of salt compared to the growth of wild type. SOS1 is a secondary active transporter coupling movement of sodium ions out of the cell using energy stored in the transplasma membrane proton gradient, thereby preventing the build-up of toxic levels of sodium in the cytosol. SOS1 is regulated by complexes containing the SOS2 and CALCINEURIN B-LIKE10 (CBL10) or SOS3 proteins. CBL10 and SOS3 (also identified as CBL4) encode EF-hand calcium sensors that interact physically with and activate SOS2, a serine/threonine protein kinase. The CBL10/SOS2 or SOS3/SOS2 complexes then activate SOS1 Na+/H+ exchange activity. We completed our studies to understand how SOS1 activity is regulated. Specifically, we asked: (1) how does CBL10 regulate SOS1 activity? (2) What role do two putative CBL10-interacting proteins play in SOS1 regulation? (3) Are there differences in the regulation and/or activity of SOS1 in plants differing in their adaptation to salinity

Identification of transcription-factor genes expressed in the Arabidopsis female gametophyte

Dongfang Wang, Changqing Zhang, David J. Hearn, Il-HO Kang, megan I. Skaggs, Karen S. Schumaker, and Ramin Yadegari are with the School of Plant Sciences, University of Arizona, Tucson, Arizona 85721-0036, USA -- Il-Ho Kang, Jayson A. Punwani, and Gary N. Drews are with the Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840, USA -- Changqing Zhang is with The Section of Molecular, Cell and Developmental Biology, University of Texas at Austin, Austin, Texas 78712-0159, USA -- David J. Hearn is with the Department of Biological Sciences, Towson University, Towson, Maryland 21252-0001, USA -- Il-Ho Kang is with the Department of Horticulture, Iowa State University, Ames, Iowa 50011-1100, USA --Jayson A. Punwani is with the Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280, USABackground In flowering plants, the female gametophyte is typically a seven-celled structure with four cell types: the egg cell, the central cell, the synergid cells, and the antipodal cells. These cells perform essential functions required for double fertilization and early seed development. Differentiation of these distinct cell types likely involves coordinated changes in gene expression regulated by transcription factors. Therefore, understanding female gametophyte cell differentiation and function will require dissection of the gene regulatory networks operating in each of the cell types. These efforts have been hampered because few transcription factor genes expressed in the female gametophyte have been identified. To identify such genes, we undertook a large-scale differential expression screen followed by promoter-fusion analysis to detect transcription-factor genes transcribed in the Arabidopsis female gametophyte. Results Using quantitative reverse-transcriptase PCR, we analyzed 1,482 Arabidopsis transcription-factor genes and identified 26 genes exhibiting reduced mRNA levels in determinate infertile 1 mutant ovaries, which lack female gametophytes, relative to ovaries containing female gametophytes. Spatial patterns of gene transcription within the mature female gametophyte were identified for 17 transcription-factor genes using promoter-fusion analysis. Of these, ten genes were predominantly expressed in a single cell type of the female gametophyte including the egg cell, central cell and the antipodal cells whereas the remaining seven genes were expressed in two or more cell types. After fertilization, 12 genes were transcriptionally active in the developing embryo and/or endosperm. Conclusions We have shown that our quantitative reverse-transcriptase PCR differential-expression screen is sufficiently sensitive to detect transcription-factor genes transcribed in the female gametophyte. Most of the genes identified in this study have not been reported previously as being expressed in the female gametophyte. Therefore, they might represent novel regulators and provide entry points for reverse genetic and molecular approaches to uncover the gene regulatory networks underlying female gametophyte development.Cellular and Molecular [email protected]

Arabidopsis CALCINEURIN B-LIKE10 Functions Independently of the SOS Pathway during Reproductive Development in Saline Conditions

The accumulation of sodium in soil (saline conditions) negatively affects plant growth and development. The Salt Overly Sensitive (SOS) pathway in Arabidopsis (Arabidopsis thaliana) functions to remove sodium from the cytosol during vegetative development preventing its accumulation to toxic levels. In this pathway, the SOS3 and CALCINEURIN B-LIKE10 (CBL10) calcium sensors interact with the SOS2 protein kinase to activate sodium/proton exchange at the plasma membrane (SOS1) or vacuolar membrane. To determine if the same pathway functions during reproductive development in response to salt, fertility was analyzed in wild type and the SOS pathway mutants grown in saline conditions. In response to salt, CBL10 functions early in reproductive development before fertilization, while SOS1 functions mostly after fertilization when seed development begins. Neither SOS2 nor SOS3 function in reproductive development in response to salt. Loss of CBL10 function resulted in reduced anther dehiscence, shortened stamen filaments, and aborted pollen development. In addition, cbl10 mutant pistils could not sustain the growth of wild-type pollen tubes. These results suggest that CBL10 is critical for reproductive development in the presence of salt and that it functions in different pathways during vegetative and reproductive development.Preview published March 15, 2016. 12 month embargo.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

An atlas of over 90.000 conserved noncoding sequences provides insight into crucifer regulatory regions

Despite the central importance of noncoding DNA to gene regulation and evolution, understanding of the extent of selection on plant noncoding DNA remains limited compared to that of other organisms. Here we report sequencing of genomes from three Brassicaceae species (Leavenworthia alabamica, Sisymbrium irio and Aethionema arabicum) and their joint analysis with six previously sequenced crucifer genomes. Conservation across orthologous bases suggests that at least 17% of the Arabidopsis thaliana genome is under selection, with nearly one-quarter of the sequence under selection lying outside of coding regions. Much of this sequence can be localized to approximately 90,000 conserved noncoding sequences (CNSs) that show evidence of transcriptional and post-transcriptional regulation. Population genomics analyses of two crucifer species, A. thaliana and Capsella grandiflora, confirm that most of the identified CNSs are evolving under medium to strong purifying selection. Overall, these CNSs highlight both similarities and several key differences between the regulatory DNA of plants and other species

Molecular Characterization of the Role of a Calcium Channel in Plant Development

A stimulus-induced change in cellular Ca2+ levels is a critical component of energy transduction in plant and animal development. Demonstrating Ca2+'s involvement in any developmental process requires identification of mechanisms that regulate these Ca2+ changes. In plants, biochemical studies have implicated the activity of Ca2+ channels in increases in cellular Ca2+ levels; however, molecular evidence for these transporters is lacking. Our studies used the mosses Physcomitrella patens and Funaria hygrometrica to establish a role for Ca2+ in hormone-induced morphogenesis and to use this developmental process to identify transporters responsible for increasing cytosolic Ca2+ levels. Using 1,4-dihydropyridines (DHPs), molecules that block Ca2+ movement through voltage-dependent channels in animal cells, we have shown that Ca2+ is important early in the transition from filamentous to meristematic-like growth that occurs in response to the plant hormone cytokinin. In addition to inhibiting moss growth (see below), these Ca2+ channel blockers prevent Ca2+ transport into moss cells (Schumaker and Gizinski, 1993) and bind specifically to two proteins in the moss plasma membrane (Schumaker and Gizinski, 1994; 1996; Dietrich et al., unpublished results). We used tandem mass spectrometry of the partially purified DHP-binding proteins with the goal of identifying the putative Ca2+ channel and providing sequence information for studies to understand channel expression, regulation, structure, and function during development. In addition, we used insertional mutagenesis to identify additional components of the pathway underlying hormone-induced morphogenesis