The SNF1-type serine-threonine protein kinase SAPK4 regulates stress-responsive gene expression in rice

Diedhiou CJ, Popova OV, Dietz K-J, Golldack D. The SNF1-type serine-threonine protein kinase SAPK4 regulates stress-responsive gene expression in rice. BMC PLANT BIOLOGY. 2008;8(1):49.BACKGROUND: Plants respond to extracellularly perceived abiotic stresses such as low temperature, drought, and salinity by activation of complex intracellular signaling cascades that regulate acclimatory biochemical and physiological changes. Protein kinases are major signal transduction factors that have a central role in mediating acclimation to environmental changes in eukaryotic organisms. In this study, we characterized the function of the sucrose nonfermenting 1-related protein kinase2 (SnRK2) SAPK4 in the salt stress response of rice. RESULTS: Translational fusion of SAPK4 with the green fluorescent protein (GFP) showed subcellular localization in cytoplasm and nucleus. To examine the role of SAPK4 in salt tolerance we generated transgenic rice plants with over-expression of rice SAPK4 under control of the CaMV-35S promoter. Induced expression of SAPK4 resulted in improved germination, growth and development under salt stress both in seedlings and mature plants. In response to salt stress, the SAPK4-overexpressing rice accumulated less Na+ and Cl- and showed improved photosynthesis. SAPK4-regulated genes with functions in ion homeostasis and oxidative stress response were identified: the vacuolar H+-ATPase, the Na+/H+ antiporter NHX1, the Cl- channel OsCLC1 and a catalase. CONCLUSION: Our results show that SAPK4 regulates ion homeostasis and growth and development under salinity and suggest function of SAPK4 as a regulatory factor in plant salt stress acclimation. Identification of signaling elements involved in stress adaptation in plants presents a powerful approach to identify transcriptional activators of adaptive mechanisms to environmental changes that have the potential to improve tolerance in crop plants

Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis

Seidel T, Schnitzer D, Golldack D, Sauer M, Dietz K-J. Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis. BMC Cell Biology. 2008;9(1): 28.BACKGROUND: The V-ATPase (VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms. RESULTS: The transmembrane topology and stoichiometry of the proteolipid VHA-c" as well as the stoichiometry of the membrane integral subunit VHA-e within the V-ATPase complex were investigated by in vivo fluorescence resonance energy transfer (FRET). VHA-c", VHA-e1 and VHA-e2, VHA-a, VHA-c3, truncated variants of VHA-c3 and a chimeric VHA-c/VHA-c" hybrid were fused to cyan (CFP) and yellow fluorescent protein (YFP), respectively. The constructs were employed for transfection experiments with Arabidopsis thaliana mesophyll protoplasts. Subcellular localization and FRET analysis by confocal laser scanning microscopy (CLSM) demonstrated that (i.) the N- and C-termini of VHA-c" are localised in the vacuolar lumen, (ii.) one copy of VHA-c" is present within the VHA-complex, and (iii.) VHA-c" is localised at the ER and associated Golgi bodies. (iv.) A similar localisation was observed for VHA-e2, whereas (v.) the subcellular localisation of VHA-e1 indicated the trans Golgi network (TGN)-specifity of this subunit. CONCLUSION: The plant proteolipid ring is a highly flexible protein subcomplex, tolerating the incorporation of truncated and hybrid proteolipid subunits, respectively. Whereas the membrane integral subunit VHA-e is present in two copies within the complex, the proteolipid subunit VHA-c" takes part in complex formation with only one copy. However, neither VHA-c" isoform 1 nor any of the two VHA-e isoforms were identified at the tonoplast. This suggest a function in endomembrane specific VHA-assembly or targeting rather than proton transport

Tolerance to drought and salt stress in plants: Unraveling the signaling networks

Golldack D, Li C, Mohan H, Probst N. Tolerance to drought and salt stress in plants: Unraveling the signaling networks. Frontiers in Plant Science. 2014;5:151.Tolerance of plants to abiotic stressors such as drought and salinity is triggered by complex multicomponent signaling pathways to restore cellular homeostasis and promote survival. Major plant transcription factor families such as bZIP, NAC, AP2/ERF, and MYB orchestrate regulatory networks underlying abiotic stress tolerance. Sucrose non-fermenting 1-related protein kinase 2 and mitogen-activated protein kinase pathways contribute to initiation of stress adaptive downstream responses and promote plant growth and development. As a convergent point of multiple abiotic cues, cellular effects of environmental stresses are not only imbalances of ionic and osmotic homeostasis but also impaired photosynthesis, cellular energy depletion, and redox imbalances. Recent evidence of regulatory systems that link sensing and signaling of environmental conditions and the intracellular redox status have shed light on interfaces of stress and energy signaling. ROS (reactive oxygen species) cause severe cellular damage by peroxidation and de-esterification of membrane-lipids, however, current models also define a pivotal signaling function of ROS in triggering tolerance against stress. Recent research advances suggest and support a regulatory role of ROS in the cross talks of stress triggered hormonal signaling such as the abscisic acid pathway and endogenously induced redox and metabolite signals. Here, we discuss and review the versatile molecular convergence in the abiotic stress responsive signaling networks in the context of ROS and lipid-derived signals and the specific role of stomatal signaling

Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex

BACKGROUND: Vacuolar H(+)-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. RESULTS: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. CONCLUSIONS: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H(+)-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1

Cellular mechanisms of environmental adaptation: Learning from non-Arabidopsis model species

Golldack D. Cellular mechanisms of environmental adaptation: Learning from non-Arabidopsis model species. Progress in Botany. 2012;74:137-151

Molecular responses of halophytes to high salinity.

Golldack D. Molecular responses of halophytes to high salinity. Progress in Botany 65. 2004:219-234

Salt-dependent regulation of chloride channel transcripts in rice

Diedhiou C, Golldack D. Salt-dependent regulation of chloride channel transcripts in rice. PLANT SCIENCE. 2006;170(4):793-800.Rice OsCLC1 homologous to voltage-dependent Cl- channels of the CLC-family was characterized to study the regulation of Cl- homeostasis under salt stress conditions. By transcript analyses, expression of OsCLC1 was found in leaves and roots. Transcriptional regulation during salt stress was compared in the salt-sensitive Cl--accumulating rice line IR29 and the salt-tolerant Cl--excluding rice line Pokkali. In response to salt stress OsCLC1 transcript levels were repressed in leaves and roots of IR29 whereas in Pokkali expression was transiently induced. Under same conditions, in IR29 mRNA levels of the Na+/H+ antiporter OsNHX1 and of the vacuolar H+-ATPase subunit OsVHA-B decreased upon salt stress whereas Pokkali showed transient stimulation of OsVHA-B transcripts. Cell-specificity of OsCLC1 transcription was analyzed by in situ PCR. In leaves, signals were detected in mesophyll cells and stomata. In addition, expression occurred in xylem parenchyma cells and in the phloem whereas in salt-treated plants transcript amounts were reduced in mesophyll cells. Our results indicate coordinated regulation of anion and cation homeostasis in salt-treated rice and suggest function of OsCLC1 in osmotic adjustment at high salinity. (c) 2005 Elsevier Ireland Ltd. All rights reserved

In the halotolerant Lobularia maritima (Brassicaceae) salt adaptation correlates with activation of the vacuolar H+-ATPase and the vacuolar Na+/H+ antiporter

Popova OV, Golldack D. In the halotolerant Lobularia maritima (Brassicaceae) salt adaptation correlates with activation of the vacuolar H+-ATPase and the vacuolar Na+/H+ antiporter. JOURNAL OF PLANT PHYSIOLOGY. 2007;164(10):1278-1288.Lobularia thaliana and may be a suitable model to identify molecular mechanisms that regulate tolerance to salt stress in plants. Under the same salt stress conditions, the accumulation of sodium was similar in shoots and roots of Lobularia maritima and Arabidopsis thaliana, whereas the sodium to potassium ratio was less in Lobularia maritima. Aquaporins, the NHX-type Na+/H+ antiporter, and the vacuolar ATPase are well established targets of regulation under salt stress that have a central role in the control of water status and cytoplasmic sodium homeostasis. Therefore, salt-dependent expression of transcripts encoding a PIP2;1 aquaporin, the Na+/H+ antiporter NHX, and V-ATPase subunit E (VHA-E) was characterized in Lobularia maritima. Transcription of LmPIP2;1 was repressed in leaves and roots by treatment with 500 mM NaCl. In contrast, salt stress stimulated the expression of LmNHX1 and LmVHA-E. Cell-specificity of the transcription of LmNHX1 was analyzed by fluorescence in situ PCR in leaf cross sections of Lobularia maritima. Expression of the gene was localized to the phloem and to mesophyll cells. In plants treated with 500 mM NaCl, transcription of LmNHX1 was stimulated in the mesophyll.. The findings indicate divergent transcriptional responses of key mechanisms of salt adaptation in Lobularia maritima and suggest distinct regulation of sodium homeostasis and water flux under salt stress. (C) 2006 Elsevier GmbH. All rights reserved