Functional responses between PMP3 small membrane proteins and membrane potential

The Plasma Membrane Proteolipid 3 (PMP3, UPF0057 family in Uniprot) family consists of abundant small hydrophobic polypeptides with two predicted transmembrane helices. Plant homologues were upregulated in response to drought/salt-stresses and yeast deletion mutants exhibited conditional growth defects. We report here abundant expression of Group I PMP3 homologues (PMP3(i)hs) during normal vegetative growth in both prokaryotic and eukaryotic cells, at a level comparable to housekeeping genes, implicating the regular cellular functions. Expression of eukaryotic PMP3(i)hs was dramatically upregulated in response to membrane potential (Vm) variability (Vm_var), whereas PMP3(i)hs deletion-knockdown led to Vm changes with conditional growth defects. Bacterial PMP3(i)h yqaE deletion led to a shift of salt sensitivity; Vm_var alternations with exogenous K⁺ addition downregulated prokaryotic PMP3(i)hs, suggesting [K⁺]-Vm_var axis being a significant feedback element in prokaryotic ionic homeostasis. Remarkably, the eukaryotic homologues functionally suppressed the conditional growth defects in bacterial deletion mutant, demonstrating the conserved cross-kingdom membrane functions by PMP3(i)hs. These data demonstrated a direct reciprocal relationship between PMP3(i)hs expression and Vm differentials in both prokaryotic and eukaryotic cells. Cumulative with PMP3(i)hs ubiquitous abundance, their lipid-binding selectivity and membrane protein colocalization, we propose [PMP3(i)hs]-Vm_var axis as a key element in membrane homeostasis.     

Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane

Yeast plasma membranes contain a small 55 amino acid hydrophobic polypeptide, Pmp3p, which has high sequence similarity to a novel family of plant polypeptides that are overexpressed under high salt concentration or low temperature treatment. The PMP3 gene is not essential under normal growth conditions. However, its deletion increases the plasma membrane potential and confers sensitivity to cytotoxic cations, such as Na(+) and hygromycin B. Interestingly, the disruption of PMP3 exacerbates the NaCl sensitivity phenotype of a mutant strain lacking the Pmr2p/Enap Na(+)-ATPases and the Nha1p Na(+)/H(+) antiporter, and suppresses the potassium dependency of a strain lacking the K(+) transporters, Trk1p and Trk2p. All these phenotypes could be reversed by the addition of high Ca(2+) concentration to the medium. These genetic interactions indicate that the major effect of the PMP3 deletion is a hyperpolarization of the plasma membrane potential that probably promotes a non-specific influx of monovalent cations. Expression of plant RCI2A in yeast could substitute for the loss of Pmp3p, indicating a common role for Pmp3p and the plant homologue.     

Isolation and characterization of maize PMP3 genes involved in salt stress tolerance

Plasma membrane protein 3 (PMP3), a class of small hydrophobic polypeptides with high sequence similarity, is responsible for salt, drought, cold, and abscisic acid. These small hydrophobic ploypeptides play important roles in maintenance of ion homeostasis. In this study, eight ZmPMP3 genes were cloned from maize and responsive to salt, drought, cold and abscisic acid. The eight ZmPMP3s were membrane proteins and their sequences in trans-membrane regions were highly conserved. Phylogenetic analysis showed that they were categorized into three groups. All members of group II were responsive to ABA. Functional complementation showed that with the exception of ZmPMP3-6, all were capable of maintaining membrane potential, which in turn allows for regulation of intracellular ion homeostasis. This process was independent of the presence of Ca(2+). Lastly, over-expression of ZmPMP3-1 enhanced growth of transgenic Arabidopsis under salt condition. Through expression analysis of deduced downstream genes in transgenic plants, expression levels of three ion transporter genes and four important antioxidant genes in ROS scavenging system were increased significantly in transgenic plants during salt stress. This tolerance was likely achieved through diminishing oxidative stress due to the possibility of ZmPMP3-1's involvement in regulation of ion homeostasis, and suggests that the modulation of these conserved small hydrophobic polypeptides could be an effective way to improve salt tolerance in plants.     

Overexpression of NtHAL3 genes confers increased levels of proline biosynthesis and the enhancement of salt tolerance in cultured tobacco cells

The Hal3 protein of Saccharomyces cerevisiae inhibits the activity of PPZ1 type-1 protein phosphatases and functions as a regulator of salt tolerance and cell cycle control. In plants, two HAL3 homologue genes in Arabidopsis thaliana, AtHAL3a and AtHAl3b, have been isolated and the function of AtHAL3a has been investigated through the use of transgenic plants. Expressions of both AtHAL3 genes are induced by salt stress. AtHAL3a overexpressing transgenic plants exhibit improved salt and sorbitol tolerance. In vitro studies have demonstrated that AtHAL3 protein possessed 4'-phosphopantothenoylcysteine decarboxylase activity. This result suggests that the molecular function of plant HAL3 genes is different from that of yeast HAL3. To understand the function of plant HAL3 genes in salt tolerance more clearly, three tobacco HAL3 genes, NtHAL3a, NtHAL3b, and NtHAL3c, from Nicotiana tabacum were identified. NtHAL3 genes were constitutively expressed in all organs and under all conditions of stress examined. Overexpression of NtHAL3a improved salt, osmotic, and lithium tolerance in cultured tobacco cells. NtHAL3 genes could complement the temperature-sensitive mutation in the E. coli dfp gene encoding 4'-phosphopantothenoyl-cysteine decarboxylase in the coenzyme A biosynthetic pathway. Cells overexpressing NtHAL3a had an increased intracellular ratio of proline. Taken together, these results suggest that NtHAL3 proteins are involved in the coenzyme A biosynthetic pathway in tobacco cells.     

Two New Group 3 LEA Genes of Wheat and Their Functional Analysis in Yeast

The group 3 late embryogenesis abundant （LEA） proteins are thought to protect cells from stresses associated with dehydration during periods of water deficit. To investigate the functions of different members of the group 3 LEA genes, we isolated and characterized two new group 3 LEA genes, namely TaLEA2 and TaLEA3, from wheat （Triticum aestivum L.） and introduced TaLEA2 and TaLEA3 into Saccharmyces cerevisiae to examine the effect of these genes on yeast cell tolerance to osmotic, salt, and cold stresses. The TaLEA2 gene encoded a protein of 211 amino acids and possessed five repeats of 11-mer amino acid motifs. The TaLEA3 gene encoded a polypeptide of 211 amino acids with nine repeated units. Overexpression of TaLEA2 and TaLEA3 improved stress tolerance in transgenic yeast cells when cultured in medium containing sorbitol, salt and-20℃ freezing treatments respectively. However, the yeast transformants with TaLEA2 seemed to be more tolerant to hyperosmotic and freezing stress than transformants with TaLEA3. This implies that a close relationship exists between function and the number of repeats of the 11- mer amino acid motif in the group 3 LEA protein.     

Rice shaker potassium channel OsKAT1 confers tolerance to salinity stress on yeast and rice cells

We screened a rice (Oryza sativa L. 'Nipponbare') full-length cDNA expression library through functional complementation in yeast (Saccharomyces cerevisiae) to find novel cation transporters involved in salt tolerance. We found that expression of a cDNA clone, encoding the rice homolog of Shaker family K(+) channel KAT1 (OsKAT1), suppressed the salt-sensitive phenotype of yeast strain G19 (Deltaena1-4), which lacks a major component of Na(+) efflux. It also suppressed a K(+)-transport-defective phenotype of yeast strain CY162 (Deltatrk1Deltatrk2), suggesting the enhancement of K(+) uptake by OsKAT1. By the expression of OsKAT1, the K(+) contents of salt-stressed G19 cells increased during the exponential growth phase. At the linear phase, however, OsKAT1-expressing G19 cells accumulated less Na(+) than nonexpressing cells, but almost the same K(+). The cellular Na(+) to K(+) ratio of OsKAT1-expressing G19 cells remained lower than nonexpressing cells under saline conditions. Rice cells overexpressing OsKAT1 also showed enhanced salt tolerance and increased cellular K(+) content. These functions of OsKAT1 are likely to be common among Shaker K(+) channels because OsAKT1 and Arabidopsis (Arabidopsis thaliana) KAT1 were able to complement the salt-sensitive phenotype of G19 as well as OsKAT1. The expression of OsKAT1 was restricted to internodes and rachides of wild-type rice, whereas other Shaker family genes were expressed in various organs. These results suggest that OsKAT1 is involved in salt tolerance of rice in cooperation with other K(+) channels by participating in maintenance of cytosolic cation homeostasis during salt stress and thus protects cells from Na(+).     

Expression of rice Ca(2+)-dependent protein kinases (CDPKs) genes under different environmental stresses

Ca(2+)-dependent protein kinases (CDPKs) play an essential role in plant Ca(2+)-mediated signal transduction. Twenty-nine CDPK genes have been identified in the rice genome through a complete search of genome and full-length cDNA databases. Eight of them were reported previously to be inducible by different stress stimuli. Sequence comparison revealed that all 29 CDPK genes (OsCPK1-29) contain multiple stress-responsive cis-elements in the promoter region (1kb) upstream of genes. Analysis of the information extracted from the Rice Expression Database indicates that 11 of the CDPK genes are regulated by chilling temperature, dehydration, salt, rice blast infection and chitin treatment. RT-PCR and RNA gel blot hybridization were performed in this study to detect the expression 19 of the CDPK genes. Twelve CDPK genes exhibited cultivar- and tissue-specific expression; four CDPK genes (OsCPK6, OsCPK13, OsCPK17 and OsCPK25) were induced by chilling temperature, dehydration and salt stresses in the rice seedlings. While OsCPK13 (OsCDPK7) was already known to be inducible by chilling temperature and high salt, this is the first report that the other three genes are stress-regulated. OsCPK6 and OsCPK25 are up-regulated by dehydration and heat shock, respectively, while OsCPK17 is down-regulated by chilling temperature, dehydration and high salt stresses. Based on this evidence, rice CDPK genes may be important components in the signal transduction pathways for stress responses. Findings from this research are important for further dissecting mechanisms of stress response and functions of CDPK genes in rice.     

Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants

Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed.     

A catalytic subunit of the sugar beet protein kinase CK2 is induced by salt stress and increases NaCl tolerance in Saccharomyces cerevisiae

Salinity is an important limiting factor in plant growth and development. We have cloned a catalytic subunit of the sugar beet protein kinase CK2 (BvCKA2) by functional expression in yeast of a NaCl-induced cDNA library. BvCKA2 was able to increase the yeast tolerance to NaCl and to functionally complement the cka1 cka2 yeast double mutant upon over-expression. Southern blot analysis indicated that, in sugar beet, the BCKA2 gene is a member of a multigene family. The mRNA levels of BvCKA2 were up-regulated in response to NaCl stress which suggests that protein kinase CK2 may be involved in the plant response to salt stress.     

Conservation of the salt overly sensitive pathway in rice

The salt tolerance of rice (Oryza sativa) correlates with the ability to exclude Na+ from the shoot and to maintain a low cellular Na+/K+ ratio. We have identified a rice plasma membrane Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the functional homolog of the Arabidopsis (Arabidopsis thaliana) salt overly sensitive 1 (SOS1) protein. The rice transporter, denoted by OsSOS1, demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of Arabidopsis. These results represent the first molecular and biochemical characterization of a Na+ efflux protein from monocots. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its Ca2+-dependent activator SOS3 were identified also. OsCIPK24 and OsCBL4 acted coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their Arabidopsis counterpart to form heterologous protein kinase modules that activated both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants of Arabidopsis. These results demonstrate that the SOS salt tolerance pathway operates in cereals and evidences a high degree of structural conservation among the SOS proteins from dicots and monocots.     

14-3-3 Protects against stress-induced apoptosis

Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3β/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3β/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3β/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3β/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called ‘autophagic death''. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3β/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins.