Difference between PM19-Containing and PM19-Absent Cell Lines - Essay Example

Until now, the function of transmembrane PM19 is still unknown. Because of its structure, its role in transcellular transport is likely. S. cerevisiae cell lines with mutated K+ channels is a good study model to determine whether PM19 has a role in K+ transport or not. In this study, the difference between PM19- containing and PM19-absent cell lines were compared.in terms of growth in media of different K+ concentrations. After 3 and 6 days after culture, it was found that trk1 mutants had poorer growth compared to the WT and tok1 mutants. However, when PM19 was inserted into these strains, the growth of trk1 mutants was then comparable with the other strains. This implies that PM19 compensated for the absence of trk1p, and thus it also acts as a high-affinity K+ transporter. In plants, since ABA, a hormone released in response to water desiccation, increases PM19 expression, the overproduction of the membrane protein allow the cells to imbibe the important K+ ion despite the low concentration in the environment. 1.Introduction The DNA sequence of an organism remains constant throughout its life despite the multitude of processes it undergoes by preventing DNA mutations that might alter genetic information, and by ensuring the structural integrity of chromosomes and their orderly transmission to progeny cells. It is necessary to prevent alterations of DNA sequence that may change information encoded in genes because it may cause dysfunction of proteins that are important in the different physiologic activities of the organism. 2. Proteins Proteins are the most significant class of molecules in biochemistry, although of course lipids and carbohydrates are also crucial for all living species whether eukaryotic or prokaryotic such as human, animal, plant and bacteria. Proteins organize and make the basics of life. They control of all actions in each known organisms, for example, the expression of the genetic code, transport of molecules, oxygen and minerals to in or out of the cells, therefore, proteins are responsible for controlling the machinery of cellular which is the phenotype of an organism (Lau, 2005). Moreover, the eukaryotic cell is surrounded by the membrane, which has the control for all substances that can enter in or out of the cell. Although the fundamental structure of membrane is made up by the lipid bilayer, the most particular functions of the plasma membrane are carried out by proteins and provides each sort of membrane a specific feature and characteristic, so under these circumstances the only part of cell that can determine whether transport of a substance is towards the outside or inside of the bilayer is the transmembrane protein (TMP) and these are responsible for most interactions between the outside environment and cell (Clements and Martin, 2002). (Wallin and von Heijne, 1998). There are four level of protein structure: Primary, Secondary, Tertiary and Quaternary structure. The first kind of proteins structure is called the primary protein structure and consists of the amino-acid sequence of the protein (Petsko and Ringe, 2004), however, primary structure of protein is not just the number of each amino acids component in the protein, but the information that depends on specific amino acid sequence as joined through peptide bonds in the chain of polypeptide. The second sort of structure in protein is called the secondary protein structure which is regular repeating shapes of the polypeptide chain (hydrogen-bonded), which is able to fold back on itself in a different of ways (Petsko and Ringe, 2004). There are two major secondary protein structures: the alpha helix and beta pleated sheet. As the name implies, alpha helix is a spring coil configuration, in which the backbone of the amino acids occupy the center and the side chains are located in the periphery. The structure is stabilized by intramolecular hydrogen bonds between the amino group of one amino acid and carboxyl moiety of the fourth amino acid away from it. On the other hand, beta pleated sheet is comprised of polypeptides aligned side-by-side. This is stabilized by the intermolecular hydrogen bonding (Ophardt, 2003). Interaction between secondary structures, form the fold of protein (tertiary structure) and quaternary dimensional shape, which is arrangement of two or more of polypeptide chain. The tertiary protein structure is the order and organization in space of secondary structures. Finally, the quaternary protein structure defines the last precise shape and function of the protein. A diversity of bonding interactions such as hydrogen bonding, disulfide bonds and salt bridges, hold the different chains into a specific geometry. In terms of quaternary structure, proteins can either be globular, such as hemoglobin and immunoglobulins, or fibrous, such as keratin. 2.1 The structure of membrane proteins Not all proteins are in an aqueous environment. Some of them contained by hydrophobic interactions inside of the membrane that form surface of cells. Biological membrane is composed of a bilayer of lipid molecules and proteins in the cells associated with the membrane are integral or peripheral. Integral, means spanning the membrane in cells and the major interaction is van der Waals interaction, with the hydrophobic core of the bilayer. These proteins can be isolated by membrane disruption. On the other hand, peripheral proteins which do not penetrate the hydrophobic core, and electrostatic is the main interaction, also the hydrophilic surfaces of the bilayer, normally involved with integral membrane and isolated of membrane by altering pH or strong salt (Petsko and Ringe, 2004). Figure 1 provides a schematic diagram of the cell membrane. 2.2 Potassium ion channels One of the protein types that is vital to cellular functions is transmembrane proteins, which allow the transfer of molecules in or out of the cell. Potassium ions (K+) are important in efficiency of cellular respiration, intracellular fluid balance and nutrient transfer (Skinner, 2005). This is why they are transported into the cells despite the already high intracellular concentration. In yeasts, they are transported into the cells by trk1, trk2 and t0k1 proteins, encoded by TRK1, TRK2and TOK1, respectively. Bertl, et al. (2003) developed yeast cell strains with mutated trk1, trk2 and/or tok1 genes to study each of the respective proteins contribution to K+ transport. In the characterization of these strains, it was found that trk1-only and trk2-only mutants were able to grow over a wide range of extracellular K+ concentrations, while those with both genes mutated grew only suboptimally. Trk1p and trk2p can thus substitute for one another, particularly but not limited to extracellular K+ concentrations ≥ 1 mM, together wih acidic pH and 1M NaCl. At lower concentrations of K+ however, trk1p becomes the main K+ transporter. Higher concentrations of NaCl also prevented the growth of strain with mutated trk1 and trk2, regardless of K+ concentration, because Na+ competes with K+ for transport. Based on these findings, trkp2 is a weakly-expressed protein that transports the ion in high K+ conditions, while trk1p is able to transport K+ even at low concentrations, ultimately enabling the cell to survive in low-K+ environment. Without these two, the cell should be grown in high K+ environment to be viable. Moreover, trk2p has also been found to transport lithium ions (Li+), aside from K+. However, in the absence of trk1p and trk2p, K+ transport can still push through, as mediated by low-affinity non-selective uptake mechanisms. Without both trk1p and trk2p, these mechanisms allow suboptimal growth of the mutants, but only at ≥ 10 mM extracellular K+ concentrations (Bertl, et al., 2003). Meanwhile, the voltage-gated tok1 has not been found to be important in cellular physiology (Bertl, et al., 2003). Interestingly, these three were found to be homologous to similarly functioning proteins in the bacterial, plant and animal cells (Rodriguez-Navarro, 2000). 2.3 PM19 The PM19 gene is largely conserved in both monocotyledonous and dicotyledonous plants, and also bryophytes and pteridophytes. The length of the Arabidopsis PM19 gene is 985 nucleotides without the poly-A tail, and it encodes 181 amino acids of putative protein (Ranford, Bryce and Morris, 2002). In addition, PM19 has been found to code for a plasma membrane hydrophobic protein of 19kd in size. About 92.8% of the Arabidopsis protein sequence is similar to the wheat PM19, in addition, there is high level of identity with two other protein sequences encoded in Arabidopsis thaliana L (Ranford, Bryce and Morris, 2002). The locations of the PM19 protein is in plasma membrane according to previous researches (Koike et al., 1997). However, the function of this protein is not yet known. According to a study by Ranford, Bryce and Morris (2002) on Hordeum vulgare, PM19 is expressed from mid-embryogenesis to maturity, with levels of PM19 mRNA decreasing during germination. Otherwise, when the seeds remain dormant by preventing germination, PM19 mRNA levels were maintained at a high level. This relation to plant’s level of maturity might be mediated by PM19’s relation with abscisic acid (ABA). Koike, Takezawa, Arakawa and Yoshida (1997) reported that PM19 expression is induced by the presence of ABA. 3. Arabidopsis  Arabidopsis thaliana is a small flowering plant from the mustard family of (Brassicaceae). It is a small size of plant compared to other plants from the family, and it became a good organism for research in plants community, because of its large number of offspring, small size and short cycle of life (The Arabidopsis Genome Initiative, 2000).  Arabidopsis has a 125-megabase genome. This is a result of whole-genome duplication and subsequent gene loss and extensive local gene duplications. There was also a lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome also contains 25,498 genes encoding proteins. Despite having a similar protein profile as that of Drosophila melanogaster and Caenorhabditis elegans, it still lacks several common protein families (The Arabidopsis Genome Initiative, 2000). 4. Statement of the problem The question regarding the function of PM19 in plants is still unanswered. With the advancements in cellular research, it is now possible to study the activity of Arabidopsis PM19 in a close relative, which is easier to manipulate and to study than plant cells. A good candidate for the study of PM19 function is yeast cells. Aside from them being eukaryotic, it can be modified to cater to the needs of the study. Since PM19 is located at the plant cell’s membrane, its role in molecule transport is likely. With K+ being one of the vital ions being transported to and from a cell, it is only logical to start here. Thus, in this study, yeast strains with or without trk1p, trk2p and/or tok1 inserted with PM19-encoding gene were observed. 5. Research Question This study aimed to answer the question, “What is the role of PM19 to intracellular K+ transport of plant cells?” 5. Objectives To help answer this question, the research was guided by the general objective: to determine the function of PM19 in plant cells To achieve this, specific objectives were: a. Insertion of PM19-encoding genes into various yeast cell lines with mutated trk1, trk2, and/or tok1 genes. b. Observe growth cells in media of different K+ concentrations c. Suggest the function of PM19 in plant cells d. Relate its function to its ABA-dependent and stage-related differential expression 6. Materials and methods 6.1 Strains of yeast (Saccharomyces cerevisiae) Different strains were used. These were PLY232 (wild type), PLY234 (trk1), PLY236 (trk2), PLY238 (tok1) PLY240 (trk1 and trk2) and PLY242 (trk1 and tok1) strains developed by the study of Bertl, et al. (2003). The genotype of PLY232, PLY234 and PLY238 are indicated in table 1. Their growth was observed on both media solid and liquid of YPD and synthetic medium with different concentrations of potassium. Table 1: Saccharomyces cerevisiae strains. Name Genotype Reference PLY232, (wild type, WT) MATa his3D200 leu2-3,112 trp1D901 ura3-52 suc2D9. Bertl et al., 2003 PLY234, (trk1) MATa his3D200 leu2-3,112 trp1D901 ura3-52 suc2D9 trk1D51. PLY238, (tok1) MATa his3D200 leu2-3,112 trp1D901 ura3-52 suc2D9 tok1D1::HIS3. 6.2 Chemicals, reagents and different materials. Table 2: List of chemicals, and reagents were used in our experiment, in table 2, with respected to providers. Names Providers Adenine Sigma Agar High Gel Strength Melford Agarose Melford Arginine Acros Organics Glucose AnalaR Histidine Duchefa Biochemie Leucine Sigma Peptone Formedium Potassium chloride, Sigma Trace elements (500 g/l boric acid, 40 g/l copper sulfate, 200 g/l ferric chloride, 400 g/l manganese sulfate, 200 g/l sodium molybdate, and 400 g/l zinc sulfate) Sigma Tris Sigma Tryptone Sigma Uracil Sigma Vitamins (2 g/l biotin, 400 g/l Ca-panthotenate, 2 g/l folic acid, 2000 g/l inositol, 400 g/l nicotinic acid, 200 g/l p-aminobenzoic acid, 400 g/l pyridoxine HCl, 200 g/l riboflavin, and 400 g/l thiamine HCl) Sigma Yeast Extract Powder Formedium 6.3 SDAP and pH Table 3: 15 mM KCl 100 M Arginine 0.174 g Glucose 2 g Agar 2 g KCl (1 M) 1.5 ml Uracil 5 mg Adenine 5mg H20 To 96 ml H3PO4 (85%) To pH 5.6 Autoclave 15 min at 120°C 100x stock solution CaCl2 0.2 mM MgSO4 0.1 ml Trace 1 ml Vitamins 1 ml His/Leu/Trp 1 ml H3PO4 (85%) 0 ul Desired pH 5.5 Measured pH 5.7 6.4 The Media and culture used: Table 4. The formulae of YPD Agar per Liter: Yeast Extract 10.0 g Peptone 20.0 g Dextrose 20.0 g Agar 15.0 g 6.4.1 The Preparation of culture in Yeast Extract-Peptone-Dextrose (YPD) both Agar (solid media) and (YPD) Broth Both YPD Agar and YPD Broth were used to propagate and maintain yeast cultures. YPD contains peptone as a source of vitamins, minerals and nitrogen, yeast extract, which encourages the organism to grow, and dextrose as source of the carbohydrate. Agar is used as a solidifying agent. 6.4.2 The Procedure of preparation in YPD the broth media YPD broth was prepared by dissolving the powder in 1 L of distilled water. Two solutions were prepared, with one added with agar. The one containing agar was heated to boil for 1 minute. Then, both broth media and agar at were autoclaved in 121°C for 15 minutes. 6.4.3 Experiment Proper The strains of yeast (Saccharomyces cerevisiae) are as listed in Table 1. Aside from YPD, the yeast strains were grown in synthetic dextrose arginine phosphate (SDAP) (Rodrigueznavarro and Ramos, 1984). All factors of the SDAP media was prepared by this order: 2 g of glucose (from a 40% w/v of stock solution) and 0.174 g of arginine were mixed to prepare a 10 mM stock. Phosphoric acid was added until pH was 6.5. 100x solution made of different elements such as vitamins, leucine, trace elements, salts and uracil, were then prepared and sterilized by filtration. It was added to the stock solution after cooling. pH was then adjusted to 5.8, On the other hand, the solid media was prepared by adding 2% w/v agar. pH was adjusted by phosphoric acid to below 4.5 (Bertl et al., 2003). 6.4.4 The examinations of yeast growth on SDAP plates Three cell strains, (1) wild type, (2) with empty pyes2 vector, and (3) PM19-containing pyes2, were each spotted on plate as follows: To discover the characterization of the growth in cells in relation to the elimination of single and multiple K+ transporters, cells were placed over 2 to 3 days at 28ºC with 15 mM KCl. Cells were inoculated into sterile tubes to a density equivalent to OD600 = 1.0 ± 0.05. Tenfold serial of dilutions were then prepared, and 2.5 ml aliquots of individual dilutions were spotted on plates of SDAP agar at 3 locations on the plate (Bertl et al., 2003). 7. Results High K+ environment The growth of the yeast strains were tested on SDAP agar plates supplemented with the highest concentration of K+ (100 mM KCl, pH 5.8) used in this study. After three days, it was observed that growth was not affected by the absence of trk1, trk2 and/or tok1 as well as presence of PM19 gene (Fig. 1). Such was still evident even in diluted aliquots of the strains (fig. 2). Fig. 1. Yeast strains grown in YPD agar with high levels of K+ (100 mM KCl). Different strains were plated, and incubated for 3 days at 28ºC. Columns 1 to 6 correspond to strains PLY232 (wild type), PLY234 (trk1), PLY236 (trk2), PLY238 (tok1) PLY240 (trk1 and trk2) and PLY242 (trk1 and tok1). All strains were able to grow effectively. Fig.2. Little growth of the yeast strains on the left plate after 3 days and obvious growing on the right plate after 6 days. K+ concentration was 100 mM. Series of yeast were diluted and prepared from wild-type and isogenic yeast strains carrying all different combinations of trk1, and tok1, and spotted on the plate of solid SDAP, at pH 5.83, The strains were grown on SDAP with 100 mM K+ and were incubated for 3 and 6 days at 28ºC, dilution series 1 to 3 correspond to strains PLY232 (wild type, WT), PLY234 (trk1) and PLY238 (tok1) respectively, and this image has been taken by scan. The first four rows were not inserted with the pyes2 vector, the middle four rows contained an empty pyes2 vector, while the last four had PM19. Medium level of K+ in media When the cells were grown for three days in media containing 15 mM KCl, it was shown that for strains not containing the PM19 gene, trk1 mutants grew poorest compared to wild type (W) and tok1-mutated strains. In contrast, trk1 mutants inserted with PM19 gene had equal, or even the best growth compared with PM19-containing W and tok1 strains (fig. 3). Comparing to that grown in 100 mM KCl, those grown in lesser KCl had smaller culture size. Looking at the same plate after six days (fig. 4), it was more evident that strains containing the pyes2 vector, either empty or with PM19, grew better than the control strains. Similar to the observation three days earlier, the growth of trk1 mutant strains improved with the insertion of PM19. Fig. 3. Different dilutions of wild type, trk1, and tok1 strains plated on SDAP agar (pH 5.8) supplemented with 15 mM KCl. The plates were incubated for 3 days at 28ºC, and columns 1 to 3 correspond to strains PLY232 (wild type), PLY234 (trk1), and PLY238 (tok1) respectively. The first four rows were not inserted with the pyes2 vector, the middle four rows contained empty pyes2 vector, while the last four had PM19. Fig.4. Wild type, trk1- and tok1 mutants’ growth in 15 mM KCl after six days. On the plate of SDAP agar with 15mM KCl (pH 5.8), strains PLY232 (wild type, WT), PLY234 (trk1), and PLY238 (tok1) were plated. The first four rows were not inserted with the pyes2 vector, the middle four rows contained empty pyes2 vector, while the last four had PM19. Growth in the absence of K+ Noticeably, the absence of K+ evidently decreased the growth of all strains tested. Although population increased as the plate was incubated longer, the strains’ culture sizes were less compared to those grown in the presence of KCl. However, similar to the pattern observed in the plate with 15 mM KCl, trk1 mutants without PM19 grew less compared with the other strains without PM19. Insertion of PM19 allowed trk1 mutants to grow at par with PM19-containing WT and tok1 mutants (fig. 5). Fig.5. Little growth of the yeast strains on the left plate (K+ = 0 mM) after 3 days and grow more on the right plate after 6 days. Series of yeast were diluted and prepared from wild-type and isogenic yeast strains carrying all different combinations of trk1, and tok1, and spotted on the plate of solid SDAP, at pH 5.83, The plates SDAP were incubated for 3 and 6 days at 28ºC, column series 1 to 3 correspond to strains PLY232 (WT), PLY234 (trk1) and PLY238 (tok1) respectively. The first four rows were not inserted with the pyes2 vector, the middle four rows contained empty vector, while the last four had PM19. 8. Discussion A Non-specific transport system at high K+ concentrations Our experiment showed that in 100 mM KCl, various mutations in trk1, trk2 and/or tok1 genes, PLY232 (wild type), PLY234 (trk1 mutant), PLY236 (trk2), PLY238 (tok1) PLY240 (trk1 and trk2) and PLY242 (trk1 and tok1), did not affect the growth of cells. This implies that a K+ transport system/s independent of trk1p, trk2p and tok1 proteins is present in S. cerevisiae. However, this can compensate for the absence of trk1p, trk2p and/or tok1 proteins only in high K+ environment, because, as discussed below, mutation resulted to decreased cell growth. It is thus possible that this system is a general transporter, most likely channeling the molecule most present in the environment. A similar finding was also found by Bertl, et al. (2003), who attributed a compensatory K+ transport to non-specific proteins. TRK1protein is important in low K+ environment In low K+ environment such as 15 and 0 mM KCl, the importance of trk1p became more evident. In such conditions, the trk1 mutant strain grew less than the tok1mutant and WT cells. Since trk1p is important in transporting K+ into the cell despite low concentration in the culture environment (Bertl, et al., 2003), the mutation seemed to result to suppression of trk1p. However, it cannot be determined whether the suppression was total or partial, since transport by other channels such as trk2p and tok1 also contributed to K+ entry. It would have been better if mutants with an isolated K+ transport protein, such as PLY240 (to isolate tok1 protein) and PLY242 (to isolate trk2p) be grown to these conditions to see how much contribution they had on K+ transport and viability in low extracellular concentrations. If these mutants’ growth is similar to that observed from trk1 mutant, total suppression of trk1 is most likely. On the other hand, the tok1 mutant was not affected by low levels of extracellular K+ because it is trk1p that facilitates K+ transport in such conditions. The role of PM19 Interestingly, insertion of PM19 into WT, trk1 mutants and tok1 mutants improved the growth of trk1 mutants in 0 and 15 mM KCl, but not of the other two strains grown. Because PM19 protein was previously described as a transmembrane protein, it is most probable that the presence of PM19 protein provided an alternate high-affinity protein that able to transport K+ into the cell even in low extracellular concentrations. In this regard, PM19 seem to have a similar function to that of trk1p. However, it is also possible that PM19 is not a specific K+ channel, but instead a general ion or cation transporter. Relation among ABA, PM19, K+, and plant development Because of the relation of PM19 to ABA and plant development, it is now important to incorporate K+ into the picture. Before proceeding, it is important to discuss first stages of plant development. The three main stages of flowering plants are ovule, ripe seed containing the embryo and seedling. In between these are processes initiated and driven by distinct hormones and proteins. Fertilization of the ovule results to the development of the embryo in the seed. Embryogenesis is the period at which this new life develops to maturity. While this mature seed waits for the moment when it will be dispersed and exposed to favorable conditions (such as adequate moisture, favourable temperature and presence of light) in order to grow on its own, it decreases metabolic activity, causing depletion of water content, thereby storing food sources. At this stage, the seed is said to be dormant (FAO, n. d.). In contrast, when all the needed resources are present, germination proceeds. In this stage, structures will emerge from the seed to allow growth on its own. This is due to the absorption of water, and subsequent swelling of the seed and splitting of the seed coat. The radicle, or the primary root, emerges and grows down the soil. Following this process is the emergence of hypocotyl, cotyledons and plumule. It must be noted, however, that there is limited time for dormant seeds to still be viable for germination. After that duration has elapsed, the seed cannot anymore become a seedling (FAO, n. d.). ABA is a plant hormone that induces and maintains state of dormancy, as well as inhibits the transition from embryonic growth to germination. Antagonistic to its effects is another plant hormone, giberrelin (GA). In a sense, ABA controls the plant’s water content because it is also implicated in stomatal pores closure to restrict transpiration. Aside from these, adjustment of metabolism to tolerate desiccation and cold temperatures, and inhibition seedlings growth are functions of ABA (Rodriguez-Gacio, Matilla-Vasquez, & Matilla, 2009). It is thus possible that PM19 expression depends on environmental to which the plant is exposed to. The increased levels of PM19 in the membrane allow the plant cell to thrive despite the unfavorable levels of water and likely K+. In dormant stages attained in response to water stress, ABA is released. Due to the presence of this plant hormone, PM19 is expressed Koike, Takezawa, Arakawa and Yoshida (1997), resulting to, as demonstrated in this study, assimilation of K+, even in low external concentrations. The relationship of stage of plant development, water stress, ABA, PM19 expression, and environmental levels of K+ is summarized in table 5. Table 5. Summary of the relationships between stage of plant development, water stress, ABA, PM19 expression, and external concentration of K+. Stage of plant development Water stress external concentration of K+ ABA PM19 expression Early embryonic growth Low Low Low Low Mid-embryonic growth High High High Late embryonic growth High High High Maturity High High High Dormancy High High High High Germination Low Low Low Low Future research prospects Another study that can stem from this research in terms of PM19 should be the analysis of the encoding gene and its protein structure. The gene’s homology to genes of other organisms known to encode K+ channels should provide more evidence as to K+ transporting function of PM19. Homology adds more proof to PM19’s function as K+ channel. However, greater evidence should come from the study of its protein structure. Its primary, secondary, tertiary and quaternary protein structures should approximate that of other well-studied K+ proteins, such as trk1p. One of the ways this can be studied is mass spectrometry. Further characterization of PM19’s function may be done as well. Since there is a possibility that PM19 may be a general instead of a specific K+ channel, the growth of PM19-inserted yeast cells with mutated transport proteins in different levels of various ion concentrations should be investigated. For example, insertion of PM19 into yeast cells with mutated Na+ channels should be grown in increasing Na+ extracellular concentrations. Because trk1 works better than the other S. cerevisiae K+ transport proteins at 1 mM KCl, it is likely that the contribution of PM19 to the total intracellular K+ transport is dependent on the concentration of K+ outside the cell, such that the proportion of PM19-transported K+ as compared to the total increases with decreasing levels of extracellular K+. Similarly, the effects of other environmental factors, such as other ions and pH, to trk1p, trk2p and PM19 may also be studied. 9. Conclusion The current project was designed to determine the effect of the plant PM19 transmembrane protein on growth of yeast strains with mutations in specific potassium transporter genes during growth on different media. After incubation of plates, it was observed that trk1p is important in K+ transport and ultimately cellular growth of yeast cells in low K+ concentrations. The ability of PM19 to maintain the growth of cells to optimum despite the absence of trk1p implied that the plant protein also acts as a high-affinity K+ channel. This breakthrough finding of PM19’s function aids in the understanding of the differences in PM19 expression based on the stage of development the seed is in. Mediating this relationship is the hormone ABA. When water is scarce, ABA is released, resulting to increased PM19 expression. It is thus suggested that the increased availability of PM19, with its high-affinity K+ channeling activity, is a compensatory action to allow the cell to imbibe K+ despite existing in suboptimal conditions. Indeed, much more should be studied in PM19, in terms of the extent of its activity and protein structure. Many studies can indeed be developed based on this research. References . BERTL, A., RAMOS, J., LUDWIG, J., LICHTENBERG-FRATE, H., REID, J., BIHLER, H., CALERO, F., MARTINEZ, P. & LJUNGDAH, P. O. 2003. 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