UNIVERSIDAD POLITÉCNICA DE MADRID ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS DEPARTAMENTO DE BIOTECNOLOGÍA MOLECULAR BASIS OF SALT TOLERANCE IN Physcomitrella patens MODEL PLANT: POTASSIUM HOMEOSTASIS AND PHYSIOLOGICAL ROLES OF CHX TRANSPORTERS. Director: Alonso Rodríguez Navarro Profesor Emérito, E.T.S.I. Agrónomos Universidad Politécnica de Madrid TESIS DOCTORAL SHADY ABDEL MOTTALEB MADRID, 2013
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UNIVERSIDAD POLITÉCNICA DE MADRID
ESCUELA TÉCNICA SUPERIOR DE
INGENIEROS AGRÓNOMOS
DEPARTAMENTO DE BIOTECNOLOGÍA
MOLECULAR BASIS OF SALT TOLERANCE IN Physcomitrella patens MODEL PLANT: POTASSIUM
HOMEOSTASIS AND PHYSIOLOGICAL ROLES OF CHX TRANSPORTERS.
Director:
Alonso Rodríguez Navarro
Profesor Emérito, E.T.S.I. Agrónomos
Universidad Politécnica de Madrid
TESIS DOCTORAL
SHADY ABDEL MOTTALEB
MADRID, 2013
MOLECULAR BASIS OF SALT TOLERANCE IN Physcomitrella patens MODEL PLANT: POTASSIUM
HOMEOSTASIS AND PHYSIOLOGICAL ROLES OF CHX TRANSPORTERS.
Memoria presentada por SHADY ABDEL MOTTALEB para la obtención del grado de Doctor por la Universidad Politécnica de
Madrid
Fdo. Shady Abdel Mottaleb VºBº Director de Tesis:
Fdo. Dr. Alonso Rodríguez-Navarro Profesor emérito Departamento de Biotecnología ETSIA- Universidad Politécnica de Madrid
Madrid, Septiembre 2013
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To my family
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ACKNOWLEDGEMENTS
This work would not have been possible without the collaboration and
inspiration of many people. First of all, I would like to express my deep appreciation to
my supervisor and mentor Alonso Rodríguez Navarro for giving me this once-in-a-
lifetime opportunity to both studying what I like most and developing my professional
career. For integrating me in his research group, introducing me to novel research topics
and for his constant support at both personal and professional level. I owe a debt of
gratitude to Rosario Haro for her unconditional help, ongoing support and care during
my thesis. For teaching me to pay attention to the details of everything and for her
immense efforts in supervising all the experiments of this thesis. I also feel grateful to
Begoña Benito for her constant help and encouragement, for her useful advice and for
always being available to answer my questions at anytime with enthusiasm and a
pleasant smile. I would like to thank a lot Blanca Garciadeblás for her constant
encouragement and help in many important experiments of this thesis… and for bearing
my constant flow of silly (and not so silly) questions during the past four years! I would
also like to thank Mª Antonia Bañuelos for her useful advices and for our interesting
conversations.
I wish to express my appreciation to my colleagues in the research group. Their
help, comments and unique expertise helped me to be accurate, thorough, and balanced
throughout the course of this work. Thank you for the great help and for all the good
laughs we shared together during the coffee breaks! Ana Claudia Ureta for her help
and funny conversations. Marcel Veldhuizen for his help and advice on many useful
shortcuts for experimental protocols! Angela Sáez for her support and understanding.
Rocío Álvarez for her dedicated help in many experiments, her constant encouragement
and for our many funny conversations. A special thanks to my dear friend Ana Fraile
for teaching me her experience on the different experimental techniques I used in this
work, for her constant help, advices and constructive critics. For being patient with my
many questions and for all the after-work activities and trips shared together!
Thanks to our neighbor “Rhizobium lab.” members, professors Tomás Argüeso,
Pepe Palacios, Juan Imperial, Luis Rey and Belén Brito… and thanks to Bea,
Carmen, David, Laura, Marta, Mónica, Rosabel for their collaboration and good
times shared during coffee and lunch breaks. A special thanks to my good friend and
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“bus partner” Anabel for all the good times, laughs and conversations we had during
our coffee and lunch breaks, in the bus, trips and everywhere!
I deeply appreciate the technical help and dedication of Dr. Pablo Melendi in
the confocal microscopy experiments.
My gratitude is extended to all my friends in the “Plant virus lab.” Antolín,
Jean-Michel, Manuel & Manuel, Nils and Pablo, and a special thanks to my “lunch
partner” Miguel Ángel for all the good laughs and for teaching me so many aspects of
the Spanish culture, typical foods, slang language, sayings and many more!
I would also like to thank a lot many people that I was lucky to know in the
Centro de Biotecnología y genómica de plantas (CBGP), and had the opportunity to
share with them many nice moments: Alessandro, Amir, Angela, Anja, Bahia,
ATPase Protein that transports a substrate driven by catalysis of ATP Amp Ampicillin AP Arginine phosphate medium ATP Adenosine triphosphate BLAST Basic local alignment search tool Bp Base pair cDNA Complementary DNA CHX Cation H+ exchanger CPA Cation proton antiporter family CTAB Cetyltrimethylammonium bromide C-terminus Carboxyl terminus d Days DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid EST Expressed sequence tag FAO Food and Agriculture Organization g Grams GFP Green fluorescent protein h Hours HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Hyg Hygromicin KEA K+ efflux antiporter KFM K+ free medium L Liter Km “Michaelis constant”: Substrate concentration at which the reaction rate is
half of Vmax LB Lysogeny Broth medium M Molar MES 2-(N-morpholino)ethanesulfonic acid min Minutes MM Minimal medium for bacteria mRNA Messenger RNA N-terminus Amino terminus OD Optical density ORF Open Reading frame PCR Polymerase chain reaction PEG Polyethylene glycol pH Negative logarithmic value of H+ concentration PM Plasma membrane PRM-B Protoplast regeneration medium for the bottom layer medium PRM-L Liquid protoplast regeneration medium PRM-T Protoplast regeneration medium for the top layer medium RNA Ribonucleic acid RNase Ribonuclease ROS Reactive oxygen species r.p.m Revolutions per minute RT-PCR Reverse transcription PCR
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SD Synthetic defined minimal medium SDS Sodium dodecyl sulfate sec Seconds TAE Tris-acetic acid-EDTA buffer TAPS N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid TBE Tris-boric acid-EDTA buffer TE Tris-EDTA buffer TGN Trans-Golgi network Tris Tris(hydroxymethyl)aminomethane Vmax Maximum rate achieved by the system at saturating substrate
concentrations w/v Weight per volume YFP Yellow fluorescent protein YNB Yeast-Nitrogen-Base YPD Yeast-extract Peptone Dextrose medium Zeo Zeocin
1. INTRODUCTION
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1. INTRODUCTION Soil salinity is a major abiotic stress causing serious problems in agriculture. It is
estimated that more than 800 million ha of land throughout the world are salt affected
(FAO 2008) of which, 32 million ha are dryland agriculture farms and 45 million ha are
irrigated lands (Munns and Tester 2008). Among the many types of salt-affected soils,
those with high sodium chloride (NaCl) levels are the most common. NaCl disrupts
metabolic processes in plants and hinders growth through four different mechanisms:
osmotic stress, inhibition of potassium (K+) uptake, toxicity to cytosolic enzymes, and
oxidative stress and cell death. Halophytes are adapted to high NaCl environments, but
this adaptation does not apply to glycophytes among which most crop plants are
included, and as a consequence crop productivity is greatly affected by NaCl (Flowers
2004). Moreover, the contamination and shortage of freshwater, progressive land
salinization and exponential increase of human population only aggravates the problem
implying that world food security is not ensured for the next generations. Thus, a
strategic and an unavoidable goal would be increasing salinity tolerance of plant crops to
secure future food supply (Flowers and Flowers 2005).
Salinity tolerance in plants is a complex polygenic trait difficult to explain in
simple biochemical or biological processes. It is predominately under control of additive
effects with large environment x genotype interaction effects where an evident trade-off
between yield and salinity tolerance is observed. Salinity tolerance in glycophytes
depends upon plant morphology, ions compartmentalization and fluxes, production of
compatible solutes, regulation of transpiration, control of ion movement and cytoplasmic
tolerance (Flowers 2004).
A special and important trait in salinity tolerance is the plants’ ability to maintain a
high cytosolic K+/Na+ ratio. Evidence shows that among the effects of high Na+
concentration in the soil, the reduction of K+ uptake as a result of the direct competition
between Na+ and K+ for uptake sites at the plasma membrane is one of the most
aggressive effects. Furthermore, high Na+ levels induce plasma membrane (PM)
depolarization, which in turn has two negative consequences on K+/Na+ ratio. First, at
the normal K+ concentration in the soil solution, PM depolarization makes passive K+
uptake through inward-rectifying K+ channels impossible in thermodynamic terms and
Fig. 6. CHX complete sequence alignment. Sequence alignment was performed using clustalW2
(http://www.ebi.ac.uk/Tools/msa/clustalw2/). Highlighted in grey and marked with M indicates
transmembrane region for PpCHX1-4 which were calculated from the TMHMM tool (CBS
prediction server, http://www.cbs.dtu.dk/services/TMHMM/) and for the AtCHX18 and AtCHX20
as indicated by Chanroj et al. 2011. In red, residues conserved in all CHX from Arabidopsis
including the Aspartic in the MVI for the cation binding site. Highlighted in black, the sequence
from the putative intron of PpCHX2.1.
Additional searches on Phytozome database also revealed that the similarity
between PpCHX1 and PpCHX2 is relatively high (76.7%), while much lower when
comparing PpCHX1 with either PpCHX3 (47.9%) or PpCHX4 (51.2%). PpCHX3 and
PpCHX4, on the other hand, are much more similar to each other (68.9%). A
subsequent BLASTP search in the NCBI databases (http://www.ncbi.nlm.nih.gov) using
PpCHX1 protein as a query sequence revealed that, among the sequenced plant species
in the database, the proteins with highest identity to PpCHX1 (58-60%) were the three
CHX proteins of the non-vascular club moss Selaginella moellendorffii (SmoCHX1-3),
followed by a 54% identity of the higher-plant black cottonwood Populus trichocarpa
PtrCHX17 (POPTR_0001s10170.1). This was also the case for BLASTP searches using
PpCHX2-4 as a query. Regarding the percentage of identity to CHX proteins of
Arabidopsis thaliana, the highest identity was found to occur with several “clade IV”
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CHX proteins (Sze et al. 2004), where AtCHX18 (AT5G41610.2) and AtCHX19
(AT3G17630.1) proteins showed 53% identity, while AtCHX17 (AT4G23700.1) and
AtCHX20 (AT3G53720.1) showed 52% and 51% of identity, respectively. This
similarity was indeed confirmed by the clustering of PpCHX1-4 together with clade IV
CHX proteins of A. thaliana by phylogenetic analysis of their sequences together with
the 28 full-length CHX proteins sequences from A. thaliana (Fig. 7).
Fig. 7. Phylogenetic tree of CHX transporters of Physcomitrella patens and Arabidopsis thaliana.
It is also worth noting that in contrast to the relatively constant number of the
CPA2 family KEA genes among early non-vascular and flowering plants (e.g. 7 PpKEA
genes in P. patens and 6 AtKEA in A. thaliana), CHX gene orthologs had seemed to
multiply during the evolution of early plants into flowering plants (e.g. 4 PpCHX genes
in P. patens and 28 AtCHX in A. thaliana) as shown in figure 8.
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Fig. 8. Phylogenetic tree of CPA2 family showing both CHX and KEA transporters in A)
Physcomitrella patens and, B) Arabidopsis thaliana.
4.2. PpCHX1 and PpCHX2 gene expression and cDNAs cloning To investigate whether the four genes were functional in normal conditions, we
performed a search in the EST database of the NCBI website
(http://www.ncbi.nlm.nih.go/, accessed in June, 2012) using the sequences of the four
CHX genes of P. patens. The search retrieved 27 ESTs for PpCHX1 and 54 for
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PpCHX2, but none for either PpCHX3 or PpCHX4. As mentioned earlier, the sequences
of PpCHX3 and PpCHX4 genes are inexistent in NCBI databases until this very
moment. This perplexing fact motivated us to investigate if PpCHX3 and PpCHX4
genes really do exist in the genome of P. patens plant material manipulated in our
laboratory, and thus, we first carried out a PCR using genomic DNA as a template
extracted from plants grown in normal conditions. DNA fragments were amplified and
detected for both genes by PCR, confirming their existence in the genome (Fig. 9).
Next, we investigated the existence of transcripts of these two genes by RT-PCR,
finding that in normal conditions of pH (from 5 to 7), or K+ and Na+ concentrations
(from 0.1 to 10 mM) the expression of the PpCHX3 or PpCHX4 genes was not
detectable. In view of these results we concentrated our study in the PpCHX1 and
PpCHX2 genes.
Fig. 9. PCR confirmation for existence of PpCHX3 and PpCHX3 genes in the P. patens genome.
Figure shows a UV irradiated agarose electrophoresis gel showing wells A and B with bands of PCR
amplified sequences of PpCHX3 and PpCHX4 genes, respectively, run in parallel to gene ladder
marker (100 bp). Wells C and D show primer controls (Table 11).
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To accurately characterize PpCHX1 and PpCHX2 expression, we monitored
both genes’ expression using a quantitative real time PCR (qRT-PCR) method. The
qRT-PCR study of the transcript expressions of PpCHX1 and PpCHX2 revealed that
both genes were expressed almost to the level of the ACT5 gene of P. patens. In
standard growth conditions, PpCHX1 showed a four-fold higher expression than
PpCHX2. The expression of PpCHX1 was constant in most growth conditions except
for a three-times reduction at pH 4.5. In contrast, the expression of PpCHX2 was lower
in standard conditions and slightly increased when the plants were stressed (Table 18).
These results suggest that both PpCHX1 and PpCHX2 are housekeeping genes whose
expressions are required in all growing conditions. Although the transcript levels
showed variability, the detected variations seem too low to have any biological
relevance.
Table 18. Real-time PCR determination of transcript expression of CHX1 and CHX2 genes of P.
patens at different pH values in the presence and absence of Na+ or K+.
Conditions PpCHX1 PpCHX2
pH 5.8 0.84 0.19
pH 5.8 - K+ 1.00 0.59
pH 5.8 + 100 mM Na+ 0.94 0.73
pH 4.0 0.34 0.62
pH 9.0 0.98 0.42
Next, we went on with cloning PpCHX1 and PpCHX2 cDNAs from plants
growing in standard conditions. As described in materials and methods chapter, we
sequenced the whole cloned cDNA (including both the 5’ and 3’ ends) to verify the
correct cDNAs sequence of both genes. The first observation we noticed is that the open
reading frame (ORF) of PpCHX1 cDNA we cloned did not correspond with the
published PpCHX1 CDS sequence (estExt_fgenesh1_pm.C_1230004) in the early JGI
database (http://genome.jgi-psf.org/Phypa1_1/Phypa1_1.home.html, accessed in
November, 2009); we detected in our cloned cDNA that the real start codon was 27 bp
upstream the annotated one and that the real Stop codon was present in an exon
Reported values are the ratio between the transporter transcript abundance and actin transcript abundance. Data represent results of two independent experiments with two replicates each.
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incorrectly excluded by the database algorithm. These two annotation errors were
corrected later on in the new phytozome database sequence of the gene transcript
(Pp1s123_30V6.1) (http://www.phytozome.net, accessed on 27/09/2012).
When cloning the PpCHX2 cDNA, we also noticed that the ORF of PpCHX2
cDNA sequence cloned did not match the published CDS sequence either
(estExt_gwp_gw1.C_1620028, JGI database); the annotated database CDS sequence
lacked 87 bp, from 1989 to 2076, that were excluded by the database algorithm.
4.3. Alternative PpCHX2 proteins The amino acid alignment of the abovementioned conceptual translation of the
PpCHX2 gene (estExt_gwp_gw1.C_1620028 in JGI database also known as
PHYPADRAFT_191426 in NCBI database) revealed that the translated polypeptide
lacked 29 residues in the C tail with reference to other conceptual translations. The
missing residues, from 656 to 694, seemed to correspond to a putative intron that might
be incorrectly eliminated by some conceptual translations. Consistent with this notion,
we found that the corresponding mRNA fragment was present in all ESTs in the
database. Furthermore, by RT-PCR, we could not detect the presence of PpCHX2
mRNAs in which this putative intron had been processed, neither in normal conditions
of pH (from 5 to 7) and K+/Na+ concentrations (from 0.1 to 10 mM) nor under high Na+
or K+ limited conditions. However, the alternative splicing of this intron could not be
absolutely excluded because some CHX transporters showed a gap coinciding with this
29-residue fragment that interrupts a region where CHX transporters keep high
sequence homology (Fig. 10). Therefore an alternative splicing in PpCHX2 at the point
described above was kept in mind throughout this study.
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Fig. 10. Alignment of alternative conceptual translations of the PpCHX2 gene with several mRNA
translations. Sequence alignment was performed using Clustal Omega
(http://www.ebi.ac.uk/Tools/msa/clustalo/). Sequences used in the alignment are PpCHX2.1
FG570725.1 of Brassica napus, EX095781.1 of Brassica rapa, Y911015.1 of Helianthus annus,
FN025549.1 of Petunia x hibrida and FS069311.1 of Solanum melongena.
We, thus, cloned the PpCHX2 cDNA that corresponds to the actual CDS
sequence in Phytozome database (Pp1s162_22V6.1) and, for the reasons discussed
above, we also constructed in vitro via a 2 step PCR as described in materials and
methods, the shorter PpCHX2.1 clone, deleting the 87 bases of the putative intron in the
PpCHX2 gene (Fig. 3 in Materials and Methods chapter).
4.4. Subcellular localization of PpCHX1-GFP, PpCHX2-GFP and
PpCHX2.1-GFP protein fusions in yeast cells and Physcomitrella patens
protoplasts To localize subcellularly the proteins of our study, we constructed expression
vectors with the three CHXs tagged at the carboxyl end to green fluorescent protein
(GFP) under the expression control of P35S promoter in the case constructs to be
expressed in yeast. For P. patens constructs, GFP was under the expression control of
the PpACT5 gene promoter, which shows an expression level similar to those of
PpCHX1 and PpCHX2 genes (Table 18), as described in materials and methods chapter.
First, we expressed the three constructs in the yeast Saccharomyces cerevisiae
mutant strain W∆6 (Haro and Rodríguez-Navarro 2003), defective in its main influx
systems of K+ TRK1 and TRK2. All three constructs failed to show a clear GFP signal
of localization confined to a specific membrane of the cell. The GFP signal appears,
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instead, to be present in all the cell; plasma membrane, cytoplasm and other internal
membranes (Fig. 11).
Fig. 11. Subcellular localization of A) PpCHX1-GFP, B) PpCHX2-GFP, and C) PpCHX2.1-GFP
fusion proteins in Saccharomyces cerevisiae yeast mutant strain W∆6.
Afterwards, we expressed the corresponding constructs in P. patens protoplasts.
In these experiments, PpCHX1-GFP showed a punctuate pattern that was compatible
with the Golgi network (Fig. 12A). Although the details of the images varied depending
on the observed protoplast, the pattern showed in figure 12A was observed in all
protoplasts showing the GFP signal. We confirmed the Golgi network localization
afterwards as the GFP signal colocalized with the GmMan1-YFP fluorescent marker of
the Golgi complex (Fig. 12B-C).
The GFP signal of PpCHX2-GFP showed a double localization, to the cell
periphery, enclosing the chloroplasts, and to internal round structures (Fig. 12D and
12G). The peripheral signal was characteristic of the plasma membrane while the
internal GFP signal appeared to correspond to the vacuolar membrane. To confirm the
latter localization we co-expressed either PpCHX2-GFP or PpCHX2.1-GFP and the γ-
TIP-YFP marker of tonoplast. These co-expressions showed that the GFP internal round
structures and YFP signals co-localized, demonstrating the tonoplast localization of
PpCHX2 (Fig. 12D-F) and PpCHX2.1 (Fig. 12G-I). Unfortunately, we could not co-
localize PpCHX2 with a positive control of plasma membrane. Therefore, the temporal
or circumstantial localization of PpCHX2 to the plasma membrane is currently only a
possibility, which does not have physiological support (see below).
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Fig. 12. Localization of PpCHX1–GFP and PpCHX2–GFP fusion proteins in protoplasts of P.
patens. Images of the green fluorescence of PpCHX1–GFP (A), PpCHX2–GFP (D) and PpCHX2.1–
GFP (G). Images of the yellow fluorescence of the GmMan1–YFP marker of the Golgi complex (B)
and the γ-TIP–YFP marker of the tonoplast (E, H). Merged images of the GFP, YFP marker
fluorescence and chloroplast fluorescence are in red (C, F, I). Images show the maximum projection
of 11 consecutive sections (A–C), 25 consecutive sections (D–F) and nine consecutive sections (G–
I). Frequency of the green fluorescence patterns: (A) pattern appeared in all protoplasts that showed
fluorescence with small differences; (D) pattern, most fluorescent protoplast showed peripheral and
internal labelings, some protoplasts showed only internal labeling and very few only peripheral
labeling; (G) pattern appeared in all protoplasts that showed fluorescence, with small differences.
4.5. Growth rescue of Escherichia coli mutants Before carrying out the functional characterization of the CHX transporters in
planta, we needed to have an idea of their mechanism of function by expressing them in
simple heterologous systems such as Escherichia coli bacterium and Saccharomyces
cerevisiae yeast. E. coli is by far the most widely used expression host for the testing
and production of recombinant proteins. Its short generation time, low cost and ease of
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use, as well as its extensive characterization make it an ideal candidate (Sahdev et al.
2008). Another extremely helpful characteristic of E. coli, and bacteria in general, is
that it has no subcellular membranes, and thus, any protein (transporter) expressed in it
will be inserted in its plasma membrane regardless of it being natively expressed in
endomembranes, which consequently facilitates the characterization of such protein
(Uozumi 2001).
Knowing beforehand that CHX proteins characterized to date in Arabidopsis
thaliana are implicated in K+ transport, we chose the TKW4205 mutant of E. coli
defective in its K+ transport systems Kdp, TrkA, and Kup (Schleyer and Bakker 1993)
which requires a high K+ concentration to grow at pH 5.5. This defect can be highly
reduced by heterologous K+ transporters in an expression level-dependent manner (Senn
et al. 2001). To test whether the PpCHX proteins transported K+, we cloned the
PpCHX1, PpCHX2, and PpCHX2.1 cDNAs into pBAD24 plasmid (Guzman et al. 1995)
under the control of the arabinose-responsive PBAD promoter. PpCHX1 partially
rescued the growth of TKW4205 at 10 μM arabinose (not shown in Figure 13) but when
increasing the arabinose concentration to 100 μM the growth of TKW4205 was
excellent at 5 mM (Fig. 13). As shown with other K+ transporters (Senn et al. 2001), K+
became toxic to E. coli when the expression level of PpCHX1 was increased by
increasing the arabinose concentration up to 13 mM (Figure 13 shows the growth
inhibition at 13 mM arabinose, 5 mM and higher concentrations of K+). However, when
K+ was decreased to 2 mM, the PpCHX1 clone grew fairly well at 13 mM arabinose but
not at 100 μM arabinose.
PpCHX2 and PpCHX2.1 partially rescued the growth of TKW4205 at 100 μM
arabinose (Fig. 13). When increasing the arabinose concentration to 13 mM, the growth
of PpCHX2 and PpCHX2.1 was excellent at 5 mM K+, slightly positive at 2 mM K+
(Fig. 13), and was inhibited at 50 mM K+.
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Fig. 13. Growth complementation tests of E. Coli mutant strain TKW4205 at low K+ pH 5.5 by
empty vector pBAD24 or PpCHX1, PpCHX2 and PpCHX2.1 constructs. A colony of fresh
transformed cells was picked and grown for approximately 3 hours in LB media supplemented 50
mM KCl. Cultures were brought to a uniform cell density and 3-fold serial dilutions were placed on
pH 5.5 MM media at 100 μM or 13 mM arabinose and varying concentrations of KCl of 2, 5, 10, 20
or 50 mM. Photos were taken after 4 d of growth at 37 ºC.
When carrying the same experiments on solid medium at pH 7.5, the empty
vector pBAD24 clone started growing at very low K+ concentrations (lower than 5 mM).
This prevented us to notice the suppression of the defective growth of the mutant in
PpCHX1, PpCHX2 or PpCHX2.1 clones compared to empty vector. However, we
noticed that only PpCHX1 caused toxicity at 100 µM arabinose concentration, while at
13 mM arabinose concentration, all CHX cDNAs caused toxicity (Fig. 14).
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Fig. 14. Toxicity of E. Coli mutant strain TKW4205 with empty vector pBAD24 or PpCHX1,
PpCHX2 and PpCHX2.1 constructs. A colony of fresh transformed cells was picked and grown for
approximately 3 hours in LB media supplemented 50 mM KCl. Cultures were brought to a uniform
cell density and 3-fold serial dilutions were placed on pH 7.5 MM media at 100 μM or 13 mM
arabinose and varying concentrations of KCl of 2, 5, 10, 20 or 50 mM. Photos were taken after 4 d of
growth at 37 ºC.
In some kinetic studies it is necessary to measure zero-trans influxes, which
cannot be performed with K+. In these studies, Rb+ might substitute for K+ if it is
transported. Therefore, for future experiments, it was convenient to know whether
PpCHX1 and PpCHX2 transported Rb+. This possibility can be tested in growth
experiments because Rb+ can substitute for cellular K+ in a large proportion without any
toxic effects. Therefore, we repeated the growth experiments with TKW4205 described
above, but using Rb+ in this case instead of K+. The results showed that both PpCHX1
and PpCHX2 rescued the growth of TKW4205 at Rb+ concentrations that were only
slightly higher than those found for K+; 10 mM Rb+ instead of 5 mM K+, at 100 μM
arabinose for PpCHX1 and 13 mM arabinose for PpCHX2 (Fig. 15). These results
demonstrated that both PpCHX1 and PpCHX2 are K+ transporters that show a notable
capacity to transport Rb+.
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Fig. 15. Growth complementation tests of E. Coli mutant strain TKW4205 at low Rb+ pH5.5 with
empty vector pBAD24 or PpCHX1, PpCHX2 and PpCHX2.1 constructs. A colony of fresh
transformed cells was picked and grown for approximately 3 hours in LB media supplemented with
50 mM KCl. Cultures were brought to a uniform cell density and 2-fold serial dilutions were placed
on pH 5.5 media at 100 μM or 13 mM arabinose and varying concentrations of RbCl of 10, 20 or 50
mM. Photos were taken after 4 d of growth at 37 ºC.
4.6. PpCHX1 and PpCHX2 complement the kha1 mutation in yeast Yeast, in particular Saccharomyces cerevisiae, share many characteristics of
E.coli bacterium that makes it a great model organism for biological and genetic studies
including the heterologous expression of many proteins; completely sequenced genome,
short life cycle, and easy genetic transformation and manipulation in the laboratory. S.
cerevisiae, however, has an additional advantage being an eukaryote and, thus,
phylogenetically less distant to plants than the prokaryote E. coli.
Several yeast mutants have been extensively used to functionally characterize
plant transporters (Dreyer et al. 1999). For example, the heterologous expression of
plant transporters in the S. cerevisiae yeast mutant W∆6 (Mat a ade2 ura3 trp1
trk1∆::LEU2 trk2∆HIS3), defective in the TRK1 and TRK2 K+ uptake systems (Haro
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and Rodríguez-Navarro 2003), is routinely performed to test K+ and Na+ influxes by the
expressed transporters. However, these mutants can usually be complemented only with
plasma membrane transporters and not by transporters of internal membranes. As
previously described in this chapter, the subcellular localization of our three cloned
CHX transporters using GFP in yeast cells did not show a clear localization to a specific
cellular membrane, being instead present in various locations including the plasma
membrane (Fig. 11). This result, together with PpCHX2p being located to the plasma
membrane in P. patens protoplasts (Fig. 12), left the window open to a possibility,
although minimal, that any of these transporters might promote K+ or Na+ influx in the
W∆6 mutant. First we tested whether PpCHX1, PpCHX2 or PpCHX2.1 suppressed the
defect of this mutant by promoting high affinity K+ uptake, and we found that none of
these transporters succeeded in suppressing the defective growth of the mutant at low
K+ concentrations when carrying out yeast drop-test experiments (Fig. 16). Also, their
implication in high affinity Na+ transport in the W∆6 mutant turned to be null, as K+-
starved yeast clones of PpCHX1, PpCHX2 and PpCHX2.1 failed to deplete micromolar
concentrations of Na+ from liquid AP media during the course of time.
Fig. 16. Tests of functional expression of PpCHX1, PpCHX2 and PpCHX2.1 in yeast mutant W∆6. Cells transformed with empty pYPGE15 vector or PpCHX1, PpCHX2 and PpCHX2.1 cDNA
constructs were grown overnight in YPD growth medium supplemented with 50 mM KCl. In the
following day, cultures were brought to a uniform cell density of 0.3 and 3-fold serial dilutions were
placed on (SD + Adenine and Tryptophan) media at pH 6.5 and varying concentrations of KCl of
100 μM, 1 mM, or 5 mM. Photos were taken after 4 d of growth at 28 ºC.
As previously reported, PpCHX1, PpCHX2 and PpCHX2.1 showed the ability
to transport Rb+. Thus, using Rb+ as an analogue of K+, we wanted to test these
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transporters ability to mediate low affinity K+ transport in the millimolar range in the
W∆6 mutant. Again, none of the three transporters promoted a faster millimolar Rb+
influx in the clones compared to the empty vector pYPGE15, being the low affinity Rb+
influx rate almost identical in all the clones (Fig. 17). This was also the case for Na+ low
affinity influx experiments.
Fig. 17. Time course of Rb+ influx measuring its internal content (nmol/mg) in S. cerevisiae strain
squares; and ∆Pphak1 ∆Ppchx2 plants, closed triangles.
Another possibility tested was whether PpCHX2 promotes low affinity Rb+
uptake. Thus, we followed the kinetic of influx at a wide range of Rb+ concentrations, 3,
5 and 10 mM, measuring the internal Rb+ concentrations in both wild-type and ∆Ppchx2
plants over the course of several hours (Fig. 27). No significant differences were
observed between both plants in low affinity Rb+ uptake.
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Fig. 27. Time course of Rb+ influx measuring internal content (nmol/mg) in P. patens plants.
Symbols: wild-type plants, open circles; ∆Ppchx2 plants, closed circles. Plants grown in normal
conditions for a week and influx tests were started by the addition of 10 mM RbCl, taking samples
during the course of 5 hours.
The tests described above were aimed to detect defective K+ or Rb+ influxes, but
the PpCHX2 transporter might mediate K+ efflux. To test this possibility, we grew wild-
type and ∆Ppchx2 plants in normal BCDNH4 medium for one week and then the plants
were thoroughly rinsed and transferred to KFM media for another week. When we
measured the decrease of K+ contents in wild-type and ∆Ppchx2 plants through the
course of several days in KFM media, we found no significant differences between both
of them.
Next, to investigate other possible functions of PpCHX2, we tested for
alterations in K+/Rb+ exchanges. For this purpose, we substituted 10 mM Rb+ for the K+
content of the culture medium. First we grew the plants in normal BCDNH4 medium for
one week, then the plants were rinsed and transferred to KFM medium with 10 mM Rb+
for a second week. During the course of the second week, we followed the external K+
concentration and the internal Rb+/K+. When plants were grown at 10 mM Rb+, the re-
uptake of the K+ lost by the plants is inhibited, and consequently a defective K+ efflux
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should result in a slower increase of external K+ of the medium in the case if ∆Ppchx2
was defective in efflux. Once again, however, we did not find differences that reveal a
defective K+ efflux. In contrast, when we recorded the Rb+/K+ ratio we found that the
ratio was slightly higher in ∆Ppchx2 plants. In general, the variability was lower in
experiments carried out with the same batch of plants –e.g. a time course experiment
(Fig. 28)– than in experiments with independent batches of plants (Fig. 29). Although
the differences were small, the statistical analyses of independent five-days exchange
experiments (Fig. 29) revealed that the Rb+/K+ ratio in ∆Ppchx2 plants was significantly
higher than in wild type plants (2.24 ± 0.46 versus 1.75 ± 0.21; n = 7; t-test, p = 0.024).
Fig. 28. Rb+/K+ exchange in wild-type and ∆Ppchx2 plants. Plants grown at 10 mM K+ for one week
then transferred to K+-free medium with 10 mM Rb+. Internal contents of Rb+ and K+ were measured
and the Rb+/K+ ratio calculated. Data points represent a time course experiment of the Rb+/K+
content; symbols: wild-type plants, open circles; ∆Ppchx2 plants, closed circles.
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Fig. 29. Means of Rb+/K+ ratios in seven independent experiments measured at the fifth day for
wild-type plants (closed bars) and ∆Ppchx2 plants (open bars). Standard errors are shown and means
are significantly different (P = 0.024) according to t-test.
In the described experiments, the K+ loss to the external medium was
determined with high precision and no differences were found between ∆Ppchx2 and
wild type plants and; on the other hand, we found that Rb+ uptake was not affected in
∆Ppchx2 plants. Thus, the defect underlying the higher Rb+/K+ ratio in ∆Ppchx2 plants
seems to be a higher retention of Rb+ in the steady state–influx versus efflux–that
determines the Rb+ content. This may result from a slower transfer of Rb+ from either
the vacuole to the cytosol, resulting in a higher vacuolar Rb+ content, or from the
cytosol to the external medium, resulting in a higher cytosolic Rb+ content.
Unfortunately, it is not currently possible to distinguish between these two possibilities
by measuring Rb+ contents.
5. Discussion
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5. DISCUSSION
5.1. Analysis in silico and cloning of CHX transporters of
Physcomitrella patens This study investigates the function of CHX transporters in P. patens and is the
first of its kind to investigate members of the CPA2 family in early non-vascular plants.
This family, comprised of CHX and KEA transporters, is the less studied family of
cation-proton antiporters in plants (Sze et al. 2004, Aranda-Sicilia et al. 2012). In the
case of CHX transporters, scarce information is known, as most functional studies on
CHX transporters are relatively recent. These are so far limited to Arabidopsis thaliana
(Chanroj et al. 2012) as well as some superficial studies on Oryza sativa rice, (Sze et al.
2004, Senadheera et al. 2009).
It is suggested from comparative genomics studies that CHX transporters have
evolved from fresh water green algae charophytes rather than from marine green algae
chlorophytes (Chanroj et al. 2012). This proposal is supported by the fact that CHX
homologs are absent in some chlorophytes, like Chlamydomonas reinhardtii or Volvox
carteli, but present in the charophyte Spirogyra pratensis (one CHX homolog). Whether
the ancestral CHX gene was horizontally transferred or not to charopytes from the
bacterial GerN or cyanobacterial NhaS4 highly similar homolog genes, probably
through endosymbiosis, is a mystery and cannot be asserted for sure. However,
comparative genomics studies between fresh water green algae (charophytes), early
non-vascular land plants (bryophytes) and flowering plants suggest that CHX genes
have suffered several events of duplication along the course of evolution from fresh
water green algae into higher plants. The first event of duplication of the ancestral plant
CHX gene probably occurred in early non-vascular plants (e.g. 3 CHXs in Selaginella
moellendorffii and 4 in Physcomitrella patens). Interestingly, subsequent duplications of
CHX genes in flowering plants raise many questions on their specialized functional
importance to flowering plants. As mentioned in the past chapter, in contrast to the
relatively constant number of the CPA2 family KEA genes among early non-vascular
and flowering plants, CHX gene orthologs have continued to multiply during the
evolution of flowering plants. For example, dicotyledonous plants have roughly double
the number of CHX genes in monocotyledonous plants (e.g. 15 CHX genes in Zea mays
and 28 in A. thaliana). Nitrogen fixating plants (legumes) have also around double the
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number of CHX genes than non-nitrogen fixating plants (e.g. Glycine max have 46
predicted CHX genes).
The abovementioned apparent duplication events of the CHX genes, in contrast
to their closely related CPA2 gene family members KEA, might suggest that CHX genes
have played crucial roles for adaptation during the evolution from marine algae to
freshwater algae, and then to land plants, as well as many specific cell-type functions in
flowering plants. This specific cell-type functions are evident, for example, in A.
thaliana, where its 28 CHX transporters are expressed either specifically or
preferentially in pollen grains (Sze et al. 2004). In fact when aligning CHX members of
A. thaliana together with the 18 members of rice and drawing a phylogenetic tree, it was
found that all rice CHX members grouped only to clades I, IV and V, with no rice CHX
transporter orthologues grouping to clades II or III (Sze et al. 2004). It is worth noting
that, 10 out of 15 AtCHX members of clades II and III are specifically expressed in
pollen, while the 5 remaining are preferentially expressed in pollen. This suggests that
the AtCHX members of clades II and III play a specific and important role in A.
thaliana pollen, one not present in pollen of monocot plants like rice. This possible role,
and the significance of this great number of specialized pollen transporters, however,
remains to be clarified knowing that only clade IV AtCHX21 and AtCHX23
transporters seem to have a significant effect on the pollen tube guidance process to the
ovule (Lu et al. 2011), while none from clades II and III have been discovered with this
function until this moment.
Besides the important functions of A. thaliana CHX transporters in pollen,
further results on other CHX transporters lead to the establishment of an emerging
model suggesting that these transporters play important roles in the cellular homeostasis
of K+. For example, AtCHX20 transporter was found to be expressed and functional in
guard cells (Cellier et al. 2004) while AtCHX17 and AtCHX13 are functional in roots
(Padmanaban et al. 2007, Zhao et al. 2008). Regarding the subcellular localization of
CHXs, although AtCHX13 (Zhao et al. 2008) and AtCHX21 (Hall et al. 2006) are
apparently functional in the plasma membrane, most CHX transporters appears to reside
in internal membranes such as prevacuolar, endoplasmic reticulum, Golgi or other
nonspecific endomembranes (Padmanaban et al. 2007, Chanroj et al. 2011, Lu et al.
2011).
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Concerning the phylogenetic relationship between P. patens and A. thaliana
CHX transporters, PpCHX1-4 transporters group to the clade IV (Fig. 7) of the
Arabidopsis thaliana CHX phylogenetic tree (Sze et al. 2004, Chanroj et al. 2012). As
expected, none of the four P. patens CHX transporters grouped to clades II and III, the
two clades comprising AtCHXs specific to A. thaliana pollen, as was the case of rice
mentioned earlier. Nevertheless, clade IV has two AtCHX members that are
“specifically” expressed in pollen, AtCHX15 and AtCHX23 (Sze et al. 2004). These,
however, are phylogenetically located at a further distance from other AtCHXs in this
cluster, as well as PpCHX1-4. It might be speculated that the presence of AtCHX15 and
AtCHX23 in clade IV suggests that both are conserved CHXs that might have served as
a basis for duplication of other members during the evolution of flowering plants (e.g.
members of clades II and III). It is also worth noting that the remaining AtCHX
members in clade IV are also expressed in pollen. However, in this case they are not
specific to pollen and are expressed in other structures including roots and leaves
(AtCHX16, 17, 18, 19, 20, and 21). In general, the relatively close phylogenetic
distance between PpCHX1-4 and AtCHX15-23 in clade IV may lead us to suspect that
these AtCHXs have been relatively conserved during evolution, given their higher
similarity to the more “primitive” CHXs of P. patens.
Another fact worth noting is that PpCHX3 and PpCHX4 are phylogenetically
more distant to PpCHX1 and PpCHX2, suggesting that they might play different roles
in P. patens. Given that we did not detect any expression of PpCHX3 and PpCHX4
genes in gametophytes, it might be suspected that these could be expressed in
sporophytes, and thus, play a possible role there. The PpCHX3 and PpCHX4 in
subclade IV are phylogentically close to AtCHX20, a K+ transporter expressed in guard
cells (Padmanaban et al. 2007), which makes the possibility that PpCHX3 and PpCHX4
play a role in P. patens guard cells a reasonable hypothesis for further investigation. It is
well documented, however, that P. patens gametophyte phase cells (protonema and
gametophores) lack guard cells which are, nevertheless, present in sporophytes (Knight
et al. 2009). In all cases, this hypothesis is rather difficult to test. On one hand, the
current RNA and DNA extraction protocols in P. patens are adapted for gametophyte
tissue (especially protonema), and when applied to sporophyte tissues
(sporangiophores), the DNA yield is very low and the RNA yield is almost non-existent.
Thus, assessing gene expression of PpCHX3 and PpCHX4 is a complicated task. If this
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shortcoming is overcome soon, future works on PpCHX3 and PpCHX4 are mandatory.
On the other hand, the sporophyte phase is too minute to work with and no current
protocols are developed to test any possible defective K+ uptake or any other abnormal
transport in P. patens mutants.
Although pollen and guard cells do not exist in P. patens protonema, the basic
cellular functions mediated by the CHX transporters, that are primary altered in these
mutants (e.g. membrane trafficking events that affect protein and cargo sorting; Chanroj
et al. 2012), are probably very similar in Arabidopsis and P. patens. Also, our results on
the localization of PpCHX1 to the Golgi complex and PpCHX2 to plasma membrane
and tonoplast seem consistent with the Arabidopsis model mentioned earlier. The
double localization of a transporter is not uncommon and was previously reported in the
HAK transporter TRH1 showing a double localization in the plasma membrane and
tonoplast (Rigas et al. 2012). Interestingly, PpCHX2.1, the shorter version of PpCHX2,
did not localize to the plasma membrane. When analyzing the PpCHX2 protein
sequence with various algorithms, we detected the existence of a low complexity region
(LCR) in the residues that we excluded (HSESLGLSKLHHSSL) to construct the
PpCHX2.1 version using the SEG algorithm program
(ftp://ftp.ncbi.nih.gov/pub/seg/seg/) (Wootton and Federhen 1996). These low
complexity sequences are sequences with overrepresentation of a few residues that
strongly indicate disorder in the protein. Such disordered regions have been shown to be
involved in a variety of functions, including DNA recognition, modulation of
specificity/affinity of protein binding, molecular threading, activation by cleavage, and
control of protein lifetimes (Dunker et al.1997). The shorter PpCHX2.1 version not
localizing to the plasma membrane like PpCHX2, thus, might suggest that the missing
residues with reference to PpCHX2 have localization determinants. Currently it cannot
be predicted whether this finding reflects a physiological process, e.g. alternative
splicing, or if it is a fortuitous experimental response. Alternative splicing in
transporters is well documented in animal cells, where it is found that it can change not
only the cellular localization of the transporter, but its transport properties as well
(Lazaridis et al. 2000). In plants, although less studied, is not uncommon either
(Takahashi et al. 2007, Mao et al. 2008, Cotsaftis et al. 2012). In any case, the missing
residues in PpCHX2.1 did not affect the functional expression of the protein in either E.
coli or yeast mutants.
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5.2. Functional analysis of PpCHXs in heterlogous systems of
expression
5.2.1. Escherichia coli To deepen our knowledge regarding the transport functions of PpCHX1 and
PpCHX2, we started by their functional study in simple heterologous systems of
expression; K+ uptake deficient mutants of Escherichia coli bacterium and
Saccharomyces cerevisiae yeast. K+ uptake deficient E. coli TKW4205 mutant has been
successfully used for expressing and studying the function of different plant transporters
such as HAK (Senn, 2001), NHAD (Barrero Gil et al. 2007), SOS1 (Garciadeblás et al.
2007), and recently CHX transporters (Chanroj et al. 2011). Overall, our study revealed
that the functional expression of PpCHX1 and PpCHX2 in the E. coli TKW4205 mutant
(Fig. 13) was similar to previous findings with AtCHX17, AtCHX20 (Chanroj et al.
2011), and AtCHX23 (Chanroj et al. 2011, Lu et al. 2011). All these transporters
improved substantially the capacity of TKW4205 to grow at low K+, which
demonstrates that all these proteins mediate K+ uptake.
According to these growth experiments PpCHX1 and PpCHX2 seem to play
identical functions in E. coli, however, they show different specific activities. This is
suggested by the different concentrations of arabinose that are required to obtain similar
responses with the two cDNAs, which implies different expression levels of the
arabinose-responsive PBAD promoter . For example, for rapid growth on 5 mM K+, the
PpCHX1 clone required 100 μM arabinose, while PpCHX2 clone required 13 mM
arabinose, which represents more than tenfold different transcript expression levels
(Guzman et al. 1995). On the other hand, a concentration of 13 mM arabinose was toxic
for the PpCHX1 clone at 5 mM and higher K+ concentrations, but suitable for PpCHX2
clone. Also, PpCHX1 clone growth at 2 mM K+ occurred at 13 mM but not at 100 μM
arabinose. This suggests that 100 μM arabinose concentration was not sufficient to
induce the expression of the adequate number of PpCHX1 transporters required to fulfill
the K+ necessities of PpCHX1 clone to grow at 2 mM K+. By the same reasoning, it is
highly likely that the slower growth of PpCHX2 and PpCHX2.1 clones at 2 mM K+
compared to PpCHX1 (Fig. 13) can be explained because of the low the number of
transporters, i.e. the low Vmax of the system. Overall, the slower growths, thus, can be
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explained by the slower rates of K+ uptake, which depends on the number of
transporters, K+ concentration, and K+ influx kinetics, i.e. Km and specific activity of the
system. These observations are important regarding the mechanism of transport
discussed below because it strongly suggests that the growth at low K+ is limited by the
kinetics and not by the thermodynamics of the system.
To understand the physiological function of CHX transporters, the study of their
functional mechanism is of crucial importance. For this issue, the mechanism
underlying the CHX mediated K+ uptake in E. coli constitutes the most basic
information. The first question to answer is whether or not they mediate “active” K+
uptake. In our experiments (Fig. 13) this uptake could be mediated by either a “passive”
K+ uniporter or by an “active” K+-H+ symporter. The possibility of an electroneutral
K+/H+ antiporter is improbable as it would mediate K+ efflux (where no growth would
be observed). To address this question, the simplest way to distinguish between the
uniport and symport mechanisms is to calculate whether the value of the membrane
potential can explain the internal/external ratio of K+ concentrations in growing cells of
E. coli. To calculate this ratio, the external K+ concentration is 2 mM, because the
PpCHX1 clone was found to grow at this concentration. In a medium without Na+, the
internal concentration of K+ in growing cells of E. coli is 211 mM (Schultz and
Solomon 1961). This concentration might be reduced at K+ limited growth rates, but not
very much, considering that the K+ content of chemostat cultures of Enterobacter
aerogenes are not greatly reduced even in the presence of NH+4 and Na+ (Tempest and
Strange 1966). According to these data, thus, an internal K+ concentration lower than
150 mM is absolutely unlikely. Consequently, this corresponds to a minimal
internal/external ratio of 75:
To get this ratio by “passive” K+ uptake, it would require a membrane potential
of -112 mV. However, E. coli cannot attain that membrane potential at pH 5.5. The
membrane potential in E. coli has been extensively studied (Padan et al. 1976,
Zilberstein et al. 1979, Felle et al. 1980, Bakker and Mangerich 1981, Kashket 1982,
Setty et al. 1983). According to these studies a likely value of the membrane potential of
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E. coli at pH 5.5 is between -90 and -95 mV. The most negative value is given in the
study of Felle et al. (1980) by microelectrode recording, reporting a measured value of -
80 mV that is corrected to -100 mV after calculating the effect of the current leakage
through the membrane seal. Accordingly, given that a membrane potential value of -112
mV cannot be attained at pH 5.5 in E. coli cells, and consequently neither the value of
75 internal/external K+ ratio, it can be concluded by these simple calculations that it is
highly unlikely that either PpCHX1 or PpCHX2 mediate a “passive” K+ uptake. Also, to
interpret these calculations, it is worth noting that a positive growth is an unequivocal
proof of fulfilling an energetic requirement, while a negative growth might be the
consequence of an influx that is too slow to support a detectable growth rate. We
discussed above that the growth of the PpCHX1 and PpCHX2 clones of TKW4205 at
low K+ depended on the arabinose concentration, which points out that growth is limited
by the kinetics (i.e. amount of transporters) rather than by the thermodynamics (i.e.
energetic of transport) of the system.
These abovementioned thermochemical calculations do not prove but strongly
suggest that PpCHX1 and PpCHX2 mediate an “active” K+ uptake. Therefore, assuming
this conclusion, the most likely mechanism of action is a K+-H+ symport in E. coli
(Rodriguez-Navarro et al. 1986). Driven by this mechanism of transport, one K+ ion can
move against its electrochemical gradient with the concomitant movement of one
proton, which may move up or down its electrochemical gradient. This same conclusion
can be applied to AtCHX20 and AtCHX17 based on similar growth tests of E. coli
transformants reported previously by Chanroj et al. (2011). These coincidences between
Arabidopsis and P. patens CHXs give strong support to the notion that a K+-H+ symport
mechanism might apply to many, if not all, CHXs. This mechanism is of crucial
important to develop a comprehensive functional model of these transporters that
explains the results obtained with the yeast mutants.
In contrast, it is worth noting that if the mechanism of the CHX transporters
were K+ uniport they would be equivalent to K+ channels (without voltage gating),
through which K+ moves but not H+. Using a different approach, the same issue has
been previously discussed by Chanroj et al. (2011) for the Arabidopsis transporters
AtCHX17 and AtCHX20. This approach was not followed in present study because of
its methodological uncertainties. First, the authors tried to extract mechanistic
conclusions from differences in transport rates at pH 7.2 and 6.2, which is a difficult
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task to achieve. Instead, these differences can be explained on kinetic basis in most
cases. For example, the toxicity of PpCHX1 and PpCHX2 at pH 7.5 (Fig. 14) cautions
about the use of neutral pHs for K+ uptake experiments in the TKW4205 strain. The
observed toxicity at pH 7.5 can be explained by a high K+ influx which, together with
the intrinsic influx of the mutant cells and the influx mediated by the CHX transporter,
cannot be controlled by the bacterial cells and consequently causing toxicity in them.
This is similar to the toxic effect discussed earlier produced at pH 5.5 and 13 mM
arabinose, at 50 mM K+ for PpCHX2 and 5 mM K+ and higher concentrations for
PpCHX1 (Fig. 13). At pH 5.5 the interpretation of the results is simpler because at this
pH the intrinsic K+ influx in the bacterial cells is negligible; for that reason the mutant
does not grow without the CHX transporters. In contrast, at pH 7.5, the intrinsic K+
influx is much higher, sufficient to support a rapid growth at 5 mM K+ and lower
concentrations (not shown, but see the excellent growth at 5 mM K+ in Fig. 14), and
exceeding the influx that can be mediated by the expression of the CHX transporters at
any K+ concentration. The second uncertainty in Chanroj et al. (2011) approach is that
the kinetic analysis of Rb+ influx performed is unlikely to provide reliable mechanistic
information. Specifically, these analyses are performed using the initial rates of Rb+
uptake, which means that they are performed when the internal concentration of Rb+ is
very low and the chemical Rb+ gradient is huge. The rate tests under these conditions,
including those performed with uncouplers, will reveal kinetic responses of the
transporters but very little of their thermodynamical dependence.
In summary, although K+-H+ symport is currently the most likely functional
mechanism of CHX transporters as proposed above, a definitive demonstration is still
pending. Most likely this demonstration will require the use of membrane vesicles
obtained from the plant membranes where the transporters are expressed. Unfortunately,
both the preparation these vesicles and the tests for K+-H+ symport in plant
endomembrane vesicles present many technical difficulties, which makes the use of
indirect approaches inevitable in the current studies on the functional mechanism of
CHX transporters.
5.2.2. Saccharomyces cerevisiae So far, much of the knowledge generated on the function of different plant
transporters has been acquired by heterologous expression in Saccharomyces cerevisiae
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yeast mutants defective in K+ and/or Na+ transport systems (Hasegawa et al. 2000,
Serrano and Rodriguez -Navarro, 2001). Our aforementioned obtained results using the
E. Coli mutant defective in K+ uptake, indicate that the CHX cDNAs used in the present
study are implicated in K+ transport, and thus, further elucidation on their transport
characteristics was required before proceeding with their study in planta. To achieve
this, we used distinct defective S. cerevisiae mutants in their K+ and/or Na+ transport
systems. We used the trk1 trk2 mutant (W∆6) defective in its K+ uptake systems (Haro
and Rodríguez-Navarro 2003) to test any possible K+ and Na+ influxes promoted by our
cloned CHX cDNAs. Also, we used the ena1-4 nha1 mutant (B3.1) defective in its K+
and/or Na+ efflux systems to test if they promote K+ and/or Na+ efflux under high K+ or
Na+ conditions (Bañuelos et al. 1998). Finally, any possible roles in regulating internal
K+ and pH homeostasis were studied using ena1-4 nha1 kha1 mutant (LMB01)
(Maresova and Sychrova 2005).
Although the GFP localization of PpCHX2p in W∆6 yeast mutant was present in
the plasma membrane (Fig. 11), it wasn’t exclusively confined to it as in the case of P.
Patens protoplasts (Fig. 12). Thus, we wanted to test the possibility of any existing
activity of the PpCHX2 transporter in the plasma membrane. The trk1 trk2 W∆6 mutant
has been routinely used in the past years to characterize several high affinity K+ or Na+
transporters such as HAK (Garciadeblás et al. 2007, Benito et al. 2012). Our results
showed no complementation of any of the P. Patens CHX transporters used in our study
either in high or low affinity K+ or Na+ uptake. It is worth noting that W∆6 mutant has
the inconvenient that it is only successfully complemented with plasma membrane plant
transporters, as described in many previous works. This was also clearly shown in the
CHX family where the plasma membrane AtCHX13 transporter successfully suppressed
the defective growth of trk1 trk2 mutant (Zhao et al. 2008) while the endomembrane
transporter AtCHX17 did not (Cellier et al. 2004, Maresova and Sychrova 2006). Many
explanations, although non-conclusive, can be considered to explain why PpCHX2 is
inactive at the plasma membrane. These might include a faulty protein processing,
missing components required for the transporter activation, or retention of the majority
of the protein in internal organelles such as the endoplasmic reticulum. Based on our
results, it is risky to draw any conclusions regarding these possibilities. Another
disadvantage when using the W∆6 mutant, is that it is unlikely that a low affinity
transporter would suppress its defect, as it maintains a very rapid intrinsic low affinity
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K+ uptake (Santa-María et al. 1997). This could explain why we were unable to detect
any differences in the low affinity range either (Fig. 17). However, we cannot negate for
sure the possibility that these transporters could mediate low affinity K+ transport.
It was reported in previous works that the plasma membrane transporter
AtCHX21, in addition to its important role in pollen K+ homeostasis (Lu et al. 2011),
may be directly or indirectly involved in Na+ transport in planta (Hall et al. 2006). To
test whether any of the PpCHXs of our study are involved in Na+ or K+ efflux, we
expressed these in the Na+ and K+ efflux defective mutant (ena1-4 nha1) B3.1, finding
no complementation (Fig. 18). This makes the implication of the PpCHXs in either Na+
or K+ efflux, at least in yeast, highly unlikely. However, once again, these results are not
conclusive and should be taken with care due to the lack of a clear plasma membrane
localization in any of these transporters.
On the other hand, both PpCHX1 and PpCHX2 suppressed the defect of the
kha1 mutation in S. cerevisiae mutant LMB01 (Fig. 19), which coincides with
previously reported results for several Arabidopsis CHX transporters (Maresova and
Sychrova 2006, Padmanaban et al. 2007, Chanroj et al. 2011). Kha1 is a K+ transporter
residing in the Golgi apparatus membrane of S. cerevisiae. Although a single KHA1
gene mutation does not result in an evident phenotype, nevertheless, when additional
mutations in ENA1-4 and NHA1 genes are added to kha1 mutant, an evident growth
defect in media with high pH and low K+ is seen (Maresova and Sychrova 2005).
Evidence suggests, thus, that K+ is the most preferred substrate for Kha1 tranporter, as
the inability of the kha1 mutant to grow at higher pH can be suppressed by the addition
of moderate concentrations of K+. The role of Kha1 is believed to be the regulation of
intraorganellar K+ and pH homeostasis (Ariño et al. 2010). Also, it is worth mentioning
that substrate specificity of PpCHX1 and KHA1 seem to be similar, in contrast to when
comparing KHA1 with PpCHX2 or PpCHX2.1. When predicted in terms of the
observed phenotype of yeast cells growing in the presence of Na+ (Fig. 21); PpCHX1
seems to transport Na+ similar to KHA1 (Maresova and Sychrova 2005, Ariño et al.
2010).
Assuming that the KHA1 gene of S. cerevisiae encodes a K+/H+ antiporter, a
tentative interpretation of our results obtained with the heterologous expressions (Fig.
13 and 19) would be that CHX transporters mediate K+-H+ symport in E. coli and K+/H+
antiport in S. cerevisiae. However, an alternative explanation would be that K+-H+
133
symporters and K+/H+ antiporters fulfill similar functions in endosomal compartments
and that these two mechanisms show a certain degree of functional redundancy and
reciprocal substitution. This means that a K+-H+ symporter could substitute for KHA1,
even if KHA1 mediates K+/H+ antiport (see models of action below). This reasoning
would not only apply to CHX transporters but also to PpHAK2, which also suppresses
the kha1 mutation of S. cerevisiae (Haro et al. 2013). The basis of this notion is that
these two mechanisms participate in the pH control of organelles.
5.3. Proposed models of action for PpCHXs NHX, HAK, and CHX transporters are probably present in the membrane of
most organelles (see below in section 5.4). Therefore, the possibility that the functional
mechanisms associated to these transporters might be either K+-H+ symport or K+/H+
antiport raises the question about how these two mechanisms can participate in the pH
control of the organellar lumen. In the most likely model the organelle pH is established
by the steady state that results from H+ pumping into the organelle and return to the
cytosol, in parallel with K+ and Cl- conductances (Demaurex 2002, Paroutis et al. 2004,
Casey et al. 2010, Ohgaki et al. 2011). H+ pumping and a parallel influx of anions
would decrease the organelle pH to very low values but the effective control of the pH
requires the return of H+ to the cytosol. H+ pumping is mediated by the electrogenic V-
ATPase while the return of H+ can be mediated by multiple systems: H+ passive leaks
and fluxes coupled to Cl- or K+ fluxes. In plant cells a specific type of pyrophosphatase
might cooperate with the H+ pump V-ATPase (Segami et al. 2010). The question raised
above refers to K+ coupling and can be addressed with a simple model including the
pump, the coupled H+ efflux, and a K+ channel, if necessary (Fig. 30). Assuming that
the membrane potential drives all the other movements, a simple calculation shows that
both a K+/H+ antiporters and a K+-H+ symporter are similarly effective to return H+ from
the organelle lumen to the cytosol, assuming the existence of a K+ channel for K+
recirculation. If the equilibrium is reached, which is the limit of the gradient that can
generate the system, the organelle pH could be higher than the cytosolic pH (ΔpH
would be 1 for a ΔΨ of -60 mV). It is worth noting that the three couplings: antiport
plus channel, symport plus channel, and antiport plus symport would be similarly
effective to return H+ to the cytosol (Fig. 30) but they would be associated to different
K+ contents.
134
Fig. 30. Alternative models of action for PpCHXs. Coupling of: a K+-H+ symporter with a K+
channel (a), a K+/H+ antiporter with a K+ channel (b), and a K+-H+ symporter with a K+/H+ antiporter
(c). The equations for the systems in equilibrium are used to calculate the ∆pH that the system can
attain. ∆Ψ denotes the membrane potential, negative in the cytosolic side; for calculations, 2.3 RT/F
= 60 mV.
5.4. Functional analysis of PpCHXs in Physcomitrella patens To analyze the function of PpCHX1 and PpCHX2 in plant cells, we disrupted
these genes and also constructed the ∆Pphak1 ∆Ppchx2 double mutant. PpCHX1
localized to the Golgi complex and ∆Ppchx1 plants showed no growth defects when
growth was tested in a large variety of conditions of pH, and K+, Na+, and Ca2+
concentrations. This suggests that the function of PpCHX1 may be redundant with other
transporters. As already discussed, the mechanism of the redundant transporters might
be either K+-H+ symport or K+/H+ antiport. PpHAK3 also localizes to Golgi (Haro et al.
2013), which makes it a candidate substitute of PpCHX1. Most likely there are also
other candidates. In Arabidopsis, for example, NHX5 and NHX6 are associated with
135
Golgi and TGN (Bassil et al. 2011) and two NHX transporters in P. patens show high
sequence homology with AtNHX5 and AtNHX6 (Chanroj et al. 2012). The Golgi
complex is an important metabolically active organelle performing important functions
in the cell including modifying, sorting, and packaging protein and lipid
macromolecules for cell secretion or use within the cell. A strict and precise K+ and pH
homeostasis is crucial for the functioning of this organelle, thus, it might be possible
that CHX, HAK, and NHX transporters are working in parallel in the Golgi complex
performing similar functions. Consequently, due to this possible functional redundancy,
it was expected beforehand that single ∆Ppchx1 knockout mutant plants of P. patens
would hardly display obvious growth phenotypes even under stress conditions.
In the case of ∆Ppchx2 plants, no defects were found that connect CHX2 to K+
(or Rb+) uptake and the same conclusion applies to ∆Pphak1 ∆Ppchx2 plants as well
(Fig. 26). Furthermore, the involvement of PpCHX2 in K+ uptake through the plasma
membrane can be ruled out considering the phenotype of ∆Ppchx2 plants regarding its
morphological appearance under normal or stress conditions, in addition to the simple
Rb+ influx tests we carried out (Fig. 26 and 27). On the other hand, when ∆Ppchx2
plants were exposed to Rb+ they showed an increased Rb+/K+ ratio but the same K+ loss,
which implies a higher cellular retention of Rb+. Considering that Rb+ influx was not
increased by the mutation, the increased Rb+ content of ∆Ppchx2 plants must be the
consequence of either a slower Rb+ efflux through the plasma membrane or a slower
vacuole-to-cytosol Rb+ transfer, which results in a higher vacuolar Rb+ retention in both
cases. Because we did not detect differences in K+ contents, and the differences in Rb+
contents between ∆Ppchx2 and wild-type plants were small, the most likely hypothesis
is that PpCHX2 mediates K+ and Rb+ movements in parallel with other transporters that
exhibit a higher K+/Rb+ discrimination either in the plasma membrane or in the
tonoplast.
Because PpCHX2-GFP localized to the tonoplast and plasma membrane, either
or both of the functions proposed above for PpCHX2 are possible. The functional
difference between these two is mechanistic because Rb+ efflux (K+ efflux in
physiological conditions) across the plasma membrane must be Rb+/H+ antiport while
vacuole-to-cytosol Rb+ transfer (K+ transfer in physiological conditions) must be either
Rb+ uniport or Rb+-H+ symport assuming that the vacuole has low pH, high K+ content,
136
and a weak membrane potential that is positive with reference to the cytosol.
Considering these observations and the previous discussion about the mechanism of
CHX transporters, the most likely possibility is that PpCHX2 mediates K+ (or Rb+)-H+
symport in the tonoplast. If this hypothesis is true, it is highly probable that PpCHX2
contributes to the cell K+ homeostasis by supplying the cytosol with K+, if it were
functional in tonoplast. In all cases, it is surprising to detect an evident effect by
knocking-out one single vacuolar transporter gene, bearing in mind the redundancy of
protein function in plants as well as the enormous adaptive plasticity of plant responses
(Maathius 2010). Indeed, other transporters (like members of HAK/KUP/KT family) or
channels might fully substitute PpCHX2 for K+ transport, however in this case, only
partially for Rb+ transport.
Unfortunately, taking into account the large volume of the vacuole, the technical
tools to distinguish from a slightly higher Rb+ content in the cytosol or in the vacuole
between mutant and wild type plants are practically inexistent. Therefore, at the current
level of knowledge, a precise conclusion cannot be reached and it is doubtful that it can
be reached from biochemical experiments. Most likely, the identification of the
functions of both PpCHX1 and PpCHX2 will come from a genetic approach, by
constructing double or triple mutants that show clear defects. Nevertheless, functional
redundancy is a common phenomenon in transporters that most probably will make
these future studies a difficult task. This phenomenon was clearly demonstrated in the
case of CPA2 family members AtCHX21 and AtCHX23, where no apparent phenotype
is present in their single mutants but highly evident in double mutants, although both
localizing to different membranes (Lu et al. 2011). This also appears to be the case in
KEA transporters of Arabidopsis, where kea1 kea2 double mutant shows a clear
phenotype in contrast to kea1 or kea2 single mutants (Kees Venema, Personal
communication). Functional redundancy in other transporter families is not uncommon
either, as in the case of NHX1 and NHX2 (Barragán et al. 2012). It is unlikely, thus, that
P. patens double mutant ∆Ppchx1 ∆Ppchx2 might show a clear phenotype, given that
PpCHX3 and PpCHX4 might also be playing a role in such redundancy. Moreover, the
problem of this approach is that the number of “non-CHX” transporters that can mediate
K+/H+ exchange and K+/H+ exchange or K+-H+ symport is high, and the possibility of
obtaining triple or quadruple mutants may not be always possible. As discussed above,
the presence of HAK and NHX transporters in endomembranes might substitute
137
PpCHX functions, but also if PpCHX2 were functional in plasma membrane as a K+/H+
antiporter, it might be replaced by the PpENA1 K+ ATPase in Physcomitrella (Fraile-
Escanciano et al. 2009). Thus, this complicates even more the detection of any abnormal
phenotype, even when multiple mutations are achieved. In addition to the previous
discussion about NHX transporters in the Golgi complex, P. patens have five NHX
transporters showing high sequence homology to the NHX1-4 transporters of
Arabidopsis (Chanroj et al. 2012), which play vital K+/H+ exchanges in vacuoles
(Barragan et al. 2012, Chanroj et al. 2012).
All in all, it is apparent that K+ homeostasis in plants is extremely complex and
several transporter families are involved in the pH homeostasis of organelles by
mediating either K+/H+ antiport or K+–H+ symport. Although these systems might not
be essentially redundant considering their functional conditions, they might replace each
other in mutant plants.
138
6- CONCLUSIONS
141
6. Conclusions 1. The CHX family of transporters has suffered numerous events of duplication
and diversification during the course of evolution of non-vascular plants into
flowering plants. Physcomitrella patens has four CHX genes; two of them are
expressed at normal levels (PpCHX1-2) while the remaining two show very low
levels of expression (PpCHX3-4).
2. Despite the phylogenetic distance between Arabidopsis and P. patens, the
functional basis of CHX transporters in both species seems to be very similar.
As in the case of other Arabidopsis CHX transporters, PpCHX1 localizes to the
Golgi complex while PpCHX2 localizes to vacuolar compartments and plasma
membrane.
3. Both PpCHX1 and PpCHX2 are implicated in K+ transport. They successfully
rescued the defective phenotype of yeast mutants unable to grow at low K+ and
alkaline pH, and also rescued the phenotype of E. coli mutants defective in their
K+ influx transporters.
4. Experiments carried out in E. coli mutants strongly suggest that PpCHX1 and
PpCHX2 mediate “active” K+ uptake, which might be a K+-H+ symport. K+-H+
symporters and K+/H+ antiporters are both effective for controlling the pH of the
organellar lumen.
5. The in planta studies carried out in this thesis suggest the implication of
PpCHX2 in transporting Rb+ from either the vacuole to the cytosol or from the
cytosol to the external medium. Our knowledge on the function of PpCHX1 is
still lacking.
6. Taken together, present and other studies about CHX, HAK, and NHX
transporters in P. patens and Arabidopsis provide compelling evidence about the
existence of a complex series of K+ transporters in plant organelles. Although
these systems might not be essentially redundant considering their functional
conditions, they might replace each other in mutant plants.
7. Future works on the construction of double mutants involving two different
transporter families seems the only way to unravel the complexity of the
individual functions of endomembrane K+ transporters in plant cells.
142
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