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Life, 63(10): 864–872, October 2011
Critical Review
Aluminum Stress and its Role in the Phospholipid Signaling Pathway
in Plants and Possible Biotechnological Applications
Wilberth Poot-Poot and Soledad M. Teresa Hernandez-Sotomayor
Unidad de Bioquı´mica y Biologı´a Molecular de Plantas, Centro de Investigación Cientı´fica de Yucatán, Calle 43, No. 130,
Chuburná de Hidalgo, C.P. 97200 Me´rida, Yucatán, Me´xico, Me´xico
Summary
An early response of plants to environmental signals or abiotic
stress suggests that the phospholipid signaling pathway plays a pivotal role in these mechanisms. The phospholipid signaling cascade
is one of the main systems of cellular transduction and is related to
other signal transduction mechanisms. These other mechanisms
include the generation of second messengers and their interactions
with various proteins, such as ion channels. This phospholipid signaling cascade is activated by changes in the environment, such as
phosphate starvation, water, metals, saline stres, and plant–pathogen interactions. One important factor that impacts agricultural
crops is metal-induced stress. Because aluminum has been considered to be a major toxic factor for agriculture conducted in acidic
soils, many researchers have focused on understanding the mechanisms of aluminum toxicity in plants. We have contributed the last
fifteen years in this field by studying the effects of aluminum on
phospholipid signaling in coffee, one of the Mexico’s primary crops.
We have focused our research on aluminum toxicity mechanisms in
Coffea arabica suspension cells as a model for developing future
contributions to the biotechnological transformation of coffee crops
such that they can be made resistant to aluminum toxicity. We conclude that aluminum is able to not only generate a signal cascade in
plants but also modulate other signal cascades generated by other
types of stress in plants. The aim of this review is to discuss possible
involvement of the phospholipid signaling pathway in the aluminum
toxicity response of plant cells. Ó 2011 IUBMB
IUBMB Life, 63(10): 864–872, 2011
Keywords
aluminum toxicity; phospholipid signaling; Coffea arabica.
INTRODUCTION
There is a great diversity of external signals to which cells
must respond. Independent of the simplicity or complexity of the
Received 9 May 2011; accepted 6 July 2011
Address correspondence to: S. M. Teresa Hernández-Sotomayor;
Calle 43 No. 130 Col, Chuburná de Hidalgo, C.P. 97200, Mérida,
Yucatán, México. Fax: 152 999 9813900. E-mail: [email protected]
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.550
cell type, the biochemical and molecular mechanisms to which
cells need to respond are universal. Throughout different developmental stages, cells are susceptible to a variety of external signals
from both the environment and neighboring cells. Indeed, cells
must be ready to respond to these signals, and they do so through
different types of universal signal transduction mechanisms.
Plant cells are no different from the most complex eukaryotic cells. However, because of their sessile nature, plants must
respond to drastic environmental changes to survive, and they
must adjust their growth and developmental behaviors in
response to daily and seasonal environmental changes in a
timely manner. An intriguing and important question in our
understanding of a plant’s developmental programs and environmental responses involves the types of strategies and mechanisms that plant cells use for the transmission and integration of
various developmental signals.
Phospholipids are a major and vital component of all biological membranes and play a key role in processes, such as signal
transduction, cytoskeletal rearrangement, and membrane trafficking. Genetic studies using Arabidopsis thaliana confirm that
changes in phospholipid homeostasis profoundly affect plant
growth and development. For example, the over-accumulation
of phosphatidylinositol-4,5-bisphosphate (PIP2) and inositol1,4,5-triphosphate (IP3) is characteristic of sac9 mutants, which
show a constitutive stress response (1).
The route of phosphoinositides is one of the most important
in plant signaling, and as such, they are located in all cell membranes; there is evidence to suggest that phosphatidylinositolspecific phospholipase C (PI-PLC) is one of the component in
this pathway involved in stress responses. Furthermore, changes
in phosphoinositide levels have been characterized in a number
of different plant species, and the stimulation of this signaling
pathway is involved in many different plant reactions to environmental factors, such as drought, cold, salinity, and pathogen
attack (2). PIP2 turnover is stimulated by the drought hormone
abscisic acid in the stomata of Vicia faba (3) and by osmotic
stress in A. thaliana cell cultures (4).
ALUMINUM STRESS AND POSSIBLE BIOTECHNOLOGICAL APPLICATIONS
Phospholipid metabolism is also affected by the metal aluminum (Al), with the most important physiological consequence
of Al-toxicity being a cessation of root growth and changes in
root morphology; this suggests that the root cytoskeleton is a
target structure. This article discusses aluminum toxicity in
plants and the impact that this metal has on phospholipid cell
membranes of important agronomical plants in Mexico, namely
Coffea arabica.
Aluminum Toxicity in Plants
Aluminum (Al) is the most abundant metallic constituent in
the crust of the earth; only the elements oxygen and silicon,
which are both nonmetals, are more abundant. Al is never found
as a free metal but is commonly found as aluminum silicate or
as a silicate of aluminum mixed with other metals, such as sodium, potassium, iron, calcium, or magnesium. These silicates
are not useful ores because the process of extracting Al from
them is chemically difficult and expensive. Bauxite, an impure
hydrated aluminum oxide, is the commercial source of Al and
its compounds (5).
Al-toxicity is an important growth-limiting factor for plants
in many acidic soils with a pH below 5.0. However, this toxicity can occur at pH levels as high as 5.5 (6, 7). This problem is
particularly serious in strongly acidic subsoils that are difficult
to lime (8), where the challenge has been intensified by ongoing
heavy applications of acid-forming nitrogenous fertilizers. High
subsoil-acidity (Al-toxicity) reduces plant-rooting depth,
increases susceptibility to drought, and decreases the use of subsoil nutrients (5).
At low soil-pH levels, solubilized Al ions (mainly in the
phytotoxic form of Al) severely inhibit root elongation. Thus,
Al-toxicity is a serious problem that causes decreased plant
growth in acidic soils around the world (9). Indications that Al
interferes with signal transduction pathways in cells have been
observed (10).
Plants showing symptoms of aluminum toxicity are more
sensitive to changes in environmental conditions, and this sensitivity can be caused by aluminum-mediated effects on any number of signal transduction cascades. Al accumulation is localized
primarily at the root apex, suggesting that Al interacts with
actively dividing and expanding cells. Among Al-toxicity symptoms, the main responses include inhibition of root growth and
induction of callose (b-1,3-glucan) synthesis after a short-term
treatment with Al. Both events have been related to oxidative
stress induced by Al treatment (11), but the mechanism of signaling in response to Al remains unclear. The cumulative data
on Al interactions indicate that Al has a significant effect on
different signal transduction pathways in plants, such as phosphoinositide (12) and protein phosphorylation pathways, and
that anion channels may participate in these interactions by
excreting organic acids as an Al-tolerance mechanism.
In most plant species, especially Al-sensitive and crop species, Al uptake is limited mainly to the root system, where it
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accumulates predominantly in the epidermis and the outer cortex (13, 14). However, there are many plant species that accumulate considerable amounts of Al in their shoots (15). These
plants, frequently called hyperaccumulators, are mainly woody
plants from tropical or subtropical regions, such as some species
native to the region of central Brazil. Tea plant (Camellia sinensis), hydrangea and members of the Rubiaceae family are classic examples of hyperaccumulator plants (14).
On the other hand, there is unfortunately not much information in the literature related to the mechanism, cellular localization, or chemical form of the Al that accumulates in these
plants. In tea leaves, most Al is chelated to the catechin group
of polyphenols and, to a lesser extent, to phenolic and organic
acids (16). In hydrangea leaves, Al is found as a complex with
citrate (17), and in the hyperaccumulator plant Melastoma
malabathricum Al is found bound to oxalate.
Likewise, it has been suggested that the rapid inhibition of
root growth by Al treatment indicates more rapid signal transduction processes may be involved in causing this response. In
particular, special attention has been paid to the phosphoinositide-associated transduction pathway because early research
with animal cells indicated that cellular mechanisms of Al-toxicity could involve interactions between Al and components of
the pathway (18).
Yakimova et al. (19) evaluated the effects of aluminum on
signal transduction involving phosphoinositides and their possible relationship with cell death in tomato plants. The results
suggested that low concentrations of heavy metal ions stimulate
both PLC and phospholipase D (PLD) signaling pathways,
which lead to the production of reactive oxygen species (ROS)
and subsequent cell death executed by caspase-like proteases.
Thus, this study demonstrated that the phospholipid signaling
pathway is considered to be one of the important plant-signaling
mechanisms involved in cell death. However, it remains difficult to determine the signaling pathways and where and how
plants accumulate Al to counteract its toxic effects if the mechanism for how cells sense the presence of this metal is
unknown.
Genes Expressed in the Presence of Aluminum
In recent years, considerable evidence has emerged in the literature that Al promotes oxidative stress in plant cells, although
certain conditions are required for this to occur. Whether or not
Al-induced oxidative stress is a primary or secondary effect is
still a matter of debate. However, lipid peroxidation has been
frequently observed as an early symptom. The Al-induced genes
encoding proteins that function to overcome oxidative stress
(e.g., glutathione S-transferase, peroxidase, blue copper-binding
protein, phenylalanine ammonia lyase, 1,3-b-glucanase, and cysteine proteinase) have been previously reported (18, 20). In
addition, it was shown that the expression of these Al-induced
genes in transgenic Arabidopsis plants conferred Al-tolerance
and enhanced oxidative stress (21).
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The first gene controlling Al31 resistance in plants was isolated from wheat (22). The Triticum aestivum aluminum-activated
malate transporter (TaALMT1) gene encodes a member of the aluminium activated malate transporter (ALMT) family that consists of membrane-bound proteins (23). TaALMT1 functions as
an Al31-activated anion channel, releasing malate from root cells
(24). It has also been shown that several other members of the
ALMT family contribute to Al31 resistance in cereal and noncereal species in a similar manner. These discoveries were exciting
at the time because it appeared as though a single gene family
controlled Al31 resistance in a diverse range of species (25).
However, the model soon required revision after major resistance genes in sorghum and barley were mapped and
sequenced. Aluminum resistance in these species relies on citrate efflux, and the proteins involved are not ALMTs but members of a completely different family of proteins called the multidrug and toxic compound extrusion (MATE) family. The
MATE family of transporter proteins is a large and diverse
group present in both prokaryotic and eukaryotic cells. Many of
these proteins appear to function as secondary carriers for the
removal of small organic compounds from the cytosol (26, 27).
Studies on the heterologous expression and homology of the
ALMT1 and MATE genes, in addition to the exploitation of
available information on the physiology and genetics of resistance in other species, have uncovered additional resistance
genes (28). This approach has helped identify candidate resistance genes in Arabidopsis (29), Brassica napus (30), rye (31),
wheat (32), sorghum (33), and maize (34).
Aluminum and Coffee
The Rubiaceae family contains 500 genera, but Coffea is by
far the most commercially significant genus in the family. Over
100 species have been described within Coffea L., and at least
25 major species likely exist, all of which are indigenous to
tropical Africa and certain islands in the Indian Ocean. All Coffea species are woody but range in size from small shrubs to
large trees over 10 m tall. Despite this diversity, only two species, both from Africa, are grown commercially on a large scale
as follows: Coffea arabica L. (Arabica type coffee) and Coffea
canephora Pierre ex Froechner (Canephora or Robusta type coffee) (35, 36). C. arabica is a major crop worldwide and is the
most heavily traded commodity apart from oil, accounting for
4% of the total world food trade. Coffee production has been
negatively affected by factors such as disease and aluminum
toxicity, the mitigation of which will require research into disease susceptibility, photosynthetic efficiency, water utilization,
and tolerance to both soil acidity and Al content.
Coffee is often grown in acidic soils that have high Al31
levels. Al31 is an interchangeable form that can be released
into the soil, making it very accessible to plants (37). Al31 also
produces toxic effects in plants (38–40). Aluminum can be
transferred to plants from the soil, which can modify regulation
of the soil dynamics. Therefore, an understanding of the con-
centrations and dynamics of heavy metals in soils is necessary
to determine the adequate properties and conditions needed for
optimal agricultural working conditions (41, 42).
Suspension Cells as a Model Tool
In vitro cell and tissue cultures from plants may provide an
adequate system in which to perform studies on metal toxicity.
An embryogenic-suspension cell line of Coffea arabica var.
Catuai was obtained from a dispersed callus. The callus was originally obtained from the cotyledonary leaves of zygotic embryos
from seeds cultured in vitro in Murashige–Skoog media at half
the normal ionic strength and at a pH of 4.3. Under these conditions, the ability to grow in the presence of aluminum was diminished (Fig. 1) (43). Using this cell line as a model, we have
focused on searching for the signaling pathway associated with
growth inhibition that results from Al-mediated toxicity.
Al Effect on Phospholipid Signaling
In coffee cells, Al is known to produce growth inhibition at
different concentrations. Al has also been related to different
biochemical processes involving membrane phospholipids as
well as several enzymes, such as PLC and PLD and the second
messenger phosphatidic acid (PA). The breakdown of PIP2 into
IP3 and diacylglycerol (DAG) by the action of PLC plays an
important role in signal transduction pathways (44–47). IP3 and
DAG produced by PIP2 hydrolysis act as second messengers,
triggering the release of Ca21 from internal stores and activating protein kinases, respectively (45). In addition, PIP2 is important for the regulation of cytoskeletal dynamics, vesicle trafficking and ion transport; a change in the phospholipid composition can noticeably affect cell function (46).
However, increasing experimental evidence has suggested
that this pathway is not as simple as was first proposed. For
example, levels of PIP2 are also regulated by different lipid kinases and lipid phosphatases. In addition, PIP2 regulates the activity of several enzymes and participates in the regulation of
membrane trafficking. DAG can be a substrate for DAG kinase
(DGK), in turn generating PA, and IP3 can also be phosphorylated by inositol kinases to generate IP6 (46), among others.
In previous studies focused on changes of lipids in response
to Al-toxicity, Zhang et al. (48) found that when roots were
treated with Al for 1 day, no effects were observed on the general lipid composition. In contrast, they also reported that Al
treatment for 3 days induces modifications in the patterns of
phospholipids, free sterols, fatty acids, and triacylglycerols.
Other reports have shown that Al affects PLC activity (49) and
interactions with enzymatic catalytic metal binding sites, specific membrane lipids, and ion channels (50).
Because of the suggestion that PLC is a main target for Altoxicity, our group has been studying the effect of AlCl3 on the
different components of this pathway, namely PLC and lipid kinases (51), using suspensions of C. arabica cells as a model (43).
Two main effects were seen when cells were treated with AlCl3.
ALUMINUM STRESS AND POSSIBLE BIOTECHNOLOGICAL APPLICATIONS
867
Figure 1. Cell suspensions of C. arabica were grown in Murashige–Skoog media at half the normal ionic strength, subcultured for
14 days, coated with gold particles and then observed under the scanning electron microscope. (A) and (B) control cells, (C) cells
treated with 100 lM of AlCl3 for 30 min and (D) cells treated with 100 lM of AlCl3 for 1 h.
In periods as short as 1 Min, Al-exposed cells displayed up to a
twofold increase in their PLC and IP3 formation activities. Over
longer periods, PLC activity was inhibited by more than 50%. It
is important to note that this is the first report describing PLC
activation by Al exposure. The activity of phosphatidylinositol 4kinase (PI 4-K), phosphatidylinositol phosphate 5-kinase (PIP 5K), and DGK increased when cells were incubated in the presence
of different concentrations of AlCl3 (51). These results strongly
support the theory that Al disrupts the metabolism of membrane
phospholipids, regulating not only PLC, but also other enzymes
that have key roles in signal transduction pathways.
PLD is ubiquitous in plants and hydrolyzes the terminal
phosphodiester bond of phospholipids, in turn generating a free
head group and PA. PA itself acts as a signaling molecule and
is the precursor of additional regulatory molecules, including
DAG pyrophosphate, lyso-PA, and arachidonic acid (46).
We prelabeled cells with 32Pi and assayed for 32P–PA formation in response to Al31. Treatment of the cells with either AlCl3
or Al(NO3)3 for 15 min inhibited the formation of PA. To test
how Al31 affects PA signaling, we used the peptide mastoparan-7
(mas-7), which is known as a very potent stimulator of PA formation. Al31 inhibited the mas-7-mediated induction of PA levels,
both before and after incubation with Al31. The PA involved in
signaling is generated by two distinct phospholipid signaling pathways: via PLD or PLC and via DGK (52). By labeling with 32Pi
for short periods of time, we found that PA formation was inhibited by almost 30% when the cells were incubated with AlCl3,
suggesting involvement in the PLC/DGK pathway. The effects of
incubating the cells with the PLC inhibitor U73122 on PA formation were similar to those seen in response to AlCl3. In vivo PLD
activation by mas-7 was reduced by Al31. These results suggest
that PA formation is prevented through the inhibition of PLC activity, and these data provide the first evidence for the effects of
Al-toxicity on PA production (52).
While the existence of a plant receptor specific for Al31 has
not been demonstrated, enhanced expression of a cell wall-associated kinase receptor upon Al treatment has been found (53).
Perhaps the signal transduction cascade(s) activated by Al and
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the regulation of different pools of PA may be reflected in Altoxicity as well as in Al tolerance. Whether or not the PA that
forms by the two different routes has the same composition of
fatty acids and the same target within the cell remains unknown.
Aluminum and the Protein Phosphorylation
Signaling Pathway
Protein phosphorylation plays an important role in the regulation
of various biological processes in plants (54, 55). Protein phosphorylation has also been demonstrated to provide a signal transduction
pathway for mediating extracellular stimuli in cells (56).
The aspect of Al-toxicity and signal transduction that has
been most intensely studied to date involves phosphoinositide
turnover, but protein phosphorylation is known to be one of the
post-transduction modifications that regulates physiological
events at the cellular level. Genetic, biochemical, and pharmacological analyses have shown that protein kinases and protein
phosphatases play important roles in environmental stress
responses. Studies examining the effect of Al on protein phosphorylation were not available until the last decade. In 2001,
two reports examining the role of protein phosphorylation in
Al-toxicity were published (57, 58).
Suspension cells of C. arabica were incubated with increasing
concentrations of AlCl3 (200–1,000 lmol L21), and an in vitro
phosphorylation reaction with cell extracts was performed. No
changes in the proteins present in extracts from cells treated with
AlCl3 were detected as compared with the proteins in extracts from
untreated cells. However, the protein phosphorylation patterns did
change. Phosphorylated proteins with molecular masses of 18, 31,
and 53 kDa increased dramatically after in vivo treatment of cells
with AlCl3. When AlCl3 was added to the reaction mixture, no differences in phosphorylation patterns were observed (57).
Although Al-induced organic acid exudation has been extensively documented [for a review, see ref. 38], signal transduction
pathways that lead to this event are less well understood. Osawa
and Matsumoto (58) have suggested that protein phosphorylation
is required for signal transduction in Al-activated malate efflux
from wheat root apex, likely through phosphorylation and subsequent activation of anion channels (58). In this study, treatment
with the protein kinase broad-range inhibitor K-252a blocked Alinduced malate efflux at the root apex, and in-gel kinase assays
with myelin basic protein, a substrate specific to mitogen-activated
protein kinases (MAPK), showed that Al treatment induces the
activation of a 48-kDa protein kinase. This putative MAPK was
rapidly and transiently activated, and its activity increased from
0.5 to 5 min after Al exposure and subsequently diminished after
5 min. In addition, the activity of the 48-kDa kinase was approximately 10-fold higher after treatment with Al than without, and
the Al-induced activation was lost within 5 min. The authors suggested that Al transiently activates this protein kinase quickly
enough to precede the initiation of malate efflux (58). However,
whether or not the 48 kDa protein kinase is directly involved in
the pathway for malate efflux remains unknown. This kinase
appears to play an essential role in transduction of the Al signal
and expression of resistance mechanisms in the root apex of Alresistant wheat (58).
Aluminum has been shown to be able to induce a rapid and
transient activation of a putative 58 kDa MAPK protein in coffee suspension cells (59). Although this cell suspension culture
showed a basal level of malate exudation (60), no direct evidence suggested that activation of the 58 kDa MAPK-like protein is related to Al resistance in coffee. The oxidative burst
evoked by Al may directly affect MAPK signaling cascades
(61). In Arabidopsis, H2O2 activates the MAPKs MPK3 and
MPK6 via the MAP kinase kinase kinase Arabidopsis NPK1
(Nicotiana protein kinase 1)-like protein kinase 1 (ANP1) (62).
Indeed, overexpression of the ANP1 gene in transgenic plants
resulted in increased tolerance to cold, heat shock, and salinity
(62). H2O2 also increases expression of the nucleotide diphosphate (NDP) kinase 2 in Arabidopsis (63). The overexpression
of AtNDPK2 reduced H2O2 accumulation and enhanced tolerance to multiple stresses, including cold, salt, and oxidative
stress. The effects of NDPK2 may be mediated by the MAPKs
MPK3 and MPK6, as NDPK2 can interact with and activate the
MAPKs. Therefore, these studies clearly suggest that abiotic
stresses (including those from Al) induce ROS generation,
which in turn activates MAPK signaling pathways.
ROS can also affect other signaling pathways. Kawano et al.
(64) have observed that Al-induced ROS activates Ca21 influx at
the plasma membrane, leading to an increase in the cytosolic
Ca21 concentration. Briefly, the authors reported on aluminuminduced O2l– accumulation via membrane-bound nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase (optimal
[AlCl3] 5 6 mM), and, following an acute spike of O2l–, a gradual increase in the cytosolic free Ca21 concentration ([Ca21]c)
was detected. This increase in [Ca21]c was exclusively a consequence of Al-induced ROS production. However, the Al concentration that was optimal for O2l– was inhibitory for [Ca21]c,
implying that high concentrations of Al are inhibitory not only to
cation channels but also to the H2O2-induced influx of Ca21 (64).
These data also suggest that aluminum may induce Ca21-dependent signaling events at the beginning of treatment, but a gradual
increase in Aln1 activity in cells eventually turns off this signal.
An increased cytosolic Ca21 concentration could activate other
proteins such as PLD. PA is a second messenger that enhances
the activity level of proteins, including protein kinases and phosphoenolpyruvate carboxylase (46, 65). Because PA is a wellknown activator of MAPK cascades in response to a variety of
environmental stresses (65), a possible relationship between PA
levels and MAPK regulation by Al may contribute to the Al-resistance response, as proposed in a recent report (52).
RNA Interference and its Potential to Elucidate Signaling
Networks
Increased attention has been given to RNAi (RNA interference) as a potential therapeutic agent in the treatment of various
ALUMINUM STRESS AND POSSIBLE BIOTECHNOLOGICAL APPLICATIONS
869
Figure 2. Model of the Al31 effect on PA formation. Al31 inhibits PLC and decreases DAG availability, reducing the amount of
DAG phosphorylation to PA. This inhibition affects PA signaling levels by impairing the response of PA target proteins to signal
cascades. The PA formed by PLD could lead a signaling cascade for Al31 toxicity tolerance by releasing organic acids through anionic channels activated by phosphorylation that involves MAPK signaling.
diseases in humans (66). To date, the results of its application
for the treatment of diseases such as cancer and viral infections
(66, 67) and those that affect the eye (68), nervous system (69),
and bones[0] (70) have been significant and substantial. However, one limitation of the application of RNAi as a therapeutic
agent in any disease is the strategy for liberation of the small
molecules of siRNA (small interfering RNA). In this respect,
changes have been made to these small molecules, ranging
from the alteration of the phosphate backbone of the siRNA to
the use of a complex involving lipids (71).
Much of the progress in the application of RNAi has been
achieved in animal cells, and this knowledge has also been
applied to plants. RNAi in plants is primarily used to determine
the role of genes involved in different metabolic pathways with
the aim of improving nutrient content and reducing the production of toxins (72).
Because plants are sessile organisms, they must find ways to
detect and respond to external stimuli and to convert these stimuli into internal signals. As mentioned earlier, one of the first
signaling pathways involved in converting a vast majority of
environmental stimuli into signaling pathways is the phosphoinositide pathway. Some of the enzymes involved in this path-
way have been observed to be regulated by many environmental
factors, including changes in osmolarity, salinity (73), oxidative
stress, metals, and pathogens (74).
Two common methods for the characterization of genes
include the selection of mutants with desired phenotypes and
the insertion of a transgene into the chromosome through
genetic transformation. Although valuable, these methods are
very time-consuming. However, the application of RNAi as a
tool for assessing gene function using cell suspensions or protoplasts in combination with transient transformation assays can
specifically facilitate the silencing of a large number of genes
in a short period of time (75).
A protoplast is a plant cell that has been completely removed
from the cell wall using mechanical and/or enzymatic
approaches. In 1960, Cocking was the first to isolate protoplasts
from higher plants by enzymatic methods. Using this method, it
is currently possible to easily obtain protoplasts of whole organs
and plant tissues in culture. These cellular systems are currently
used as tools in concert with approaches, such as mutagenesis,
selection, genetic transformation or fusion, somatic hybridization
(76), assessment of gene function by RNAi, and the introduction
of foreign DNA to study protein localization, ion channels,
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POOT-POOT AND HERNANDEZ-SOTOMAYOR
transport processes, cell division, and morphogenesis. Therefore,
protoplasts can be used as a cellular model for RNAi experiments to answer the following questions regarding the phosphoinositide signal transduction pathway:What is the effect of transient PLC silencing on the activity of other phospholipases?
Does a protein signal transduction pathway regulate phospholipids in response to the RNAi-mediated silencing of PLC?
What happens to the activity of lipid kinases, such as PI4-K
and PIP5-K, after the silencing of PLC or the silencing of the
entire family?
RNAi has been used in greater depth to study phosphoinositide signaling in animals and has demonstrated that some
enzymes, such as the lipid kinase phosphatidylinositol 3-kinase
(PI3-K), are involved in the proliferation of cancer cells. However, in plants, interest in the study of phosphoinositides has
been generated as a result of the enzymatic components and
second messengers that are involved in processes, such as vesicular trafficking and pathogen attack, among others. Enzymes
such as PLD and myo-inositol-1-phosphate synthase have been
silenced in plants (including tomato and soybean) to determine
their functions (77, 78).
Therefore, we propose the use of RNAi to evaluate the function of each of the enzymes in the phosphoinositide signal
transduction pathway and the levels of their product, PA, in
response to different stimuli by silencing one, two or all of the
enzymes in this pathway.
Model for the Effects of Al on the Different Signal
Transduction Pathways
The possible effects of Al on signal transduction pathways
are represented in Fig. 2. The effects of Al could occur in one
of two ways as follows: (1) Al could interact directly with a receptor (R) on the membrane surface, or with the membrane
itself, to initiate a secondary messenger cascade that then regulates the activity of different enzymes, such as PLC, PLD, lipids, or protein kinases, followed by a consequent activation of
an anion channel or (2) Al enters into the cytoplasm and regulates the different enzymes as mentioned earlier, affecting anion
channels directly or indirectly via secondary messengers. The
regulation (up or down) of second messenger production may
lead to protein phosphorylation through the activation of protein
kinases, such as MAP kinases, cyclin dependent kinases, or
others that may affect transcription factors and gene expression.
Further studies will be required to corroborate this model and
improve the understanding of aluminum-affected metabolic
events in plants, specifically to elucidate the elements that constitute the Ca21 activated MAPK cascade and the role of phospholipid signaling in Al resistance in plants.
PERSPECTIVES
Despite the progress in recent years, many important questions
and challenges remain in the field of intracellular plant signaling.
The aim of this study was to note the importance of the effect of
Al on different signal transduction pathways, such as phosphoinositide and protein phosphorylation pathways. Although Mexico is
a major crop producer, it must deal with economic problems,
including providing enough food to the population. Several groups
in Mexico are focused on the study of different plants stresses in
both models, such as Phaseoulus vulgaris or maize, and in crops
with economic importance, such as the producers of secondary
metabolites. However, research is also needed in crops, such as
coffee, because the income of several thousand families depends
on their production. However, whether or not the regulation of
enzymes, such as PLC, PLD, lipid kinases, and protein kinases, is
attributable to post-transcriptional regulation or other types of biochemical regulation remains to be determined.
The use of genomic tools such as RNAi may provide
answers that will allow us to transform crops, including coffee,
and induce resistance to Al or other stressors. Much effort will
be required once there is a biological understanding of how
plants respond to abiotic stress, and then even more effort will
be needed to convince the Mexican authorities to accept the
appropriate use of genetically modified organisms and to support basic science research in Mexico. While we anticipate this
effort, let us enjoy a good cup of coffee in the morning.
ACKNOWLEDGEMENTS
This work was supported by CONACYT grant 98352. Critical
review by Armando Muñoz-Sanchez and the technical work of
Eduardo Domı́nguez-Domı́nguez and Angela Ku with the electron microscope are greatly appreciated.
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