Directory UMM :Data Elmu:jurnal:P:PlantScience:Plant Science_BioMedNet:021-040:

trends in plant science
Reviews

Inositol signaling and plant growth
Jill M. Stevenson, Imara Y. Perera, Ingo Heilmann, Staffan Persson and Wendy F. Boss
Living organisms have evolved to contain a wide variety of receptors and signaling pathways
that are essential for their survival in a changing environment. Of these, the phosphoinositide
pathway is one of the best conserved. The ability of the phosphoinositides to permeate both
hydrophobic and hydrophilic environments, and their diverse functions within cells have
contributed to their persistence in nature. In eukaryotes, phosphoinositides are essential
metabolites as well as labile messengers that regulate cellular physiology while traveling
within and between cells. The stereospecificity of the six hydroxyls on the inositol ring provides the basis for the functional diversity of the phosphorylated isomers that, in turn,
generate a selective means of intracellular and intercellular communication for coordinating
cell growth. Although such complexity presents a difficult challenge for bench scientists, it is
ideal for the regulation of cellular functions in living organisms.

T

he challenge for today’s scientists is to maintain a broad perspective of interactive signaling pathways, while dissecting
their parts sufficiently to render them understandable. Our
goal is to stimulate the reader to consider the phosphoinositide (PI)

pathway as a functional component of a complex growth response
and to move beyond the reductionist’s perspective of confining
PI signaling to the generation of a transient, Ins(1,4,5)P3-induced
Ca21 oscillation. (For additional coverage of plant PIs and the
enzymes involved in their metabolism see Refs 1,2.)

Most plant responses to external stimuli involve a change in
growth and therefore membrane biogenesis. Membrane trafficking
and signaling are inexorably linked in regulating cellular metabolism and controlling growth. To coordinate these processes, evolution appears to have capitalized on the stereospecificity of the
PIs. During membrane trafficking, individual inositol phospholipids on the vesicle surface specify functional information like
cogs on a wheel. As vesicles traffic from the endoplasmic reticulum (ER) to the plasma membrane, and from the ER to the vacuole
and retrograde pathways, inositol phospholipids attract specific
proteins necessary for budding, docking and fusion3,4. Similarly,
the presence of a multitude of polyphosphorylated inositol lipids
and soluble inositol phosphates imparts specificity to signaling
pathways by recruiting and activating signaling proteins into
functionally distinct complexes. In the following sections, the
major inositol lipids (Fig. 1) and their potential roles in membrane
biogenesis, signaling and plant growth are briefly described.

wortmannin has been shown not only to inhibit PtdIns-3-kinase,
but also to bind and inhibit PtdIns-4-kinases8.
Even though its activity is low in vitro, PtdIns-3-kinase is
clearly essential for growth because the antisense expression of
the gene for PtdIns-3-kinase results in transgenic plants that grow
poorly, if at all9. This apparent incongruity might be explained
in part by recent work showing that the soybean PtdIns-3-kinase
co-localizes with nuclear transcription sites, suggesting that
PtdIns(3)P is important for regulating transcription10.
Prevalent in animal cells, but not in plants or yeast are
two additional families of PI-3-kinases, the class I and class II
PI-3-kinases1. Unlike the Vps34p-type PtdIns-3-kinase, the class
I and II PI-3-kinases can phosphorylate PtdIns, PtdIns(4)P, PtdIns(5)P
and PtdIns(4,5)P2 to produce PtdIns(3)P, PtdIns(3,4)P2,
PtdIns(3,5)P2 and PtdIns(3,4,5)P3, respectively. The physiological relevance of the class II PI-3-kinases is not understood8. The
class I PI-3-kinase and its stereospecific lipid products are
involved in signaling pathways that are activated via Ras and
phosphotyrosine-coupled plasma-membrane receptors. The fact
that the class I PI-3-kinase and PtdIns(3,4,5)P3 appear to be absent
in plants is consistent with the lack of plasma-membrane-associated tyrosine-receptor kinases11. In summary, the molecular and

biochemical data support the thesis that essential biochemical
functions of nuclear and ER-localized PtdIns-3-phosphate have
been evolutionarily conserved between plants and animals,
whereas the evolution of plasma-membrane signaling is specific
to the organism, controlling responses to stimuli and ensuring
survival under changing environmental conditions.

Biosynthesis and function of PtdIns(3) P

Biosynthesis and function of PtdIns(4) P

The first evidence for the exacting requirements for stereospecific
isomers of phosphatidylinositol (PtdIns) phosphate in membrane
trafficking was revealed when the gene for yeast PtdIns-3-kinase,
VPS34, was shown to be essential for trafficking of hydrolytic
enzymes to the vacuole5. The plant PtdIns-3-kinase is related to the
yeast Vps34p, which uses only PtdIns as a substrate. In plants,
PtdIns-3-kinase activity is associated with nodule formation during
symbiotic nitrogen fixation1,6. The temporal increase in PtdIns-3kinase activity in root nodules, where the peribacteroid membrane
is being formed, and the selective inhibition of vacuolar trafficking

by wortmannin in tobacco cells1,7, are consistent with the yeast
data, in that PtdIns(3)P is vital for vesicle trafficking. However, little is known about the plant enzyme and additional characterization of these systems is warranted. This is especially true because

PtdIns(4)P is important for membrane biogenesis and vesicle
trafficking from the ER to the Golgi and plasma membrane4. For
example, PtdIns-4-kinase activity is necessary for vesicle formation and stimulated secretion of catecholamines in chromaffin
cells, and for regulated exocytosis of the small synaptic vesicles
from nerve terminals12. Exploiting yeast genetics to demonstrate
the necessity of PtdIns(4)P in membrane trafficking and to
elucidate the physiological roles of the distinct PtdIns-4-kinase
isoforms has corroborated these findings.
In an elegant mutant screen, yeast mutants were identified that
were compromised in various steps of intracellular lipid transport13. One mutant that was unable to transport phosphatidylserine
(PS) from the ER to the Golgi was isolated and found to accumulate PS in the ER, with diminished phosphatidylethanolamine

Inositol phospholipids as regulators of growth

252

June 2000, Vol. 5, No. 6

1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01652-6

trends in plant science
Reviews
(PE) formation in the Golgi in spite of norO
mal PS-decarboxylase activity. The mutant
C O CH2
defect was complemented by STT4, the
C O CH
O
yeast ortholog (functional homolog) of the
O HC O P O –
a isoform of PtdIns-4-kinase (PtdIns4Ka).
H
OH O
6 OH
The rescue of the yeast PS transport mutant
2
1 4

indicates the importance of phosphoinosi5
OH
HO
OH
tides, specifically PtdIns(4)P generated
3
from PtdIns4Ka, in intracellular aminophospholipid transport from the ER to the
PtdIns
Golgi. PIK1, the yeast ortholog of the b
isoform of PtdIns-4-kinase (PtdIns4Kb),
was not identified in the complementation
O
O
screen of .10 000 transformants, suggestC O CH2
C O CH2
ing that it would not complement the
C O CH
C O CH
O
O

aminophospholipid transport mutant.
O HC O P O –
O HC O P O –
Additional data supporting non-overlapH
H
OH O
OH O
6 OH
6 OH
ping roles for the PtdIns-4-kinase isoforms
2
2
O
1 4
1 4
has recently been reported in yeast secretory
5
5
–
OH

OH
HO
O P O
OH
O
3
3
mutants14. Although overexpressing the
–
O
P
O
O
gene for PtdIns4Ka did not rescue the yeast
O
SEC14 secretory mutant phenotype, increasing PtdIns(4)P levels, either by overPtdIns(4)P
PtdIns(3)P
expressing the gene for PtdIns4Kb, or by
eliminating PtdIns(4)P phosphatase activity
did14. This implies that the location and rate

O
O
of PtdIns(4)P turnover is important for
C O CH2
C O CH2
secretion and that the two PtdIns-4-kinase
C O CH
C O CH
O
O
isoforms, a and b, are functionally distinct
O HC O P O –
O HC O P O –
H
H
and generate different pools of PtdIns(4)P.
OH O
OH O
6 OH
6 OH

2
2
In animals and yeast, PtdIns4Kb appears to
O
O
O
1 4
1 4
5
5
be more involved in sustaining the structural
–
–
O P O–
OH
O P O
O P O
O
O
3

3
integrity of the cytoskeleton and Golgi, and
O
O
O
O P O–
O P O–
plays a vital role in secretion from the Golgi
O
O
apparatus15,16.
PtdIns(4,5)P2
In plant cells, PtdIns-4-kinase activity is
PtdIns(3,4)P2
associated with the plasma membrane,
nucleus, endomembranes, cytosol and the
O
cytoskeleton1. This wide distribution in
C O CH2
C O CH
subcellular compartments implies that disO
O
O HC O P O –
tinct pools of PtdIns(4)P are targeted
O P O–
H
OH O
OH O
6 OH
differentially to or synthesized in these
6 OH
2
2
subcellular compartments and might perO
O
O
1 4
1 4
5
5
–
form distinct physiological roles as they
O P O–
O P O–
HO
O P O
O
OH
3
3
O
O
clearly do in yeast.
O
O P O–
In spite of a wide range of subcellular
O
locations reported, only two PtdIns-4-kinase
Ins(1,4,5)P3
PtdIns(3,5)P2
genes have been cloned from any organ8
ism . The gene for PtdIns4Ka is the larger
Trends in Plant Science
of the two and it encodes a 200–230 kDa
Fig. 1. The phosphoinositide stereoisomers known to exist in plants. Abbreviations:
polypeptide. This isoform contains a pleckIns(1,4,5)P3, inositol (1,4,5) trisphosphate; PtdIns, phosphatidylinositol; PtdIns(3)P, phosstrin homology (PH) domain, a poorly conphatidylinositol-3-monophosphate; PtdIns(3,4)P2, phosphatidylinositol (3,4) bisphosphate;
served 100 amino acid motif that binds
PtdIns(3,5)P2, phosphatidylinositol (3,5) bisphosphate; PtdIns(4)P, phosphatidylinositol-4polyphosphorylated inositol lipids and
monophosphate; PtdIns(4,5)P2, phosphatidylinositol (4,5) bisphosphate.
thereby targets the protein in which it resides
17
to the membrane . The gene for PtdIns4Kb
is smaller and encodes a polypeptide of
~110–126 kDa, and does not have a PH domain. Both genes have alternative splice variant or proteolytic fragment of one of the
been cloned from Arabidopsis, one encoding a polypeptide of known isoforms. Antibodies generated to the C-terminal third
205 kDa (AtPtdIns4Ka)18 and the other encoding a polypeptide of of AtPtdIns4Ka, which includes the catalytic, PH and lipid-kinase
126 kDa (AtPtdIns4Kb)19. However, as in other systems, a small unique domains, crossreact with all three isoforms of the proteins
molecular weight (65 kDa) PtdIns-4-kinase has been found20. It in carrot suspension culture cells and Arabidopsis seedlings,
remains to be determined if the small molecular weight plant en- indicating significant sequence similarity (J.M. Stevenson and
zyme is an as yet unknown PtdIns-4-kinase isoform, or if it is an W.F. Boss, unpublished).
June 2000, Vol. 5, No. 6

253

trends in plant science
Reviews
In spinach, the putative PtdIns4Kb isoform and a 65 kDa
isoform co-purify with the plasma membrane20, whereas the
PtdIns4Ka isoform is most prevalent in the microsomal F-actin
fraction18. The differential localization of the PtdIns-4-kinase
isoforms implies that the lipid kinases will dictate the location of
the different PtdIns(4)P pools and that distinct isoforms will have
non-overlapping functions, as in yeast.
An added means of regulating PtdIns(4)P distribution within
cells was revealed in studies of the PH domain of PtdIns4Ka. The
PH domain of Arabidopsis PtdIns 4-kinase, AtPtdIns4Ka, binds
to PtdIns(4)P in preference to other polyphosphoinositides and
phospholipids18. Binding to the product of the reaction might
enable the enzyme to retain the PtdIns(4)P in a specific intracellular pool and dictate its fate by awaiting competitive binding by a
PtdInsP kinase, PtdIns transfer protein, actin-binding proteins or
other vesicle-trafficking machinery. Clearly, regulation of the
PtdIns(4)P pools within the cell is important, and determining the
function of the PtdIns-4-kinase isoforms in plants will bring new
insights as to their individual contributions to membrane trafficking
and signaling.
Biosynthesis and function of PtdIns(4,5) P2

As the precursor of the second messengers Ins(1,4,5)P3 and diacylglycerol, PtdIns(4,5)P2 plays a pivotal role in signaling. In addition, PtdIns(4,5)P2 is important for the regulation of cytoskeletal
dynamics, vesicle trafficking and ion transport. PtdIns(4,5)P2
interacts with many actin-binding proteins, including profilin, gelsolin, a-actinin and cofilin21,22, and overexpression of a PIP-5kinase type I a isoform in COS-7 cells results in a massive increase
in actin polymerization23. PtdIns(4,5)P2 also plays a role in vesicle
formation and in endocytosis24. PtdIns(4,5)P2 modulates proteins
involved in vesicle trafficking, such as the small G protein ADPribosylation factor 1 (Ref. 25) and phospholipase D (Ref. 26).
PtdIns(4,5)P2 is involved in both anterograde and retrograde
vesicle trafficking. PtdIns(4,5)P2 and PtdIns(4)P have been
shown to stimulate vesicle budding in reconstitution assays with
coat proteins24. Endocytosis of G-protein-coupled receptors by a
complex formation of b-arrestin, AP-2 and clathrin is dependent
on PtdIns(4,5)P2 turnover27,28. The requirement for PtdIns(4,5)P2
turnover for retrograde trafficking was underscored recently
by studies with synaptojanin knockout mice. Mice that lacked the
inositol polyphosphate 59phosphatase, synaptojanin, had increased PtdIns(4,5)P2 levels, and their inability to dephosphorylate PtdIns(4,5)P2 for synaptic vesicle recycling proved to be fatal29.
Similarly, PtdIns(4,5)P2 accumulation can lead to defects in trafficking from the lysosome to the extracellular space. Individuals
suffering from Lowe’s syndrome lack the lysosome-associated
inositol polyphosphate 59phosphatase, OCRL-1 (Lowe’s oculocerebrorenal syndrome), which leads to a two- to threefold increase
in PtdIns(4,5)P2 levels in kidney cells and eventually results in
renal failure30.
PtdIns(4,5)P2 can regulate ion transport by modulating the
activity of integral membrane proteins, such as the P-type
ATPases1, and the ATP-sensitive K1 channel31. PtdIns(4,5)P2 can
also mediate the subcellular location and activity of proteins
recruited to the membrane via their PH domains during signaling,
such as phospholipase C d1 and bARK (β-adrenergic receptor
kinase) associated with G-protein-coupled receptors17. In this
capacity, PtdIns(4,5)P2 might act as a nucleation site for protein
scaffolding. However, plant isoforms of PLCd identified so far do
not contain a PH domain.
As with PtdIns(4)P, the myriad of functions of PtdIns(4,5)P2
can be dictated by a family of PIP kinases that differ in their localization, regulation and substrate specificity. Two major types of
254

June 2000, Vol. 5, No. 6

mammalian PtdIns phosphate kinases (each containing three isoforms) have been characterized. The type I enzymes phosphorylate PtdIns(4)P preferentially, whereas the type II enzymes have a
higher affinity for PtdIns(5)P [in either case the end product will
be PtdIns(4,5)P2]. In addition, both enzymes use PtdIns(3)P as a
substrate in vitro21. This is probably also the biosynthetic route for
PtdIns(3,4)P2 and PtdIns(3,5)P2 in plants1.
Yeast contain two PtdInsP kinase orthologs. MSS4, which
encodes the major PtdIns(4)P(5)-kinase, is an essential gene that
was first identified as a multicopy suppressor of the PtdIns-4kinase STT4 (Ref. 8). Mss4p is localized in the plasma membrane
and conditional mutants exhibit disruption of cell polarity and the
actin cytoskeleton32,33. The second yeast PtdIns phosphate kinase
ortholog, FAB1 is a PtdIns(3)P(5)-kinase involved in regulating
vesicle trafficking and vacuolar morphology and function. Fab1p
has a functional alliance with Vps34p and is responsible for phosphorylating PtdIns(3)P to generate PtdIns(3,5)P2 which accumulates in yeast in response to hyperosmotic shock5. The PtdIns(3,5)P2
isomer has not been functionally characterized in plants. In some
plants, there is an increase in the PtdIns(3,5)P2 isomer in response
to osmotic stress34, whereas in others, the PtdIns(4,5)P2 isomer increases35. Whether these are truly species differences or differences
in methods or the physiological status of the cells remains to be seen.
PtdInsP kinase activity is associated with different subcellular
fractions, including the ER, plasma membrane, cytoskeleton and
the nucleus1,8. The distribution of the kinases changes in response to
agonist stimulation in vivo, suggesting that location of the enzyme
is the key to its regulation. Phosphorylation of the kinases has been
proposed as a reversible means of affecting both their activity and
localization21. The different types of PtdInsP kinases and their
regulated translocation might generate different cellular pools
of PtdIns(4,5)P2. Recent studies offer intriguing evidence for
the presence of such dynamic and functionally distinct pools of
PtdIns(4,5)P2 in plants36,37. One method of visualizing PtdIns(4,5)P2
within unstimulated cells is to express the cDNA encoding a fusion
protein of green fluorescent protein (GFP) with the human
PLCd–PH domain, which binds with higher affinity to PtdIns(4,5)P2
than any other polyphosphorylated inositol lipid. This fusion protein was first used in plant cells to identify PtdIns(4,5)P2 within
membranes of pollen tubes and to block pollen-tube growth37.
Whether the observed effects on pollen-tube growth resulted from
the PH domain binding to PtdIns(4,5)P2 or Ins(1,4,5)P3 remains to
be determined. The PLCd–PH domain binds to Ins(1,4,5)P3 with an
affinity that is eight times higher than its affinity for PtdIns(4,5)P2
(Ref. 17). Nonetheless, the overexpression of the human PLCd–PH
domain is a valuable tool for blocking both PtdIns(4,5)P2 and
Ins(1,4,5)P3 metabolism.
Although the levels of PtdIns(4,5)P2 in plants are low, there is
good correlative evidence of changes in Ins(1,4,5)P3 levels in
response to various stresses2 and for the up-regulation of the activity
of the enzymes involved in the synthesis and hydrolysis of PtdIns
(4,5)P2 (Refs38,39). To date, one Arabidopsis PtdInsP kinase cDNA
has been characterized and the corresponding recombinant polypeptide was shown to have PtdInsP kinase activity38,40. In addition, several more putative genes have been annotated from the Arabidopsis
genome-sequencing initiative. If these multiple members of the
PtdIns phosphate kinase family prove to have as much functional
complexity as their animal counterparts they will probably define
unique microdomains for regulating subcellular metabolism.
Ins(1,4,5)P3: a means of coordinating growth

The microdomains of PtdIns(4,5)P2 localized throughout the
membranes establish a network of initiation sites for generating
rapid, transient increases in Ins(1,4,5)P3 in response to stimuli.

trends in plant science
Reviews

Angle of bending (degrees)

pmol Ins(1,4,5)P3 / g FW

Although the sequence of events leading
up to stimulus-mediated Ins(1,4,5)P3 proIncrease in invertase
duction has not been delineated in plants,
on the lower side
Increase
in
auxin
any stimulus that increases cytosolic Ca21
on the lower side
in a PtdIns(4,5)P2 microdomain should, in
5000
50
Ins(1,4,5)P3
theory, activate plant PLCs to produce a
4000 oscillations
transient increase in Ins(1,4,5)P3 (Ref. 2).
40
It is not surprising that a transient increase
Ins(1,4,5)P3 on
3000
in Ins(1,4,5)P3 occurs in plants in response
30
lower side
to many stimuli. Although increases in
2000
Ins(1,4,5)P3 levels might be a result of iniGravitropic
20
tiating reversible responses, such as guardbending
1000
10
cell closure, as sessile organisms, plants
Ins(1,4,5)P3 on upper side
must be able to distinguish between transient
0
perturbations and a persistent stress that
0
2
4
6
8
10
15
36
48
would require a growth response. This
leads to the question, is there evidence that
Presentation time
long-term increases in Ins(1,4,5)P3 levels
Time (h)
are associated with plant-cell growth? The
37,41
Persisting gravistimulus
answer is ‘yes’. In both tip-growing cells
and elongating cells of gravistimulated
Trends in Plant Science
maize pulvini42, the data indicate a role for
PtdIns(4,5)P2 turnover in regulating cell
Fig. 2. Transient and long-term changes in inositol (1,4,5) trisphosphate [Ins(1,4,5)P3]
elongation.
levels in the gravistimulated maize pulvinus. Starting at 10 s, transient oscillatory changes in
Ins(1,4,5)P3 occur in both the upper and lower sides of the pulvinus over the first 2 h of graviIn maize pulvini, between 2 and 4 h of
stimulation (red bar). Over the first 2–8 h of gravistimulation, there is a gradual long-term
gravistimulation are required to induce a
increase in Ins(1,4,5)P3 levels on the lower side (unbroken red line) compared with the
bending response. That is, it takes at least
upper side (broken red line) and preceding gravitropic bending. Bending (green) is
2 h in a horizontal position for the cells
first detectable around 8 h and reaches a maximum at 48 h. The blue arrows indicate
on the lower side of the pulvinus to be comthe timing of the increases in auxin and invertase levels on the lower side of the pulvinus
mitted to elongate, even if the plant is
(W. Zhao, J.C. Long and G.K. Muday, unpublished).
returned to a vertical position. When maize
plants are placed horizontally, there is a
rapid (10 s), transient fivefold increase in
Ins(1,4,5)P3 levels in the tissue on the lower side of the pulvinus. alone (unlike the ER-localized channels in animal cells) would be
Over the next 2 h, these levels increase alternately in both the unable to sustain Ca21 oscillations48. Functionally, therefore, the plant
upper and lower tissue. Between 2 and 8 h, Ins(1,4,5)P3 levels Ins(1,4,5)P3 receptor might be most similar to the subtype 3
increase on the lower side by up to six times more than in the receptor in animals, suggesting that a sustained increase in
vertical control and then decrease as bending of the pulvinus Ins(1,4,5)P3 would be a requisite to maintain Ins(1,4,5)P3-mediated
becomes visible (Fig. 2). One interpretation of these data is that Ca21 oscillations.
the early fluctuations in Ins(1,4,5)P3 (prior to 2 h) are a part of the
Homology-based approaches to clone the elusive plant
process that coordinates the cells within a tissue as they make a Ins(1,4,5)P3 receptor have been largely unsuccessful, suggesting
commitment to elongate. After the presentation time (2 h), but that the plant and animal receptors might not share much sequence
prior to visible growth (8 h), the sustained increase in Ins(1,4,5)P3 similarity. The only putative plant Ins(1,4,5)P3 receptor that has
levels might predict the growth response. If this is true, then the been purified and characterized biochemically to date appears to be
increase in Ins(1,4,5)P3 should precede measurable increases in much smaller in size compared with any of the animal counterparts
auxin (Fig. 2; Refs 43–45). Significantly, the sustained increase in and its activity is enhanced by inositol phosphate metabolites49.
PI metabolism might reflect a need for the inositol lipid turnover
In plant cells, the proteins involved in terminating the
associated with increased membrane biogenesis and cytoskeletal Ins(1,4,5)P3 signal by hydrolyzing Ins(1,4,5)P3 are also different
restructuring, as well as an increase in Ins(1,4,5)P3-mediated Ca21 from those in animal cells. In plants, both in vivo50 and in vitro51
release to initiate and sustain cell elongation.
data indicate that Ins(1,4,5)P3 is hydrolyzed by both inositol
If Ins(1,4,5)P3-mediated Ca21 oscillations are involved in the polyphosphate 19phosphatase (InsP19ptase) and InsP59ptase to
growth response, plants might require a sustained increase in form Ins(4,5)P2 and Ins(1,4)P2, respectively; whereas, in animal
Ins(1,4,5)P3 to generate these oscillations. The duration of cells InsP59ptase is the first enzyme in the hydrolytic pathway.
Ins(1,4,5)P3-mediated Ca21 oscillations will depend, in part, on The plant InsP19ptase (IMP) has been cloned and characterized52.
the properties of the receptor and the rate of Ins(1,4,5)P3 metab- Furthermore, at least ten InsP ptase cDNAs have been identified
olism; both of which appear to differ in plants and animals. in Arabidopsis on the basis of sequence similarity (G.E. Gillaspy,
Animals have evolved to contain at least three isoforms of pers. commun.). Because inositol is an essential metabolite syntheIns(1,4,5)P3 receptors that have been cloned and characterized46. sized de novo by dephosphorylation of Ins1P by an InsP19ptase, it
Although the animal receptors share ~70% amino acid identity, is crucial that all of the InsP ptase isoforms are identified and
they differ in their affinity for Ins(1,4,5)P3 and show varying sus- characterized and that the different metabolic roles are delineated.
ceptibility for feedback inhibition by Ca21 (Ref. 47). The major Theoretically, altering the production of selective isoforms
Ins(1,4,5)P3-sensitive Ca21 store in plants appears to be the vacu- will affect the duration of the Ins(1,4,5)P3 signal and could
ole48. Electrophysiological studies indicate that the vacuolar Ca21 thereby affect the rate of cell growth without eliminating de novo
channel is not Ca21 regulated, which implies that this channel synthesis of inositol.
June 2000, Vol. 5, No. 6

255

trends in plant science
Reviews
charged analog, is commonly used as a
protein-synthesis inhibitor. Furthermore,
any neomycin that does enter the cell will
RNA transport
not bind PtdIns(4,5)P2 with high enough
out of nucleus
affinity to displace membrane proteins that
Nuclear
InsP6
are already bound.
transcription
Although sustained increases in
S
S
Ins(1,4,5)P3 and Ca21 can predict growth,
i
i
g
g
continuous elevation of Ca21 could also,
Trafficking to plasma membrane
n
n
PLC
PtdIns3K
PtdIns4K
PtdInsPK
over time, decrease growth and even lead
a
a
PtdIns(3)P
PtdIns(4,5)P2
PtdIns
PtdIns(4)P
to apoptosis57. It is therefore not surprising
l
l
Ins(1,4,5)P3
that Ins(1,4,5)P3 decreases in the maize
i
i
n
n PtdIns(3,5)P2
pulvinus
when bending is detectable and
InsP 59Ptase
g
g
when cells are beginning to elongate (8 to 10
Endocytosis
Osmotic
h after gravistimulation). One mechanism to
Ca2+
Trafficking
retrograde trafficking
stress
decrease Ins(1,4,5)P3 levels and yet sustain
to vacuole
PtdIns(4,5)P2 turnover would be to switch
from a predominantly PLC-mediated PtdInsCell expansion
(4,5)P2 pathway to phosphatase-mediated
Trends in Plant Science
PtdIns(4,5)P2 metabolism as cell enlargement ensues. There are not enough data from
Fig. 3. The dynamic interplay between the stereospecific phosphoinositide (PI) isomers and
either pollen tube or the maize pulvini
cell growth. In addition to generating inositol (1,4,5) trisphosphate [Ins(1,4,5)P3]-induced
to determine which pathway predominates
Ca21 oscillations, PIs have been reported to affect membrane biogenesis, cytoskeletal strucduring cell elongation. Clearly, an increase
ture, ion transport, and RNA synthesis and transport. Temporal shifts in PI metabolism can
occur with growth and development. For example, phospholipase-C-mediated phosin retrograde trafficking would be an
phatidylinositol (4,5) bisphosphate [PtdIns(4,5)P2] turnover provides Ins(1,4,5)P3 for initiatefficient means of meeting the increased
ing signaling and providing a substrate for InsP6 biosynthesis. PtdIns(4,5)P2 turnover by
demands for tonoplast biogenesis and
Ins 59phosphatase might favor cell expansion by increasing retrograde trafficking important
for secretion of new plasma membrane
for increased demands of an expanding tonoplast membrane. Because the PIs influence so
and polysaccharides as the cell enlarges.
many cellular processes, the steady-state profile of the lipid isomers and their metabolites
Although further biochemical and molecushould reflect the physiological status of the cell.
lar studies are essential before we can
determine if PtdIns(4,5)P2-mediated pathways are crucial for regulating retrograde
In addition, phosphorylation of Ins(1,4,5)P3 affects the duration trafficking or cell enlargement in plants, inarguably, inositol
of the Ins(1,4,5)P3 signal. In yeast, InsP6, which results from the phospholipids provide a source of regulatory metabolites that
phosphorylation of Ins(1,4,5)P3, is essential for transport of RNA could coordinate both intracellular and intercellular growth
out of the nucleus53. Although phytate metabolism has been studied responses. The potential shifts in PI metabolism associated with
for years with regard to seed development and embryo for- initial signaling and cell growth are shown in Figure 3.
mation54, there is little information on the roles of the complex
The broader implications of a change in lipid metabolism are
InsP isomers in signaling49 and regulating plant-cell growth. (For intriguing and provide a wealth of material for future studies. We
comparison see http://dir.niehs.nih.gov/dirlst/shears.htm where de- have only touched on the complexities of inositol metabolism in
velopments from the animal literature are continuously updated.)
plants. Virtually nothing is known about the functional role of the
If a sustained increase in Ins(1,4,5)P3 is necessary for a growth scyllo-inositol stereoisomer of PIs (Ref. 58). Until recently, it was
response, then inhibiting PtdIns(4,5)P2 turnover should inhibit always assumed that the inositol lipids identified in plant systems
growth. Molecular studies that overexpress the human PLCd–PH were PtdIns, PtdIns(3)P, PtdIns(4)P and PtdIns(4,5)P2. We now
domain to prevent PtdIns(4,5)P2 hydrolysis or to bind Ins(1,4,5)P3 know that there are PtdIns(3,4)P2, PtdIns(3,5)P2 and possibly some
suggest that PtdIns(4,5)P2 metabolism is essential for pollen-tube as yet uncharacterized stereoisomers that are involved in signaling
growth37. Furthermore, treatment with the PLC inhibitor, U73122, and membrane trafficking. These isomers will need to be considreduced gravitropic bending by 60% and abolished the long-term ered when interpreting past and future data. Inositol conjugates
increase in Ins(1,4,5)P3 levels on the lower side of oat pulvini that can affect hormone-stimulated reactions and inositol metabo(I.Y. Perera et al., unpublished). Although U73122 appears to lites that serve as osmolites within the cytosol of osmotically
be effective at inhibiting recombinant PLC activity in vitro stressed cells must also be considered in the overall pathway54.
and abscisic acid-induced Ca21 increases in guard cells in vivo55,
the mechanism of action and the degree of specificity of the What does the future hold?
compound are not known. Presumably, U73122 inhibits all As we begin to combine biochemical and molecular genetic apisoforms of PI–PLC, and it will take a molecular genetic approach proaches to understand physiological responses on a macro scale
to separate PLC-mediated signaling from growth-related pro- it will be important to characterize the changes in inositol signalcesses such as membrane biogenesis. Caution should be exercised ing as plants mature and respond to changes in nutrient flux and
when interpreting data from other putative PLC inhibitors2. One environmental conditions36. Crucial to interpreting these data will
example is neomycin, a positively charged aminoglycoside and be an understanding of the subcellular localization of the proteins
protein-synthesis inhibitor, which also inhibits PLC-mediated and lipids. To this end, the recent development of useful tools
hydrolysis of PtdIns(4,5)P2 when added at a 1:1 ratio to liposomes such as the fluorescently labeled inositol phospholipids59 and
in vitro, but is ineffective in vivo2,56. Most of the neomycin added GFP-fusion proteins of lipid-binding domains will help to identify
will bind to the cell wall, which is why kanamycin, a less positively microdomains where signaling events are occurring. In addition,
Preceding and during the initial stages of cell growth

256

June 2000, Vol. 5, No. 6

trends in plant science
Reviews
further insight will be gained from genomic analyses, and the
study of the physiology and biochemistry of transgenic plants
with altered PI metabolism. We anticipate that these studies will
reveal essential information about the role of inositol lipids as key
regulators of growth.
In summary, living organisms have exploited membrane lipids,
using them as regulatory metabolites and second messengers. As
sessile organisms, plants need a system to determine whether a
stimulus is transient or sustained before making a commitment to
grow. Once committed, the plant must coordinate a growth
response both within cells and throughout the tissue. The multiple
stereoisomers of the phosphoinositides, provide both water and
lipid-soluble metabolites that can diffuse within and between
cells, creating microdomains that can coordinate and transmit
information. Our challenge is to understand the key events that
orchestrate the symphony of interacting pathways that result in
stimulus-induced growth.
Acknowledgements

We apologize to the authors who could not be cited because of limited reference space. We would like to acknowledge the support
of the National Science Foundation (MCB-9604285), the National
Aeronautics and Space Administration (NAGW-4984) and the
North Carolina Agriculture Research Service to W.F.B., and a
DAAD fellowship HSPIII financed by the German Federal Ministry
of Education, Science, Research and Technology to I.H. We also
acknowledge Bjørn Drøbak, Steve C. Huber, Gloria K. Muday and
Glenda Gillaspy for sharing their unpublished results.
References
1 Drøbak, B.K. et al. (1998) Phosphoinositide kinases and the synthesis of
polyphosphoinositides in higher plant cells. In International Review of
Cytology (Vol. 189) (Jeon, K.W., ed.), pp. 95–130, Academic Press
2 Munnik, T. et al. (1998) Phospholipid signaling in plants. Biochim. Biophys.
Acta 1389, 222–272
3 Corvera, S. et al. (1999) Phosphoinositides in membrane traffic. Curr. Opin.
Cell Biol. 11, 460–465
4 Roth, M.G. (1999) Lipid regulators of membrane traffic through the Golgi
complex. Trends Cell Biol. 9, 174–179
5 Wurmser, E.A. et al. (1999) Phosphoinositide 3-kinases and their FYVE
domain-containing effectors as regulators of vacuolar/lysosomal membrane
trafficking pathways. J. Biol. Chem. 274, 9129–9132
6 Hong, Z. and Verma, D.P.S. (1994) A phosphatidylinositol 3-kinase is induced
during soybean nodule organogenesis and is associated with membrane
proliferation. Proc. Natl. Acad. Sci. U. S. A. 91, 9617–9621
7 Matsuoka, K. et al. (1995) Different sensitivity to wortmannin of two vacuolar
sorting signals indicates the presence of distinct sorting machineries in tobacco
cells. J. Cell Biol. 130, 1307–1318
8 Fruman, D.A. et al. (1998) Phosphoinositide kinases. Annu. Rev. Biochem. 67,
481–507
9 Welters, P. et al. (1994) AtVPS34, a phosphatidylinositol 3-kinase of
Arabidopsis thaliana, an essential protein with homology to a calciumdependent lipid binding domain. Proc. Natl. Acad. Sci. U. S. A. 91,
11398–11402
10 Bunney, T.D. et al. Association of phosphatidylinositol 3-kinase with nuclear
transcription sites in higher plants. Plant Cell (in press)
11 Lease, K. et al. (1998) Challenges in understanding RLK function. Curr.
Opin. Plant Biol. 5, 388–392
12 Wiedemann, C. et al. (1998) An essential role for a small synaptic vesicleassociated phosphatidylinositol 4-kinase in neurotransmitter release.
J. Neurosci. 18, 5594–5602
13 Trotter, M.J. et al. (1998) A genetic screen for aminophospholipid transport
mutants identifies the phosphatidylinositol 4-kinase, Stt4p, as an essential
component in phosphatidylserine metabolism. J. Biol. Chem. 273, 13189–13196

14 Hama, H. et al. (1999) Direct involvement of phosphatidylinositol 4phosphate in secretion in the yeast Saccharomyces cerevisiae. J. Biol. Chem.
274, 34294–34300
15 Walch-Solimena, C. and Novick, P. (1999) The yeast phosphatidylinositol-4OH kinase Pik1 regulates secretion at the Golgi. Nat. Cell Biol. 1, 523–525
16 Godi, A. et al. (1999) ARF mediates recruitment of PtdIns-4-OH kinase-b and
stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex. Nat. Cell Biol. 1,
280–287
17 Lemmon, M.A. et al. (1996) PH domains: diverse sequences with a common
fold recruit signaling molecules to the cell surface. Cell 85, 621–624
18 Stevenson, J.M. et al. (1998) A phosphatidylinositol 4-kinase pleckstrin
homology domain that binds phosphatidylinositol 4-monophosphate. J. Biol.
Chem. 273, 22761–22767
19 Xue, H-W. et al. (1999) A plant 126-kDa phosphatidylinositol 4-kinase with a
novel repeat structure. J. Biol. Chem. 274, 5738–5745
20 Westergren, T. et al. (1999) Phosphatidylinositol 4-kinase associated with
spinach plasma membranes. Isolation and characterization of two distinct
forms. Plant Physiol. 121, 507–516
21 Hinchliffe, K.A. et al. (1998) PIPkins, their substrates and their products: new
functions for old enzymes. Biochim. Biophys. Acta 1436, 87–104
22 Staiger, C.J. et al. (1997) Profilin and actin-depolymerizing factor: modulators
of actin organization in plants. Trends Plant Sci. 7, 275–281
23 Shibasaki, Y. et al. (1997) Massive actin polymerization induced by
phosphatidylinositol-4-phosphate 5-kinase in vivo. J. Biol. Chem. 272, 7578–7581
24 Toker, A. (1998) The synthesis and cellular roles of phosphatidylinositol 4,
5-bisphosphate. Curr. Opin. Cell Biol. 10, 254–261
25 Randazzo, P.A. (1997) Functional interaction of ADP-ribosylation factor 1
with phosphatidylinositol 4,5-bisphosphate. J. Biol. Chem. 272, 7688–7692
26 Wang, X. (1999) The role of phospholipase D in signaling cascades. Plant
Physiol. 120, 645–651
27 Gaidarov, I. and Keen, J.H. (1999) Phosphoinositide-AP-2 interactions required
for targeting to plasma membrane clathrin-coated pits. J. Cell Biol. 146, 755–764
28 Jost, M. et al. (1998) Phosphatidylinositol-4,5-bisphosphate is required for
endocytic coated vesicle formation. Curr. Biol. 8, 1399–1402
29 Cremona, O. et al. (1999) Essential role of phosphoinositide metabolism in
synaptic vesicle recycling. Cell 99, 179–188
30 Majerus, P.W. et al. (1999) The role of phosphatases in inositol signaling
reactions. J. Biol. Chem. 274, 10669–10672
31 Shyng, S-L. and Nichols, C.G. (1998) Membrane phospholipid control of
nucleotide sensitivity of KATP channels. Science 282, 1138–1141
32 Desrivieres, S. et al. (1998) MSS4, a phosphatidylinositol-4-phosphate
5-kinase required for organization of the actin cytoskeleton in Saccharomyces
cerevisiae. J. Biol. Chem. 273, 15787–15793
33 Homma, K. et al. (1998) Phosphatidylinositol-4-phosphate 5-kinase localized
on the plasma membrane is essential for yeast cell morphogenesis. J. Biol.
Chem. 273, 15779–15786
34 Meijer, H.J.G. et al. (1999) Hyperosmotic stress induces rapid synthesis of
phosphatidyl-D-inositol 3,5-bisphosphate in plant cells. Planta 208, 294–298
35 Pical, C. et al. (1999) Salinity and hyperosmotic stress induce rapid increases
in phosphatidylinositol 4,5-bisphosphate, diacylglycerol pyrophosphate, and
phosphatidylcholine in Arabidopsis thaliana cells. J. Biol. Chem. 274,
38232–38240
36 Heilmann, I. et al. (1999) Changes in phosphoinositide metabolism with days
in culture affect signal transduction pathways in Galdieria sulphuraria. Plant
Physiol. 119, 1331–1339
37 Kost, B. et al. (1999) Rac homologues and compartmentalized
phosphatidylinositol 4,5-bisphosphate act in a common pathway to regulate
polar pollen tube growth. J. Cell. Biol. 145, 317–330
38 Mikami, K. et al. (1998) A gene encoding phosphatidylinositol-4-phosphate
5-kinase is induced by water stress and abscisic acid in Arabidopsis thaliana.
Plant J. 15, 563–568
39 Hirayama, T. et al. (1995) A gene encoding a phosphatidylinositol-specific
phospholipase C is induced by dehydration and salt stress in Arabidopsis
thaliana. Proc. Natl. Acad. Sci. U. S. A. 92, 3903–3907

June 2000, Vol. 5, No. 6

257

trends in plant science
Reviews
40 Satterlee, J.S. and Sussman, M.R. (1997) An Arabidopsis phosphatidylinositol 4phosphate 5-kinase homolog with seven novel repeats rich in aromatic and glycine
residues (Accession no. AF01938) (PGR 97–150). Plant Physiol. 115, 864
41 Franklin-Tong, V.E. et al. (1996) Growth of pollen tubes of Papaver rhoeas
is regulated by a slow-moving calcium wave propagated by inositol
1,4,5-trisphosphate. Plant Cell 8, 1305–1321
42 Perera, I.Y. et al. (1999) Transient and sustained increases in inositol
1,4,5-trisphosphate precede the differential growth response in gravistimulated
maize pulvini. Proc. Natl. Acad. Sci. U. S. A. 96, 5838–5843
43 Rosen, E. et al. (1999) Root gravitropism: a complex response to a simple
stimulus? Trends Plant Sci. 4, 407–412
44 Philippar, K. et al. (1999) Auxin-induced K1 channel expression represents an
essential step in coleoptile growth and gravitropism. Proc. Natl. Acad. Sci.
U. S. A. 96, 12186–12191
45 Kaufman, P.B. et al. (1995) Hormones and the orientation of growth. In
Plant Hormones: Physiology, Biochemistry and Molecular Biology
(Davies, P.J., ed.), pp. 547–571, Kluwer
46 Patel, S. et al. (1999) Molecular properties of inositol 1,4,5-trisphosphate
receptors. Cell Calcium 25, 247–264
47 Taylor, C.W. (1998) Inositol trisphosphate receptors: Ca21-modulated
intracellular Ca21 channels. Biochim. Biophys. Acta 1436, 19–33
48 Sanders, D. et al. (1999) Communicating with calcium. Plant Cell 11, 691–706
49 Dasgupta, S. et al. (1996) Interaction of myoinositoltrisphosphate-phytase
complex with the receptor for intracellular Ca21 mobilization in plants.
Biochemistry 35, 4994–5001
50 Brearley, C.A. et al. (1997) Metabolic evidence for PtdIns(4,5)P2-directed
phospholipase C in permeabilized plant protoplasts. Biochem J. 324, 123–131

51 Drøbak, B.K. et al. (1991) Metabolism of inositol (1,4,5) trisphosphate by a soluble
enzyme fraction from pea (Pisum sativum) roots. Plant Physiol. 95, 412–419
52 Gillaspy, G.E. et al. (1995) Plant inositol monophosphatase is a lithiumsensitive enzyme encoded by a multigene family. Plant Cell 7, 2175–2185
53 York, J.D. et al. (1999) A phospholipase C-dependent inositol polyphosphate
kinase pathway required for efficient messenger RNA export. Science 285, 96–100
54 Loewus, F. and Murthy, P.P. (2000) myo-Inositol metabolism in plants. Plant
Sci. 150, 1–19
55 Staxen, I. et al. (1999) Abscisic acid induces oscillations in guard-cell
cytosolic free calcium that involve phosphoinositide-specific phospholipase C.
Proc. Natl. Acad. Sci. U. S. A. 96, 1779–1784
56 Chapman, K. (1998) Phospholipase activity during plant growth and development
and in response to environmental stress. Trends Plant Sci. 3, 419–425
57 Berridge, M.J. et al. (1998) Calcium – a life and death signal. Nature 395, 645–648
58 Pliska-Matyshak, G. et al. (1997) Novel phosphoinositides in barley aleurone
cells, additional evidence for the presence of phosphatidyl-scyllo-inositol.
Plant Physiol. 113, 1385–1393
59 Tuominen, E.K. et al. (1999) Fluorescent phosphoinositide derivatives reveal
specific binding of gelsolin and other actin regulatory proteins to mixed lipid
bilayers. Eur. J. Biochem. 263, 85–92

Jill M. Stevenson, Imara Y. Perera, Ingo Heilmann,
Staffan Persson and Wendy F. Boss* are at the Botany Dept,
North Carolina State University, Raleigh, NC 27695, USA.
*Author for correspondence (tel +1 919 515 3496;
fax +1 919 515 3436; e-mail wendy_boss@ncsu.edu).

Patchy stomatal conductance:
emergent collective behaviour
of stomata
Keith A. Mott and Thomas N. Buckley
Until recently, most scientists have tacitly assumed that individual stomata respond independently and similarly to stimuli, showing minor random variation in aperture and behaviour.
This implies that stomatal behaviour should not depend on the scale of observation. However,
it is now clear that these assumptions are often incorrect. Leaves frequently exhibit dramatic
spatial and temporal heterogeneity in stomatal behaviour. This phenomenon, in which small
‘patches’ of stomata respond differently from those in adjacent regions of the leaf, is called
‘patchy stomatal conductance’. It appears to represent a hitherto unknown type of emergent
collective behaviour that manifests itself in populations of stomata in intact leaves.

D

o stomata act independently regardless of the conduct of
those around them? Or, do neighbouring stomata dictate
their behaviour? Fifteen years ago, most stomatal physiologists would have assumed that stomata act independently, but recent
discoveries now suggest that stomatal behaviour dictated by that of
neighbouring stomata is also possible. Certainly, anyone studying
stomatal responses at the level of the intact leaf would agree that
stomata are complex and unpredictable, withstanding even the most
determined efforts to understand their behaviour. Doubtless, this is
partly because of the many environmental parameters that stomata

258

June 2000, Vol. 5, No. 6

respond to, and to the level of interaction among these responses.
However, it now appears that some of this unpredictability might
result from complex interactions between stomata, leading to a
form of emergent behaviour that is inherently difficult to predict.
This phenomenon, which we term ‘patchy stomatal conductance’,
has been the subject of several recent exhaustive reviews1–3.
This review will therefore focus on three aspects of the issue:
hydraulic interactions as a mechanism for patchiness, the importance of scale in stomatal dynamics, and patchiness as an emergent
collective property of stomata.

1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01648-4