Download 5. Possible outcomes and significance

Justification of the collaborative proposal
INTERACTION BETWEEN AUXIN AND LIGHT SIGNALING IN SEEDLING GROWTH AND DEVELOPMENT
OF LEAF ANGLE IN CORN
Abbreviations: ABP = auxin-binding protein; B = blue light; BM = basal medium; FR = far-red light; D = dark; DIDS = 4,4’diisothiocyanato-stilbene-2-2’-disulfonic acid; IAA = indole-3-acetic acid; IAA-94 = R(+)-methylindazone;indanyloxyacetic acid-94; MES =
2-[N-Morpholino]ethanesulfonic acid; NAA = 1-naphthalene acetic acid; NPA=N-1-naphthylphthalamic acid; NPPB = 5-nitro-2-(3phenylpropyl amino)-benzoic acid; PAT = polar auxin transport; PCIB = 2-(4-chlorophenoxy)-2-methyl-propionic acid; R = red light; SITS
= 4-acetomido-4’-isothiocyanato-stilbene-2-2’-disulfonic acid; TEA+ = tetraethylammonium; TIBA = 2,3,5-triodobenzoic acid; W = white
light; WT = wild-type; 9-AC = anthracene-9-carboxylic acid.
1. INTRODUCTION
Plant responses to neighbor proximity.
Plants are able to sense neighbor proximity by a mechanism based on their ability to detect changes in R/FR
ratio within the canopy, while R/FR signals are perceived by different phytochromes and transduced to trigger
morphological changes in plants (Smith 1982, 1995). It was shown that the expression of the Athb-2 gene is
reversibly regulated by changes in the R/FR ratio, and that overexpression of Athb-2 induces processes is
regulated by auxin (Carabelli et al. 1996; Steindler et al. 1999). The authors also suggest that a FR-rich light
regime produces a reorientation of the auxin transport stream (Morelli and Ruberti 2000, 2002, see for review).
The role of auxin, light, and molecular links between light and auxin signaling pathways in regulation of shoot
and leaf growth were recently reviewed (Tian and Reed 2001; Chen 2001; Dengler and Kang 2001; Halliday
and Fankhauser 2003). To avoid shade, plants respond to close neighbor proximity with morphological
changes such as stimulation of elongation growth, reduced branching, and a redistribution of leaves to the top
of the canopy (Morgan and Smith 1979). Grass plants, including corn, respond to dense planting by holding
their leaves more erect. However, how light and auxin interact in the regulation of leaf angle in corn remains to
be resolved. Modern hybrids of corn (Zea mays L.) have been selected for their ability to maintain production in
dense plantings, i.e. where there are likely to be low R/FR conditions. One of the interesting phenotypic
characters of modern hybrids is their more upright leaf in comparison with the older, density sensitive varieties.
It is believed that maize upright leaves may result in tolerance of modern corn hybrids to neighbors, and higher
yield in dense planting. On the basis of our previous results (Fellner et al. 2003) we hypothesize that
development of upright leaves in 3394 plants is due to a reduced responsiveness to auxin, caused by reduced
numbers of auxin receptors. Our hypothesis may be supported by our recent results on analysis of auxininduced expression of ABP1 and ABP4 genes in 307 and 3394 hybrids (Fellner et al., 2004 submitted), and by
our preliminary results on analysis of loss-of-function mutants in ABP1 and ABP4 genes (see below).
Light-regulated leaf growth.
Leaf growth is regulated by light acting via phytochrome and other photoreceptor signaling pathways (Downs
1955; Van Volkenburgh and Cleland 1980; Dale 1988). Interestingly, even in fully greened leaves, cell
expansion is regulated by non-photosynthetic photosystem including phytochromes (Smith 1995; Stahlberg
and Van Volkenburgh 1999). Cellular mechanisms of light-stimulated leaf expansion involve enhanced proton
efflux from mesophyll and epidermal cells, and in epidermal cells blue and red light increase proton efflux from
by separate mechanisms (Van Volkenburgh et al. 1990; Van Volkenburgh 1999, see for review). It has been
proposed that blue light stimulates the proton efflux by direct interaction between blue light receptor and the
pump (Elzenga 1997). Red light-induced stimulation of the proton pump may involve indirect modulation of
calcium and potassium channels (Staal et al. 1994; Elzenga et al. 1997). Unlike blue light, red-induced
acidification is inhibited by far-red light (Staal et al. 1994). A calcium-dependent K+ channel has been described
in growing epidermal cells of pea (Elzenga and Van Volkenburgh 1994). Recently, Stiles et al. (2003) found
that for still dividing cells at the base of tobacco leaves, light-stimulated growth depends on K+ uptake, and is
inhibited by the potassium channel blocker TEA. It was also reported that in Populus light stimulates leaf
expansion via developmentally regulated ion fluxes across the plasma membrane (Stiles and Van Volkenburgh
2002).
Auxin-regulated growth.
Auxin regulates three essential events in cell development: division, and elongation. It has been demonstrated
that endogenous as well as externally supplied IAA stimulates growth of intact maize coleoptiles (Baskin et al.
1986; Iino 1995; Haga and Iino 1998). Epstein et al. (1980) reported that corn kernels supply coleoptile tip with
auxin in a conjugate form from which free IAA is released by specific enzymes and moved from the coleoptile
tip to the elongation zone via polar transport (Goldsmith 1977; Lomax et al. 1995). Auxin-induced coleoptile
growth is accomplished by cell elongation whereas the number of cells in the coleoptile remains constant
(Philippar et al. 1999). The relationship between the IAA level and the growth rate is complex since the level of,
and sensitivity to, auxin is regulated during plant growth (Trewavas 1981, Hanson and Trewavas 1982;
Trewavas and Cleland 1983; Hall et al. 1985; Haga and Iino 1997, 1998; Zazimalova and Napier 2003, see for
1
review). The underlying mechanisms of cell elongation still remain a subject of debate, while distinct
interpretations of auxin-induced growth have been proposed (Hager et al. 1971; Rayle and Cleland 1972, 1992;
Hoth et al. 1997; Claussen et al. 1997; Philippar et al. 1999; Bauer et al. 2000). A model for auxin-induced
growth in six phases has been proposed (Becker and Hedrich 2002). Under this model, auxin action is initiated
in phase 2, which involves the transcription of very early genes such as ZMK1 and H+-ATPases. It results in
enhanced pumping, hyperpolarization of the membrane, acidification of the apoplast, and thereby activation of
voltage- and proton-activated Kin+ channels (e.g. ZMK1). The increase in turgor and cell wall loosening drives
cell expansion, which is typical for following phase 3, and is explained by the acid-growth theory (Becker and
Hedrich 2002, Hager 2003, and references therein). In contrast to excised segments, in intact plants on a
longer time scale, growth of various plant organs is inhibited by exogenous auxin (Boerjan et al. 1995; King et
al. 1995; Fellner 1997; Thomine et al. 1997). Data of several reports suggest the involvement of anion
channels in auxin signal transduction in the auxin-induced inhibition of growth (Marten et al. 1991;
Zimmermann et al. 1994; Keller and Van Volkenburgh 1996; Thomine et al. 1997).
Interaction of light with auxin during elongation.
Plants growing in the light are shorter than those developing in dark or shade, and the inhibition of elongation
during photomorphogenesis is nicely evident especially very early after seed germination. The mechanism of
light-induced growth inhibition is not yet fully understood. Several distinct mechanisms are involved, including
light-induced changes in hormone homeostasis. Various studies have shown correlation between light
responses and auxin levels or polar auxin transport (PAT) (Tian and Reed 2001, see for review). It has been
shown that light reduces intensity of PAT in etiolated coleoptile segments (Huisinga 1964, 1967; Naqvi 1975;
Iino 1982a; Fellner et al. 2003) and reduces the content of free IAA in etiolated maize seedlings (Bandurski et
al. 1977; Iino 1982b). An important role of light and PAT in photomorphogenesis of corn seedlings is also
deduced from the knowledge of how light-induced decrease in auxin transport is involved in elongation of
mesocotyl (Van Overbeek 1936; Vanderhoef and Briggs 1978; Iino 1982a; Jones 1990; Jones et al.1991;
Barker-Bridgers et al. 1998). The interaction of auxin with light may also involve light-reduced responsiveness
to auxin. Jones et al. (1991) reported that R reduces the abundance of auxin-binding protein 1 (ABP1), a
putative auxin receptor that controls cell expansion (Jones et al. 1998). It was also reported that expression of
ABP1 in maize seedlings is much less in light- than in dark-grown plants (Im et al. 2000).
Auxin receptors – auxin binding proteins.
The auxin signal transduction pathway remains unclear although molecular genetic studies resulted in the
identification of a number of transcription factors and novel proteins involved in protein degradation by the
ubiquitin-proteosome pathway (Kepinski and Leyser 2002, see for review). One of the primary steps of auxin
signaling is binding of auxin to a receptor, an auxin-binding protein (ABP). Many such proteins have been
identified (Jones 1994; Napier et al. 2002). Among them ABP1 is a high-affinity auxin receptor mediating cell
expansion (Jones et al. 1998). Constitutive over-expression of ABP1 in maize cells resulted in larger cells and
this effect was auxin dependent, consistent with ABP1 having an auxin receptor function (Im et al. 2000).
Several studies demonstrated that ABP1 acts at the plasma membrane (Barbier-Brygoo et al. 1989; Leblanc et
al. 1999). On the other hand, the predominant localization of ABP1 is in the endoplasmic reticulum lumen
(Jones and Herman 1993). This could mean that only a small percentage of the total pool is needed to be
active at the plasma membrane (Klämbt 1990) suggesting that the amount of ABP is not the rate-limiting
component and that the total ABP abundance is irrelevant to its action. This is consistent with results of Im et
al. (2000). In order to dissect the role of ABP1 and other members of the ABP gene family, the authors used a
reverse genetic approach of screening an F1 population of maize plant mutagenized with the Mutator elements
(Mu). They isolated and characterized loss-of-function mutants in maize ABP1 and ABP4 genes, and revealed
that the mutants did not show obvious phenotypic aberration. The authors suggest functional redundancy in
ABP gene family in maize and an existence of compensatory mechanisms because in the abp4 mutant, ABP1
protein level was significantly elevated relative to WT (Im et al. 2000).
In addition to cell expansion auxin induces cell division. Analysis of the alf4-1 mutant in Arabidopsis
provided evidence that the two auxin-triggered responses are distinctly separable (Celenza et al. 1995). The
alf4-1 mutant was insensitive to auxin-promotion of lateral root initiation, but root elongation was still inhibited
by auxin in the mutant. In tobacco BY-2 cells auxin at low concentration induced elongation without cell
division, whereas at high concentration, BY-2 cells divided (Hasezawa and Syono 1983; Nagata et al. 1992). It
raises the question of how auxin at different concentrations can trigger different responses. It could be that the
auxin receptor has two different binding sites, high-affinity for elongation and low-affinity for division. ABP1 has
only a single high-affinity binding site, and thus does not fit this criterion. In addition, it was shown that ABP1
antisensed line lacks auxin-induced cell elongation but retains auxin-induced cell division (Chen et al. 2001 a,
b). So, it is quite possible that an ABP1 mediates only cell elongation, whereas another unknown receptor
directing cell division exists. Evidence was provided that auxin-regulated cell division through a low-affinity
auxin receptor involves a heterotrimeric G-protein (Zaina et al. 1990; Ishida et al. 1993; Christensen et al.
2000).
2
2. PREVIOUS RESULTS (Fellner et al. 2003; Fellner et al. 2004, submitted)
Modern corn hybrids developing upright leaves have been selected for their ability to maintain productivity in
dense planting. We have previously tested the possibility that one physiological consequence of the selection
of the modern hybrid, 3394, for increased crop yield includes changes in response to light and auxin.
The modern corn hybrid 3394 is resistant to light-induced growth inhibition and to auxin.
We found that light-induced inhibition of elongation in etiolated corn seedlings was relatively less in 3394 than
in an older hybrid 307. However, it did not correlate with the similar extent of the reduction of endogenous IAA
and PAT by R or FR in etiolated 307 and 3394 seedlings (Fellner et al. 2004, submitted). Light also reduces
auxin receptivity by reduction of auxin-binding proteins (ABPs), putative auxin receptors (Jones et al. 1991;
1998; Im et al. 2000). We hypothesized that the two hybrids differ in their receptivity (meaning number of
receptors; Firn 1986) rather than in affinity to auxin (meaning receptor affinity) (Fellner et al. 2004, submitted).
Recently, we found that 3394 cells were insensitive to auxin- and light-induced hyperpolarization of the plasma
membrane, and expression analysis of ABP1 and ABP4 genes revealed upregulation of ABP4 by auxin and
light in 307, but not in 3394. Altogether, our results suggest that auxin interacts with light in regulation of growth
and development of corn seedlings, and we showed that the modern corn hybrid is affected in this interaction.
We hypothesize that ABP4 may integrate the auxin and light signaling pathways in corn seedlings by negative
regulation of ABP1 (Fig. 1). We also hypothesize that in the modern corn hybrid 3394, ABP4 is “mutated”,
which affects the interaction between ABP4 and ABP1, and thus it results in the 3394 phenotypes (Fellner et al.
2004, submitted).
Fig. 1. A working model showing the effect of auxin, light, and their interaction on
expression of ABP4 gene, its interaction with ABP1, and possible role of the ABPs
in seedling growth.
In etiolated seedlings expression of ABP4 is low, which results in a high amount
of ABP1 protein, high binding of endogenous auxin, and consequently elongation
of etiolated seedlings (not shown). Light or exogenous auxin can overexpress ABP4.
In addition, light reduces amount of endogenous auxin and intensity of PAT. High
ABP4 expression can negatively regulate the level of ABP1 protein. This results in
low binding of endogenous auxin (relative to dark and absence of exogenous auxin),
and leads to less growth of intact seedlings exposed to exogenous auxin or light.
We hypothesize that in the modern corn hybrid 3394, the ABP4 is “mutated”, which
would result in only basal transcription of the gene in etiolated seedlings, and in reduced
responsiveness of the gene to exogenous auxin and light. The 3394 plants can
therefore respond much less than 307 to various stimuli, which involve ABP4 and ABP1.
Arrows and T-bars represent positive and negative effects, respectively. Hormone
binding is shown by a line ending with a dot.
Exogenous
auxin
ABP4
ABP1
Growth
Light
[IAA]
+
intensity
of PAT
Auxin and light regulate development of leaf angle in corn.
In the corn leaf, at the junction of the blade and the sheath, a specialized structure is formed, called the auricle
(Fig. 2). The growth of the auricle at the edges thus allows the blade to bend down (outward) and provides for
its free lateral movement without tearing (Kiesselbach 1980; Freeling and Lane 1994; Howell 1998). We
reported that auricle
growth was proportionally associated with the size of leaf declination,
which is to say the smaller leaf declination occurred for leaves with
Leaf
declination
shorter auricle dimension at the blade margin. Etiolated corn
seedlings developed vertical leaves relative to light-grown seedlings,
while 3394 leaves were even more upright than in 307. We found that
NPA reduces auricle growth and thus makes leaves more vertical
and, as for elongation responses, 3394 plants were resistant to the
Blade
Auricle
effect of NPA. The results support our hypothesis that development of
length
more upright leaves in etiolated 3394 plants is a consequence of
Auricle
reduced responsiveness of leaf tissues to auxin. We found that light
Sheath
promotes auricle growth and thus makes leaves more horizontal, and
that NPA can abolish the light-induced auricle growth and
proportionally reduces leaf declination in 307. It suggests that light
regulates auricle growth and leaf declination by controlling PAT.
Fig. 2. Diagram showing leaf declination (angle)
and auricle length measured in corn seedlings.
The fact that light inhibits PAT similarly in etiolated 307 and 3394 seedlings, but leaves in the modern hybrid
are more erect than in the older line could reflect reduced responsiveness of etiolated 3394 seedlings to auxin,
in comparison with 307. It is consistent with our hypothesis that 3394 may have a defect in an auxin receptor.
3
Leaf angle (degrees)
40
The abp mutants show changes in leaf angle development
(Fellner, unpublished data).
To support our hypothesis that auxin- and light-induced development
20
of leaf angle is mediated by auxin-binding proteins, we characterized
10
abp mutants (Mutator-tagged lines) with respect to leaf angle
development. In contrast to observations of Im et al. (2000) (see
0
Introduction), we found strong phenotypic differences between WT
and single abp1, abp4, or double abp1/abp4 mutants. Leaf angle in
abp1 mutants was less than in WT, whereas abp4 mutants had
Genotype
leaves with greater declination than WT (Fig. 3). Im et al. (2000)
found that ABP1 was detected in WT, not in the abp1 mutant, but in
4-7 times the level in abp4 mutant indicating that elimination of the ABP4 gene activates ABP1 expression.
Altogether, these results support our hypothesis that differential leaf angle in 307 and 3394 may have a basis in
differential amount of or activity of ABPs. However, the fact that declination of leaves in the double mutant
abp1/abp4 was greater than in WT (Fig. 3), while ABP1 was not detected in the double mutant (Im et al. 2000)
makes the role of ABPs and their cross-talk in leaf angle development puzzling.
double B2/K1
double B11/K1
abp4-K5
abp4-B2/K1
abp1-B2/K1
WT
abp1-B2
30
Fig. 3. Leaf angle of the abp1, abp4 and double
abp1/abp4 mutants, and corresponding WT grown
in the greenhouse. Leaf angle, measured as a
declination from vertical was determined with a
protractor.
3. OBJECTIVES
The auxin-binding protein ABP1 appears to function as a receptor in various signal transduction pathways.
However, it is unlikely that ABP1 mediates all auxin response pathways because a number of auxin-mediated
responses are not affected by the application of anti-ABP1 antibodies. It is supported by the fact that other
members of the ABP gene family have been identified, such as ABP4, ABP5, or ABP57, while their role in plant
growth and development remains to be determined. The long-term objective of our work is to contribute to
understanding the role of auxin and light in growth and development of leaves. Our previous data indicate that
auxin, in interaction with light, contributes to regulation of leaf angle development in corn. Preliminary
phenotypic characterization of the abp mutants suggests that ABP1 and/or ABP4 may be involved and interact,
with each other, in development of leaf angle. To the best of our knowledge, there has been no
investigation so far of the mechanism(s) by which auxin may regulate leaf angle. Therefore, in this
project, we are focusing on study of the role of the ABP genes in auxin- and light-mediated
development of leaf angle in corn. This project will test several basic hypotheses:
1) Differential amounts of ABP transcripts and/or differential capacities/activities of auxin-binding proteins in the
abp mutants cause differential leaf angle development, i.e. ABPs interact in regulation of leaf angle
development in corn.
2) ABPs may mediate and/or interact in mediating rapid auxin-induced electrical responses such as activity of
H+-ATPase, K+ and anion channels, involved in regulation of leaf auricle growth.
3) Differences in phenotypic traits between modern and older corn hybrids, including leaf angle development,
are caused by differential light-dependent activities of ABPs in target tissues, and consequently by altered
auxin- and light-induced growth responses.
To investigate whether and how ABPs are involved in regulation of leaf angle development, we will measure
leaf angle in the abp mutants and estimate how development of leaf angle correlates with levels of ABP
transcripts and/or with activities of corresponding proteins. The relationship between ABPs and auxin- and
light-induced growth responses in abp mutants will be determined. To investigate whether and how ABPs
interact in mediating rapid auxin-induced electrical responses, protoplast swelling and apoplastic acidification
as a function of both exogenous auxin and activity of ABPs will be studied in the abp mutants and WT plants.
In addition, expression of the ZmK1 gene encoding K+ inward channels will be determined in the abp mutants
in response to exogenous auxin. In parallel, effect of inhibitors of the plasma membrane H +-ATPase, inhibitors
of K+-channels, and anion channel blockers on auxin-induced growth responses in abp mutants will be tested.
On the basis of previous results, we plan to compare promoter structures of ABP4 gene in 307 and 3394
hybrids, and to analyze ABP1 amounts and activities in seedlings of 307 and 3394 hybrids. Expression analysis
of ABP5, another member of the ABP gene family, in the hybrids and abp mutants will be performed to
elucidate ABP5’s role in plant growth and development and in possible interaction with ABP1, ABP4, and light.
Expression of ABP1, ABP4, and ABP5 genes will also be studied in phytochrome-deficient mutant elm 1 in
maize (Sawers et al. 2002) to investigate possible role of phytochromes in ABP-mediated growth responses.
Expression of maize phytochrome genes PHYA, PHYB, PHYC (Sheehan et al. 2004) will be compared in the
modern and older corn hybrids to find out whether differential light-induced responses in 307 and 3394 may
have basis in potentially differential levels of phytochrome transcripts.
4
4. EXPERIMENTAL
Maize (Zea mays L.) strains:
a) The Pioneer Hi-Bred collection of F1 maize (Zea mays L.) plants mutagenized by means of the Robertson’s
Mutator transposable element system (Bennetzen 1996) was screened for Mu-containing alleles of ABP1 or
ABP4 genes (Im et al. 2000). Following abp mutants have been selected and will be used in our project: single
mutants abp1 (B2 alleles and B2/K1 alleles), abp4 (B2/K1 alleles and K5 alleles), double mutants abp1abp4
(B2/K1 background and B11/K1 background), and WT, i.e. near isogenic line. All mutant seeds are a gift from
Alan Jones (The University of North Carolina).
b) Hybrids 307 (a double-cross), 3306 (a single-cross), and 3394 (a single-cross) were provided by Pioneer HiBred, Intl., (Des Moines, Iowa); commercially released in 1930, 1960, and 1990, respectively.
c) Phytochrome-deficient mutant elm1 (elongated mesocotyl1) (Sawers et al. 2002) of maize will be kindly
provided by Thomas P. Brutnell (Cornell University, Ithaca, NY).
Plant growth conditions. Corn plants will grow in soil (in vivo) or on the basal medium (in vitro) similarly as
described in Fellner et al. (2003).
A) ANALYSIS OF LOSS-OF-FUNCTION MUTANTS IN ABP1 AND ABP4 GENES.
According to our hypothesis that altered growth and responses to auxin and light in 307 and 3394 seedlings
are a consequence of differential amount of ABPs and of differential expression of ABP4, we expect that abp
mutants will exhibit altered auxin- or light-induced growth responses in comparison to WT. We will therefore
study growth of abp mutants along with corresponding WT in dark and light and in the absence and presence
of exogenous auxin.
Study of light- and auxin-related growth responses, and determination of activity of PAT.
Growth responses of intact plants. Seedlings will germinate and grow in conditions in vitro in dark or
light (R, FR) in the presence or absence of NAA, NPA, or PCIB as described in Fellner et al. (2003). After 7
days, lengths of coleoptile, mesocotyl, and roots will be measured with a ruler. Elongation zones of coleoptile,
mesocotyl, and whole leaf auricle will be examined for number, size, and orientation of epidermal cells using
either acrylic impressions of epidermis or peels prepared as described in Gallagher and Smith (1999).
Segment elongation. Growth of segments excised from mesocotyl and leaves will be monitored as
described in Fellner et al. (2003). Briefly, segments will be excised from etiolated mesocotyl and leaf auricles
and incubated in the absence or presence of NAA in dark for 24 hrs. Afterwards, lengths of the segments will
be measured with a ruler.
Polar auxin transport assay. Segments excised from mesocotyl of etiolated seedlings grown in vitro,
and from leaf sheath and blade of etiolated plants grown in conditions in vivo will be used. The effect of light
(R, FR) and NPA on activity of PAT will be studied as described by Fellner et al. (2003).
Leaf angle development in intact plants and determination of ABP transcripts and amount of ABP1.
Resistance to auxin and light of young 3394 seedlings was associated with development of upright leaves in
etiolated as well as light-grown seedlings, and with the lack of auxin- and light-reduced upregulation of ABP4.
We hypothesize that development of more vertical leaves in 3394 is a consequence of a defect in ABP4
expression, and thus altered interaction between ABP4 and ABP1 (Fellner et al. 2004, submitted). Here we will
measure development of leaf angle in abp mutants along with expression of ABP1 and ABP4 genes especially
in the leaf auricle. The amount of ABP1 only will be determined since there is no specific antibody against
ABP4 (A. Jones, personal communication). Since light enhances leaf angle (Fellner at al. 2003), but reduces
amount of ABP1 transcript and protein (Im et al. 2000) we would like to know how light affects ABPs
expression and ABP1 amount in leaf auricle, and how it relates to leaf angle development. In addition,
expression of ABP5 gene (Schwob et al. 1993) in the abp mutants will be studied in relation to leaf angle
development.
Leaf growth and angle development. Plants will grow in soil in dark, under R, FR, or under W. Leaf
growth and leaf angle development in intact seedlings will be measured as described in Fellner et al. (2003).
RT-PCR will be carried out using a one step RT-PCR kit according the manufacture’s instructions
(InVitrogen). Total RNA will be extracted from different tissues using an RNA extraction kit (RNeasy Plant
Minikit) supplemented with RNase-free Dnase during extraction. First-strand cDNA will be synthesized from
total RNA (1g) using oligo (dT)20 primer and using ThermoScript RT-PCR System. cDNA will be amplified with
forward and reverse primers that specifically amplify the ABP1, ABP4, and ABP5 genes (Fellner et al. 2004,
submitted). An internal standard will be carried out with primers that amplify a constitutively expressed maize
actin1 gene (ACT1) (Shah et al. 1983).
Northern blot analysis. The methods described in Im et al. (2000) will be used. Isolated total RNA (10
g) will be fractioned on 1.2% agarose gels in the presence of 3% formaldehyde and transferred to Nytran
membrane. Hybridization to ABP1-, ABP4-, and ABP5-gene specific probes and washings will be done as
described in Cheng et al. (1996).
Western blot analysis. The methods described in Im et al. (2000) will be used to determine level of
ABP1. Microsomal proteins of auricles will be extracted as described (Shimomura et al. 1999; Chen et al.
5
2001a). Proteins will be run in a SDS-polyacrylamide gel, electroblotted onto nitrocellulose and incubated with
maize anti-ABP1 polyclonal antibodies (Jones and Herman 1993; Napier et al. 1988). ABP1 will be detected
using goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase. The blots will be visualized
using the SuperSignal® West Pico chemiluminescent substrate (PIERCE).
Monitoring of internal auxin levels.
Content o endogenous free auxin (IAA) will be measured in elongation zones of coleoptile, mesocotyl, and
whole leaf auricle in various hybrids at various treatments (light, auxin). For routine screening IAA will be
extracted and determined using HPLC with fluorimetric detection as described in Zazimalova et al. (1995).
Data will be verified by LC-MS.
Effect of blockers of K+ channels or anion-channels on auxin-controlled growth responses of abp
mutants.
Auxin-induced growth of coleoptiles strictly depends on the presence of potassium and is suppressed by K+
channel blockers (e.g. TEA+) suggesting that inwardly rectifying K+ channels play a crucial role in auxin action
(Claussen et al. 1997; Philippar et al. 1999). Antibodies directed against ABP are able to mimic K+-channel
activation in guard cells (Thiel et al. 1993). In contrast to isolated segments, in intact plants of various species
exogenous auxin inhibits elongation, while anion-channel blockers (e.g. 9-AC) could suppress the inhibitory
effect of auxin suggesting a role of anion channels in auxin signaling (Thomine et al. 1997). Using the abp
mutants we will study whether ABP1 and/or ABP4 are involved in auxin-regulated growth by engaging activity
of K+ and anion channels.
Etiolated seedlings of the abp mutants will grow in conditions in vitro in the absence or presence NAA
and TEA+, or NAA and 9-AC. As necessary, alternate blockers including Ba2+, DIDS, SITS, IAA-94, NPPB, or
niflumic acid may also be used. Coleoptile and mesocotyl elongation, or root formation and elongation, will be
measured as described above. Auxin-induced growth of excised coleoptile and mesocotyl segments will also
be investigated in the absence or presence of the channel blockers as described above.
Study of auxin-induced swelling.
Protoplasts of corn coleoptile and Arabidopsis hypocotyls respond to auxin with a rapid change in volume and
this process depends on plasma membrane K+ channel (Keller and Van Volkenburgh 1996). Steffens et al.
(2001) provided evidence that the auxin signal for protoplast swelling is perceived by extracellular ABP1. Using
abp mutants we will investigate whether ABP4 may interacts with ABP1 in auxin-induced swelling, and if the
processes involve activity of K+ channels. Special interest will be focused on response of protoplasts isolated
from leaf auricle tissues, and correlation among auricle growth, leaf angle development and ability of auxin to
induced swelling in abp mutants.
Protoplasts will be isolated from coleoptile, mesocotyl, and auricle tissues as described by Steffens et
al. (2001). The swelling experiments will take place in the washing solution. The osmolality of all solutions
employed will be adjusted to an osmolarity found suitable for swelling. Protoplast images will be acquired using
a microscope and an automatic camera. Effects of NAA and TEA+ on protoplast volume will be investigated by
adding the effectors to the bath medium.
Auxin-induced expression of ZmK1 gene in etiolated segments and intact seedlings.
Auxin-induced cell elongation during sustained growth rates requires activation of the transcription of the K+
channel gene ZmK1 encoding a member of the AKT1 subfamily of plant K+ channels (Philippar et al 1999).
Auxin increases the channel density in the plasma membrane and promotes K+ uptake (Bauer et al. 2000).
However, evidence was not provided that ABPs mediate auxin-induced expression of the ZmK1 gene.
Growth experiments. Auxin-induced growth of segments in coleoptile and mesocotyl of etiolated
seedlings will be studied as described above. Secondly, growth of intact mutant seedlings in vitro in the
absence or presence of auxin will be measured. Finally, etiolated corn seedlings grown in soil will be sprayed
with NAA, and leaf angle and growth of auricles will be determined. Afterwards, incubated segments, in vitro
seedlings, and leaf auricles will be collected for isolation of total RNA. RNA will be used in RT-PCR
experiments or Northern blot analyses.
Gene expression. ZmK1 gene specific forward and reverse primers will be used for RT-PCR as
described above. Northern blot analysis will be performed according to standard protocols with a cDNA probe
specific for the ZmK1 gene (Philippar et al. 1999; Bauer et al. 2000).
Effects of auxin on apoplastic acidification in etiolated abp mutants.
Cell elongation in the coleoptile and mesocotyl of maize seedlings is controlled by auxin. In the target cells,
auxin stimulates activity and synthesis of the plasma membrane H +-ATPase resulting in membrane
hyperpolarization and apoplastic acidification (Felle et al. 1991; Hager et al. 1991; Lohse and Hedrich 1992).
ABPs were shown to activate the membrane H+-ATPase in the presence of auxin (Rűck et al. 1993; Steffens et
al. 2001; Kim et al. 2001).
Auxin-induced apoplastic acidification will be measured in abp mutant tissues to find out the role of
ABP1 and ABP4 in this response. Correlation with auxin-induced growth responses of the mutants will be
6
traced with special interest focused on leaf auricle tissues. Apoplastic pH as a function of exogenous auxin will
be measured using a combination pH electrode as described in Stahlberg and Van Volkenburgh (1999).
Measurement of auxin-induced electrical responses in etiolated mutant seedlings.
Auxins rapidly initiate electrical events on the plasma membrane thought to be involved in early auxin action
(Blatt and Thiel 1993). Furthermore, as discussed above, indirect evidence suggests that ABP1 mediates at
least some of these events. In order to dissect the role of ABP1 and ABP4, we will investigate changes in
membrane potential and activity of K+ channels in abp mutants by using conventional electrophysiological
methods (Stahlberg and Van Volkenburgh, 1999) and patch clamp techniques (Hamill et al. 1981; Bauer et al.
2000), respectively. In the experiments excised tissues from the etiolated mutant seedlings will be used,
including coleoptiles and mesocotyls from 7-d-old seedlings grown in vitro and auricle tissues from 2nd and 3rd
leaves in 14-d-old plants grown in conditions in vivo.
Membrane potential will be measured with conventional electrophysiological glass microelectrodes,
filled with 300 mM KCl, inserted by micromanipulator into epidermal or cortical cells of coleoptile, mesocotyl,
and auricle. Tissues will be mounted into a perfusion chamber on a microscope stage, which contains the
reference electrode. The mounted tissues will be continuously perfused with the incubation solution (10mM
KCl, 1mM CaCl2, and 1mM MES/BTP (pH 6.0) and the microelectrode will be inserted under microscopic
control. The Ag/AgCl reference electrode will be connected to the bath solution with a liquid junction reference
electrode and filled with 300 mM KCl (Stahlberg and Van Volkenburgh, 1999). The microelectrodes will be
pulled from borosilicate glass capillaries (Kwik-Fil; World Precision Instruments, Saratosa, Fl) and will have tips
with resistance ranging between 10 and 30 M, and tip potential of less than 10 mV. After impalement, cells
will be required to show a steady Vm value for at least 30 min before various effectors will be applied. The effect
of NAA, TEA+, and ortho-vanadate on membrane potential will be tested. Fussicoccin application will be
included as a positive control (apoplastic acidification).
Patch clamping in the whole-cell mode will be used for study of changes in activity of K+ channels. Ion
currents across the plasmamembrane will be measured on corn protoplasts using established patch clamp
electrophysiological methods (Hamill et al. 1981: Elzenga and Van Volkenburgh 1997a, b; Philippar et al. 1999;
Bauer et al. 2000). For whole-cell measurements, the standard bath and pipette solution will contain 30 mM Kgluconate, 20 mM CaCl2, 1 mM MgCl2, and 10mM mes/Tris (pH 5.6) (600-630 mOsmol) and 150 mM Kgluconate, 2 mM MgCl2, 10 mM EGTA, 2 mM MgATP, and 10 mM Hepes/Tris (pH 7.2) (630-650 mOsmol),
respectively. Protoplasts of corn coleoptile, mesocotyl, and auricle cells will be studied in a whole-cell
configuration, whereby the cytoplasm is perfused with pipet solution, and plasmamembrane is exposed to
known concentrations of ions on both sides; pipet solution on the inside, and bath solution on the outside.
Voltage steps will be applied and current passing across the membrane measured so as to construct
current/voltage (I/V) relationships. Currents will be attributed to ion-specific channels by ion-substitution
experiments (for example substitution of K-gluconate will remove any current contributed by Cl-), by calculation
of reversal potential as determined from I/V relationships and Nernst potentials of ions in the solutions, tailcurrent analysis (Elzenga and Van Volkenburgh 1997a), and by the effect of ion-specific channel inhibitors as
described above. The effect of NAA and TEA+ on whole-cell currents will be tested.
Protoplast isolation will be carried out by a method described by Wang and Iino (1997). Protoplasts will
be prepared from cortical and epidermal cells from elongation zones (see above) of coleoptile, mesocotyl, and
from leaf auricle tissue.
B) PHYSIOLOGICAL, GENETIC, AND MOLECULAR ANALYSIS OF OLDER AND MODERN CORN
HYBRIDS.
In this project part older hybrids, 307 and 3306, and the modern hybrid 3394 will be used as experimental
plants.
Analysis of ABP4 gene promoter in the corn hybrids.
Our previous results (Fellner et al. 2004, submitted) let us to hypothesize that the promoter of ABP4 may be
altered or “mutated” in the modern corn hybrid 3394. Since the promoter sequence of ABP4 gene in corn is
known (Schwob et al. 1993), we will check whether 307 and 3394 differ in the sequence of the ABP4 promoter.
For that, we will design specific primers and perform PCR to amplify the promoter region of ABP4 gene. The
PCR product will be cleaned using Qiagene PCR Cleaning kit and sequenced directly.
Determination of ABP1 protein in etiolated and light-grown seedlings.
Plants will be grown in dark, under R, FR, or W. Analysis of ABP1 will be performed by Western blot as
described above for abp mutants.
Activity of auxin-binding proteins.
Plasma-membrane-enriched preparation will be prepared from various maize hybrids showing differences in
leaf angle development, and in response to light and auxin, and auxin-binding activities/capacities will be
determined in these preparations by classical radioligand-binding assay (Ray et al. 1977). Ultrafiltration will be
used for the separation of free from bound ligands (Zazimalova and Kutacek, 1985). The experiments will be
7
performed in collaboration with Eva Zazimalova, Institute of Experimental Botany, Academy of Sciences of the
Czech Republic, Prague.
Effects of auxin and K+-channel blockers on the auxin-induced protoplast swelling.
Etiolated seedlings grown in vitro and plants grown in soil will be used. Coleoptile, mesocotyl, and leaf auricles
will be used for protoplast isolation. Protoplast isolation and measurement of protoplast diameter changes will
be performed as described for abp mutants. Auxin and inhibitors of K+ channels will be added to the bath
medium and differences between hybrids in the swelling responses will be determined.
Auxin-induced expression of ZmK1 gene in hybrid plants.
Total RNA will be isolated as described above for abp mutants. Mesocotyls of etiolated seedlings grown in vitro
in the absence or presence of auxin, and leaf auricles dissected from etiolated plants grown in soil and sprayed
with NAA will be used. Expression of ZmK1 gene will be studied using RT-PCR and Northern analysis as
described above.
Expression of ABP5 genes in etiolated and light-grown hybrid seedlings.
On the basis of our previous results (Fellner et al. 2003) we hypothesize that etiolated seedlings in modern
corn hybrids contain fewer of ABPs than older hybrids. Recently we found that expression of ABP4 gene, but
not of ABP1, is upregulated by auxin and light in 307 hybrid. In contrast, expression of ABP4 in 3394 is low and
is not affected by auxin or light (Fellner et al. 2004, submitted). Expression analysis of ABP5 (Schwob et al.
1993), another member of ABP gene family, will be studied in the hybrids to highlight its possible role in
seedling growth and leaf angle development, and in possible interaction with ABP1 and/or ABP4.
Plants will grow in dark, R, FR, and W in conditions in vitro or and in vivo. Mesocotyl segments from
seedlings grown in vitro, and auricles from leaves of plants grown in soil will be used for isolation of total RNA.
RNA isolation, RT-PCR, and Northern blot analysis will be performed as described above for abp mutants.
C) STUDY OF INTERACTION BETWEEN AUXIN-BINDING PROTEINS AND PHYTOCHROMES.
We previously found that the modern corn hybrid 3394 is resistant or less sensitive to auxin- and light-induced
responses of etiolated seedlings than the older hybrid 307 (Fellner et al. 2004, submitted). In comparison to
307, 3394 lacks up-regulation by auxin and light of ABP4 expression. To determine possible role of
phytochromes in ABP4 expression and ABPs-mediated growth responses, expression of ABP1, ABP4, and
ABP5 genes will be studied in phytochrome-deficient mutant elm1 in corn (Sawers et al. 2002). Consequently,
we will study expression of genes coding for maize PHYA, PHYB, and PHYC (Sheehan et al. 2004) in the
hybrids and abp mutants. The experiments would answer the question whether differential light-induced
responsiveness in 307 and 3394 may have basis in differential levels of phytochrome transcripts. In addition,
we will study whether or not expression of PHY genes can be, similarly like ABP4, regulated by auxin.
Expression analysis of ABP and PHY genes will be studied by RT-PCR and Northern blot analysis and
performed as described above and in Sheehan et al. (2004), respectively.
5. POSSIBLE OUTCOMES AND SIGNIFICANCE
One possible outcome of the proposed experiments is that the phenotype of modern corn hybrids will be partly
explained by reduced expression of ABP4 and/or by reduced level of ABP1. Morphological phenotype, such as
erect leaf display in modern hybrids versus more horizontally held leaves in older hybrids, is a result of cell
growth regulation. In the case of modern hybrids, the cells at the outer edges of the auricle expand less, as do
elongating cells in coleoptile, mesocotyl and leaf sheath, especially in response to auxin. We hope to be able to
show that the modern hybrids have changes in promoter sequence, relative to 307 and that 3394 and 307
differ in levels of ABP1. We also want to show that ABP5 may function as a compensatory factor for a loss of
ABP1/ABP4, at least in some responses. Although leaf display is a major phenotypic difference between older
and modern corn hybrids, it is probably true that this is only an indicator of a more fundamental difference
allowing modern plants to remain highly productive in increasingly dense plantings. Plants detect neighbors,
and thus plantation density, by sensing FR via phytochromes, and invoking growth responses enabling them to
escape shade or otherwise cope with density stress. As reviewed in the Introduction, it is well recognized that
phytochrome signaling interacts with auxin-mediated regulatory pathways to control growth responses. Using
phytochrome-deficient mutant elm1 in corn we hope to be able to show that phytochromes mediate light effect
on regulation of expression of ABP genes and/or amount of ABPs.
6. TIMELINE AND DIVISION OF LABOR
The collaborative project between Czech Republic and USA will take place over 4 years. Experiments on
Czech side will be conducted by the applicant, Martin Fellner, one post-doc, one technician, and one graduate
student. The applicant and post-doc will carry most of the experiments described above. A trained technician
will be hired to carry out phenotype descriptions of experimental plants, leaf angle measurements, and will do
basic laboratory work. The applicant, post-doc, and technician will teach the methods to the graduate student
who will perform growth, genetic, and molecular biology experiments. On the American side, applicant
8
Professor Elizabeth Van Volenburgh and her group will perform electrophysiological experiments described in
the project.
YEAR1
- study of growth responses to auxin, light, and K+ channel blockers in the abp mutants (Fellner)
- determination of activity of polar auxin transport in abp mutants (Fellner)
- determination of leaf angle and activity of ABPs in abp mutants (Fellner, Zazimalova)
- determination of ABP gene expression in the mutants (Fellner)
- manage method(s) for protoplast isolation from corn seedlings and leaves (Van Volkenburgh)
YEAR 2
- measurement of endogenous free auxin levels in the abp mutants (Fellner)
- description of effect of auxin and K+- and anion-channel blockers on protoplast swelling in the mutants and
hybrids (Van Volkenburgh)
- analysis of ABP4 promoters in the hybrids (Fellner)
- determination of ZmK1 gene expression in the abp mutants and hybrids (Fellner)
YEAR 3
- measurement of auxin-induced electrical responses in etiolated mutant seedlings (Van Volkenburgh)
- determination of amount of ABP1 in corn hybrids (Fellner)
- determination of activity of ABPs in corn hybrids (Fellner, Zazimalova)
YEAR 4
- investigation of auxin-induced apoplastic acidification in the abp mutants and hybrid plants (Van Volkenburgh)
- study of ABP gene expression in phytochrome-deficient mutant (Fellner)
- determination of amounts of PHYA, B, C transcripts in corn hybrids and abp mutants (Fellner)
7. REFERENCES TO PROJECT DESCRIPTION
Bandurski RS, Schulze A, Cohen JD (1977) Biochemical and Biophysical Research Communications 79: 1219-1223;
Barbier-BrygooH, Ephritikhine G, Klämbt D, Gishlan M, Guern J (1989) Proc Natl Acad Sci USA 86: 891-895; BarkerBridgers M, Ribnicky DM, Cohen JD, Jones AM (1998) Planta 204: 207-211; Baskin TI, Briggs WR, Iino M (1986) Plant
Physiol 81: 306-309; Bauer CS, Hoth S, Haga K, Philippar K, Aoki N, Hedrich E (2000) Plant J 24: 139-145; Becker D &
Hedrich R (2002) Plant Mol Biol 49: 349-356; Bennetzen (1996) Curr Top Microbiol Immunol 204: 195-229; Blatt MR &
Thiel G (1993) Ann Rev Plant Physiol Mol Biol 44: 543-567; Boerjan W, Cervera M-T, Delarue M, Beeckman T, Dewitte
W, Bellini C, Caboche M, Van Onckelen H, Van Montagu M, Inzé D (1995) Plant cell 7: 1405-1419; Carabelli M, Morelli G,
Whitelam G, Ruberti I (1996) Proc Natl Acad Sci USA 93: 3530-3535; Celenza JL Jr., Grisafi PL, Fink GR (1995) Genes
Dev 9: 2131-2142; Chen JG (2001) J Plant Growth Regul 20: 255-264; Chen JG, Shimomura S, Sitbon F, Sandberg G,
Jones AM (2001a) Plant J 28: 607-617; Chen JG, Ullah H, Young JC, Sussman MR, Jones AM (2001b) Genes Dev 15:
902-911; Cheng W, Im KH, Chourey PS (1996) Plant Physiol 111: 1021-1029; Christensen SK, Dagenais N, Chory J,
Weigel D (2000) Cell 100: 469-478; Claussen M, Lüthen H, Blatt M (1997) Planta 201: 227-234; Dale JE (1988) Annu Rev
Plant Physiol Mol Biol 39: 267-295; Dengler N & Kang J (2001) Curr Opinion Plant Biol 4: 50-56; Downs RJ (1955) Plant
Physiol 30: 468-475; Elzenga JTM (1997) Plant Physiol 144: 1474; Elzenga JTM, Staal M, Van Volkenburgh E (1997) J
Exp Botany 48: 2055-2060; Elzenga JTM & Van Volkenburgh E (1994) J Membr Biol 137: 227-235; Elzenga JTM & Van
Volkenburgh E (1997a) Plant Physiol 113: 1419-1426; Elzenga JTM & Van Volkenburgh E (1997b) Planta 201: 415-423;
Epstein E, Cohen JD, Bandurski RS (1980) Plant Physiol 65: 415-421; Felle H, Peters W, Palme K (1991) Biochim
Biophys Acta 1064: 199-204; Fellner M (1997) Search for selection screens of auxin-response mutants in Arabidopsis
thaliana.Genetic and physiological characterization of the first isolated mutants. PhD thesis, Gif-sur-Yvette, France:
University of Paris-South XI; Fellner M, Horton LA, Cocke AE, Stephens NR, Ford ED, Van Volkenburgh E (2003) Planta
216: 366-376; Fellner M, Ford ED, Van Volkenburgh E (2004, submitted); Firn RD (1986) Physiol Plant 67: 267-272;
Freeling M & Lane B (1994) In M Freeling, V Walbot, eds, The Maize Handbook. Springer-Verlag, New York, Inc., pp 1728; Gallagher K & Smith LG (1999) Development 126: 4623-4633; Goldsmith MHM (1977) Annu Rev Plant Physiol 28:
439-478; Haga K & Iino M (1997) Aust J Plant Physiol 24: 215-226; Haga K & Iino M (1998) Plant Physiol 117: 1473-1486;
Hager A (2003) J Plant Res 116: 483-505; Hager A, Menzel H, Kraus A (1971) Planta 100: 47-75; Hager A, Debus G,
Edel HG, Stransky H, Serrano R (1991) Planta 185: 527-537; Hall JL, Brummell DA, Gillespie J (1985) New Phytol 100:
341-345; Halliday KJ & Fankhauser C (2003) New Phytol 157: 449-463; Hamill OP et al. (1981) Pflugers Arch 391: 85100; Hanson JB & Trewavas AJ (1982) New Phytol 90: 1-18; Hasezawa S & Syono K (1983) Plant cell Physiol 24: 127132; Hoth S, Dreyer I, Dietrich P, Becker D, Müller-Röber B, Hedrich R (1997) Proc Natl Acad Sci USA 94: 4806-4810;
Howell SH (1998) Molecular Genetics of Plant Development. SH Howell, ed, Cambridge University Press, pp. 137-168;
Huisinga B (1964) Acta Bot Neerl 13: 445-487; Huisinga B (1967) Acta Bot Neerl 16: 197-201; Iino M (1982a) Planta 156:
197-201; Iino M (1982b) Planta 156: 388-395; Iino M (1995) Plant Cell Physiol 36: 361-367; Im KH, Chen JG, Meeley RB,
Jones AM (2000) Maydica 45: 319-325; Ishida S, Takahashi Y, Nagata T (1993) Proc Natl Acad Sci USA 90: 1115211156; Jones AM (1994) Annu Rev Plant Physiol Plant Mol Biol 45: 393-420; Jones AM (1990) Plant Physiol 93: 11541161; Jones AM, Cochran DS, Lamerson MA, Evans ML, Cohen JD (1991) Plant Physiol 97: 352-358; Jones AM &
Herman E (1993) Plant Physiol 101: 595-606; Jones AM, Inn KH, Savka MA, Wu MJ, DeWitt NG, Shillito R, Binns AN
(1998) Science 282: 1114-1117; Keller CP & Van Volkenburgh E (1996) Planta 198: 404-412; Kepinski S & Leyser O
(2002) Plant Cell, Supplement: S81-S95; Kiesselbach TA (1980) The structure and reproduction of corn. University
Nebraska Press, Lincoln, USA, pp 16-25; Kim YS, Min JK, Kim D, Jung J (2001) J Biol Chem 276: 10730-10736; King JJ,
Stimart DP, Fisher RH, Bleecker AB (1995) Plant Cell 7: 2023-2037; Klämbt D (1990) Plant Mol Biol 14: 1045-1050;
Leblanc N, David K, Groslaude J, Pradier JM, Barbier-Brygoo H, Labiauet S, Perrot-Rechenmann C (1999) J Biol Chem
274: 28314-28320; Lohse G & Hedrich R (1992) Planta 188: 206-214; Lomax TL, Muday GK, Rubery PH (1995) In PJ
Davies, ed, Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer Academic Publishers, Dordrecht,
The Netherlands, pp 509-530; Marten I, Lohse G, Hedrich R (1991) Nature 353: 758-762; Morelli G & Ruberti I (2000)
9
Plant Physiol 122: 621-626; Morelli G & Ruberti I (2002) Trends Plant Sci 9: 399-404; Morgan DC & Smith H (1979) Planta
145: 253-258; Nagata T, Nemoto Y, Hasezawa S (1992) Int Rev Cytol 132: 1-30; Napier RM, David KM, PerrotRechenmann C (2002) Plant Mol Biol 49: 339-348; Napier RM, Venis MA, Bolton MA, Richardson LI, Butcher GW (1988)
Planta 176: 519-526; Naqvi SM (1975) Z Pflanzenphysiol 76: 379-383; Philippar K, Fuchs I, Lüthen H, Hoth S, Bauer CS,
Haga K, Thiel G, Ljung K, Sandberg G, Böttger M, Becker D, Hedrich R (1999) Proc Natl Academy Sci USA 96: 1218612191; Ray PM, Dohrmann U & Hertel R (1977) Plant Physiol. 59: 357-364; Rayle DL & Cleland RE (1992) Plant Physiol
99: 1271-1274; Rayle DL & Cleland RE (1972) Planta 104: 282-296; Rűck A, Palme K, Venis MA, Napier R, Felle HH
(1993) Plant J 4: 41-46; Sawers RJH, Linley PJ, Farmer PR, Hanley NP, Costich DE, Terry MJ, Brutnell TP (2002) Plant
Physiol 130: 155-163; Schwob E, Choi S-Y, Simmons C, Migliaccio F, Ilag L, Hesse T, Palme K, Soll D (1993) Plant J 4:
423-432; Shah DM, Hightower RC, Meagher RB (1983) J Mol Appl Genet 2: 111-126; Sheehan MJ, Farmer PR, Brutnell
TP (2004) Genetics 167: 1395-1405; Shimomura S, Watanabe S, Hiroaki I (1999) Planta 209: 118-125; Smith H (1982)
Annu Rev Plant Physiol Plant Mol Biol 33: 481-518; Smith H (1995) Annu Rev Plant Physiol Plant Mol Biol 46: 289-315;
Staal M, Elzenga JTM, Van Elk AG, Prins HBA, Van Volkenburgh E (1994) J Exp Botany 45: 1213-1218; Stahlberg R &
Van Volkenburgh E (1999) Planta 208: 188-195; Steffens B, Feckler C, Palme K, Christian M, Böttger M, Lüthen H (2001)
Plant J 27: 591-599; Steindler C, Matteucci A, Sessa G, Weimar T, Ohgishi M, Aoyama T, Morelli G, Ruberti I (1999)
Development 126: 4235-4245; Stiles KA & Van Volkenburgh E (2002) J Exp Botany 374: 1651-1657; Stiles KA, McClintick
A, Van Volkenburgh E (2003) Planta 217: 587-596; Thiel G, Blatt MR, Fricker MD, White IR, Millner P (1993) Proc Natl
Acad Sci USA 90: 11493-11497; Tian Q & Reed JW (2001) J Plant Growth Develop 20: 274-280; Thomine S, Lelièvre F,
Boufflet M, Guern J, Barbier-Brygoo H (1997) Plant Physiol 115: 533-542; Trewavas A & Cleland R (1983) Trends in
Biochem Sci 8: 354-357; Trewavas A (1981) Plant Cell Environ 4: 203-228; Vanderhoef LN & Briggs WR (1978) Plant
Physiol 61: 534-537; Van Overbeek (1936) Rec Trav Bot Neerl. 33: 333-340; Van Volkenburgh E (1999) Plant Cell
Environ 22: 1463-1473; Van Volkenburgh E & Cleland RE (1980) Planta 148: 273-278; Van Volkenburgh E, Cleland RE,
Watanabe M (1990) Planta 182: 77-80; Wang X & Iino M (1997) Plant Physiol 114: 1009-1020; Zaina S, Reggiani R,
Bertani A (1990) Plant Physiol 136: 653-658; Zažímalová & Kutáček M (1985) Plant Growth Regul 3: 15-26; Zažímalová E
& Napier RM (2003) Plant Cell Rep 21: 625-634; Zažímalová E, Opatrny Z, Březinová A, Eder J (1995) J Exp Bot 46:
1205-1213; Zimmermann S, Thomine S, Guern J, Barbier-Brygoo H (1994) Plant J 6: 707-716.
10