Plant Molecular Biology 56: 367–380, 2004.
Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.
367
Differential expression profiles of growth-related genes
in the elongation zone of maize primary roots
Michal Bassani1, Peter M. Neumann2 and Shimon Gepstein1,*
1
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000 Israel (*author for correspondence; e-mail gepstein@tx.technion.ac.il); 2Plant Physiology Laboratory, Division of Environmental,
Water and Agricultural Engineering, Faculty of Civil and Environmental Engineering, Technion-Israel
Institute of Technology, Haifa 32000, Israel
Received 21 July 2004; accepted in revised form 20 September 2004
Key words: elongation zone, gene expression in situ hybridization, root elongation, water deficit
Abstract
Growth in the apical elongation zone of plant roots is central to the development of functional root
systems. Rates of root segmental elongation change from accelerating to decelerating as cell development
proceeds from newly formed to fully elongated status. One of the primary variables regulating these
changes in elongation rates is the extensibility of the elongating cell walls. To help decipher the complex
molecular mechanisms involved in spatially variable root growth, we performed a gene identification study
along primary root tips of maize (Zea mays) seedlings using suppression subtractive hybridization (SSH)
and candidate gene approaches. Using SSH we isolated 150 non-redundant cDNA clones representing root
growth-related genes (RGGs) that were preferentially expressed in the elongation zone. Differential
expression patterns were revealed by Northern blot analysis for 41 of the identified genes and several
candidate genes. Many of the genes have not been previously reported to be involved in root growth
processes in maize. Genes were classified into groups based on the predicted function of the encoded
proteins: cell wall metabolism, cytoskeleton, general metabolism, signaling and unknown. In-situ hybridization performed for two selected genes, confirmed the spatial distribution of expression shown by
Northern blots and revealed subtle differences in tissue localization. Interestingly, spatial profiles of
expression for some cell wall related genes appeared to correlate with the profile of accelerating root
elongation and changed appropriately under growth-inhibitory water deficit.
Introduction
The development of higher plants is dependent on
the continuous mining of water and essential
mineral nutrients from the soil by a growing root
system. Plant root systems are built up by the
growth of individual roots and their elongation is
the result of cell division and cell expansion (Silk,
1992; Beemster and Baskin, 1998, 2000). Root
elongation in maize is achieved through accelerating and decelerating cell expansion within the
elongation zone and decreases to zero at the end of
the elongation zone. Similar spatial patterns of
growth distribution have been described in
Arabidopsis and may be universal for flowering
plants (Van der Weele et al., 2003).
The elongation zone in relatively robust maize
roots is 10 mm long. Cell expansion occurs along
files of cells in the elongation zone. The cell files
resemble ‘cellular assembly lines’. New cells are
continuously produced by ongoing meristematic
divisions and each cell in the cellular line is more
developed than the one beneath it (Carmona and
Cuadrado, 1986; Schiefelbein et al., 1997).
Developing young cells are displaced through the
entire elongation zone in a few hours. Segmental
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analysis of relative growth rates in the tip-region of
primary roots of maize seedlings indicates accelerating elongation of cells and tissues approximately 0.1–0.5 cm from the apex and subsequent
deceleration in the region 0.5–1.0 cm from the
apex. Root elongation then ceases (Silk, 1992; Fan
and Neumann, 2004). Under water defict the
elongation zone is shortened but it is not known
how this is associated with changes in gene
expression. We therefore investigated this point.
Physiological and biochemical studies have
shown that root growth requires co-ordinated
water and solute uptake for development of turgor
pressure and irreversible expansion of cell walls
(Cosgrove, 1987, 1993; Neumann, 1995). Micropressure–probe analyses of cell turgor pressure
distribution along the elongation zone of maize
roots undergoing steady-state growth have
revealed that the spatial distribution of turgor along
the root tip is relatively constant. Thus, the spatial
differences in root elongation rates appear to be
regulated by local differences in cell wall extensibility (Spollen and Sharp, 1991; Pritchard, 1994).
Dramatic changes in the expression profiles of cell
wall regulatory genes are therefore likely to occur
along the length of the elongation zone. We report
here on an investigation of the tempero-spatial
development of gene expression in isogenic populations of root cells found in adjacent segments
along the length of the growing zone.
Studies investigating the regulation of root
growth at the level of gene expression have traditionally used a candidate gene approach which
samples predetermined genes in isolation from the
entire repertoire of mRNA transcripts. For
example, Wu et al. (2001) studied the spatial pattern of expression of expansin genes which encode
cell-wall loosening proteins. They showed four of
five alpha and beta expansins were specifically
expressed in the growing region. Recent study
identified the root-specific expansin GmEXP1
whose expression levels were specifically high in
the elongating region of soybean roots. Over
expression of this gene caused accelerated root
growth in tobacco (Lee et al., 2003). In contrast to
the GmEXP1 gene, the expression levels of the
extensin gene, SbHRGP3, were low in the elongating region and specifically high in fully elongated cells in the maturation zone. This suggested
a role for extensins in the cell maturation process
(Lee et al., 2003). Similarly Hukin et al., (2002)
showed that two plasmamembrane intrinsic genes
which encode aquaporin proteins associated with
transmembrane water transport were more highly
expressed in younger regions than in older regions
of the root elongation zone. Recently, it has been
shown that the transcription factor DELLA proteins that repress growth mediate the effect of
auxins, gibberellins and ethylene on root cell
expansion (Fu and Harberd, 2003). Since the
elongation process is a complex one involving
transitions of growing root cells between accelerating, decelerating and fully elongated stages of
development, multiple differences in gene expression are likely to be involved in its regulation.
This was confirmed by Birnbaum et al., (2003)
who performed a remarkably comprehensive
mapping of gene expression in the Arabidopsis root
tip using microarrays. Multiple differences in gene
expression can also be usefully investigated using a
suppression subtractive hybridization (SSH) technique (Diatchenko et al., 1996; Gepstein et al.,
2003). This genomic approach is based primarily
on the suppression PCR technique and combines
normalization and subtraction in a single procedure. This technique enriches differentially
expressed sequences by selective amplification. The
major advantage of SSH technology is the
enrichment of rarely transcribed clones with a
much higher sensitivity than other methods of
differential screening. For example, Gepstein et al.
(2003) used SSH to reveal a large number of nonabundant and novel senescence-associated genes
that have not been previously reported in the
literature.
We used SSH to compare elongating and
adjacent fully elongated tissues of maize roots and
thereby identify genes whose expression is preferentially upregulated during root elongation. This
report concerns 41 genes (out of 150 non-redundant SSH clones identified) for which upregulated
expression in elongating root tissues has been
confirmed by Northern blots and in some cases,
visualized by in-situ hybridization. After identification, root growth-related genes (RGGs) and
selected candidate genes were clustered according
to their putative functioning: cell-wall metabolism,
intracellular signaling, cytoskeleton structure,
general metabolism or unknown. Finally, we
investigated possible matching between expression
profiles of RGGs and the spatial changes in root
elongation profiles under control and water deficit
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conditions. The aims of this research were to
determine: (1) Whether the expression of known or
unknown root growth-related genes is differentially regulated along the length of the maize root
elongation zone. (2) To specifically look for differential expression of genes which may be related
to the regulation of cell-wall extensibility.
Materials and methods
Growth of plants
Seeds of maize (Zea mays L. cv. 647) were germinated on filter paper wetted with 0.5 mM CaCl2
at 27 ± 2 °C in the dark. After 4 days, seedlings
with primary roots about 1.5 cm long were grown
hydroponically in a growth chamber. The roots of
plants were inserted through holes in rectangular
polystyrene plates floating in trays containing
continually aerated 0.1 strength nutrient solution.
Relative humidity in the growth chamber varied
between 35% by day and 60% at night. Light in
growth chamber, provided by mixed incandescentfluorescent lamps during a 12 h photoperiod was
150 lmol m)2 s)1 PAR and temperature was
27 ± 2 °C (Bogoslavsky and Neumann, 1998).
After growth for 60 h, the root segments were
cut at various distances from the tip: 1–10 mm
(entire elongation zone), 1–5 mm (accelerating
growth zone), 5–10 mm (decelerating growth
zone), and 10–15 mm (non-elongating zone). The
first 1 mm section (0–1 mm) which included the
root cap and tip meristematic tissues was
discarded. The segments were immediately frozen
in liquid nitrogen and stored at )70 °C until use
for RNA extraction.
Water deficit was obtained by transferring
seedlings after 12 h of growth to a continually
aerated 0.1 strength nutrient solution with the
osmolyte polyethylene glycol (PEG) 6000 and
extra 1 mM CaCl2. In order to minimize hyperosmotic shock the PEG was added gradually: 2 h
at )0.2 MPa, 2 h at )0.4 MPa and 44 h at
)0.5 MPa. The root segments were then cut at
various distances from the tip 1–3 mm (accelerating growth zone), 3–6 mm (decelerating growth
zone), 6–9 mm and 9–12 mm (fully elongated
zone). The segments were immediately frozen in
liquid nitrogen and stored at )70 °C until use.
Relative segmental elongation rate
Relative segmental elongation rate (RSER) along
the root elongation zone was measured by digital
photography on primary roots about 7 cm long, as
in Fan and Neumann (2004). The surface of the
root elongation zone was gently blotted and
marked with Indian ink using a device consisting
of 12 silk threads (0.06 mm diameter) spaced at
equal distances of about 1 mm. The marked roots
of whole seedlings were mounted upright in a glass
container in 150 ml of aerated solution. After 1 h
of equilibration a series of digital pictures of the
marked roots were taken at 0.5 h intervals using a
digital camera (Olympus C-5050). The resolution
was 92 pixels per mm at 3 cm from the root. The
relative elongation rate of each segment (RSER)
was determined as (lnLt ) lnL0) L0 )1 t )1, where
L0 is the initial distance between marks and Lt is
the final distance after time t. Distance between
marks and elongation profiles were obtained
with a customized program in Matlab (Highperformance numeric computation and visualization
software-version 6.5). This fitted a vertical line to all
the marks and determined the mid points of the
marks. The distances between the marks were then
calculated from pixel counts before and after a
30 min interval. Measurement error, based on
equivalent assays of millimeter markings on a thin
steel ruler, was less than 1%.
Isolation of RNA and RNA gel blot analysis
Total RNA was prepared according to Hajouj
et al. (2000) from Zea mays roots. RNA was separated on 1.0% (w/v) agarose–formaldehyde gels
and blotted to nylon filters (NytranN, Schleicher
& Schuell). cDNA clones were used as specific
primers. Labeling was performed using the Rediprime DNA labeling system (RPN1633/1634,
Amersham). After an overnight hybridization the
membrane was washed, visualized after 2 h with
phosphor imager, and exposed to X-ray film.
Suppression subtractive hybridization
Two SSH were performed with the PCR-Select
cDNA Subtraction kit (Clontech Laboratories
Inc., Palo Alto, CA) as described by the manufacturer. The first was performed with RNA that
was obtained from roots of well watered seedlings.
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Two micrograms of mRNA from the region of 1–
10 mm from the root tip (tester) and 2 lg of
mRNA from 10 to 15 mm from the root tip (driver) were used. The second SSH was performed
with RNA that was obtained from roots of
seedlings that grew 48 h under water stress
conditions. Two micrograms of mRNA from the
region of 1–7 mm from the root tip (tester) and
2 lg of mRNA from 7 to 12 mm from the root tip
(driver) were used. The PCR products generated
by the SSH were cloned into the pGEM- easy
vector using the T-cloning kit (Promega).
Sequencing and homology analysis
Nucleotide sequence of each insert was determined
by Sequencing Services, Macrogene (Korea).
Sequence homology was analyzed using the
BLAST program.
In-situ hybridization
Root tips 1.3 cm in length were cut, fixed with 4%
formaldehyde and 0.25% glutaldehyde in PBS
buffer (pH 7.0) and embedded in Paraplast Plus
(Sigma–Aldrich). Sections (10 lm) were cut with a
microtome and placed on SuperFrost Plus slides
(Menzel-Glaser, Germany). Because the cDNA
was cloned into pGEM either T7 or SP6 polymerase was used to generate a digoxigenin-labeled
RNA probe from the linearized plasmid. In situ
analysis was performed with digoxigenin-labeled
RNA probe added to the sliced root and the
overnight hybridization temperature was 50 °C.
Prehybridization treatment, hybridization conditions and post-hybridization treatment were performed as described previously by Langdale (1994).
The tissue image and blue staining of the resulting
signal was visualized by applying alkaline phosphatase-conjugated antidigoxigenin antibodies and
enzyme substrate (Roche Molecular Biochemicals).
The hybridization signal was then photographed.
RGG144 and RGG279 cloning
cDNA was synthesized from 0.5 lg total RNA, that
was obtained from the zone 1–10 mm from the root
tip, using oligo(dT) as a primer and reverse transcriptase (M-MULV reverse transcrip tase, Stratagene). Partial RGG144 and RGG279 cDNAs
were amplified using RGG144-f (50 -TACAC-
CGCCATCAAGGGAGA-30 ), RGG144-r (50 -TAGTTCTGGGACACTGTTCTTGC-30 ), RG-G279-f
(50 -CTCCTCCGAGAACTCCATGCTC-30 ), and
RGG279-r (50 -CTGCGAACAAAAACCTTGCTC
-30 ) primers. The PCR products were cloned separately into pGEM-easy vector, sequenced, and were
used for the in-situ hybridization procedure.
XET and H+ATPase cloning
cDNA was synthesized from 0.5 lg total RNA,
that was obtained from the zone 1–10 mm from
the root tip, using oligo(dT) as a primer and
reverse transcriptase (M-MULV reverse transcriptase, Stratagene). Partial XET and H+ATPase cDNAs were amplified using XET-f (50 GGGCGCAGGCCAGCTACATGAT-30 ), XET
-r (50 -CATATATCCGCCGAACTTATGCCG
-30 ), H +ATPase-f (50 -ATGGGTGGGCTC
GAGGA GATCAA-30 ), and H +ATPase-r (50 CCATGAC AGCGGGTTCCACAT GAA-30 )
primers. The PCR products were cloned separately into pGEM-easy vector, sequenced,
and were used for the Northern blot analysis.
Results
Segmental profile of root elongation
Relative segmental elongation rates were determined along the zone of elongation at the root
apex (Figure 1). The elongation zone is seen to
extend ca. 10 mm behind the tip. In order to
further investigate the relationships between
localized changes in growth rate and gene
expression, we divided the elongation zone into a
zone of accelerating elongation 1–5 mm behind
the tip and a zone of decelerating elongation
5–10 mm behind the tip. The zone from 10 to
15 mm behind the tip represented fully elongated
tissues.
Identification of root growth related genes
by the subtraction suppression method
To identify genes whose products are preferentially
involved in the process of root growth, SSH was
carried out. Two SSHs were performed. The first
was performed with RNA that was obtained from
roots of well watered seeds. The driver cDNA was
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Functional grouping of genes related
to root growth
Figure 1. Spatial distribution of relative segmental elongation
rates (RSER) along apex of primary roots of maize seedlings
(means ± SE, n ¼ 5).
synthesized from a mRNA population isolated
from fully elongated tissues, 11–15 mm behind the
tip of the Zea mays root. The tester cDNA was
produced from mRNA isolated from the entire
elongation zone situated 1–10 mm behind the root
cap. The second SSH was performed with RNA that
was obtained from roots of seedlings that grew 48 h
under growth inhibitory water deficit. In this case,
the driver cDNA was synthesized from a mRNA
population isolated from fully elongated tissues, 7–
12 mm behind the tip. The tester cDNA was produced from mRNA isolated from the entire elongation zone situated 1–7 mm behind the tip. These
two zones were expected to contain transcripts
representing a broad spectrum of genes involved in
regulating root elongation.
Around 550 recombinant cDNA clones were
isolated by the SSH method and DNA sequence
analysis was performed for 400 clones. Around
250 cDNA sequences showed high homology to
EST/mRNA sequences available in public databases, and 150 cDNA clones were found as nonredundant (see supplementary material). Seventy
five percent of the cDNA clones that were analyzed by Northern blot were confirmed as ‘true’
differentially expressed genes. The remainder was
false positives (data not shown). We selected 41
clones representing transcripts whose steady state
levels were relatively up-regulated in the elongating tissues for further examination (Table 1).
Clones that were obtained from the second SSH
(under water deficit conditions) were given numbers with the suffix B.
Out of selected 41 cDNA clones, 19 clones were
annotated to known sequences with predicted
functions in Zea mays, although most of them
have not been previously reported to be associated with root growth. Functions for 18 cDNA
clones of unknown functions in Zea mays (in the
updated NCBI database www.ncbi.nlm.nih.gov),
were predicted from homologies to genes of other
genomes. An additional four cDNA clones showed
homologies to mRNAs sequences of Zea mays
found in the databases but had unknown functions. To confirm the differential gene expression
represented by the cDNA clones isolated with
SSH, Northern blot comparisons of levels of
transcripts along the elongation zone and in the
adjacent fully elongated zone were performed.
Furthermore, in-situ hybridization was carried out
to determine whether spatially variable patterns of
expression would be observed for 2 arbitrarily
selected genes.
We have designated the differentially expressed genes in the elongation zone as RGGs.
The identified RGGs whose differential expression was verified by Northern blot were classified
into five functional groups: (1) cell-wall metabolism, (2) cytoskeleton; (3) general metabolism;
(4) signaling; (5). unknown function.
In-situ hybridization of root growth-associated
genes in the elongation zone of the root
Spatial distribution of expression of the two genes
and localization within different root tissues in the
elongation zone were investigated by in situ
hybridizations. The two genes investigated were:
RGG144 (similar to root specific protein RCc3)
and RGG 279 (ABA-inducible gene for glycinerich protein). We found differential spatial distribution of both RGG144 and RGG279 (Figure 2).
RGG144 was mainly expressed in the region of
accelerating growth at 2–4 mm above the root
cap. It was specifically expressed in cortical and
stelar tissues. RGG279 was also expressed in the
region of accelerating growth between 0.5 and
3 mm from the root cap. In this case, expression
was preferentially localized in the tip meristem,
the epidermal layer and in the stele.
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Table 1. List of 41 sequences of cDNA clones isolated by the SSH, that were also confirmed subsequently by Northern blots to be
differentially expressed in the elongation zone. Homology analysis was performed using the BLAST database, and the possible roles in
root growth were assigned according to functional groups. XET and H+ ATPase were not isolated by the SSH and were amplified
directly from cDNA. The suffix B indicates clones that were obtained by the SSH from roots under water stress.
Clone no.
Accession no.
Homology
Species
Cell wall metabolism
–
–
RGG6
RGG8B
RGG22
RGG105
RGG152
RGG161
U15964
X85805
U89897
AY109400
AF225411
AY105847
AF082347
X98245
XET (Xyloglucan endo-transglycosylase)
H+ ATPase
Golgi associated protein se-wap41
Similar to beta glucosidase
Exoglucanase precursor (exg1)
Similar to proton pump interactor
C13 endopeptidase NP1 precursor
Annexin p35
Zea mays
Zea mays
Zea mays
Oryza sativa
Zea mays
Arabidopsis
Zea mays
Zea mays
Cytoskeleton
RGG15B
AY104735
Oryza sativa
RGG41
RGG160
AY103587
L10633
Similar to putative microtubule
associated protein
Similar to actin
Beta-6 tubulin (tub6)
General metabolism
RGG29
AY104186
Oryza sativa
RGG62B
AY103938
RGG130B
AY104575
RGG145
RGG162
RGG170B
RGG218
RGG240B
RGG289
RGG305
RGG357
RGG469B
L13431
AY103583
M84164
AB016064
AY103927
AF439723
X55981
AY103658
X12872
Similar to 26s proteosome regulatory triple-A
ATPase subunit 1
Similar to protease inhibitor/seed storage/lipid
transfer protein (LTP)
Similar to putative ATP synthase delta chain,
mitochondrial precursor
Glyceraldehyde-3-phosphate dehydrogenase
Similar to s-adenosyl-l-homocysteine hydrolase
Chitinase A
Mitochondrial phosphate transporter
Similar to vacuolar ATPase
Methionine synthase
Enolase (2-phospho-D -glycerate hydrolase)
Similar to aspartate aminotransferase cytoplasmic
Anaerobically regulated gene for
fructose bisphosphate aldolase
Signal associated genes
RGG7B
RGG16B
RGG34B
RGG215
RGG279
RGG326
RGG346
RGG371
RGG485B
Unknown function
RGG24B
RGG120
RGG137
RGG144
RGG147
RGG159
Hordeum vulgare
Zea mays
Arabidopsis
Oryza sativa
Zea mays
Catharanthus roseus
Zea mays
Zea mays
Arabidopsis
Zea mays
Zea mays
Oryza sativa
Zea mays
AY103545
AY110951
AB042268
M35388
X12564
U06108
X77396
AF136826
AY105672
Similar to shaggy-related protein kinase gamma
Similar to putative ethylene forming enzyme
ZmPR6 response regulator 6
Histone H3
ABA-inducible gene for glycine-rich protein
B37 QM protein
Calmodulin (CaM1)
Elongation factor 1 alpha
Similar to zinc finger (C2H2) protein family
Oryza sativa
Arabidopsis
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Arabidopsis
AY107535
AY104135
AY104682
AY105972
AY109100
X62455
Similar to expressed protein
Similar to putative transmembrane protein
Unknown
Similar to root specific protein RCc3
Unknown
Cytoplasmic ribosomal protein s13
Arabidopsis
Oryza sativa
Zea mays
Oryza sativa
Zea mays
Zea mays
373
Table 1. Continued.
RGG175
RGG178
RGG304
AY103659
AY104278
AY109323
RGG358B
X92422
RGG385
AY107458
Unknown
Similar to 40s ribosomal protein s14
Similar to endomembrane protein
EMP70 precursor isolog
Ubiquitin/ribosomal protein
S27a fusion protein
Unknown
Zea mays
Oryza sativa
Arabidopsis
Zea mays
Zea mays
Figure 2. Localization of the RGG279 and RGG144 mRNA in roots of maize seedlings by in situ hybridization: (A) Hybridization
with antisense probe for RGG279. (B) Hybridization with sense probe for RGG279. (C) Hybridization with antisense probe for
RGG144. (D) Hybridization with sense probe for RGG144. (E) Enlargment of the zone 1 to 1.7 mm from the root cap from Figure A.
(F) Enlargment of the zone 2.3 to 3 mm from the root cap from figure C.rc, root cap; e, epidermis; v, vascular cylinder; m, meristerm;
c, cortex.
Differential expression of genes along elongating
and fully elongated zones of the root
Since the root elongation zone consists of regions
of accelerating or decelerating elongation (Figure 1) and is followed by fully elongated tissues, it
was of interest to determine whether specific genes
might be preferentially associated with acceleration deceleration or cessation of root elongation.
Selected RGGs from Table 1 plus additional candidate gene probes for proton pumping ATPase, and
xyloglucan endo-transglycosylase, were classified
according to their spatial association with accelerating or decelerating regions of elongation and the
adjacent fully elongated region, by Northern blot
analyses of segments respectively located 1–5, 5–10
and 10–15 mm from the root tip (Figure 3).
Spatial analysis of the expression profile of the
selected genes along the three zones indicated the
existence of five distinct profiles. Two expression
profiles were dominant. The first dominant
expression profile, representing 12 RGGs, displays
an enhanced expression in the accelerating zone
and gradual reduction in expression along the
decelerating and non-elongating zones. The second
dominant profile represents seven genes that have
relatively high expression in the accelerating region
and relatively low and equal expression in both
decelerating and non-elongating zones. Candidate
genes related to wall metabolism (H+ ATPase and
XET) revealed this profile. These profiles, with
highest expression during rapidly accelerating
elongation, suggest a close association between
accelerating growth and the expression of these
particular genes. Similarly, the relatively down
regulated expression in decelerating and fully
elongated regions could indicate that the level of
these genes products becomes growth limiting
further from the tip. The third expression profile,
representing three genes, RGG41, RGG62B and
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Figure 3. Special analysis of the distribution of expression of
selected genes indicates five distinct profiles. Total RNA was
derived from three regions of the root: E* Region of accelerating elongation 1–5 mm from the root cap. E** Transition and
decelerating region 5–10 mm from the root cap. F: Fully
elongated region 10–15 mm from the root cap.
RGG130B, shows lower levels of expression in
the decelerating zone, while in the other two regions
their expression is relatively high. The fourth
expression profile represents three genes, RGG15B,
RGG120B and RGG137, that have relatively high
expression in both the decelerating and the nonelongating zones. Only one gene, RGG8B, belongs
to the fifth profile. It has relatively high expression
only in the decelerating region. The last two profiles
suggest that these genes may be involved in deceleration of elongation and in cell maturation.
Effect of water deficit on gene expression
in the elongation zone
The segmental growth profile of the root elongation zone is known to shorten dramatically during
Figure 4. RNA blot analysis of root growth related genes in
different root tip regions under water stress: Total RNA derived
from four different regions of the root (1–3, 3–6, 6–9 and
9–12 mm from the root cap) after 48 hours water deficit.
water deficits. Accelerative growth is maintained in
the region 0–3 mm behind the tip and growth
between 3 and 10 mm behind the tip is increasingly
inhibited by water deficit (Silk, 1992; Fan and
Neumann, 2004). It was therefore of interest to
determine whether the effect of water deficit on
profiles of elongation correlated with changes in
profiles of gene expression. To this end the elongation zone was divided into four shortened segments at 1–3, 3–6, 6–9 and 9–12 mm from the tip
(Figure 4). A comparison was made between the
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accelerating zone in the control experiment
(1–5 mm from the root cap) and the shortened
accelerating zone under water deficit (1–3 mm
from the root cap). The other zones were similarly
compared. The region 9–12 mm from the tip of
both control and water stressed plants represents a
fully elongated region as does the region 6–9 mm
from the tip in water stressed plants.
Genes like RGG7B, RGG16B, RGG34B,
RGG105, RGG279, RGG485B, XET, and H+
ATPase that were strongly and exclusively
expressed in the accelerating region (1–5 mm from
root cap) of control plants without water deficit
(Figure 3), were also expressed most strongly in
the shortened accelerating region (1–3 mm from
root cap) under water deficit (Figure 4). Gene
expression in the growth-inhibited or non-growing
regions between 3 and 12 mm from the tip of
water stressed plants was comparatively reduced.
Genes like RGG6, RGG144, RGG152 and
RGG240B whose expression in well watered
plants appeared to decrease progressively at
increasing distances from the root tip had an
equivalent but shortened pattern of expression
after 48 h of water deficit. Interestingly RGG8B
was specifically up-regulated in the decelerating
region, in both the well watered control experiment (5–10 mm from the tip) and in the 48 h water
deficit experiment (3–6 mm from the tip). Thus,
profiles of growth and expression were apparently
related under both control and water deficit
conditions.
Discussion
This study was aimed at elucidating genes associated with the regulation of root and particularly
cell wall elongation. We first investigated the differential expression of genes in the 9 mm long
zone of root elongation by comparison with the
region just behind the elongation zone containing
fully elongated tissues. In addition, we tried to
determine whether genes associated with cell
wall metabolism or other growth related functions
might be preferentially located in regions of
accelerating or decelerating growth rate located 1–
5 and 5–10 mm, respectively behind the root cap.
Note that the tip meristam region was discarded.
Using the SSH technique we discovered known
and unknown genes that were preferentially
expressed in the root elongation zone. In all, we
found 19 clones that were annotated to known
sequences with predicted functions in Zea mays.
Most of these had not been previously reported to
be associated with root growth. Functions for
another 18 cDNA clones of unknown functions in
Zea mays were predicted from homology to genes
of other genomes. Four cDNA clones showed
homologies to mRNAs sequences of Zea mays
found in the databases but all were of unknown
functions. Location profiles for two additional
candidate genes known to be associated with
growth (XET, H+ ATPase) were also determined.
For the discussion below, genes which were
found to be preferentially expressed in the elongation zone and which may be connected to root
growth regulation were divided into five functional
groups according to the putative activities of their
protein products. These were: cell-wall metabolism, cytoskeleton, general metabolism, signaling,
or unknown activity. In addition, the specific
distribution of gene expression in regions of
accelerating root growth (1–5 mm from the
root cap), decelerating growth, (5–10 mm from
the root cap) and root maturation (10–15 mm
from root cap) were considered. It is possible that
not all the gene expression that was found to be
up-regulated in the elongation zone was directly
involved in cellular elongation.
Cell-wall metabolism
Changes in cell wall extensibility appear to be a
primary factor regulating the changing profile of
segmental growth rates in the root elongation zone
(e.g. reviews by Pritchard, 1994; Neumann, 1995).
In our SSH library we found six genes that have a
role in cell-wall metabolism and structure and we
also determined the expression profiles of several
candidate genes such as XET and H+ATPase. A
most interesting gene RGG105 is an unknown
mRNA in the Zea mays genome. However, it has
homology (BLASTX, score: 231, E value: 2e)59)
to a novel protein called PPI1 from Arabidopsis
which is able to modulate the plasma membrane
H+ ATPase activity but does so by binding to a
site different from the 14-3-3 binding site
(Morandini et al., 2002). RGG105 could therefore
have a role in switching on and off cell wall
acidification by proton pumping ATPases as do
the better known 14-3-3 proteins (Palmgren, 2001).
376
It is well established that plant cells enlarge
faster when wall pH is reduced below approximately 5.5; this ‘acid-growth’ behavior is also
characteristic of isolated walls and coincides with
the concept of wall-loosening enzymes with a
low pH optimum (Rayle and Cleland, 1992;
Bogoslavsky and Neumann, 1998). The Northern
blots in Figure 3 show that RGG105 is primarily
expressed in the 1–5 mm region of the maize root
tip where root elongation is accelerating and little
expression is found in the region 5–15 mm from
the tip where root elongation decelerates and then
stops. Importantly, the correlation between
expressions of RGG105 in tissues showing accelerating expansion is maintained even when growth
is selectively inhibited by water deficit and it also
matches the expression profiles shown by us for
proton pumping ATPase (Figure 4). Peters and
Felle (1999) showed apparent correlations between
profiles of root surface acidification and root
growth in the elongation zone of maize roots. Fan
and Neumann (2004) showed similar correlations
between segmental growth rate, proton fluxes and
pH in epidermal cell walls, in well watered roots
and roots under water stress. It is therefore likely
that profiles of increasing and decreasing acidification and segmental growth rate are related to the
location and activities of PM H+ ATPase and its
RGG105 activator.
The plant cell wall contains an array of proteins
potentially able to modify matrix polysaccharides
so as to facilitate wall loosening. These cell wall
proteins offer many possibilities for modulating cell
wall expansion (Cosgrove, 2001). One such cell wall
enzyme located by us is RGG22, an exo-b-D -glucanase. In maize it was purified from cell walls of
developing shoots (Kim et al., 2000). b-glucan
accumulates during cell elongation and becomes the
major cellulose cross-linking glycan. The hydrolysis
of b-Glucan by exo- (and endo) glucanases was
considered to be necessary for cell expansion in
grasses (Hoson and Nevins, 1989). Our finding that
exoglucanase gene expression was up-regulated
mainly in the accelerating region of the maize root
elongation zone supports the hypothesis that the
activity of this enzyme is intimately involved in
regulating the process of cell elongation.
RGG152 which was up-regulated in the accelerating region annotated to a gene called nucellain.
Nucellain in Barley is a homolog of the dicot
vacuolar processing proteinase which has been
shown to be located in the walls of all barley
nucellar cell types (Linnestad et al., 1998). Proteinases have been suggested to modify cell wall
polypeptides during growth (Van der wilden et al.,
1983; Varner and Lin, 1989).
Two other wall associated genes that were upregulated in the accelerating region were RGG6
and RGG161. RGG6 matched with a Zea mays
golgi associated protein, se-wap41. It is a reversibly
glycosylated polypeptide (RGP). RGPs are
thought to be involved in wall polysaccharide
metabolism (Darvill et al., 1980; Hayashi, 1989;
Brummell et al., 1990; Driouich et al., 1993;
Staehelin and Moore, 1995). RGG161 annotated to
a gene called annexin p35. Annexins are a family of
proteins of conserved sequence that associate with
phospholipid membranes in the presence of Ca2+
and may have a role in exocytosis (Creuzt, 1992;
Battey and Blackbourn, 1993; Raynal and Pollard,
1994; Battey et al., 1996). Plant annexins can also
regulate the activity of enzymes involved in
ATPase, peroxidase and (1!3)-b-glucan (callose)
synthesis (Andrawis et al., 1993; McClung et al.,
1994; Gidrol et al., 1996).
It is of interest that expression of two other wall
metabolizing enzymes, xyloglucan endo trans
glycosylase (XET) and b glucosidase (RGG8B)
were respectively up-regulated in regions 0–5 and
5–10 mm behind the tip. XET is known to be
associated with wall loosening but it is difficult to
see how specific up-regulation of a hydrolytic
enzyme like beta glucosidase in the decelerating
region could be associated with decreases in wall
extensibility. Nevertheless, a recent report by
Chanliaud et al. (2004) indicates that polysaccharide removal by glucosidases may cause cell wall
stiffening in vitro. A similar wall stiffening effect
resulting from increased b glucosidae activity
in vivo would reduce wall extensibility and hence
elongation growth.
Finally, a comparison of the spatial distribution of four selected genes found by us to be
up-regulated in the maize elongation zone showed
a similarity to the spatial distribution of the
homologous genes in Arabidopsis roots (Birnbaum
et al., 2003). These genes are: RGG8B whch is
homologous to Arabidopsis beta glucosidase
At3g18070; RGG22 which is homologous to the
exoglucanase At5g20950; RGG105 whch is
homologous to the proton pump interactor
At4g27500. In addition to these wall related genes,
377
RGG485B is
At3g02790.
homologous
to
zinc
finger
Signal associated genes
RGG279 was annotated to a gene called ABAinducible gene for glycine-rich protein (Gomez
et al., 1988). Using the in-situ hybridization procedure, we found that RGG279 was expressed in
the 0.5–3 mm region above the root cap. It was
localized in the tip meristem, epidermal layer and
in the stele. Variations in tissue localization of root
tip genes were also shown by Birnbaum et al.
(2003) in Arabidopsis.
For the in-situ hybridization we performed RTPCR with specific primers for RGG279 and the
sequence of the PCR product showed perfect
homology to the expected sequence. However, it
contained only 8 glycine molecules, instead of 42
molecules (i.e. a total length of 59 bp was missing).
RGG279 has strong homology (72% sequence
identity) to SaGRP from Sinapis alba (Heintzen
et al., 1994). SaGRP transcripts were found predominantly in young, growing tissue and in meristematic regions. Only the full spliced transcripts
contain the uninterrupted ORF for the functional
glycine-rich RNA binding protein. Specific splicing factors have been described regulating their
own expression by controlling alternative splicing
of their primary transcripts (Boggs et al., 1987;
Zachar et al., 1987; Bell et al., 1988) and RGG279
might conceivably interact with its own RNA.
Another signal associated root gene revealed by
our investigation was RGG7B. RGG7B is an
unknown mRNA in maize but has homology to
SHAGGY-related protein kinase. In higher plants,
the SHAGGY-like genes are present as small gene
families. GSK3/SHAGGY-like protein kinases
have been shown to play diverse roles in signal
transduction and in development (Piao et al., 2001;
Charrier et al., 2002; Perez-Perez et al., 2002).
RGG16B, was annotated to a putative ethylene
forming enzyme. Ethylene plays an important role
signaling many aspects of plant growth, development and responses to the environment (Yang and
Hoffman, 1984; Theologis, 1992) and has been
specifically associated with root (and shoot)
growth responses to water stress (Sharp and
LeNoble, 2002).
Finally, RGG485B is an unknown mRNA in
maize, but has high homology to a C2H2 zinc
finger from Arabidopsis. Another transcription
regulatory protein is RGG326 that has homology
to B37 QM protein. Their possible signaling roles
in the regulation of root elongation remain to be
determined.
Cytoskeleton related genes
Microfilaments (polymerized actin) and microtubules (polymerized tubelin) are considered to be
major components of the cytoskeleton and their
possible roles in plant cell division and elongation
have been extensively reviewed (Goddard et al.,
1994; Baluska et al., 1998; Barlow and Baluska,
2000). Our findings therefore support and extend
previous conclusions about the involvement of
cytoskeleton in maize root elongation.
Genes related to general metabolism
RGG289 and RGG162 are related to methionine
metabolism and can therefore play a major role in
methylations of a large variety of acceptor molecules, such as cell wall components, lipids, polysaccharides, nucleic acids, proteins, and secondary
plant products, including those involved in regulation of cell growth, cell division, and cell morphology (Hepburn et al., 1987). Other genes in this
category include RGG218, a mitochondrial phosphate transporter and RGG240B which shows
high homology to vacuolar ATPase from Arabidopsis and functions in transport and house keeping
roles (Ratajczak, 2000). A single gene with putative proteolytic functions RGG29 is also found in
this category.
Genes with unknown function
Using the SSH approach we also came across
several unknown genes. Either no homology was
found for these genes using the NCBI database or,
despite homology to other genes, their function is
still unclear. Two examples are RGG144 and
RGG304. RGG144 is similar to root specific
protein RCc3 from Oryza sativa that was previously found to be expressed in the root cap and in
the elongation zone but not in the apical meristerm
and zones of cell division (Xu et al., 1995). We
performed in-situ hybridization in order to locate
RGG144’s expression in maize and got similar
results. It was expressed mainly along 2–4 mm
378
region above the root cap, and specifically
expressed in the cortex adjacent to the procambium
and stele. This finding corroborates the efficacy of
the techniques used in this investigation.
RGG304 is similar to endomembrane protein
EMP70 precursor isolog from Arabidopsis.
According to the MIPS database (http://
mips.gsf.de/proj/thal/db/index) it may be a channel or a pore transporter since it has nine predicted
transmembrane domains. Finally, RGG137 is a
unique unknown gene that was up-regulated in the
decelerating and fully elongated regions. Its upregulation may therefore be somehow involved in
the deceleration of growth in the 5–10 mm region.
Effect of water deficit on gene expression
in the elongation zone
We confirmed the correlations between location of
gene expression and root elongation rates by
examining the expression of the same genes under
growth inhibitory water deficit. After 48 h of water
deficit, the altered profiles of gene expression were
still related to elongation. Thus, genes associated
with the zone of accelerating growth rate in
control roots were similarly associated with the
shortened zone of accelerating growth (1–3 mm
behind the tip) in roots under water deficit.
Expression of these genes was comparatively reduced in adjacent regions (3–6 and 6–9 mm from
the tip) where segmental growth rate was inhibited
by water deficit. Furthermore, one gene that was
found to be associated with the zone of decelerating growth rate in the control roots was similarly
associated with the shortened zone of decelerating
growth in water deficit treated roots.
Conclusions
By using the SSH technique, Northern blots,
in-situ hybridization and candidate genes, we showed
the occurrence and identity of genes that were
expressed mainly or exclusively in elongating root
tissues. The spatial patterns of gene expression in
root tips are expected to be complex since the
expanding root cells change position and growth
rate during their rapid passage through the cell
elongation zone. However, by dividing the maize
root tip into three distinct regions (representing
tissues with accelerating, decelerating or fully
elongated cells) we discovered, apparently for the
first time, several interesting genes which showed
unique profiles of expression and may be involved
in processes regulating root growth rates. In particular, we were able to show that genes for a
plasma membrane proton pumping ATPase and a
comparatively rare proton pump activator were
strongly expressed in the zone of accelerating
elongation where acid induced wall loosening is
thought to facilitate rapid growth. The fact that
many of the genes that we investigated also
showed preferential expression in the (shortened)
zone of accelerating elongation after 48 h of
growth inhibitory water deficit, suggests an equally
vital role for their gene products in growth maintenance under water deficit conditions. The gene
identification and spatial localization presented
here provide useful targets for functional analysis
and a basis for conceptual models of growth
regulation in roots.
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