ABBS 2005,38(06): Rice GTPase OsRacB: Potential Accessory Factor in Plant Salt-stress Signalling

Abstract        As the sole ubiquitous
signal small guanosine triphosphate-binding protein in plants, Rop gene plays
an important role in plant growth and development. In this study, we focus on
the relationship between the novel rice Rop gene OsRacB and plant salt
tolerance. Results show that OsRacB transcription is highly accumulated
in roots after treatment with salinity, but only slightly accumulated in stems
and leaves under the same treatment. Promoter analysis showed that OsRacB
promoter is induced by salinity and exogenous salicylic acid, not abscisic
acid. To elucidate its physiological function, we generated OsRacB sense
and antisense transgenic tobacco and rice. Under proper salinity treatment,
sense transgenic plants grew much better than the control. This suggests that
overexpression of OsRacB in tobacco and rice can improve plant salt
tolerance. But under the same treatment, no difference could be observed
between OsRacB antisense plants and the control. The results indicated
that OsRacB is only an accessory factor in plant salt tolerance.

Key words        OsRacB; rice; salt
tolerance; promoter; transgenic analysis

Accumulation of salts in
irrigated soil is a primary factor in depressing yield in crop production. Previous
studies showed that products of some stress-inducible functional genes could
directly counteract this detrimental condition. Transfer of these genes into
plants confirmed their protective roles in stress adaptation. But the
effectiveness of an individual gene is normally rather small [1,2]. It was
generally thought that salt stress was regulated by a complex signaling
network, and modulation of signaling regulators would be a promising method for
improving plant salt stress tolerance. So it is thrilling to study the
molecular mechanisms of salt tolerance underlying signal transduction, although
little has been known on the topic until now [1].

Small guanosine
triphosphate (GTP)-binding proteins are pivotal molecular switches in signal
transduction. They are normally divided into five families, including Ras, Previous studies showed
that Rops participate in signaling to a series of physiological processes
including actin cytoskeleton remodeling, secondary wall formation,
intracellular Ca2+ gradients establishment, regulation of polar
cell growth, production of reactive oxygen intermediates, and modulation of
hormone signaling and gene expression. During these studies, the function of
Rop in biotic stress response was noted. The importance of Rops in defence
mechanisms has been shown in at least three plant micro-interactions, such as
OsRac1 function in the rice-rice blast fungus and rice-rice bacterial leaf
blight systems [12–14], and
HvRacB action in the barley-barley powdery mildew fungus system [5,14,15]. But
little is known about its function in abiotic stress response, except for one
study of Rop action in transient flooding endurance [16]. So it is significant
to study how Rop acts in abiotic stress response such as salinity resistance.

During our previous
work, a novel rice Rop gene OsRacB was isolated from a young ear cDNA
library [17]. Structural analysis and in vitro GTP binding tests
identified OsRacB as a new member of the Rop family [17,18]. OsRacB
encoded a putative protein of 197 amino acids. Its transcription unit was
2930 bp in length, consisting of seven exons and six introns. Genomic Southern
blotting revealed that OsRacB was a low abundance gene. Reverse
transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that OsRacB
was ubiquitously expressed in various tissues, but the expression level in
stems and young ears was much higher than that in other organs. OsRacB
showed very high amino acid homology to maize ZmRacB (99.5%), which can induce
superoxide production when expressed in a mammalian system (NIH 3T3 cells)
[19], and barley HvRacB (99.0%), which was involved in processes supporting
parasitic entry into epidermal host cells [5,14,15]. In this paper, we analyzed
OsRacB‘s expression pattern, promoter regulation and transgenic plant
phenotype under salinity stress. Results suggest that OsRacB might play
an accessory role in plant salt tolerance.

Materials and Methods

Plant growth and stress
treatment

Seeds of late ripening
japonica rice cultivar (cv.) Nongken 58 were obtained from Prof. Tong-Min MOU (In addition, seeds were
germinated for 3 d and grown in nutrient solution comprising 0.1% (V/V)
Hyponex (water) (Hyponex Co., Copley, USA) for 20 d under normal conditions at
28 ºC and 14 h light/10 h darkness. For osmotic stress treatment, 20-day-old
(three-leaf-old) rice seedlings were grown in nutrient solution containing

Semiquantitative RT-PCR
analysis

Following the procedure
provided with the SuperScript II kit (Gibco BRL), reverse transcription of
total RNA was carried out using Oligo(dT)12–18 (Invitrogen). A set
of primers specific for the rice actin gene actin1 (X15865), 5‘-CATGCTATCCCTCGTCTCGACCT-3‘
and 5‘-C GCACTTCATGATGGAGTTGTAT-3‘, were synthesized and used in
RT-PCR as internal control. Amplification was carried out under the following
conditions: 94 ºC for 5 min; 94 ºC for 1 min, 54 ºC for 0.5 min, 72 ºC for 0.5
min, 25 cycles; 72 ºC for 7 min. OsRacB was amplified by RT-PCR, based
on its specific primer pair 5‘-TTGCTTTGCTCCTCCTTCAACCTT-3‘ and 5‘-GCCACGACTTGTCAGTCACACG-3‘.
PCR thermocycling profile was 94 ºC, 60 ºC and 72 ºC, 1 min each step, for a
total of 26–28 cycles.
PCR products were analyzed through the Gel-Doc 2000 system (Bio-Rad).

5‘-Deletion
analysis of OsRacB promoter

Based on previous
research [17], we isolated OsRacB promoter and subcloned it into
pBluescriptII SK(+) vector (Stratagene). Four 5‘ deletion OsRacB
promoter fragments were constructed by PCR for promoter element analysis. They
were covered from –1241 to +175,
–721 to +175, –481 to +175, –281 to +175. 5‘ deletion vectors p1301-BPn-b-glucuronidase (GUS) were constructed by cloning these fragments into XbaI/BglII
sites of pCAMBIA1301 instead of its original constitutive cauliflower mosaic
virus (CaMV) 35S promoter. To construct the negative control vector p1301-CK-GUS,
pCAMBIA1301 was digested with XbaI and BglII to delete the
original 35S promoter, and circularized by blunt end ligation after turning
adhesive ends to blunt ends. Both p1301-BPn-GUS, pCAMBIA1301 and p1301-CK-GUS
vectors were separately transformed into Agrobacterium tumefaciens
EHA105 by electroporation and introduced into tobacco cv. NC89. Transformed
tobaccos were screened by 50 mg/L hygromycin and confirmed by Southern blot and
RT-PCR analysis. Excised leaves from transgenic plants were tested for GUS
activity using quantitative fluorometric assay [20]. During the treatments,
leaves were cut into flakes approximately

Construction of
OsRacB binary vectors and plant transformation

Sense and antisense OsRacB
vectors, pWMSB and pWMAB, were constructed by cloning full-length OsRacB
cDNA into the KpnI/BamHI site or XbaI/BamHI sites,
respectively, of plant expression vector pWM101, in sense or antisense
orientation under the control of enhanced CaMV 35S promoter. Using the Agrobacterium-mediated
method [21,22], these OsRacB vectors were separately introduced into
tobacco cv. NC89 and rice cv. Nongken 58 cells. In addition, blank vector
pWM101 was introduced into the plants as the control. PCR and Southern
hybridization were sequentially used to verify transgenic plants. Transformed
calli were selected by hygromycin resistance, and regenerated. In a growth
chamber (16 h light/8 h darkness, 28 ºC), transgenic tobaccos were separately
grown in

Estimation of OsRacB
transgenic plants salt tolerance

In a growth chamber (16
h light/8 h darkness, 28 ºC), sterilized T1 transgenic tobacco seeds were
germinated on 1/2 MS agar medium [23] containing 0–At the same time, T1
transgenic rice seeds were germinated for 3 d and separately transplanted in

Results

OsRacB is highly
accumulated in roots upon exposure to salt stress

The conserved

Identification of
promoter sequence that regulates expression of OsRacB

Using OsRacB
intron IV (1957–2265 nt) and
the 5‘-coding region (1–327 nt) as
specific probes, we isolated OsRacB promoter by screening a rice genomic
DNA library [17]. Analyzed by PLACE software (http://www.dna.affrc.go.jp/PLACE/),
some putative regulation elements were found, such as cytoskeleton-related
element (I-box), To clarify its
regulation pattern in plants, different 5‘ deletion fragments of OsRacB
promoter were isolated by PCR and inserted in transgenic vector pCAMBIA1301
instead of the original CaMV 35S promoter. These vectors were named p1301-BPn-GUS
(with n representing A, B, C or D). Fig. 2 shows all deletions
and their locations with respect to the structure of OsRacB genomic
clone. To eliminate the original GUS background in transgenic plants,
p1301-CK-GUS vector was constructed with original 35S promoter deleted to act
as a negative control. All of these constructs and the positive control
pCAMBIA1301 were introduced into tobacco cv. NC89 by means of Agrobacterium-mediated
transformation. Transgenic plants were regenerated from hygromycin-resistant
transformants. Excised leaves from transgenic plants were tested for GUS
activity using quantitative fluorometric assay [20].

Functional studies
showed that many plant Rop genes were regulated by

Phenotypic and genetic
analysis of transgenic tobacco transformed with sense and antisense OsRacB

To clarify the
physiological role of OsRacB in plants, OsRacB cDNA was
introduced into tobacco cv. NC89 cells in sense as well as antisense
orientation under CaMV 35S promoter control by means of Agrobacterium-mediated
transformation [Fig. 4(A,B)]. Six cDNAs encoding Rop protein
homologs were isolated in tobacco [33–35], which
are highly homologous to OsRacB. Heterologous expression of OsRacB
in tobacco plants should influence the expression of endogenous Rop homologs.
NtRac5, the highest homology tobacco Rop protein to OsRacB (91.4%), can
regulate active oxygen species production by negative regulation of NtrbohD, an
oxidase involved in active oxygen species production upon elicitation [33].

In order to simplify
PCR-based molecular analysis of our transgenic plants, we designed a special 5‘
primer based on the hyp gene sequence. The integration of hyp-35S
P-OsRacB-cDNA in both types of transformants was confirmed by genomic PCR,
without simultaneously amplifying endogenous Rop-related tobacco genes [Fig.
4(C,D)]. Twelve independent transformants with OsRacB in sense
orientation, fourteen plants transformed with OsRacB in antisense
orientation and six vector control plants were regenerated for further
analysis. In order to minimize the interference of tissue culture, nine aseptic
regenerated NC89 plants were obtained. Expression levels of these OsRacB
transgenes were demonstrated by RT-PCR (data not shown). RT-PCR analysis
confirmed that 12 transgenic tobacco plants transformed with OsRacB
sense constructs expressed the foreign gene at mRNA level.

Several biological
characters, such as shoot development, weight of 1000 seeds, seed vitality and
germination capacity, were compared between all types of T0 and T1 tobaccos.
Compared to vector control plants and regenerated NC89, OsRacB-sense
plants showed no differences except plant height (Table 1; Fig. 5).
OsRacB-antisense plants showed distinct differences in height, stem
perimeter and seed weight relative to control plants (Table 1; Fig. 5).
This implied that expression changes in the OsRacB homolog affects
tobacco shoot development, especially in plant height. Antisense expression of
MS-rac1 cDNA in transgenic tobacco plants can also cause such growth
inhibition and dwarfing [36].

To test whether OsRacB
played a role in abiotic defence reactions, all types of tobacco plants were
treated with salinity. Fig. 6 shows that three tobaccos grew similarly
in basic culture medium (1/2 MS) and low concentration salinity medium (

Overexpression of OsRacB
cDNA in transgenic rice plants causes salt tolerance

To analyze OsRacB roles
in rice in vivo, same transgenic vectors were also introduced into rice
cv. Nongken 58 cells in sense as well as antisense orientations, as had been
done in tobacco. Genomic PCR and Southern blot analysis confirmed the
successful integration of OsRacB into chromosomes (data not shown). A
highly sensitive RT-PCR, in which the number of amplification cycles was
controlled, was developed to detect OsRacB expression modification in
transgenic rice leaves. As shown in Fig. 7, we succeeded in modifying OsRacB
expression levels in rice cv. Nongken 58 by transgenic technology.

Based on previous
studies, our insights were focused on how the altered OsRacB expression
affected the salinity tolerance of rice plants. In Fig. 8, sense T1
transgenic rice treated with salinity stress grew much better than the control,
suggesting that overexpression of OsRacB in rice can also partly improve
salinity tolerance, as had been shown in tobacco. So it was inferred that OsRacB
plays a role in the signal pathway of plant salt tolerance. There was no
difference between OsRacB antisense plants and controls in their growth
under salinity treatment, neither in transgenic tobacco nor in rice, which
indicated that OsRacB does not play an indispensable role but is an
accessory factor in plant salt tolerance. In addition, during the analysis of T0
and T1 transgenic rice, there were many anomalous growths and abortions in
sense and antisense transgenic rice, which indicated that abnormal expression
of OsRacB could induce lethality.

Discussion

As the most major crop
in the world, a better understanding of stress signaling in rice has an
enormous impact. In the present study, we identified a Rop protein, OsRacB,
which acts as an accessory regulatory component in salt stress responses.
Phylogenetic analyses showed that OsRacB belongs to Group IV of the Rop
family [37]. Another rice Rop gene, OsRacD, and Arabidopsis Rops AtRop1
to AtRop6, also belong to this subgroup [4, 38]. In this group, most
Rops are involved in actin dynamics regulation, which controls polar growth and
root hair development. Other Rops were found to act in the production of active
oxygen species, which can induce programmed cell death. But these similar
proteins have not been attributed to salinity responses. This is the first
report to suggest that Rops plays a role in salt tolerance signal pathways in
plants.

Plants execute a wide
range of physiological and metabolic processes to cope with adverse
environmental conditions, such as high salinity or water deficit. Many genes
have been found to respond to salt stress, but little is known about the
signaling events involved in the mechanisms that make plants tolerant to high
salt concentrations. It is popularly considered that the signaling pathway of
salt tolerance is an intricate web of interconnecting signal networks rather
than a collection of parallel but separate pathways. During the signal net,
some phytohormones such as One early response to salinity
stress in plant cells is a transient increase in cytosolic Ca2+, derived from either an influx from apoplastic
space or release from internal stores
[39,40]. Several lines of evidence suggested that many proteins related with Ca2+ release,
such as calmodulin, calcineurin B-like proteins and calcium-dependent protein
kinases, were involved in stress signal transduction [41]. In rice, OsCDPK7 has
proved to act upstream of one small GTP-binding protein, rabStimulated vesicle
transport under salt stress is also an important physiological process to
altered growth conditions, based on rapidly adjusted biochemistry of the lytic
compartment [44]. Under salt stress, vacuolar volume and activity of vacuolar
ATPase are often increased. Rab protein, the only known small GTP-binding
protein involved in salt tolerance signal transduction, participated in
regulating intracellular vesicular transport by activation of vesicle formation
and fusion at the endoplasmatic reticulum, Golgi apparatus and plasma membrane.
Studies in mammalian cells suggest that biogenesis and dynamics of endosomes
and lysosomes, which share some functional and biogenetic similarities to plant
vacuoles, involve Salt stress also causes
induction of oxidative stress. Under salt stress, stomatal closure triggered by
In addition, research
showed that G-proteins and mitogen-activated protein kinase (MAPK) cascades are
also related to salt resistance. In plants, G-proteins regulate phospholipase C
activity, which could modulate expression of many functional genes involved in
stress signaling, such as LEA-like genes. It has been proved that the a-subunit of G protein acts upstream of Rop protein
OsRacAs signaling proteins,

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