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https://www.abbs.info ISSN 0582-9879 |
Induced Expression
of the Gene for NADP-malic Enzyme in Leaves of Aloe vera L. under Salt
Stress
SUN Shu-Bin,
SHEN Qi-Rong, WAN Jian-Min1, LIU
Zhao-Pu*
( College of Resources and
Environmental Science, Nanjing 210095, China;
1Institute of Rice, Nanjing Agricultural University, Nanjing 210095, China
)
Abstract cDNA fragment for NADP-malic enzyme, catalyzing
the reversible oxidative decarboxylation of L-malate to produce CO
pyruvate and NADPH, was isolated from the leaves of a 2-month-old Aloe
vera L., The level of expression of NADP-ME mRNA and accumulation of NADP-ME
(AvME) protein under salt stress conditions were compared between a tolerant
aloe, Aloe vera L. and a sensitive aloe, Aloe saponarea Haw.
The results suggested that both the expression of the gene and the accumulation
of the protein were induced in the two kinds of aloe, and the strength was
related to the degree of salt tolerance. Northern blot analysis revealed that
the gene for NADP-malic enzyme in Aloe vera L.(AvME) was induced
by high salt, dehydration, and exogenous abscisic acid (ABA), but not by cold
treatment. To further confirm whether the synthesis of AvME protein was induced
with hours of treatment, Western blot analysis of the samples was conducted.
The results indicated that the induction of AvME protein expression was obvious
after 48 h at high salt and the level was increased with the hours of treatment.
Key words aloe; NADP-ME; gene expression; salt tolerance
NADP-malic enzyme (EC 1.1.1.40) (NADP-ME) acts in a wide range of metabolic
pathways in both plants and animals[1]. It can catalyze the oxidative decarboxylation
of malate to pyruvate: malate+NADP+→pyruvate+CO
In plants, two forms of this enzyme are known to occur and both play important
metabolic roles. The cytosolic form is thought to participate in the regulation
of intracellular pH[2, 3] or in the provision of reducing power that can be
used in processes that require NADPH[4]. The chloroplast stromal form is found
specifically in the bundle sheath chloroplasts of NADP-malic enzyme-type C
plants, and this enzyme plays a key role in photosynthesis by providing CO
for fixation in the Calvin cycle in bundle sheath cells[4].
At least two chloroplast isoforms of NADP-ME have been characterized in NADP-ME-type
C
to bundle sheath chloroplasts of green leaves where it is involved in malate
decarboxylation during C
properties of this kind of isoform were widely studied and the primary structure
of the proteins was deduced from the nucleotide sequence of its cDNA[1, 5].
The cytosolic isoform was purified from etiolated leaves and its kinetic parameters
were studied[6].
Aloe vera L. is an ‘obligate’ monocot crassulacean acid metabolism
(CAM) plant, and possesses NADP-ME activity, which is responsible for the
decarboxylation that occurs during CAM photosynthesis[7]. NADP-MEs in CAM
plants can be classified as either photosynthetic or non-photosynthetic[8],
and NADP-malic enzyme cDNA induced by salt stress has been described in the
CAM-inducible plant M. crystallinum[9] but not in aloe.
In order to study how salt affects aloe growth and development at the molecular
level, a cDNA fragment for NADP-ME from the leaves of Aloe vera L.
was cloned and the gene expression under stresses in aloe was investigated.
1 Materials and Methods
1.1 Materials
Seedlings of Aloe vera L. were provided by Hainan Taiyangcheng Tissue
Culture Factory in China and grown hydroponically in a greenhouse at the College
of Resources and Environmental Science of Nanjing Agricultural University,
China. The 2-month-old seedlings were treated under various conditions as
follows: plants were grown in solution containing 2 mmol/L ABA and 300 mmol/L
NaCl, respectively. For dehydration stress treatment, the plants were grown
at limited water. Low-temperature stress was imposed by transferring the plants
directly to 10 °C. The harvest leaves of the plants were frozen in liquid
nitrogen and stored at －80 °C for further analysis.
1.2 RT-PCR and DNA sequencing
A pair of primers, 5′-agtggctactctttgttgcg-3′ and 5′-gcagtgtaaagagagagctt-3′
were designed from conserved regions of known Aloe arborescens, M.
crystallinum, Flaveria species NADP-ME genes. RT-PCR was carried
out according to the procedure described in the RT-PCR Kit (TaKaRa). The Perkin
Elmer Cetus model 9600 DNA thermal cycler in our laboratory was used. The
PCR parameters consisted of 30 s at 94 °C for denaturing, 30 s at 60 °C for
annealing and 90 s at 72 °C for extension for 30 cycles and a final extension
step of 7 min at 72 °C. The PCR products were analyzed on 1.2% agarose gel
and visualized with ethidium bromide staining. A 496 bp cDNA fragment was
amplified from the total RNA of Aloe vera L. The PCR fragment was cloned
into a pGEM-T Easy vector (Promega).
DNA sequence was determined by Shanghai United Gene Limited Company. Nucleotide
and amino acid sequences were analyzed with the DNAman software system.
1.3 Determination of tolerance to NaCl and NADP-malic enzymatic activity
Tolerance to NaCl was determined as follows. The 2-month-old seedlings of
five aloe strains (Aloe saponaria Haw, Aloe nobilis, Aloe ferox
Miller, Aloe arborensis and Aloe vera L.) were grown hydroponically
in greenhouse in 300 mmol/L NaCl solution, and in distilled water as control.
The numbers of surviving plants were counted.
NADP-malic enzymatic activity was assayed by the malate-dependent reduction
of NADP at 340 nm[10]. The reaction mixture (1.0 ml) contained 50 mmol/L Tris-TES
(pH 7.5), 5 mmol/L MgCl
initiated by addition of L-malate to a final concentration of 5 mmol/L
and carried out at 30 °C. One unit of enzyme was defined as the amount of
enzyme that catalyzed the reduction of 1 μmol of NADP per min under these
conditions.
1.4 RNA isolation and Northern blotting
Total RNA was extracted from leaves of 2-month-old seedlings of Aloe vera
L. during control, salt, dehydration, exogenous abscisic acid (ABA) and cold
treatment by the method of improved acid-guanidinium thiocyanate[11]. Thirty
micrograms of total RNA were denatured and resolved on 1.2% agarose gels containing
formaldehyde[12]. After electrophoresis, the RNA was blotted onto nylon membrane
(Hybond-N, Amersham) by capillary transfer in 20×SSC overnight. The RNA was
fixed by baking for 2 h at 80 °C under a vacuum. The cDNA fragment encoding
the gene for NADP-ME in Aloe vera L. (AvME) was labeled by random priming
with [32P] dCTP according to the manufacturer’s specifications for Northern
blots (Boehringer Mannheim, Indianapolis, IN) and then exposed to X-ray film
at －80 °C overnight. Blots were rehybridized using EF-1α gene (kindly provided
by Peng JL) to ensure that equal amounts of total RNA were present in each
lane.
1.5 Western blotting analysis
Crude extracts from the leaves of 2-month-old Aloe vera L. under 300
mmol/L NaCl were used for protein analysis. The amount of total protein in
the crude extract was estimated using a protein assay kit (Bio-Rad). SDS-PAGE
was carried out by the methods of Laemmli[13]. After electrophoretic separation,
proteins on the gels were electro-blotted onto a nitrocellulose membrane for
immunoblotting according to the work of Burnette[14]. After blocking free
protein binding sites for 1 h in 1 g/L fat-free milk powder dissolved in phosphate
buffered saline (PBS), anti-maize 72 kD-NADP-ME IgG (diluted 1∶1000) which
was affinity-purified according to the method of Plaxton[15] was used for
detection. Bound antibodies were visualized by linking to alkaline phosphatase-conjugated
goat anti-rabbit IgG according to the manufacturer’s instructions (Promega).
2 Results
2.1 Isolation and sequencing of a cDNA fragment for ME of Aloe vera
L.
The 496 bp of NADP-ME cDNA fragment was obtained from the leaves of 2-month-old
Aloe vera L. by RT-PCR. The nucleotide sequence data will appear in
the GenBank Nucleotide Sequence Databases under the accession No. AY179511.
The nucleotide sequence and the deduced amino acid sequence of the cDNA clone
were compared with those of known plant NADP-MEs.
Fig.1 Comparison of the deduced amino acid sequence of AvME with those
of other known plant NADP-MEs
AvME is aligned with rice ME and Zea mays ME. Hyphens have been introduced
to maximize the identity to other sequences. Highly conserved regions, I and
II, which may be involved in binding to NADP, are boxed.
The deduced amino acid sequence was highly homologous to those of AME1 (98%,
Accession No. AB016804.1), AME2 (90%, Accession No. Ab005808.1), OsME (88%,
Accession No. AP002836), ZmME (85%, Accession No. AY040616.1) (Fig. 1). It
has been reported that the three regions are involved in NADP binding[16，17].
Two of the three regions (binding box I, II) were also conserved in this clone
as shown in Fig. 1.
2.2 Expression of NADP-ME gene and the activity of NADP-ME
protein in aloe under salt condition
Fig.2 Comparison of tolerance to NaCl
The 2-month-old aloe seedlings were hydroponically treated in 300 mmol/L NaCl
solution. Survival ratio = number of surviving plants/number of planted plants.
As shown in Fig. 2, Aloe saponarea Haw was sensitive to NaCl and did
not survive after treatment for 100 h with 300 mmol/L NaCl. However, Aloe
vera L. was tolerant to NaCl under the same conditions as above and its
survival ratio after 100 h treatment was much higher than that of the other
four kinds of aloe. Therefore, Aloe vera L. was considered as salt
tolerant aloe and Aloe saponarea Haw as salt sensitive aloe.
To investigate whether the expression of NADP-ME gene and the accumulation
of NADP-ME protein induced by high-salt treatment are related to salt tolerance
in the aloe plant, Northern blot analysis was performed and the activity of
NADP-ME protein was measured in a tolerant aloe, Aloe vera L., and
a sensitive aloe, Aloe saponarea Haw under high-salt stress. As shown
in Fig. 3(A), the NADP-ME mRNA was induced after 12 h salt treatment, and
its level increased up to 48 h in both kinds of aloe, but the level of expression
of NADP-ME gene in Aloe vera L. was higher than in Aloe saponarea
Haw. These observations suggested that the expression of NADP-ME gene and
the accumulation of NADP-ME protein were strongly induced in both kinds of
aloe under high-salt conditions, and the intensity induced by high-salt treatment
was related to the degree of salt tolerance in aloe. Similarly, as shown in
Fig. 3(B), the activity of NADP-ME protein began to increase until 24 h after
the initiation of treatment. The activity increased steadily for a further
96 h. But the activity of NADP-ME protein in Aloe vera L. was higher
than that in Aloe saponarea Haw. Furthermore, we also observed that
the activity of NADP-ME protein followed the accumulation of NADP-ME mRNA
in aloe under salt stress.
Fig.3 Comparison of the level of expression of NADP-ME gene and the activity
of NADP-ME
Protein under 300 mmol/L salt treatment between a salt-sensitive aloe ( Aloe
saponarea Haw) and a salt-tolerant aloe ( Aloe vera L. ).
EF-1αwas used as an internal control.
2.3 Analysis of the effects of high salt, dehydration, exogenous ABA and
cold on the expression of AvME, and NADP-ME induction by high salt
To investigate the expression of AvME under several types of stress,
Northern blot analysis was performed. As shown in Fig. 4, the expression of
AvME was induced by salt (300 mmol/L), dehydration and exogenous ABA
(2 mmol/L) treatments, but not by cold (10 ℃) treatment. Moreover, the accumulation
of AvME mRNA reached the peaks at different treatment time.
Fig.4 Northern blot analysis of AvME mRNA
(A) salt (300 mmol/L NaCl); (B) dehydration; (C) exogenous abscisic acid (ABA,
2 mmol/L); (D) cold (10 °C).
The salt-induced strength of the AvME mRNA was highest among several
stresses. It was clearly induced within 12 h after the start of salt treatment,
and its level increased up to 72 h and then decreased. The AvME mRNA
was also induced within 6 h after the start of dehydration and its level increased
up to 12 h and then decreased. Rapid accumulation of AvME mRNA in response
to exogenous ABA was observed within 3 h and continued for 12 h, and the accumulation
was earlier than that in response to dehydration stress. It has been proposed
that the biosynthesis of ABA is induced by water deficiency and such an increased
level of endogenous ABA would then induce the expression of AvME in
Aloe vera L. under water stress. It also can be seen from Fig.4 that
AvME mRNA was not induced by cold treatment. This is consistent with
its biological property in aloe.
Approximately 30 μg of total RNA is loaded in each lane. Time indicates the
number of hours after the initiation of the treatment prior to the isolation
of RNA. In the upper panel, a representative pattern of Northern blot is shown;
error bars indicate SD of the mean. Results are the mean of three different
determinations from independent samples. EF-1α is an elongation factor, which
is highly conserved in animals and plants. Here it was used as internal control.
To investigate whether the synthesis of NADP-ME protein under salt stress
was induced over time, Western blot analysis of the samples was conducted.
As shown in Fig. 5, all the lanes loaded with extracts from leaves yielded
one major band of approximate 72 kD in un-induced and induced aloe leave tissue.
The induction of the synthesis of this protein was clearly shown after 48
h at high salt, and the intensity of the protein band reacted with the antibody
increased with the time of treatment. These changes paralleled the changes
in the transcription of AvME during salt stress.
Fig.5 Western blot analysis of NADP-ME in Aloe vera L. with varied hour
treatment by high salt (300 mmol/L) exposure
(A) Coomassie blue staining to ensure equal protein amounts in the lanes;
(B) Western blot analysis. The molecular massed of the marker were indicated
on the left side. Time indicates the number of hours after the initiation
of the treatment prior to the extraction of protein.
3 Discussion
Plants are exposed to many types of environmental stresses. Among these, salinity
and drought is the most important problem that limits plant growth. Plants,
in turn, have evolved many different physiological and biochemical strategies
for coping with water stress imparted by saline or drought conditions. Such
strategies include the synthesis and accumulation of organic osmolytes, enhanced
Na+/H+ antiporter and H+-ATPase activities
at the tonoplast and plasma membranes for ion sequestration, enhanced expression
of water channel genes and changes in photosynthetic metabolism[18, 19]. In
this report, characterization of a cDNA clone for NADP-ME from aloe provides
a starting point for reversing the salt-tolerant mechanism within the plant.
Northern and Western blot data indicated that in aloe, NADP-ME gene was strongly
induced by high salt (Fig. 4 and Fig. 5). The increases in transcript levels
and translation levels of NADP-ME in leaf tissue during salt stress suggest
that this gene is responsible in part for salt tolerance in aloe.
Aloe is a crassulacean acid metabolism (CAM) plant with a specialized photosynthetic
pathway[20－22]. Under this system, stomata are only open during the night
to minimize the loss of water by transpiration, and carbon dioxide is absorbed
at night by a CO
with the breakdown of stored carbohydrates into phosphoenolpyruvate (PEP),
atmospheric CO
(PEPC) to form C
vacuoles. The following day, malate is released from the vacuoles into the
cytosol and decarboxylated to form pyruvate and CO
NADP-malic enzyme. NADP-ME is, therefore, one of the key enzymes in this pathway,
which contributes to strong salinity-tolerance and dehydration-tolerance in
aloe.
The mechanism of Na+ uptake into plant cells across the plasmademma is not
yet clear. It might be mediated by K+ channel and non-selective cation channel[24].
To balance the influx of positive charge, organic anions, principally malate,
and chloride accumulated[25]. Malate is synthesized in the cytosol from starch
stored by a unique, regulated form of phosphoenolpyruvate carboxylase[26].
The fate of malate during this period is unclear, but several lines of evidence
support the hypothesis that a form of NADP-ME may facilitate malate degradation[27].
In plants, the final level of stress-induced expressed genes is determined
not only by transcription rates, but also by post-transcriptional mechanism
such as selective translation or transcript stabilization. There has been
indirect evidence that post-transcriptional events are involved in regulation
of gene expression during stress condition[28]. In this study, Western analysis
revealed that the expression of AvME protein was also strongly induced. This
finding suggests that AvME protein synthesis is under transcriptional control
although salt stress may also affect the stability of AvME transcript in
vivo.
Studies are now in progress to isolate other key genes in photosynthesis
pathway in aloe plant responsible for the activation of transcription by salt
stress. Meantime, the promoter sequences that confer salt-stress-specific
expression are analyzed. Future studies in our laboratory introduce these
important photosynthesis genes in aloe into the C
facilitate to better decipher the mechanisms of signal transduction leading
to this activation. Furthermore, they will improve photosynthesis and crop
yield in C
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Received: December 30, 2002Accepted: March 4, 2003
This work was supported by a grant from the National High Technology Research
and Development Program of China (863 Program), Marine Technology Projects（No.
2001AA627040）
*Corresponding author: Tel, 86-25-4396678; Fax, 86-25-4396678; e-mail, [email protected]