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 CO2, 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+CO2+NADPH+H⁺. 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 C4 plants, and this enzyme plays a key role in photosynthesis by providing CO2 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 C4 plants[5, 6]. The C4-specific isoform is restricted to bundle sheath chloroplasts of green leaves where it is involved in malate decarboxylation during C4 photosynthesis. The structural and kinetic 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 MgCl2 and 0.5 mmol/L NADP. The reaction was 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 CO2-concentrating mechanism[23]. At night, concomitant with the breakdown of stored carbohydrates into phosphoenolpyruvate (PEP), atmospheric CO2 is primarily fixed by phosphoenolpyruvate carboxylase (PEPC) to form C4 acids (mainly malate) which are stored in the vacuoles. The following day, malate is released from the vacuoles into the cytosol and decarboxylated to form pyruvate and CO2 through the activity of 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 C3 plants. These works will facilitate to better decipher the mechanisms of signal transduction leading to this activation. Furthermore, they will improve photosynthesis and crop yield in C3 plants.

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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]