Plant Biotechnology
Molecular Biotechnology and Genetics
EJB Electronic Journal of Biotechnology ISSN: 0717-3458
© 2001 by Universidad Católica de Valparaíso -- Chile
BIP RESEARCH ARTICLE

Identification of drought-responsive transcripts in peanut
(Arachis hypogaea L.)

Ashok K. Jain*
Plant Biotechnology Laboratory
301 South Perry Paige Bldg.
Division of Agricultural Sciences
Florida Agricultural and Mechanical University
Tallahassee, FL 32307, USA
Tel: (850) 561-2219
Fax: (850) 599-3119
E-mail: ashok.jain@famu.edu

Sheikh Mehboob Basha
Plant Biotechnology Laboratory
301 South Perry Paige Bldg.
Division of Agricultural Sciences
Florida Agricultural and Mechanical University
Tallahassee, FL 32307, USA
Telephone: (850) 561-2218
Fax: (850) 599-3119
E-mail: mehboob.sheikh@famu.edu

C. Corley Holbrook
United States Department of Agriculture
Agricultural Research Services - South Atlantic Area
Nematodes, Weeds, and Crops Research Unit
Coastal Plain Experiment Station
P.O. Box 748, Tifton GA 31793, USA
Tel: (912) 386-3176
Fax: (912) 386-3437
E-mail: Holbrook@tifton.cpes.peachnet.edu

* Corresponding author

Financial support: The research was supported by Grant No. 96 38814 2869 from the United States Department of Agriculture.

Keywords: Arachis hypogaea L., differential display, drought response, gene expression, peanut, water stress.


BIP Article

Peanut, Arachis hypogaea, a legume, is commercially grown in many subtropical and tropical regions of the world and is endemic to southeastern regions of the United States. Peanut is rich in oil (50-60%) and protein (24%) and is the third most important source of vegetable protein and provides approximately 11% of the word's protein supply. It is the staple food crop in some developing countries of the world and is mainly consumed in confectionery form in the United States.

Over half of the peanut crop in the United States is not irrigated. In many peanut-producing areas in the world, irrigation is unavailable or impractical. However, when crop is grown under water stress condition it became susceptible to pre-harvest aflatoxin contamination. Aflatoxins are toxic and carcinogenic secondary metabolites produced by a common soil fungus (Aspergillus flavus and A. parasiticus). Due to high carcinogenic nature of aflatoxins, the U.S. Food and Drug Administration does not permit food products containing more than 20 ppb aflatoxin for human consumption. Pre-harvest aflatoxin contamination is a recurrent and common occurrence in those peanuts grown under non-irrigated conditions, exposed to prolonged drought and elevated soil temperature conditions. Aflatoxin contamination does not occur in peanuts grown under irrigated conditions. Also proper irrigation of peanuts during drought decreases the severity of aflatoxin contamination. Prevention of pre-harvest infection of peanut by these fungi and/or the elimination of aflatoxin contamination is an international priority. Since 1970's, peanut breeders and researchers have been trying to develop a peanut cultivar resistant to pre-harvest aflatoxin contamination. However, it turned out to be extremely difficult to achieve this goal.

One of the major problems in finding the source of resistance to aflatoxin contamination is the lack of rapid and reliable screening techniques. Peanut genotypes show tremendous variability in the rate of infection and contamination, even for a single genotype within one field condition. Therefore, screening large number of genotypes to find the source of resistance would not only be difficult but also be deceptive. Recent research demonstrated that drought tolerant peanut genotypes have some degree of resistance to aflatoxin contamination and generally display lower rates of pre-harvest aflatoxin contamination. Because of this observation it is believed that drought tolerant peanut lines may possess some degree of resistance to aflatoxin contamination. Therefore, selecting for tolerance to drought stress has practical advantage and provides an indirect selection method to peanut breeders for searching the resistance to aflatoxin contamination. The current methods for screening peanut genotypes to drought tolerance are based on visual observations and are predominantly dependent on morphological characters such as leaf temperature, root size, comparative yield and biomass production. These traits are often influenced by environmental factors, and screening in one environment may not reflect the true genetic potential for drought tolerance. On the other hand, the widely used DNA based genetic/molecular markers to analyze complex traits and identifying a Quantitative Trait Loci (QTLs) such as Restriction Fragment Length Polymorphism (RFLP), Restriction Landmark Genome Scanning (RLGS), micro satellite, Random Amplification of Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP) or Florescent in situ Hybridization (FISH) have shown limited polymorphism in peanut and have proved inadequate and incapable of identifying unique bands for use as molecular markers in peanut. Therefore, the unavailability of reliable tools to screen peanut genotypes for drought tolerance is the major hurdle in the genetic improvement of peanut for drought tolerance and/or aflatoxin contamination.

The mechanism of drought response has been investigated in the model plant, Arabidopsis thaliana. However, in peanut, little is known about the physiological and molecular events regulating gene expression under drought conditions. It is important to analyze drought-responsive gene expression in water-stressed and irrigated peanuts, as it may increase our understanding of the molecular mechanism of water stress and the role of differential gene expression in drought tolerance. The aim of the present study was to examine the differential expression of transcripts under drought stress and irrigated conditions. Specific transcripts uniquely affected due to water stress can be used as markers for selecting drought tolerant lines. We have also focused on isolating and identifying genes suppressed due to drought in leaves from seedlings because it is not possible to grow peanuts to a mature stage without microbial contamination and the microbial RNA may lead to false gene expression profiles. Initially, we have focused on those transcripts that are turned-off or down-regulated during water stress. The expression of these down-regulated transcripts in a drought tolerant and a susceptible genotype was compared to identify putative transcripts that can be used as molecular markers in screening peanut genotypes for drought tolerance.

To understand the molecular mechanism and differential gene expression under irrigated and water stress conditions, we have applied a highly sensitive technique namely Differential Display Reverse Transcribed - Polymerase Chain Reactions (DDRT - PCR) which is capable of identifying and isolating those genes that are differentially expressed in various cells or under altered conditions. Forty five days old seedlings of peanut cultivar, Florunner were subjected to water stress for 15 days. Total RNA from the leaf tissue of stressed and non-stressed plants was isolated and complimentary DNA molecules were synthesized in vitro. DDRT-PCR were performed using nine 5' Arbitrary primers (represented as P1, P2, P3.) and nine 3' Anchored oligo-dT primers (represented as T1, T2, T3.). The DDRT-PCR products from stressed and non-stressed samples were resolved side by side on a sequencing gel to compare qualitative and quantitative differences in the gene expression. A total of 21 primer combinations (for example P1/T1) were tested. DDRT enabled us to identify differentially expressed transcripts between water stressed and irrigated peanut seedlings. A total of 1235 bands with normal to high level of expression were observed in irrigated samples, compared to 950 bands in stressed samples. Most of the transcripts showed quantitative effect leading to over-expression or suppression of genes following water stress. In addition, transcripts that are turned-on or turned-off in response to water stress have also been identified. Primer combination P1/T1; P1/T6; P2/T1; P2/T2 and P4/T4 were highly effective in amplifying the suppressed transcripts (genes turned-off following water stress), whereas P1/T3; P1/T9 and P5/T5 amplified newly expressed transcripts (genes turned-on following water stress). It is clear that the transcripts suppressed by water stress may be critical for effective functioning of plant defensive mechanism, since irrigated peanuts do not show aflatoxin contamination.

The differentially expressed transcripts were collectively named PTRD (Peanut Transcripts Responsive to Drought). We have identified a total of 43 PTRD, which were significantly altered due to water stress. Sixteen of the severely affected PTRDs were analyzed for their expression under drought stress condition, 12 of them were completely suppressed following prolonged drought, two were down-regulated, and two were up-regulated under drought stress conditions. The 12 of the completely suppressed transcripts were studied further to compare differences in their expression between drought-tolerant and drought-susceptible peanut lines, following three weeks of drought stress. PTRD-1, -10, and -16 expressed for longer period in tolerant line compared to the susceptible lines. In conclusion, the transcripts identified in this study have great potential as molecular markers for screening and selecting peanut lines with drought tolerant characteristics.

Supported by UNESCO / MIRCEN network
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