DNA Fidelity: Expression of a Monocot Promoter in a Dicot Plant

The knowledge generated from the identification of plant promoters has been very important for plant biotechnology development. The use of promoters in transgenic plants allows a reasonable level of regulating protein expression. With the application of reporter genes, such as gusA (uidA,) the production of a colored protein, β-glucuronidase, can be detected and measured both qu-alitatively and quantitatively, and the activity of the promoter can be assessed. In this work we use a promoter of an abundant banana fruit protein gene Musa acuminata Acidic Chitinase class III a monocot species, to drive expression of gusA in a dicot species, like tomato. We evaluated the monocot promoter capabilities by localizing and quantifying β-glucuronidase (GUS) expression through fluorometric assays during tomato fruit ripening. Our results suggest that this promoter could be used for specifically strong fruit protein expression in dicot plants.


Introduction
Fruit ripening is a complex metabolic process involving changes in color, flavor, texture, and aroma that are catalyzed by highly regulated specific enzyme activities. The onset of ripening involves the expression of specific genes, and the expression specificity lies in the genes promoter region. Although chitinases are abundant proteins found in a wide variety of plants, the presence of chitin has not been reported in higher plants. Since chitin is the major structural compo-nent of fungal cell walls, it has been proposed that chitinases serve as defense proteins with antifungal activity [1]. Chitinases are reported to be induced in higher plants by several different types of stress [2] [3]. Many plant chitinases are expressed although at a low level constitutively [1]. Some evidence exists for the development regulation of chitinase expression in specific tissue and all defined stages during plant development [4] [5] [6]. The abundant 31 KDa banana pulp protein is homologous to class III chitinases [7]. There are several reports about chitinase genes expression during fruit ripening like avocado [8] [9] [10], pineapple [5], grapes [11] and pears [12]. However, in contrast to the ripening associated PR-proteins studied in some fruits, banana acidic chitinase decreases in abundance during ripening [7]. Although it is possible that this banana chitinase serves a protective role during fruit development, an alternate hypothesis is that it serves as a storage protein in this tissue [7] [13]. One of the most abundant proteins in tamarind seeds is an acidic class III chitinase based on its abundance accumulation without any pathogenesis-related stimulus, temporal regulation, amino acid composition, and very low enzyme activity; this 34 KDa protein designated "tamarinin" physiologically serves as the major storage protein [4]. In this work, we isolated a fragment of 2.1 Kb of the promoter of the banana acidic chitinase class III (MaChIII), and using GUS reaction as reporter gene detected its expression in transgenic tomato fruit (Solanum lycopersicum).

Genomic Library Construction and Screening
Banana genomic library for Musa acuminata cv Giant Nain was constructed with DNA isolated from immature green leaves, using the EMBL3 vector (Stratagene) [14]. Approximately, 2 × 10 6 primary plaques from the genomic library were plated, blotted and hybridized with 32 P labeled banana p31 cDNA [7]. Three successive hybridizations identified positive clones, from which DNA was isolated by restriction mapping. The genomic clone, Musa acuminata acidic chitinase class III was subcloned into plasmid Sport (Invitrogen) and sequenced by Sanger Method (GenBank: AY525367.1).

pGPT-31G Vector Construction
The 2.1 Kbp 5' region of banana acidic chitinase class III (MaChIII) gene was obtained from genomic DNA clone. The NcoI site at -1739 bp from the start codon was removed by cutting with NcoI and filling the ends with Klenow enzyme. Then, a NcoI site was created at the start codon by PCR. The 2.1 Kbp Bam-HI-NcoI fragment containing the MaChIII promoter was fused to the GUS gene with the CaMV 35S 3' region from pRTL2-GUS [15] and the expression cassette was inserted into pGPTV-Kan [16] to make pGPT-31G vector (Figure 1(A)).

Tomato Plant Transformation
Agrobacterium-mediated transformation of tomato cotyledons (variety Tanksley American Journal of Plant Sciences TA234TM2R) was performed according to Frary [17], except those seeds were sterilized by soaking in 70% ethanol for 2 min before rinsing in sterile water and washing in a mixture of 10% bleach and 1% Tween-20 for 10 min. The seeds were rinsed three times in sterile distilled water before plating on half-strength MS medium [18] (half-strength MS: 50 mg·L −1 myo-inositol, 2 mg·L −1 thiamine HCl, 0.5 mg·L −1 pyridoxine HCl, 0.5 mg·L −1 nicotinic acid, 10 g·L −1 sucrose and 8 g·L −1 Difco bacto agar, pH 5.8). Plantlets were regenerated on medium containing kanamycin at 300 mg/L.

Analysis of β-Glucuronidase (GUS) Expression by Fluorometry
We made fluorometric GUS assay following the method described by Jefferson [19]. Samples of leaves and fruits tissues were assayed. Transgenic tomatoes fruits were pick up at three ripening stages; green, brake and red (ripen). Fruits were divided in pericarp and placenta tissues. The volume equivalent to 20 μg of protein was incubated with 1 mM MUG buffer at 37˚C for 90 min. The enzy-

Analysis of p31 Promoter Expression in Tomato Plants
The banana acidic chitinase promoter vector pGPT-31G was introduced into A. tumefaciens LBA4404 by electroporation. Transformants were selected on LB medium containing kanamycin and confirmed by PCR. Using pGPT-31G vector Tomatoes cotyledons were transformed using Agrobacterium system. Four transgenic plant lines were obtained; we chose the line GPT31G-2 for the next experiments. Transgenic line was grown under greenhouse conditions and grown over a period of six months. Tomato total DNA was obtained and used for PCR assays to verify the MaChIII promoter incorporation in the tomato genome ( Figure 1(B)).

Fluorometry Analysis of β-Glucuronidase (GUS) Expression
Samples from pericarp and placenta of transgenic tomato fruits of GPT31G-2 line at three stages of tomato fruit ripening; green, brake and ripen, were collected. We made fluorometric assay in these three tomato ripening tissues and leaves samples. Data was quantified, and then arrayed in column graphs plotting the pmol/min/mg values obtained for each sample. We can observe that the higher expression is specified in the pericarp and placental regions at tomato brake stage. Low values were obtained in pericarp and placenta tissues of green and ripen stages of tomato fruit ripening. Fluorometric leaves values are lower than obtained from fruit tissues (Figure 2(A)).   [7]. The expression of the banana acidic chitinase class III promoter in a dicot model plant like tomato is interesting and suggests that this sequence gives organ specificity to the coding region and that is associated to fruit development too. The turn off the expression of the gene in presence of ethylene is similar, when climacteric ethylene production occurs during fruit ripening, the promoter turns off the expression of the gene in pericarp and placenta tissue. An interesting observation is the funiculus intense expression during three ripening stages, this result suggests that this acidic chitinase could be involved in seed development.

Histochemical Fruit GUS Staining Assay
Banana express abundantly this gene in the green pericarp like tomato, both fruits before of climacteric fruit stage.

In Silico p31 Promoter Analysis
The sequence of the 5' flanking promoter DNA of the MaChIII was determined.
The likely start codon is located at position 2153. The putative cis-element of this promoter was analyzed using PLANT Care program and Table 1

Discussion
Although chitinases are a family of antifungal proteins, the precise functions of individual members in this family and their expressional mechanism are still largely unknown. From deduced amino acid sequence of MaChIII only three of the five amino acids necessary for chitinase activity are conserved. We reported previously that the abundance of MaChIII decreased as ripening proceeded. The MaChIII antibody recognized a single 31 KDa polypeptide in banana pulp that was not present in peel, corm meristem or root tissues. These results indicated that this chitinase is fruit-specific and its physiological role is not for plant protection, but as a storage protein in banana pulp [7]. It is possible that this promoter response is associated in a tissue-specific way in tomato fruit [20], as found with the regulation of ethylene biosynthesis in the different tomato fruit tissues [21]. Another interesting observation is the very specific GUS staining of the fruit vascular tissue in the three stages of ripening. This suggests that this promoter could be carpel-specific. More studies are necessary since banana fruit is a parthenocarpy fruit.
In silico analysis shows that this promoter presents cis-acting elements principally related to stress, development and phyto-regulators response does not have pathogen cis-acting elements, like W Boxes present in chitinase promoters reported in other plants with antifungal activity [22]. The GUS staining expression of MaChIII in tomato fruit showed similar behavior like banana fruit, its expression goes down during fruit ripening and the higher expression was in green and breaking pericarp and placenta of preclimacteric tomato fruit.

Conclusion
Taken together our results suggest that expression of this promoter is developmentally regulated rather than pathogen-induced. It gives organ specificity expression during fruit ripening, and at least, this fruit promoter should have sequences that are conserved between the two genome systems (monocots and dicots plants) during fruit ripening and development. It is possible that in the evolution of plants promoter sequences could be conserved too. This promoter