Harvested broccoli (Brassica oleracea L. cv. Hartland) head accumulate ammonia during postharvest storatge. Glutamine synthetase (GS; EC 22.214.171.124) is the primary enzyme responsible for assimilating ammonia in plants. GS catalyses the ATP-dependent conversion of glutamate to glutamine utilizing ammonia as a substrate. Treatment of asparagus with phosphinothricin (PPT), a potent inhibitor of GS in plants, results in a rapid accumulation of ammonia in the spear tips and a reduction of shelf-life. Ammonia accumulated only in floral sections of branchlets during late senescence. Ammonia accumulation is thought to be a factor contributing to the perishability of harvested asparagus spears and occur due to alterations in GS activity after harvest. As ammonia is thought to be toxic to plant cells, the changes in ammonia content and GS gene expression in both floret and branchlet portions of the broccoli were studied in order to further understand the factors influencing ammonia assimilation during storage. Asparagus spears like leaf cells of other higher plant species, contain two isoforms of GS that are distinguished by their subcellular position: GS-1 is only present in the cytosol, whereas GS-2 is exclusively detectable in the chloroplast matrix. Little is known about the regulation of GS gene expression in plant, especially broccoli or the significance of multiple GS family members. Expression of some of these members is localized to the phloem elements, cotyledones and senescing leaves[5,7,8], suggesting a role in N uptake, translocation and mobilization. Although both the GS-1 and GS-2 cDNA clones have become available for many mono- and dicotyledonus species, for broccoli they have not been isolated yet. In the study one partial cDNA clone was isolated and sequenced that showed high homology with other GS in the databases and was used as a probe for northern blot analysis of total GS without separation of cytosolic and plastid forms.
MATERIALS AND METHODS
Plant materials: Broccoli heads were harvested from farmers field in Kagawa prefecture, Japan during October, 2002. Heads were immediately brought to the laboratory (Faculty of Agriculture, Kagawa University, Japan) and stored at 20°C in perforated plastic bags for up to 4 days in darkness. After storage treatment heads were cut into two portions (designated as florets and branchlets), weighed and immediately stored at -80°C until needed for GS and ammonia assay and total RNA extraction.
Enzyme extraction: Approximately 5 g sample from each portion were homogenized under ice cold condition (ca. 0-4°C) with 1% polyvinylpolypyrrolidone (PVPP), propotional to the sample weight and 1 g of sea sand in Buffer A by using a mortar and pestle. 1 ml extraction buffer g-1 fresh weight of plant materials was used. Extraction was performed according to the method of Hurst and Clark, in which buffer A contained 50 mM Tris-HCl (pH 7.6) 10 mM MgSO4, 1 mM EDTA, 1 mM dithiothreitol (DTT), 12 mM 2-mercatoethanol, 5 mM glutamate and 100 ml L-1 glycerol. The homogenate was squeezed through a four-layer cotton cloth and the filtrate was centrifuged at 11000xg for 10 min. The residual tissues were re-extracted in 5 ml buffer A and dialyzed with 40 times dilution of the same buffer for 1 h and then centrifuged. The resulting supernatant were mixed together and used for the enzyme assay.
GS and ammonia assay: GS was measured in a total volume of 1.0 ml. The assay mixture contained 80 mM L-glutamate-Na, 500 mM tricine-KOH buffer ( pH 7.0 ), 600 mM NH2OH, 200 mM MgSO4.7H2O, 10 mM diethylenetriamine pentaacetic acid ( DTPA ), 80 mM ATP and 800 mM mercaptoethanol. After incubating at 35°C for 8 min, the reaction was stopped by adding 1 ml ferric reagent ( 25 ml FeCl3.6H2O, 50 ml HCl and 20 ml TCA ). GS activity was measured using a double beam spectrophotometer (Shimadzu model UV-150-02 ) at 540 nm.
For assay of ammonia 5g sample from each portion were extracted with 10% trichloroacetic acid and centrifuged at 11000xg for 10 min as described by Kun and Kerrney. Ammonia content of supernatant was determined from a triplicate 500 μl sample by adding 200 μl of 0.5 M Tris-HCl buffer (pH 8.0), 100 μl of 0.1 M 2-oxoglutarate solution (pH 7.4), 30 μl of 8 mM βNADH solution and 150 μl of distilled water. The absorbance was recorded at 340 nm against a reagent blank.
RNA extraction: Total RNA was extracted according to the Hot Borate methods of Wan and Wilkins.
Amplification of poly (A)+ RNA by RT-PCR: The first strand cDNA was synthesized from 2 g of the total RNA by reverse transcriptase with Oligo-(dT) primer according to the instruction of SuperScriptTM Preamplification System for First Strand cDNA synthesis (BRL, Tokyo, Japan). PCR was performed in a total volume of 25 μl containing the first strand cDNA reaction products, 10xPCR buffer, MgCl2, dNTP, First Start Taq DNA Polymerase (Roche) and primers. The primers (5'-TCNAGCACNGGGCARGCNCC-3' as the sense primer and 5'-TCRTTNCCYTCNCCRTANGC-3' as the antisense primer) were designed and synthesized on the basis of amino acid domains conserved in various GS genes. The Sal 1 and Not 1 restriction site sequences were also included at 5'-end of the sense and antisense primer, to facilitate cloning of PCR product. The PCR procedure started with 10 min at 95°C and was carried out 35 cycles of 30 at 95, 50 and 72°C and 10 min at heating at 72°C with ASTEC Program Temperature Control System PC-700. The PCR products were confirmed by agarose gel electrophoresis.
Cloning and Sequencing of cDNA: The amplified cDNA was ligated to the
plasmid pSPORT 1TM and cloned into Escherichia coli (DH-5α)
Not 1 and Sal 1-cut (BRL, Tokyo, Japan). Sequencing was performed
by the Cycle sequencing methods using GATCR-Bio Cycle sequencing
Kit and a DNA sequencer GATC 1500 Long-Run system (GATC GmbH, Konstanz, Germany).
||Changes in activity and expression of Glutamin synthetase
in floret and stem portion of stored broccoli during storage at 20°C.
(A) Changes in Glutamin synthetase activity. Each point represents the mean
of three replicats and bars show SE about mean (when larger than symbols).
(B) Northern blot analysis for Glutamin synthetase gene. Equal loading of
RNA was confirmed by staining a gel with ethidium bromide
||Nucleotide sequence and deduced amino acid sequence of the
cDNA clone corresponding to pBR-GS. The predicted amino acid sequence is
given in signle-letter code for each amino acid. The arrows indicate the
positions of degenerated primers (sense, antisense) used for RT-PCR. Numbering
refers to total nucleotide residues on each line
||Comparison of the amino acid sequences broccoli (AB125110),
Brassica napus(Y12458), lotus (AF459587), kidney bean (X04002), maize
(D14578) and European grape (X94320) by multi alignment. The amino acid
residues are numbered at the beginning and end of the sequences on each
line. Asteriks denote the amino acid residues those are identical. Dashes
in amino acid sequences represent gaps introduced to maximize alignment
of the polypeptides.
||Phylogenetic tree of the alignment of pBR-GS deduced amino
acid sequence with other GS gene in the database. Protein sequences were
aligned using the UPGMA method of GENETIX-MAC software. The GenBank accession
numbers are shown in the parentheses
Sequence data analysis: Sequence analysis was performed using computer software from the GENETYX-MAC Version 7. Homology searches with the Genebank and the EMBL databases were performed using the Homology program in the software. The phylogenetic tree was also constructed with the UPGMA method in the software.
Preparation of the digoxigenin (DIG)-UTP-labeled RNA probe: The cloned RT-PCR product including the encoded region of GS gene was cleaved by Not 1 and Sal 1 from the pSPORT 1 vector that had been amplified in Escherichia coli (DH-5α) and it was purified and recovered by gel electrophoresis. Antisense DIG-labeled RNA probe was prepared according to the instructions DIG RNA Labeling kit (Bohringer Mannhaim) using SP6 RNA polymerase.
Northern blot hybridization: Ten μg total RNA was subjected to electrophoresis on a 1.0% agarose (Type II) gel that containing 20 x MOPS and 37% formaldehyde. After electrophoresis for 30 min, RNA was visualized with ethidium bromide under UV light to confirm equal loading of RNA in each lane. RNA was transferred to a positively charged nylon membrane Hybond-N+ (Amersham) by capillary action with 20 x SSC and then after drying the membrane RNA was fixed with UV. The membrane was prehybridized at 50°C with 5xSSPE, 5xDenharts solution, formamide and 10% SDS for 3 h. Hybridization was performed at 50°C using the gene specific DIG-labeled RNA probe for 24 hours using the same prehybridization buffer. After hybridization, the membrane was washed twice with 2xSSPE containing 0.1% SDS for 10 min at room temperature, once with 1xSSPE containing 0.05% SDS for 15 min at 65°C and once with 0.2xSSPE for 10 min containing 0.05% SDS for 15 min each. The membrane was also washed with buffer A containing maleic acid and tween 20 at room temperature was blocked with 2% blocking reagent in maleic acid buffer for 30 min. Subsequently, the membrane was incubated with anti-digoxygenin-AP, fab fragments (Bohringer Mannhaim) in blocking buffer for 30 min. Signals were detected by color reaction using 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt as the substrate.
GS activity: Figure 1 shows GS activities in the floret and branchlet portions of the broccoli during storage at 20°C. The GS activity increased in the florets of the broccoli after 12 h and declined to about 20% of the initial level by 96 h, whereas that in the branchlets increased gradually until 96 h. GS showed significantly higher activities in the florets than the branchlets of the broccoli.
Isolation and identification of cDNA clone: The cDNA pBR-GS (accession no; AB125110) is a partial clone encoding a harvest-induced transcript from branchlets. The encoded mRNA is 695 bp long (Fig. 2) and is highly homologus to the gene of other plant (Fig. 3). The pBR-GS sequence is 78.7% identical to GS from rice cytosol (AB037597), 78.7% identical to GS from rice shoot (X14245) and 77.4% identical to GS from maize (D14578) (Table 1). Allowing for conservative amino acid substitutions, the similarities are 84.0, 84.4 and 83.5% for the rice cytosole, maize and Europian grape sequences, respectively.
Expression of GS gene: The pBR-GS was used as a probe for northern blot
analysis to determine the levels of transcripts for GS. The levels of transcripts
for GS increased slightly after harvest in both florets and branchlets (Fig.
1) and correlated with enzyme activity of branchlets. Expression of GS gene
in the floret was clearly observed at 0, 72 and 96 h of storage and remained
detectable throughout experimental period.
||Ammonia contents in the floret and branchlet portion of broccoli
during storage at 20°C. Each point represents the mean of three replicates
and bars show SE about mean (when larger thatn symbols)
||Percentage of nucleic acid and amino acid identity between
Glutamine synthetase from broccoli and other plants in database
But the expression in the branchlets was barely detectable that may reflect
in the much lower GS activity in that portion. A phylogenetic tree (Fig.
4) was generated from the alignment of the deduced amino acid sequences
of pBR-GS and other GS gene in the database. The pBR-GS (AB125110), GS from
rice root (X14244) and Brassica napus (Y12458) strongly clustered together
in a subgroup belonged to the monocotyledon, having closest relationship with
lotus GS gene.
Ammonia contents: A general increase in ammonia content for floret and branchlet portions of the broccoli was observed after harvest (Fig. 5). Ammonia accumulation in the florets increased to about 9 times of initial level after 72 h storage and reached about 14 times of initial level after 92 h, whereas that in the branchlets increased slightly. Higher (almost 14 times) accumulation of ammonia was found in the floret portion than the branchlet portion of the broccoli.
Ammonia is toxic to plant cells at high concentrations and is normally assimilated if produced within the plant. The accumulation of ammonia in senescing leaves has been shown to coincide with almost complete disappearance of GS. In this study the trend, in which GS activity declined in the floret portions of the broccoli was observed with an increased in ammonia accumulation to about 14 times of initial level after 92 h storage period. The level of transcripts for total GS fluctuated in the floret portions but that was clearly higher in the floret portions than in the branchlet of the broccoli. It seemed that the floret portions of broccoli is more perishable than branchlets portion.
Phylogenetic analysis of GS sequences has revealed the existence of at least two major branches that contain characteristic conserved amino acid sequences, monocotyledon and dicotyledon. The pBR-GS belonged to the subgroup dicotyledon as it was highly homologus is to Brassica napus GS and was closely related to GS from lotus.
In conclusion, the increase in ammonia contents and decrease in total GS activity in the floret portions of the broccoli is a response to significant stress after harvest. Perhaps as a consequence of general metabolic decline occurring at this time or may be the consequence of autophagic process induced by carbohydrate depriviation.