Isolation and Characterization of Hydroxyproline-rich Glycopeptide Signals in Black Nightshade Leaves *

A gene encoding a preprohydroxyproline-rich systemin, SnpreproHypSys , was identified from the leaves of black nightshade, Solanum nigrum (Sn), which is a member of a small gene family of at least three genes that have orthologs in tobacco ( NtpreproHypSys), tomato ( SlpreproHypSys) , petunia ( PhpreproHypSys ), potato ( PhpreproHypSys ), and sweet potato ( IbpreproHypSys ). SnpreproHypSys was induced by wounding and by treatment with methyl jasmonate (MeJA). The encoded precursor protein contained a signal sequence and was post-translationally modified to produce three hydroxyproline-rich glycopeptide signals (HypSys peptides). The three HypSys peptides isolated from nightshade leaf extracts were called Sn HypSys I (19 amino acids with 6 pentoses), Sn HypSys II (20 amino acids with 6 pentoses), and Sn HypSys III (20 amino acids with either 6 or 9 pentoses) by their sequential appearance in SnpreproHypSys . The three Sn HypSys peptides were synthesized and tested for their abilities to alkalinize suspension culture media, with synthetic Sn HypSys I demonstrating the highest activity. Synthetic Sn HypSys I was capable of inducing alkalinization in other Solanaceae cell types (or species), indicating that structural conformations within the peptides are recognized by the different cells/species to initiate signal transduction pathways, apparently through recognition by homologous receptor(s). To further demonstrate the biological relevance of the Sn HypSys peptides, the early defense gene lipoxygenase D was shown to be induced by all 3 synthetic peptides when supplied to excised nightshade plants. acid (5’-CATCAATTGTTGCTCA GGCTA-3’) and PI-II-R (5’-GCAATCCAGAAGATGGACAAG-3’). Ten µg of total RNA isolated with TRIZOL reagent (Invitrogen) was treated with DNase I (New England Biolabs), and 2 µg was reverse-transcribed using DyNAmo cDNA Synthesis Kit (Finnzyme) with random hexamer. The qPCR was performed using DyNAmo HS SYBR Green qPCR Kit (Finnzyme) and Mx3500P QPCR Systems (Stratagene). Elongation factor 1 α ( EF-1 α ) gene was also amplified as an internal control using EF1 α -F (5’-ACCACTGGTGGTTTTGAAGC-3’) and EF1 α -R (5’-ACGACCAACAGGGAC AGTTC-3’) primers.


INTRODUCTION
In tomato (Solanum lycopersicon) leaves, two structurally similar but genetically unrelated peptide signals, called systemin (Pearce et al., 1991) (abbreviated SlSys) and hydroxyproline-rich glycopeptides (Pearce and Ryan, 2003) (abbreviated SlHypSys I, II, and III) are wound-inducible and activate the expression of protease inhibitors and other defense-related genes in response to herbivore attacks. The systemin peptide is derived from a precursor called prosystemin (McGurl et el., 1992) that lacks a leader sequence and is synthesized in the cytoplasm of phloem parenchyma cells (Narvaez-Vazquez et al., 2004). In contrast, the three hydroxyproline-rich glycopeptides are derived from a single precursor in tomato leaves (Pearce and Ryan, 2003) that does contain a leader sequence and is synthesized through the secretory pathway and sequestered in the plant cell walls (Narvaez-Vazquez et al., 2005). Tomato plants produce both systemin and HypSys peptide signals in response to wounding and they work cooperatively to produce a strong systemic response (Narvaez-Vazquez et al., 2007). In contrast, tobacco plants lack prosystemin and produce only HypSys glycopeptides (Pearce et al., 2001A).
HypSys glycopeptides were first isolated from tobacco (Nicotianam tabacum, Nt), facilitated by the development of a suspension cell assay for detection of bioactive peptides (Scheer andRyan, 1999, Pearce et al., 2001A). It had previously been shown that when systemin was added to tomato suspension cell cultures and bound to its receptor, it had the ability to rapidly alkalinize the media via the blockage of a proton pump in the cell membranes (Felix andBoller, 1995, Schaller andOecking, 1999). A medium alkalinization response was also measured when 1 ml aliquots of tobacco suspension cells were treated with small aliquots (1-10μl) of HPLC purified fractions of tobacco leaf extracts. The active compounds were identified as HypSys I and II (Pearce et al., 2001A). By utilizing the alkalinization assay, HypSys glycopeptides were also found in tomato (Pearce and Ryan, 2003), petunia (Pearce et al., 2007) and sweet potato (Chen et al., 2008).
The specific role of the HypSys glycopeptides has recently been investigated. In cultivated tobacco (Nicotianum tabacum), when preproHypSys is overexpressed, the plants were resistant to attack by Helicoverpa armigera larvae (Ren and Lu, 2006). The preproHypSys gene expression level was also found to increase to wounding, methyl 7 Analysis of the cDNA sequences of hydroxyproline-rich systemin precursors from tobacco, tomato, and petunia revealed a common 30-base sequence encoding the putative propeptidase splice site (Figure 1). This region was utilized as a probe for Northern blot analysis of nightshade RNA, revealing a single, strong band at approximately 750 bases (data not shown). The conserved region was utilized as a primer in 3' and 5' RACE experiments for isolation of a cDNA from nightshade whose deduced amino acid sequence had proline-rich regions, similar to sequences from members of the preproHypSys family of defense proteins (Figure 2A). The cDNA has a coding region of 159 amino acids, containing the highly conserved propeptidase splice site, three proline-rich regions, and a C-terminal 'SY' motif, similar to other preproHypSys precursor proteins (Matsubayashi andSakagami, 2006, Farrokhi et al., 2008). The cDNA coded for a precursor with high homology to SlpreproHypSys, with 63.4% amino acid sequence identity ( Figure 2B) and was designated S. nigrum preprohydroxyproline-rich systemin (SnpreproHypSys). Additionally, the locations and amino acid sequences of all three of the proline-rich regions were similar to SlpreproHypSys.

SnpreproHypSys RNA levels are induced by wounding
SnpreproHypSys and nightshade prosystemin (SnproSys) cDNA specific probes were employed in RNA gel blot analysis to determine whether the respective mRNAs are wound inducible. A SnproSys specific probe was used as a positive control. Three week old nightshade plants were repeatedly wounded across the mid vein of the fifth and sixth leaves (from the apical top), and total RNA was extracted from the unwounded systemic upper leaves at different time intervals after wounding. Time course analysis of RNA samples were performed using labeled probe specific to each of the two cDNAs (Fig.   3A). The HypSys precursor mRNA for SnpreproHypSys showed induction in the upper, unwounded systemic leaves due to wounding of lower leaves, reaching a maximum induction at 6 h. Accumulation of Snprosys mRNA due to wounding maximized at 8 h.
A higher level of the precursor transcript of SnpreproHypSys as well as SnproSys mRNA persisted even after 24 h in systemic leaves of wounded plants, compared to the leaves from unwounded plants (data not shown).

SnpreproHypSys mRNA levels are induced by methyl jasmonate
Methyl jasmonate (MeJA) is a powerful inducer of defense genes in plants (Farmer andRyan, 1992, Wasternack, 2007). Three weeks old nightshade plants were sprayed with MeJA in a closed container and total RNA was extracted at different time intervals. Northern blot analysis revealed a strong induction of both SnpreproHypSys and SnproSys mRNA following MeJA treatment (Fig.3B). The maximum level of SnpreproHypSys mRNA was attained at 2 h and remained high 4 h after treatment, whereas induction of SnproSys mRNA continued to increase through 8 h.

SnpreproHypSys is a member of a small gene family
DNA gel blots were prepared by digesting total genomic DNA isolated from a single nightshade plant with 3 different restriction enzymes, BamHI, EcoRI, and XbaI.
These enzymes do not have any internal site present within the SnpreproHypSys cDNAs, which were used as a probe in Southern blot analysis; consequently the number of bands in each lane of the autoradiogram would indicate the copy number of the SnpreproHypSys gene. Autoradiograms generated by hybridization of the DNA blot with a 32 P-labeled SnpreproHypSys specific probe showed the presence of three major bands and a minor band in each lane (Fig. 4), indicating that SnpreproHypSys is a member of a small gene family with at least two additional genes.

Isolation of Hydroxyproline-rich Systemins from Nightshade Leaves
A search for mature, bioactive peptides from nightshade leaves was initiated by generating callus on solid media and subsequently transferring to liquid media for utilization in the alkalinization assay. We extracted the leaf preparation as described in Materials and Methods and separated the fractions on reversed-phase HPLC. The fractions were assayed for alkalinizing activity as described in Materials and Methods using tobacco (Nt), tomato (Solanum peruvianum, Sp) and nightshade (Sn) suspension cells (Fig. 5). Interestingly, the first peak, designated peak 1, responded strongly in the alkalinization assay with tomato suspension cells, whereas peak 2 had a stronger response with tobacco suspension cells. Both of these peaks induced alkalinization equally with the nightshade cells. Peak 3 was non-responsive in both the tobacco and the tomato suspension cell assay but gave a similar response to peaks 1 and 2 with nightshade suspension cells. Upon further purification, Peak 1 was separated into two fractions and designated peaks 1a (minor peak) and 1b (major peak) (data not shown). The four activity peaks were further purified as described in Materials and Methods, however, only minute quantities of peak 1a were obtained and this peak was directly subjected to mass spectral analysis. The final HPLC purifications of peaks 1b, 2 and 3, with their alkalinizing peaks (insets) are shown in Figure 6. Estimates of yield were determined from the peak areas as follows: peak 1b, 91 pmoles, peak 2, 55 pmoles, and peak 3, 53 pmoles. Amino acid sequence and mass spectral data were obtained ( Figure 7A). The amino acid sequences for all three peptides were found within the isolated cDNA for SnpreproHypSys (Fig 2A). Peaks 1a and 1b, designated SnHypSys IIIa and IIIb by location in SnpreproHypSys ( Figure 2A) were found to differ only in the number of pentoses attached to the peptide backbone, with SnHypSys Ia containing nine pentoses and SnHypSys Ib containing six pentoses. Peak 2, designated SnHypSys I contained six pentoses, as did peak 3, designated SnHypSys II ( Figure 7A). The positions of the peptides within the cDNA are shown in Figure 7B.

HypSys peptides
Because of the low yield of the SnHypSys peptides, synthetic peptides lacking the carbohydrate moieties were synthesized for activity experiments as previously done for other HypSys peptides (Pearce et al., 2007, Chen et al., 2008. Although some synthetic HypSys peptides have demonstrated little or no ability to alkalinize suspension cell media, others have alkalinizing activity in the high nM to low μΜ range. Synthetic SnHypSys I, II, and III were compared with SlHypSys I for their abilities to induce medium alkalinzation in nightshade, tobacco, and tomato cells (Figure 8). The SlHypSys I peptide induces medium alkalinization in all solanaceous species tested at nanomolar concentrations (Pearce and Ryan, unpublished results). Of the 3 nightshade peptides, SnHypSys I was able to induce medium alkalinization in all 3 types of cell cultures with a half maximal response in nightshade cells of about 2.5 μM, comparable to SlHypSys I.
The native SnHypSys I also induced medium alkalinization in all 3 types of cell cultures ( Figure 5, peak 2) and its position in the cDNA matches SlHypSys I ( Figure 2B).

Lipoxygenase D induction in response to synthetic SnHypSys peptides
To study the biological relevance of the 3 HypSys peptides for defense in nightshade plants, the defense-related lipoxygenase D (LoxD) gene was utilized in wounding and excised plant assays (Heitz et al., 1997) To establish that LoxD was involved in defense responses in nightshade, young nightshade plants were wounded and the relative RNA expression levels of the gene was analyzed with real time RT-PCR. The relative LoxD expression levels increased at 0.5 h, peaking in wounded leaves after 1 h with a 170-fold increase in expression compared to unwounded control plants, before declining to a 25-fold increased expression at 4 h ( Figure 9A). The unwounded upper leaves of the wounded plant (systemic response) followed the same temporal pattern, but expression was much lower, peaking at a 10-fold increased expression after 1 h.
Since synthetic SnHypSys I was the most active of the 3 HypSys peptides in the alkalinization assay (Figure 8), this peptide was utilized to establish a concentration range for peptide response in excised nightshade plant experiments. SnSys was also supplied at lower hormonal concentrations that had previously been established for a response in tomato plants (Pearce et al., 1991, Constabel et al., 1998. Merely excising and supplying In addition to testing Lox D expression levels, two other early genes, EEF53 (phospholipase) and F1L3.3 (jasmonate ZIM-domain protein), were evaluated for expression levels after supplying SnHypSys I for 1 h. Expression levels of EEF53 were induced 3.7 fold while F1L3.3 expression levels were induced 3.9 fold over control levels ( Figure 9E).
The late genes for protease Inhibitors 1 and 2 were also evaluated for expression levels after supplying SnHypSys I through the cut stem. After 4 h, the expression of Inh 1 was 1.4-fold while Inh 2 was 2.1-fold higher than control levels. Increasing the incubation time beyond 4 h caused a large increase in control values and expression levels at later time intervals could not be evaluated.

DISCUSSION
The hydroxyproline-rich systemin family of defense glycopeptides has recently expanded to include regulation of pathogenesis-related genes with the discovery of the characteristics in this study of S. nigrum, a species closely related to the agriculturally important species S. lycopersicon and S. tuberosum.
A gene was identified from S. nigrum that encoded a precursor protein with identity to the hydroxyproline-rich glycopeptide family of defense genes. Previously isolated members of this family were obtained by first extracting a large quantity of leaf material and purifying bioactive fractions through several HPLC columns and sequencing the glycopeptide, which was then used for designing a degenerate primer for PCR and cloning. Recently, a cDNA was isolated from petunia (PhpreproHypSysI) by both isolation of the glycopeptides and utilizing a conserved thirty nucleotide region at the propeptidase splice site of both the tomato and tobacco cDNAs for primer design ( Figure   1). Here, we were able to use just the consensus sequence of the 30mer region to obtain a preproHypSys cDNA without first isolating the glycopeptides. This method should be of use in isolating other members of the HypSys family of genes.
The isolated cDNA contained three sequences that were potential HypSys peptides ( Figure 2A). These sequences had a central region that was proline-rich, basic charges towards their amino termini, and a glutamine or glutamic acid at the carboxy termini: similar to previously isolated HypSys peptides (Matsubayashi andSakagami, 2006, Farrokhi et al., 2008). The cDNA termination codon was preceded by a sequence coding for -QASY, similar or identical to homologous preproHypSys C-terminal sequences from other species. The SnpreproHypSys cDNA coded for a protein of 159 amino acids in length, similar in size to the homologous tomato cDNA (146 aa) and had 63% identity with the tomato sequence ( Figure 2B). The amino acid sequences around the proteolytic processing sites for the glycopeptide sequences were identical to the tomato cDNA, indicating a conservation of the processing machinery.
Wounding of the lower leaves of nightshade plants caused an increase in RNA levels of SnpreproHypSys in the upper unwounded leaves ( Figure 3A). The response was minimal at 4 hrs but was very strong between 6-10 h. In contrast, SlpreproHypSys and PhpreproHypSys have been shown to be strongly induced at 4 h in the upper leaves (Pearce andRyan, 2003, Pearce et al., 2007). SnproSys RNA levels were slightly higher at 2-4 h than zero time controls but a maximal response was not observed until 6-8 h.
Again, this is in contrast to SlproSys that has been shown to obtain maximal RNA levels in the upper unwounded leaves of wounded plants by 3 h (McGurl et el., 1992). In general, the systemic wound response time of Solanum nigrum appears to be somewhat slower than tomato and petunia. Recently, it was shown that there is a marked decrease in relative prosysytemin levels 30-90 minutes after wounding and application of oral secretion from Manduca sexta (Schmidt and Baldwin, 2006). Although we did not test these early time points, the delay in prosystemin expression shown here may be due to an initial down-regulation.
Nightshade plants were incubated in the presence of methyl jasmonate vapors and RNA was extracted and subjected to Northern analysis ( Figure 3B). An increase in SnpreproHypSys was detectable after 1 h, peaking at 4 h before decreasing. Early detection of increased preproHypSys RNA levels to MeJA has been found with both tomato and petunia (Pearce andRyan, 2003, Pearce et al., 2007). ProSys levels were detectable at 4 h and continued to increase with time through 8 h, similar to results found with tomato (Pearce and Ryan, 2003). These results indicate that SnpreproHypSys is both wound inducible and methyl jasmonate inducible in the same temporal pattern as other hydroxyproline-rich systemin precursors.
An attempt to purify all three of the putative glycopeptides found within the cDNA by the same extraction methods utilized for HypSys peptides from tomato and tobacco revealed only 2 alkalinizing peaks when N. tabacum or Solanum peruvianum cells were utilized in the alkalinization assay. It was only after S. nigrum cells were cultured that a third alkalinizing peak was found ( Figure 5). The three peaks were SnHypSys II contains 20 amino acids with 6 pentoses. Interestingly, when SnHypSys II was purified and analyzed by MALDI-MS, it contained 5 more amino acids than the homologous HypSys in tomato. The tomato HypSys (SlHypSys III), has an aspartic acid at position 15 (instead of the glutamic acid in SnHypSys II) followed by a proline, a bond that is labile in weak acid. Previously, SlHypSys III MALDI-MS was thought to contain 10 pentoses with one unsaturated bond on a 15 amino acid backbone.
With the new nightshade mass spectral data, a re-examination of the SlHypSys III mass spectral data reveals that it contains 6 pentoses. Upon mild acid hydrolysis (a treatment used to strip the pentoses off of the peptide backbone for peptide mass analysis), Nightshade systemin (SnSys) had previously been tested for its ability to induce protease inhibitors in an excised tomato plant assay (Constabel et al., 1998). However, nightshade plants were found to be sensitive to excision and the control levels were too high to obtain data (Constabel et al., 1998) In addition to testing Lox D expression levels, two other early genes, EEF53 (phospholipase) and F1L3.3 (jasmonate ZIM-domain protein), involved in jasmonate production and jasmonate signaling, respectively, that had previously been shown to be upregulated by both methyl jasmonate and insect feeding in S. nigrum (Schmidt and Baldwin, 2005) were evaluated for expression levels after supplying SnHypSys I for 1 h. Both of these genes had significantly higher expression levels, strongly suggesting a defense role for SnHypSys I. On the other hand, the expression levels of the late genes, Inhibitor 1 and 2, were not significantly increased at 4 h and longer incubation times after excision led to high control values. A definitive statement as to whether these end product proteins of the octadecanoid signaling pathway in other Solanaceae family members can not be made for S nigrum in this study.

Alkalinization Assay
Suspension cells were maintained in Murashige and Skoog medium as previously described (Scheer and Ryan, 1999), but excluding buffer. Instead, the medium was adjusted to pH 5.6 with KOH. Cultures were maintained by transferring 3 ml of cells to 45 ml of media every 7 d and shaking at 160rpm. Tobacco and nightshade cells were used for assays 3-5 d after transfer. Tomato cells were used 4-7 d after transfer. One hour before assaying for alkalinizing activity, a flask of cells was aliquoted into 24-well cell-culture cluster plates (1 ml/well) and allowed to equilibrate at 160 rpm. Aliquots of HPLC fractions or purified peptide (1-10 μl) were added and after 20 min the pH was recorded.

Peptide Isolation
Nightshade plants were grown in peat pots under greenhouse conditions for approximately 4 weeks. The plants were sprayed with methyl jasmonate as previously collected and the alkalinizing activity was assayed as described above using 10 μl of each fraction per ml of Solanum nigrum (Sn) cells. The activity was found at or near the void and these fractions were pooled and lyophilized. The yield was 1.7 g. Seven hundred fifty mg was dissolved in 6 ml 0.1% TFA/H 2 O for preparative reversed phase C18-HPLC. After centrifugation and filtration, the sample was loaded onto a preparative column in three sequential runs (218TP1022, 10μm, 22x250mm, Vydac, Hesperia, CA) with a flow rate of 4 ml/min. After 2 min, a gradient was applied from 0-40% acetonitrile/0.1% TFA over 90 min. The absorbance was monitored at 225 nm. One min fractions were collected and 10 μl aliquots were used with 1ml of nightshade, tobacco and tomato cells to determine alkalinizing activity. Three main activity peaks were detected along with the late eluting RALF peptide peak (Pearce et al., 2001B). Fractions 42-44 (peak 1), fractions 47-49 (peak 2) and fractions 58-60 (peak 3) were pooled and lyophilized. The yields were 18.5 mg, 21 mg, and 18.4 mg, respectively. The alkalinizing activity peaks were subjected to strong cation exchange chromatography (SCX) on a poly-SULPHOETHYL Aspartamide column (5 μm, 4.6x200 mm, The Nest Group, Southborough, MA). The column was equilibrated in 5 mM potassium phosphate, pH 3, in 25% acetonitrile. Each peak was loaded onto the column in 1 ml buffer and after 2 min, a 90 min gradient was applied to 40% elution buffer (5 mM potassium phosphate, 1 M potassium chloride, pH 3, in 25% acetonitrile) for peaks 1 and 2, and 60% elution buffer for peak 3. Absorbance was monitored at 225 nm. A flow rate of 1 ml/min was employed and 1 min fractions were collected. Five μl aliquots were used to determine activity in the cell assay. Peak 1 eluted as a doublet in fractions 71-72 (peak 1a) and fractions 74-75 (peak 1b). Peak 2 activity eluted in fractions 61-63 and peak 3 activity eluted in fractions 64-65. Activity peaks were pooled, lyophilized, and further purified by reversed phase C18 chromatography at pH 6 (column 218TP54, 5 μm, 4.6 x 250 mm, Vydac). The samples were dissolved in 1ml column equilibration buffer, 10 mM potassium phosphate, pH 6, and after centifugation, the supernatants were applied to the column with a flow rate of 1ml/min. After 2 min, a 90 min gradient was applied to 40% elution buffer consisting of 10 mM potassium phosphate, pH 6, in 50% acetonitrile, except in the case of peak 3 where 60% elution buffer was utilized. Absorbance was monitored at 220 nm and 5 μl aliquots were used to determine alkalinizing activity. The peak 1a activity eluted in fractions 49-50, peak 1b in fractions 45-49, peak 2 in fractions

Isolation of SnPreproHypSys cDNA
The cDNAs that were isolated from tobacco and tomato were found to contain a homologous region between the sequence coding for the signal peptide and the nucleotide sequence coding for the hydroxyproline-rich systemins. A degenerative 30 base pair oligonucleotide primer was synthesized for amplification of potential defense signals in other Solanaceous species.

N=G/C/T/A, R=G/A
The primer was used in 3'RACE PCR-PCR (Ambion, Austin, TX) to amplify a product that was subsequently cloned by TOPO (Invitrogen), sequenced and found to have homology to tomato prepro-hydroxyproline-rich prosystemin (SlpreproHypSys). To obtain the complete sequence, 5'RACE-PCR was performed using the reverse complement of an internal sequence that overlapped the 3'-RT-PCR product by 307bp: 5'-CATGATCGTGCTTCC-CACCAACTC-3' (NS inner). The overlapping sequence was homologous.

Mechanical Wounding of Plants
Three The leaf samples were immediately frozen in liquid nitrogen and kept at -80° C until used.

MeJA Treatment
Three week old plants were treated by spraying the plants with solutions of 125μL of MeJA in 500ml of double distilled H 2 O containing 0.1% Triton X-100, or 0.1% Triton X-100/H 2 O. The leaf samples were collected for time-course experiments at 0, 1, 2, 4, 6, and 8h after spraying and immediately frozen in liquid nitrogen and kept at -80° C until used.

Northern-Blot Analysis
Leaves of treated and control plants (3 weeks old) were removed and immediately frozen in liquid N 2 and stored at -80 °C until extraction. Each sample consisted of approximately 500 mg leaf material consisting of at least one leaf each from three independent plants. The leaf material was ground to a fine powder in a mortar and pestle with liquid N 2 and total RNA was isolated with TRIZOL reagent (Invitrogen) according to the manufacturer's protocol. Total RNA was quantified and 15μg of each sample was fractionated by electrophoresis on a 1.2% formaldehyde agarose gels, blotted on Hybond N membranes (Amersham Biosciences), and hybridized with [ 32 P]dCTP-labelled specific probes at 65 °C. Ethidium bromide stained rRNA bands were used to monitor equal loading. Following hybridization, membranes were washed twice with 2X SSC/0.1% SDS for 10 min each at 55 °C, followed by two washes each with 0.5X SSC/0.1% SDS for 10 min and two washes with 0.1X SSC/0.1% SDS for 5 min each at 65°C. Membranes were exposed to x-ray film at -80°C, from 4 h to 24h depending on the cpm recorded in a GM counter after the final wash.

Southern-Blot Analysis
Genomic DNA was isolated from young leaves from a single plant of black nightshade according to the CTAB method described by Doyle and Doyle (1990). DNA samples were restriction digested with Bam HI, Eco RI, HindIII and XbaI, size fractionated on a 0.8% agarose gel, and Southern blotted onto a Hybond N+ membrane (Amersham Biosciences). The blots were hybridized to [ 32 P]dCTP labeled specific probes.            Fifteen mg RNA was blotted onto nylon membranes and probed with either SnpreproHypSys or SnproSys cDNA. Ethidium bromide stained rRNA served as a loading control. B, Total RNA was isolated from leaves at different time intervals following spraying with MeJA. Fifteen mg RNA was blotted onto a nylon membrane and probed with either SnpreproHypSys or SnproSys cDNA. Ethidium bromide stained rRNA served as a loading control.    A crude nightshade extract was fractionated by C18 reversed-phase HPLC with an acetonitrile gradient as described in Materials and Methods. Ten ml aliquots were added to 1 ml of suspension cultured tobacco (N. tabacum, Nt), tomato (S. peruvianum, Sp), and nightshade (S. nigrum, Sn) cells. After 20 min, the pH of the media was recorded. Peaks 1, 2, and 3 were pooled and further purified.   The three alkalinizing peaks from Figure 5 were further purified through a series of HPLC steps as described in Materials and Methods. Final purification was performed on narrow-bore C-18 HPLC with acetonitrile gradients as described in Materials and Methods. Five ml samples were added to 1ml of S. nigrum cells and the pH of the media was recorded after 20 min. The active fractions were analyzed by N-terminal sequencing and mass spectroscopy.   Alkalinization activities of synthetic SnHypSys I, II, and III with S. nigrum (Sn), N. tabacum (Nt), and S. peruvianum (Sp) suspension cells. Ten ml of each peptide were added to suspension cells to give final concentrations of 250 nM, 2.5 μM, and 25 μM as described in Materials and Methods. After 20 min, the pH of the media was recorded. SlHypSys I was used as a positive control. Each bar represents the average of 3 separate experiments. Error bars indicate standard error of the mean.