Introduction

Purified secondary metabolites are usually obtained through extraction from whole plants or plant tissue. However, large-scale plant tissue culture is an attractive alternative to the traditional method of production. The use of bioreactors has led to the development of technologies suitable for large-scale in vitro plant propagation. Currently, various plant species are propagated using bioreactors for biomass production as well as large-scale micropropagation (Escalona et al. 1999, 2003; Ibaraki and Kurata 2001; Paek et al. 2001; Chakrabarty et al. 2003). For superior practical use of bioreactor cultures, research aimed at improving the physical and chemical environments—such as increased air exchange, increased photosynthetic photon flux, and optimized CO2 content—are necessary (Paek et al. 2001).

Sugarcane propagation in temporary immersion bioreactors (TIBs) has been optimized for large-scale plant production (Lorenzo et al. 1998), including the crucial acclimatization step (Rodriguez et al. 2003). An increased amount of secondary metabolites, mostly phenolic compounds, has been observed in sugarcane micropropagated in TIBs (Arencibia et al. 2008). It is known that physical wounding (such as those produced during plant culture manipulation) triggers several important changes in plant metabolism. One of the most important changes induced after wounding is the accumulation and oxidation of phenolic compounds, which is seen as tissue-browning (Falco et al. 2001).

Previous research confirmed that phenolic production by sugarcane in TIBs (despite being considered an undesired trait in conventional tissue culture) induced remarkable vigor in sugarcane micropropagated plants, which displayed a more robust performance when they were transferred to field conditions (Arencibia et al. 2008). In these plants, upregulation of genes related to cell metabolism and development, plant defenses, phenylpropanoids, methyl jasmonate response, ethylene, oxidative burst, and auxin pathways suggested that those phenolic metabolites might act as molecules eliciting systemic acquired resistance phenomena (Arencibia et al. 2008).

The present study was undertaken to investigate if sugarcane phenolic metabolites produced in CO2-rich TIBs elicitate a type of induce resistance in a heterologous species. In this case, the Solanum lycopersicum–Ralstonia solanacearum pathosystem was selected as a model because it is a systemic disease of agronomic importance in which the reliable phenotypic symptoms appear in a relative short time period, providing a more efficient diagnosis. Data that corroborates this hypothesis might pave the way for the evaluation of the effectiveness of these phenolic compounds in different plant–microbe interactions. Additionally, the findings may add value to the sugarcane micropropagation industry using the TIBs technology for large-scale byproduct development.

Materials and Methods

Biological materials.

In vitro sugarcane plants (Saccharum officinarum cv. Badila) were obtained by sterile meristem culture and micropropagated according to the method of He et al. (1994). Cultures were maintained under a combination of both natural light and cool-white fluorescent tubes (four tubes of high-output 54 W, 990 mm length, 6,500 K, with an initial lumen output of 5,000 each) at a light intensity of 110 mE m−2 s−1, under a 12-h photoperiod, at 27°C. Three subcultures were conducted each fourth week. Fifteen days after the last subculture, vigorous and pathogen-free plants were further selected for experiments in TIBs.

High quality, certified pathogen-free tomato seeds (S. lycopersicum cv. YA SHE 2301) were obtained from Nanning Liangli Seeds Development Co., Ltd., People’s Republic of China. This tomato cultivar is considered susceptible to bacterial wilt (Yang et al. 2004). Tomato seeds were germinated in sterile soil with daily irrigation under greenhouse conditions. Forty-five-d-old plants were used for the pathogen challenge. Four days before transplanting into containers, the soil was sterilized by sitting in the sun for 24 h followed by treatment with the microbicide Longkejun SC 20% (Thiodiazole-copper) at 1.25%, for 48 h while under plastic film. A R. solanacearum strain was kindly supplied by Miss Guining Zhu at the Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences. Bacterial cells were cultured and maintained according to Yang et al. (2004).

Metabolite production in temporary immersion bioreactors.

Fifteen in vitro sugarcane plant were transferred to Pyrex® glass Erlenmeyer flasks (capacity 5 l, Neck OD 57 mm, Flask OD 215.9 mm, height 330 mm) containing 1 l of shoots micropropagation medium: MS (Murashige and Skoog 1962) salts, 5.5 mM myo-inositol, 1.1 μM 6-BAP, 7.3 μM PBZ, and adjusted to pH 5.6. Combinations of sucrose (30 g/l, 20 g/l, 10 g/l, 5 g/l) and CO2 (0.4%, 0.2%, 0%) were tested. Immersion frequency was 3 min every 3 h. Cultures were maintained at 27°C under a combination of both natural light and cool-white fluorescent tubes at a light intensity of 110 mE m−2 s−1. Culture medium was collected over a period of 25 d. Experiments had three replicates and were repeated at three different times.

Phenolic compound concentration determination.

The concentration of phenolic compounds was measured in the TIBs treatments as described (Loaiza-Velarde et al. 1997; Campos-Vargas et al. 2005) for each of the treatment combinations tested. Briefly, 1 ml of sugarcane micropropagation culture medium was mixed with 1 ml methanol (HPLC grade). Absorbance was read at 320 nm (potential browning) and 437 nm (soluble o-quinones) using an UV–VIS spectrophotometer (PharmaSpec UV-1700, Shimadzu, Kyoto, Japan). Measurements from three replicates at three different times were combined, and results were subjected to an ANOVA. Five percent of LSD values were calculated when significant treatment differences were detected.

Northern blot analysis.

Approximately 1 g of fresh tissue from in vitro-cultured sugarcane plants was harvested and immediately frozen in liquid N2. This tissue was ground to a fine powder using a pre-cooled pestle and mortar. The ground tissues were subjected to RNA extraction using a commercial kit (Maxwell® 16, Promega, Madison, WI). The RNA concentrations were quantified by determining the 260:280 and 260:230 ratios using a spectrophotometer (PharmaSpec UV-1700, Shimadzu). Total RNA (10 μg) was separated by electrophoresis on a 1.2% agarose/0.4 M formaldehyde gel and transferred to a Hybond N+ nylon membrane (Amersham-Pharmacia, Amersham, UK). Probe fragments AG603 (448 bp), which show similarity (E value 4e−68) to phenylalanine ammonia-lyase (PAL) of S. officinarum (accession number EF189195), and AG609 (270 bp), which shows similarity (E value 6e−24) to ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) of Saccharum hybrid cultivar (accession number CF576750), were previously isolated by cDNA-AFLP (Arencibia et al. 2008). Probes were made from PCR-amplified fragments using the ReadyPrime DNA labeling kit (Amersham-Pharmacia) with radioactive phosphorous (32P). The 18S rRNA transcript was used as a loading control. Blots were hybridized and washed according to standard procedures (Sambrook et al. 1989).

Biological activity.

To study the biological activity of sugarcane metabolites during tomato–R. solanacearum interactions, controlled experiments were conducted under greenhouse conditions (24–26°C temperature, 14 h photoperiod, 90% humidity). A total of 0.5 ml (OD at 320 nm higher than 1.9) of sugarcane metabolites produced in micropropagation medium with either 20 g/l sucrose (S20) or 30 g/l sucrose (S30) was sprayed on tomato plants using one of two methods: (1) 48 h before transplanting and pathogen inoculation or (2) directly after transplanting and pathogen inoculation. After pathogen inoculation, the metabolite treatments were repeated once per d for 3 d. At the time of transplanting, R. solanacearum inoculation was conducted by submerging the plant roots in 1 × 108 cfu/ml bacterial suspension for 3 min. To avoid other pathogenic bacterial interferences, seedlings were transplanted to pots containing previously sterilized soil. Control treatments were: (1) plants with neither pathogen inoculation nor metabolite treatment (positive control) and (2) pathogen-inoculated plants without metabolite treatment (negative control). Three separate experiments containing five plants per treatment were conducted. Daily irrigation and temperatures of 24–26°C were maintained during the experiment. The tomato bacterial wilt disease incidence was evaluated every day as the number of wilted leaves per plants, according to Yang et al. (2004). \( {\hbox{Disease}}\;{\hbox{index}} = \left[ {\sum {\left( {{\hbox{number of withered leaves in each grading standard}} \times {\hbox{grading standard}}} \right)/\left( {{\hbox{the total number sample leaves}} \times {9}} \right)} } \right] \times {1}00 \). Grading standard (0∼5): 0, without symptoms; 1, one leaf was wilted; 2, two leaves were wilted; 3, half of the leaves were wilted; 4, all the leaves were wilted besides the top; and 5, total necrosis.

RT-PCR analysis for systemic acquired resistance induction.

RNA was extracted from tomato leaves pooled from separate replicates. Extractions were performed using TRIzol reagents (Invitrogen, Carlsbad, CA). One gram of the pooled, frozen leaf tissue was ground to a fine powder in the presence of liquid nitrogen, and then mixed with 10 ml TRIzol. The RNA sample concentrations were quantified by determining the 260:280 and 260:230 nm ratios by spectrophotometry and further confirmed on a 1.2% agarose/0.4 M formaldehyde gel. Of high quality RNA, 20 ng were added as template to each reaction using the Enhanced Avian HS RT-PCR kit (Sigma, St. Louis, MO). For specific RT-PCR amplification, genes involved in different plant defense pathways were selected and the oligonucleotide primers were designed using sequences from the GenBank public databases (www.ncbi.nlm.nih.gov). Information on primer source, nucleotide sequence, and PCR annealing temperatures are listed in Table 1.

Table 1. Selected pathogen-inducible genes for RT-PCR
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The PCR reaction mixture consisted of 3 μl of first strand cDNA, 5 μl of 10× PCR buffer, 5 μl of 25 mM MgCl2, 1 μl of dNTPs (10 mM each), 1 μl each of 10 μM primers (forward and reverse), 1 U of Taq DNA polymerase (Invitrogen), and ultrapure water (Sigma) to 50 μl. PCR reactions were conducted with the following parameters: 94°C for 3 min, 30 cycles of 94°C for 30 s, annealing according to specific gene (Table 1) for 30 s, 72°C for 1 min followed by a final incubation at 72°C for 10 min. RT-PCR products were electrophoretically separated on a 1% agarose gel and stained with ethidium bromide.

Results

Production of phenolic metabolites.

The sugarcane plants grown in TIBs supplemented with either 20 or 30 g/l sucrose displayed a strong green color under CO2-rich conditions. Spectrophotometer analyses, which indicated that the highest increase in phenolic-related metabolites, were obtained when the air was enriched to 0.4% CO2 and the media was supplemented to 20 and 30 g/l sucrose (Fig. 1A ). Phenolic production in the 0.2% CO2 environment was not significantly different as compared to the control treatments (30 g/l sucrose without CO2). Treatments combining 20 g/l sucrose with 0% CO2-enrichment displayed a slight decrease in the absorbance values, which could be explained by the use of non-optimal carbon concentrations.

Figure 1.
figure 1

Absorbance readings to determine phenolic concentrations during sugarcane micropropagation under combinations of sucrose (30 g/L, 20 g/L) and CO2 (0.4%, 0.2%, 0%). (A) 320 nm, which measures potential browning. (B) 437 nm, which measures soluble o-quinones. Values represent the mean ± SD (standard deviation) of three replicates measured at three different times.

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The phenolic compounds (as measured at 320 nm) increased 4.6- and 6.3-fold in the 0.4% CO2-rich air when combined with media supplemented to 20 and 30 g/l sucrose, respectively, during the standard 25-d sugarcane micropropagation cycle. The control treatment with 30 g/l sucrose (without CO2) displayed a 2.5-fold increase while the 20 g/l sucrose without CO2 treatment showed just a 0.78-fold increase.

Soluble o-quinones (as measured at 437 nm) increased 12.1- and 14.0-fold in 0.4% CO2 plus 20 and 30 g/l sucrose, respectively (Fig. 1B ). Only 7.6-fold increases was observed in the control treatment (30 g/l sucrose without CO2), which does not show significant differences with the 0.2% CO2 treatments. The 20 g/l sucrose without CO2 treatment displays the lowest increases (only 2.14-fold). Generally, the accumulation of soluble o-quinones during the experiment might explain the highest differences between the absorbance values at 437 and 320 nm. However, the dissimilar behavior in the 20 g/l sucrose/0% CO2 treatment could indicate that degradation of phenolics occurred, a key determinant when the available carbon became a limiting factor.

Northern blots.

Both PAL and Rubisco transcript levels were measured after culture in different CO2-rich conditions (Fig. 2) to show the transcript levels for key enzymes involved in phenylpropanoid and photosynthesis pathways.. Results showed that in 0.4% CO2 and using either 20 or 30 g/l sucrose treatments, the expression levels of both genes consistently increased after 10 d of micropropagation in TIBs, suggesting that a photomixotrophic stage occurred, allowing an increased production of phenolic metabolites.

Figure 2.
figure 2

Differential expression analysis of sugarcane plants micropropagated in TIBs to create a CO2-rich environment. Rubisco and PAL transcript expression in different CO2-rich conditions. T0 just after plant subculture, T1 10 d, T2 25 d. The 18S rRNA was used as a loading control.

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In the 0.2% CO2 treatments, a similar increase of the PAL transcripts in both the 20 and 30 g/l sucrose treatments was evident at 10 and 25 d after subculture. Rubisco transcript levels were similar at both T1 and T2 under 0.2% CO2 and 20 g/l sucrose treatment, revealing that a weakly photomixotrophic condition might have occurred. Additionally, this shows little correlation between the PAL and Rubisco activities under these culture conditions, as the PAL seems non-responsive to the increase in sucrose. The control treatment of 30 g/l sucrose without CO2 showed that both Rubisco and PAL transcripts remained at approximately the same levels throughout the experimental time course. These results confirmed the occurrence of a photomixotrophic stage in parallel with the natural production of phenolic metabolites during standard culture conditions for sugarcane micropropagation in TIBs. Meanwhile, in the treatment with 20 g/l sucrose without supplemented CO2, the Rubisco activity increased while the PAL transcripts decreased. This could indicate that in sucrose-deficient medium plants initially direct C from the CO2 to basic metabolism that will support survival. In fact, using 20 g/l sucrose, a low amount of available C for sugarcane micropropagation, plants significantly reduce both growth and multiplication rates and could die in long-term subcultures.

Biological activity.

The metabolite mixtures labeled S20 and S30 came from the 0.4% CO2-supplemented TIBs containing micropropagation medium plus 20 or 30 g/l sucrose, respectively. These extracts were selected for bioassays since they contained the highest levels of phenolic metabolites (Fig. 3) produced in parallel to sugarcane shooting. After 20–25 d of sugarcane culture, mixtures S20 and S30 were sprayed onto different 45-d-old tomato seedlings artificially infected with R. solanacearum (causal agent of bacterial wilt) at the time of transplanting. Spraying with the culture medium was conducted in two ways: (1) 48 h before (treatments I, V) and (2) directly after pathogen inoculation (treatments II, VI). Phenolic treatments (spraying) were continued for 3 d.

Figure 3.
figure 3

Sugarcane plants (cv. Badila) during micropropagation in TIBs with 0.4% CO2 and different sucrose concentrations. (A)–(C) Sugarcane plants and the culture medium after 25 d of culture under different sucrose concentrations. Scale indicates 5 cm.

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Ten days after pathogen inoculation, the disease symptoms developed rapidly in the negative control plants treated with a sucrose solution lacking phenolic metabolites (Fig. 4A , panel IV). Typical symptoms began as leaf drooping and were followed by the complete plant wilting within a few days, indicating high virulence of the R. solanacearum isolate. Infected plants treated with S20 and S30 metabolite solutions (Fig. 4A , panels I, II, V, and VI) overall showed significantly fewer disease symptoms than the negative control treatment (Fig. 4A , panel IV; Table 2). The non-inoculated plants (Fig. 4A , panel III) grew well, without disease symptoms, demonstrating the efficiency of the soil sterilization treatment.

Figure 4.
figure 4

Biological activity of sugarcane phenolic metabolite mixtures S20 and S30 on YASHE 2301 tomato seedlings artificially inoculated with R. solanacearum (causal agent of bacterial wilt). (A) 10 d post-inoculation. (B) 30 d post-inoculation. I–VI treatments: see details in Table 2. Scale indicates 10 cm.

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Table 2. Bacterial wilt incidence in YASHE 2301 tomato plants artificially inoculated with R. solanacearum
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After 30 d, the disease effects were strongly evident, as shown in Fig. 4B . Overall, the disease index between the treatments significantly differed from the infected, but non-treated, (negative) control. Since the first evaluation at day 10, tomato plants sprayed with S20 directly after pathogen inoculation displayed the lowest disease index, which was not significantly different from the positive, non-inoculated control.

The average plant height (a measure of how well the plants grew) at 30 d significantly differed between the sprayed treatments and the non-inoculated control. Better results were obtained in treatments II and V, which yielded plants with average heights of 43.4 and 42.0 cm, respectively. These heights are not significantly different from each other, but are significantly less than the non-infected positive control (Table 2).

In summary, spraying both S20 and S30 metabolite mixtures resulted in the protection of the tomato plants against bacterial wilt. Nevertheless, the plant resistance differed between the treatments. The S20 treatment resulted in the lowest disease index when applied just after pathogen inoculation (treatment II), while the S30 treatment resulted in a significantly lower disease index when applied 48 h before pathogen inoculation.

Genomic characterization of biological activity.

Tomato plants sprayed with the S20 mixture just after pathogen inoculation (treatment II) were selected for gene expression characterization since their disease index was the lowest in the plant–pathogen challenge (see Fig. 3 and Table 2). Using the primers listed in Table 1, expression profiles were analyzed for ten defense-related genes. Amplification patterns are shown in Fig. 5.

Figure 5.
figure 5

Differential expression of select defense-related genes in tomato plants (Solanum lycopersicum cv. YASHE 2301). RT-PCR analyses were conducted 0, 1, 3, 5, and 10 d following the inoculation and treatment. Panels labeled 1–10 correspond to those genes described in Table 1. RT-PCR amplifications of the actin gene were used as control.

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Transcripts of the analyzed defense-related genes were induced by S20 treatment after 1 d of challenge (TII), and the gene expression was maintained during the first 10 d. The non-treated, pathogen-free plants (C+) also showed an early differential expression of the studied transcripts, which were generally maintained through days 3 and 5. In the case of erf2 (panel 5—ethylene response), wfil (panel 8—biotic stress response), and apx (panel 9—ascorbate peroxidase), the induction was maintained during the 10 d, although the induction appeared stronger for both wfil and apx genes in the S20-treated plants.

The non-treated and inoculated plants (negative control) had elevated transcripts for only the acc4 (panel 2) and erf2 (panel 5) genes after 3 d and a weak induction for the loxD (panel 7) gene 1 d after inoculation. The rest of the studied transcripts did not show any RT-PCR amplification pattern without S20 treatment, indicating that the plants did not induce strong disease resistance.

The positive control results indicate that a defense mechanism was induced by the wounding effect occurred during seedling transplanting. This early defense response was suppressed in the pathogen-inoculated plants (negative control). Consistent upregulation of all studied genes in the infected plants treated with S20 indicate that the phenolic metabolites might induce or maintain the early plant defense response at least until 10 d after inoculation.

Discussion

This is the first report of the optimization of natural sugarcane metabolite production, mainly phenylpropanoid compounds in parallel to plant micropropagation in TIBs. Previous results by cDNA-AFLP determined that the PAL and Rubisco transcripts were differentially expressed in TIBs (Arencibia et al. 2008) The Rubisco results here show that the highest CO2 concentration studied increased the photosynthetic activity, indicating the induction of a photomixotrophic stage in the in vitro sugarcane plantlets. Both PAL and Rubisco activities increased in plants grown in the 0.4% CO2-enriched environment, indicating that improved conditions had been achieved for phenolic production. Another seven transcripts related to the phenylpropanoid pathway, including cinnamoyl-CoA reductase, chalcone synthase, caffeic acid 3-O-methyltransferase, and cinnamate 4-hydroxylase had also been identified as differentially expressed (Arencibia et al. 2008). Overall, these results corroborate the conclusion that sugarcane micropropagation in CO2-rich TIBs induces phenylpropanoid metabolism.

Considering the complexity of photosynthesis and the range of factors that determine carbon sequestration, the present work should be considered an initial step toward further research. The in vitro culture conditions must be optimized, including the crucial factors of luminosity and light quality, for the improvement of photosynthesis during plant micropropagation. In parallel, both plant species and genotype must be considered. In the present study, the maximum phenolic levels were found in 0.4% CO2-rich air with either 20 or 30 g/l sucrose; however, this might vary between sugarcane cultivars. Sugarcane is a C4 plant in which we understand that the in vitro photosynthetic efficiencies could stand to be improved. In contrast, a physiological study in TIBs-showed that micropropagated pineapple appeared to use nutrients from the culture medium as well as those from photosynthesis, suggesting a higher photomixotrophic metabolism (Escalona et al. 2003).

The contributions of CO2 supply during the proliferation and multiplication stage in media with sucrose have been extensively studied in bioreactors (Ziv 2000). The reports on the effects of CO2 enrichment suggested that increases in the plant growth during acclimatization and transplanting could be achieved (Kozai et al. 1995). High air flow rates were thought to be the contributing factor to optimum growth, rather than inhibition by either excessive oxygen, depletion of CO2, or the removal of various culture volatiles, including CO2 (Kim et al. 1991). Positive effects are plant specific, as CO2 enrichment in an illuminated bioreactor culture of Brodiaea clusters did not affect biomass growth and an increase from 0.3% to 1% CO2 gave a similar growth value (Ilan et al. 1995). However, in Cyclamen persicum Mill, high CO2 levels correlated with increased production of pro-embryogenic masses (Hvoslef-Eide and Munster 1998). Improved biomass production and increased chlorophyll and carotenoids were obtained in a mixotrophic culture of Euglena gracilis (Yamane et al. 2001). Recent results indicated that the application of photomixotrophic culture to Spirulina platensis, with stepwise addition of Se to the medium, could offer an effective and economical way for the production of high Se-enriched products such as phycocyanin and allophycocyanin (Chen et al. 2006).

In this paper, a new function of plant phenolic metabolites has been identified as elicitors or priming molecules for systemic resistance against bacterial diseases. In this particular case, sugarcane cultures in TIBs were demonstrated to be efficient producers of phenylpropanoid metabolites that, when sprayed on tomato plants, induced resistance against the pathogenic bacterium R. solanacearum, which causes a wilting disease. Tomato plants sprayed with S20 metabolites, induced by culture with 20 g/l sucrose, after pathogen inoculation displayed high resistance to bacterial wilt incidence when compared to those sprayed prior to pathogen inoculation. This resistance response was reversed when plants were sprayed with S30 metabolites. A possible explanation could be because of differences in concentration and/or composition between the S20 and S30 metabolites. A more accurate metabolomic characterization of sugarcane phenolics produced in TIBs, including different environmental conditions, remains a future task in this research.

In our experiments, differential expression analysis corroborated that defense-related genes were induced in tomato, possibly through wounds that occurred during transplanting (positive control). However, this early defense response was suppressed or delayed in the pathogen-inoculated tomato plants (negative control) and thus rendered ineffective in controlling the disease. R. solanacearum is an opportunistic and aggressive bacterium that enters roots through wounds or natural openings (Allen et al. 2005). The consistent differential expression of all studied defense-related genes in the infected plants treated with the sugarcane metabolites demonstrate that the phenolic metabolites might induce or maintain the early plant defense responses induced by wounding through handling. Overall, the tomato defense response remains induced, or on “alert,” in the phenolic-sprayed treatment, as demonstrated by the continued expression of 7 (see Fig. 5 and Table 2) of the ten studied, pathogen-related genes.

It is noticeable that the sugarcane phenolic metabolites acted as priming molecules in the tomato-bacteria interactions. Nevertheless, one key fact to be considered is that phenolic treatments on previously infected plants (showing the bacterial wilt symptoms) did not induce the plant defense response at any time. Phenolic treatments at 24 h after pathogen inoculation did not protect the tomato plants, which responded similarly to the negative controls (data not shown). This indicates that sugarcane phenylpropanoid metabolites could be used as a “preventive,” but not “curative,” means of pathogen control.

In general, natural products based on the phenylpropane skeleton (Petersen et al. 1999) have an array of important functions, which are not yet fully understood and involve a variety of ecological and physiological phenomena (Abdulrazzak et al. 2006). However, some examples of these physiological phenomena warrant mention. A study on the transcriptome changes in the phenylpropanoid pathway of Glycine max in response to Pseudomonas syringae infection showed the strong bias that soybean had toward increasing the synthesis of isoflavonoid phytoalexins concomitant with the downregulation of genes required for the synthesis of anthocyanins and proanthocyanins (Zabala et al. 2006). Although proanthocyanins are known to be toxic compounds, the cells in the soybean leaves seem to be programmed to prioritize the synthesis and accumulation of isoflavonoid and pterocarpan phytoalexins during the resistance response. Phenylpropanoid metabolites display other functions, i.e., lignification and influence on the physicochemical properties of cell walls (Boerjan et al. 2003; Merali et al. 2007), protection from UV irradiation (Booij-James et al. 2000), service as antioxidants (Mathew and Abraham 2004; Korkina 2007) and as allelochemicals (Dudareva et al. 2004), and playing a role in cell wall expansion and homeostasis control during plant development (Abdulrazzak et al. 2006).

Plants can acquire enhanced resistance to pathogens after treatment with necrotizing attackers, with non-pathogenic root-colonizing pseudomonas, or with salicylic acid, beta-aminobutyric acid, or other natural or synthetic compounds. The induced resistance is often associated with an enhanced capacity to mobilize infection-induced cellular defense responses (Conrath et al. 2002). Primed plants display faster and/or stronger activation of the various cellular defense responses that are normally induced following either attack by pathogens or insects or in response to abiotic stresses. Although the phenomenon has been known for decades, most progress in the understanding of priming was made only in recent years (Paré et al. 2005; Conrath et al. 2006).

In an elegant experiment, Arabidopsis plants were treated with the synthetic systemic acquired resistance inducer benzothiadiazole (BTH) before stimulating two prominent cellular defense responses, namely PAL gene activation and callose deposition, although BTH was essentially inactive at the immediate induction of these two induced by infection with P. syringae, wounding, or infiltration of the leaves with water. The BTH pretreatment also enhanced the production of callose. The results suggest that priming is an important cellular mechanism in acquired disease resistance (Kohler et al. 2002). Meanwhile, Arabidopsis plants treated with the non-protein amino acid beta-aminobutyric acid (BABA) developed an enhanced capacity to resist biotic and abiotic (salt and drought) stresses (Jakab et al. 2005). This BABA-induced resistance is associated with an augmented capacity to express basal defense responses, including three novel regulatory genes involved in priming for different defense responses (Ton et al. 2005). A more recent study showed that volatile organic compounds induced a specific subset of JA-inducible genes and linked these responses at the molecular level to enhanced levels of direct and indirect resistance against insect attack (Ton et al. 2007).

It has been accepted that knowledge of the biosynthetic pathways of desired compounds in plants as well as in cultures is still rudimentary, and strategies are consequently needed to develop case-by-case studies (Vanisree et al. 2004). The current research has focused on a loosely defined set of metabolites, grouped together as phenolics; although, the presence of other natural molecules cannot be disregarded. Integrated biochemical, metabolic, and genomic approaches could identify the key active molecules in the extract and the distinct receptor and downstream genes that are responsible for the plant response.

Phenolic compounds, produced during sugarcane micropropagation in CO2-enrichment TIBs, were applied to a heterologous species and shown to induce resistance in tomato plants infected with R. solanacearum, the causal agent of bacterial wilt disease. The tomato plants displayed an activated defense response, resulting in early plant protection against pathogen attack.

The control of the phenylpropanoid pathway by either conventional methods or through genetic engineering is suggested approaches for further investigation. Application of plant (sugarcane) phenolic metabolites should be tested as a potential tool for integrated management (Allen et al. 2005) of the tomato bacterial wilt (R. solanacearum) disease in tomato.