Oligosaccharins are potent biomolecules which activate defense responses and resistance in tobacco plants. However, it is not known the systemic behavior of defensive enzymes activated by these elicitors. In this work, the dynamic behavior of key defensive enzymes was evaluated in tobacco plant leaves previously treated through the roots with chitosan polymer (CH), chitosan (COS) and pectic (OGAS) oligosaccharides and Spermine (Sp). All macromolecules tested activated protein levels and defense enzymatic activity in tobacco leaves but with different response dynamics among them and depending on the biochemical variable evaluated. Defense response above control levels were detected since 12 hours after treatments and it consisted in a biphasic behavior with two peaks for PAL (EC 4.3.1.5) and β 1 - 3 glucanase (EC 3.2.1.6) enzymatic activities. The highest enzymatic levels for these enzymes were achieved at 48 hours in plantlets elicited with COS and at 72 hours for those plants treated with chitosan polymer, while the highest POD (EC 1.11.1.6) activity was detected with CH between 48 and 72 hours. These results demonstrated systemic defense activation by oligosaccharins in tobacco whose dynamic of defense response is affected by the kind of oligosaccharins tested. When applying OGAS by foliar spray on tobacco, systemic resistance against Phytoththora nicotianae was induced and plantlets were protected with the low concentration tested by 46% under the bioassays conditions performed. Moreover, enzymatic determinations on roots and leaves previous to plant-pathogen interaction showed increments above 30% of control levels for PAL and POD activities. It means that oligosaccharins activate local and systemic defense responses in plants in the absent of pathogen infection.
The cell wall is the forefront on plant-pathogen interactions. Cell wall from plants is the first barrier encountered by most pathogens; it must be degraded by them in order to allow penetration and colonization of internal tis- sues [
Pectins are one of the first targets of digestion by invading pathogens through pectinases enzymes. Oligoga- lacturonides are released when pectinases, as endopolygalacturonases and endopectatelyases, secreted by the pa- thogen degrade the homogalacturonan in the plant cell wall [
Among the first defensive responses triggered by plants, when their receptors detect the above mentioned eli- citors, are the hydrolytic enzymes as β 1 - 3 glucanases and chitinases, able to degrade pathogen cell wall poly- mers [
Pectic oligosaccharides have been implicated as signals for activating wide range of defense responses through its interaction with receptors of plant membrane and mediated by the octadecanoid and the salicylic acid accumulation pathways [
In the last decade, a collaborative work among different research groups in the subject, have demonstrated the potential of chitosaccharides (poly and oligochitosans) in the activation of defense enzymes and resistance against the pathogen of tobacco Phytophthora nicotianae [
This study provides evidences to answer the scientific questions outlined above. Firstly, the behavior of key plant defensive enzymes against pathogens was evaluated in a dynamic response study when tobacco plantlets were elicited by different oligosaccharins and plant organic compounds. In addition, it is shown how a defined mixture of pectic oligosaccharides can actually protect tobacco plants against the soilborne pathogen Phytoph- thora nicotianae by inducing plants’ own defense mechanisms.
Three amino compounds were employed as elicitors of plant defenses in a bioassay: a chitosan (CH) polymer (DA = 36.5%, DP = 794), an oligochitosan (COS) mixture (DA= 0% - 1%, DP= 5 - 9) and Spermine (Sp) (Sig- ma). As elicitor of plant defense and resistance, it was also used a mixture of pectic oligosaccharides (OGAs) commercially known as Pectimorf and obtained by enzymatic degradation of pectic acid [
Tobacco plantlets of 26 days old were gently removed from substrate, the roots washed and plantlets placed through the roots in 0.5 g∙L−1 of elicitors treatments (Chitosan polymer, chitosan oligosaccharides, pectic oligo- saccharides and Spermine) following the procedure previously describe [
After one hour of treatment, plantlets were change to glass tubes containing Hoagland solution 10% [
Plant enzymatic activities [PAL (EC 4.3.1.5), POD (EC 1.11.1.6) and β 1 - 3 glucanase (EC 3.2.1.6)] and proteins were determined in the supernatant of leaf extracts following the same procedures previously described [
In all the enzymatic determinations data were processed through a simple analysis of variance and the result- ing means were compared by the Tukey test for p < 0.05 using the statistical program SPSS for Windows©, ver- sion 11.5.
The experiment was carried out using tobacco plants of “Corojo” Cuban variety (susceptible to P. nicotianae) cultivated in a mixture of soil and organic matter 1:1 v/v under semi-controlled conditions with a light/dark re- gime of 16/8 hours and a temperature of 28˚C/23˚C, respectively.
Treatments were performed by applying two doses (0.5, 0.05 g∙L−1) of the oligogalacturonide mixture in H2Od as foliar spray. Tobacco plants of 35 days old (5 days after spraying) treated as stated before were gently removed from the substrate and placed in eppendorf tubes containing 50 times diluted nutrient solution [
Infection degree was determined in each plant using the scale of five degrees (
The true leaves and roots from 35 days old plants treated by spraying (and no used in the pathogen bioassay) were collected 5 days after treatment and ground in a porcelain mortar and pestle in presence of liquid nitrogen.
Degree | Description |
---|---|
1 | Healthy plant |
2 | Roots affected |
3 | Hypocotyls and cotyledons affected |
4 | First and second leaves pair affected |
5 | Dead plant |
Powdered leaves and roots were extracted in 50 mM sodium acetate buffer pH 5.2 containing 5 mM EDTA, 14 mM β-mercapto-ethanol and 1.0 M NaCl at the rate of 1 g per 2 ml of buffer. The extract was then centrifuged at 12,000 g for 15 minutes at 4˚C in a Sigma Microcentrifuge. The supernatant was collected in clean eppendorf tubes and stored at –10˚C for subsequent analysis. Enzymatic determinations were performed in the supernatant of leaf and roots extracts as described in the first experiment and according to [
All macromolecules tested activated protein levels and defense enzymatic activity in tobacco leaves above the control, but with different response dynamics among them and depending on the biochemical variable tested. Protein increments above control treatment on tobacco leaves were detected since 12 hours after elicitor applica- tions with all compounds evaluated (
PAL enzymatic activity was induced above control in a biphasic response by the elicitors tested, although, differentially peaking for each one (
The dynamic behaviour of β 1 - 3 glucanase activity was about similar than PAL (
POD enzymatic activity was activated above control since 12 hours of treatments by COS, Sp and OGAS (
higher at 48 and 72 hours of treatment. At similar times, the other elicitors tested caused the same behaviour, under or a little above control level.
In order to test the capacity of eliciting of induced resistance by OGAS, it was performed a five days activation bioassay studied before [
Taking into account the bioassay performed, in which plants are sprayed in their aerial parts and the infection is carried out later on through the plant roots, it is possible to conclude that the protection observed is due to the induction of systemic resistance in tobacco plants by the oligogalacturonide mixture.
The OGAS mixture caused activation of PAL and POD activities in roots and leaves of tobacco plantlets. PAL enzymatic activity on leaves was significant increased above control by 30% with the low concentration tested, while the increment of PAL activity with the highest OGAS concentration neither differed from control nor the treatment with low OGAS concentration (
For POD enzymatic activity the behaviour was similar to the PAL on roots but different on leaves. On leaves, the highest increment of enzymatic activity was activated by the high OGAS concentrations tested with signifi- cant difference regarding to the control and the low OGAS concentration tested (
Generally speaking, the results from the first experiment performed demonstrated that the organic compounds tested in this work activated systemic induced resistance in tobacco plants since, the enzymatic defences, related to induced resistance, were activated on plant leaves above control levels following several hours that roots had been treated. By first time, it was evaluated the systemic behaviour on time of protein and defence enzymes ac- tivation by these compounds in tobacco leaves.
Treatments | N | Means of Ranks | % of Infection |
---|---|---|---|
Control | 15 | 30.87 a | 100 |
OGAS 0.05 g∙L−1 | 16 | 16.94 b | 53.65 |
OGAS 0.5 g∙L−1 | 15 | 23.13 ab | 74.7 |
X2 = 12.08***.
The defence activation followed different behaviours depending on time for determination and the type of compound tested. According to results, biphasic behaviour was detected on variables evaluated, except for POD where, it was not clearly appreciated. Firstly, a lower first peak occurred at 12 hours after treatment, while a two or three times higher second peak could be detected at 48 or 72 hours after treatments and depending on the type of compound tested.
Former studies stated that induced resistance at cell level consist in, at least, two moments of defence activa- tion, an early response, where a smaller number of defence genes which are related to the recognition and a first defensive barrier against the pathogen are activated. They are also involved on the amplification and signalling of the response expressed. In addition, a second array of similar and new synthetized defence responses is acti- vated, causing the amplification of the whole plant resistance against pathogen [
With these and previous results [
All three enzymes evaluated in this study were, differentially, time activated on tobacco leaves depending on the elicitor structure type. From results, main differences for the highest peak of activity occurred, apparently, as a result of the influence of elicitor molecular weight, since, the protein contents and the enzymatic activities achieved the highest level with lower molecular weight elicitors (COS, OGAS and Spermine) about 48 hours after treatment, while for the chitosan polymer (high molecular weight) the highest increments occurred at 72 hours after treatment. Therefore, it is possible that different behaviour because of molecular weight be related with the perception at membrane level, since; low molecular weight compounds can pass easier plant coverts and wall cell to achieve receptors in membrane. Conversely, chitosan polymer must delay to achieve the mem- brane because of its polymeric structure and, probably, it could be hydrolysed by enzymes with chitosanolytic activities which are free located at the level of cell wall and apoplast [
In general, oligogalacturonide mixture induced less enzymatic activity than amino compounds; even, the bi- phasic enzymatic behaviour was much less clearly appreciated with OGAS than with the other elicitors evalu- ated. This could be the reason why the tobacco protection obtained against P. nicotianae was less effective with the same concentration ( 0.5 g ∙L−1) tested. Since low OGAS concentration caused higher protection, it is neces- sary in the next future to evaluate an experiment of dose-protection for this mixture.
Enzymatic increments on roots and leaves were not higher than 30% respect to control. This value is not so high compare to increments previously found, by using the same bioassay but, with chitosan derivatives as eli- citors [
As conclusion, this work demonstrated systemic activation of important tobacco defence markers by oligo- saccharins and confirming this defence induction in the absent of pathogen infection. As novelty, it was shown the capability of OGAS to protect tobacco plantlets against its main soilborne pathogen at nursery stage. In addi- tion, it was shown the defence enzymatic activation on tobacco leaves and roots by oligogalacturonide treat- ments. In order to appreciate the practical value of this work, next experiments with plant-pathogen interaction must be performed closer to the regular plantlets development on tobacco nurseries.
First author wants to thank the financial support to the research included in this publication by Conacyt (Mexico) and the International Foundation for Science (Project F-4446-2F). Thanks to Q. B. Francisco Soto and Q. B. Socorro Vallejo (CIAD) for their technical assistance.
DA: degree of acetylation;
DP: degree of polymerization;
CH: chitosan polymer;
COS: oligochitosans;
OGAS: oligogalacturonides or oligopectates;
Sp: spermine;
PAL: phenyl-alanine ammonia lyase;
POD: peroxidase;
PMSF: phenyl-methyl sulfonyl fluoride.