CN116803268A

CN116803268A - Application of flavonoid compound and derivative thereof

Info

Publication number: CN116803268A
Application number: CN202310767316.2A
Authority: CN
Inventors: 叶文秀; 国林领; 焦佼; 罗晓; 王继纵; 朱雅茜; 汪燕
Original assignee: Institute Of Modern Agriculture Peking University
Current assignee: Institute Of Modern Agriculture Peking University
Priority date: 2023-06-27
Filing date: 2023-06-27
Publication date: 2023-09-26

Abstract

The application discloses an application of flavonoid and derivatives thereof, which is used as a plant regulator to induce plant stomata to close. The application provides a novel application of a flavonoid compound and a derivative thereof, which is used as an inducer for inducing plant stomata to close, and the flavonoid compound and the derivative thereof can well promote the water retention and drought resistance of plants and disease control property, particularly can effectively inhibit plant diseases caused by pathogenic bacteria taking plant stomata as an invasion channel, and provide a novel thought for controlling plant diseases.

Description

Application of flavonoid compound and derivative thereof

Technical Field

The application relates to an application of flavonoid compound and derivatives thereof, belonging to the field of agriculture.

Background

Drought stress refers to a natural phenomenon that soil dryness and air humidity decrease due to lack of water, and thus external environment is unfavorable for plant growth. Drought is a multidimensional stress that can cause a series of changes in plants from phenotype, physiology, biochemistry, to molecular level, and severe drought can lead to termination of photosynthesis and metabolic disturbance, ultimately leading to death of plants. About 33% of cultivated lands in the world are in a state of insufficient water supply, and drought becomes one of the most important agricultural production adversity factors, which is the first natural disaster of China. Seasonal drought problems are commonly existed in agricultural production in China, and drought with high frequency, wide region and long duration time is frequently generated in the actual production process, so that great loss is caused to grain production. In order to reduce the influence of drought on the growth of crops and plants, the screening and cultivation of crops and plants with strong drought resistance is an effective method which is widely accepted at present. Meanwhile, the cultivation and planting of drought-resistant and water-saving plants can play a positive role in the restoration of drought environments, but the cultivation of drought-resistant varieties requires long time and does not have broad spectrum. Therefore, the broad-spectrum drought-resistant agent has practical application value in solving the drought-resistant and water-retaining problems of crops.

Plant diseases are important factors restricting agricultural production. The control of plant diseases is mainly chemical control, and at least 300 bactericide active ingredients are known to be used for controlling various plant diseases. However, due to the long-term and large-scale use of chemical bactericides, the drug resistance of plant pathogenic bacteria is increased year by year, and the sterilizing effect of the traditional bactericides is gradually weakened. Therefore, it is necessary to find new disease control strategies that can replace traditional bactericides.

The stomata are present in the epidermis of the plant's upper part, and are small holes surrounded by a pair of guard cells, and their opening and closing movements are the main means of gas exchange between plant regulation and the environment. A number of studies have shown that guard cells can induce stomatal movement in response to a variety of signals from the plant interior and environment, includingLight and CO ₂ Water stress, plant hormones and plant secondary metabolites, etc. to regulate gas exchange, transpiration water loss and to protect against microbial invasion. Studies have shown that the activity of a variety of transport proteins localized to the cytoplasmic membrane is critical for the switching movement of stomata. In the process of closing the air holes, the activation of the anion channel is a driving force, the activation of the anion channel induces depolarization of a cytoplasmic membrane, the depolarization of the cytoplasmic membrane induces activation of an outward potassium channel and inhibits the inward potassium channel, and loss of main osmotic pressure regulating ions such as potassium ions, anions and the like can cause increase of intracellular water potential to cause dehydration and atrophy of guard cells, and finally the air holes are closed. Reactive Oxygen Species (ROS) and Nitric Oxide (NO) act as second messenger molecules that play an important role in regulating guard cell transporter activity and pore closure processes.

Under drought conditions, the regulation of stomata opening and closing is a main method for controlling transpiration and loss of plants, and the moisture loss is reduced through stomata regulation, so that the normal physiological, biochemical and metabolic operations of the plants are protected. Exogenous abscisic acid ABA can reduce the transpiration and the loss of moisture by adjusting the opening and closing of air holes, and maintain the moisture content in crops. Therefore, the utilization of chemical substances to induce the pore closure is a new strategy for drought resistance and water saving of crops.

Stomata, a natural opening in the plant cuticle, is utilized by many pathogenic bacteria, including bacteria, fungi, and oomycetes, as an important pathway for invading plants to cause disease. Many important crop diseases such as leaf rust of wheat, downy mildew of grape, downy mildew of cucumber, etc. are caused by pathogenic bacteria that invade stomata specializedly. It has been found that invasive plants facing pathogenic bacteria are not disabled, and that plants are able to recognize signal molecules derived from pathogenic bacteria, known as plant immune elicitors, and induce stomatal closure to prevent invasion of pathogenic bacteria. This plant immune response is known as stomatal immunity. On the other hand, studies have shown that pathogenic bacteria are able to secrete toxins and proteins known as effectors to overcome stomatal immunity and even induce stomatal opening to successfully invade hosts such as toxins secreted by bacteria, e.g. coronatine and syringolinA, and effectors, e.g. AvrB and HopM1; oxalic acid and toxins secreted by fungi, such as Fusicoccin (FC), and the like. In conclusion, the interaction between stomata and pathogenic bacteria is an important factor for determining occurrence of diseases, and the mechanism research is a leading-edge subject in the field of plant disease resistance immunity research at present. Based on stomatal immune theory, the induction of stomatal closure by chemical substances is a recent strategy for inhibiting plant diseases. However, there are few reports of related art.

Disclosure of Invention

According to one aspect of the application, the application of the flavonoid compound and the derivative thereof is provided, and the flavonoid compound and the derivative thereof are used as an inducer for inducing plant stomata closure by plants so as to inhibit plant diseases and improve the water retention of the plants.

The application of flavonoid and derivatives thereof is characterized in that the flavonoid and derivatives thereof are used as plant regulators to induce plant stomata to close.

Optionally, the general formula of the flavonoid compound and the derivative thereof is selected from any one of the general formulas (I) to (IV);

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ 、R ₉ 、R ₁₀ Each independently selected from any one of-H, -OH, galactose, oligosaccharide, glucuronic, nitro, amino, silicon-based, halogen or C1-C6 alkanyl, C2-C6 alkenyl, C1-C5 alkoxy, hydroxyalkoxy, acyloxy, substituted or unsubstituted pyrrole groups.

Specifically, the oligosaccharide group may be any one selected from a disaccharide group, a maltose group, a lactose group, and the like.

Specifically, the glucuronic acid group may be selected from any one of glucuronic acid group and polysaccharide aldehyde acid group.

Specifically, halogen is selected from fluorine or chlorine.

In particular, the alkenyl group of C2-C6 may be selected from 3-methyl-2-butene;

the hydroxyalkoxy groups may be selected from hydroxyethoxy groups;

the acyloxy group may be selected from acetoxy groups;

the C1-C5 alkoxy group may be selected from methoxy, 3-dimethylbutoxy, wherein the alkoxy group may form a ring with the benzene ring.

The substituted pyrrole groups may be selected from

Optionally, the flavonoid and the derivatives thereof are applied as plant disease control regulators.

Alternatively, the plant disease refers to a plant disease caused by pathogenic bacteria using stomata as an invasion path. Specifically, for example, wheat leaf rust, grape downy mildew, and the like.

Optionally, the flavonoid compound and the derivative thereof are applied as drought-resistant water-retaining regulator for plants.

Optionally, the flavonoid and the derivatives thereof are used as plant fresh-keeping regulators.

Optionally, the plant is selected from any one of angiosperms, gymnosperms and ferns.

Wherein the angiosperms include monocotyledonous plants and dicotyledonous plants.

The application has the beneficial effects that:

the flavonoid compound and the derivative thereof are used as the inducing substances for inducing the closure of plant stomata, and the water retention drought resistance and disease control performance of plants can be well promoted by adjusting the plant stomata through the flavonoid compound and the derivative thereof, especially the plant diseases caused by pathogenic bacteria taking the plant stomata as an invasion channel can be effectively inhibited, and a new thought is provided for the control of the plant diseases.

Drawings

FIG. 1 is a schematic diagram showing the results of the experiment for the flavonoid-induced plant stomatal closure activity in example 2 of the present application;

FIG. 2 is a graph showing the color temperature of the whole Nicotiana benthamiana in the experimental process of example 3 of the present application;

FIG. 3 is a schematic diagram showing the temperature change of the whole tobacco strain during the experiment in example 3 of the present application;

FIG. 4 is a graph showing the temperature variation of the whole tobacco strain during the experiment in example 3 of the present application;

FIG. 5 is a chart showing pore conductance of each whole plant of Nicotiana benthamiana in the experimental process of example 3 of the present application;

FIG. 6 is a graph showing the fluorescence intensity of guard cells obtained during the experiment of example 4 of the present application;

FIG. 7 is a graph showing comparison of water retention and drought resistance activities of the test sample I-1 and the control sample in example 5 of the present application;

FIG. 8 is a graph showing comparison of water retention and drought resistance activities of the test sample I-2 and the control sample in example 5 of the present application;

FIG. 9 is a graph showing comparison of water retention and drought resistance activities of test sample I-3 and control sample in example 5 of the present application;

FIG. 10 is a graph showing comparison of freshness retaining activity of test sample I-1 and control sample in example 6 of the present application;

FIG. 11 is a graph showing comparison of freshness retaining activity of test sample I-2 and control sample in example 6 of the present application;

FIG. 12 is a graph showing comparison of freshness retaining activity of test sample I-3 and control sample in example 6 of the present application;

FIG. 13 is a graph showing the comparison of grape downy mildew inhibition activity of test sample I-1 and control sample in example 7 of the present application;

FIG. 14 is a graph showing the comparison of grape downy mildew inhibition activity of test sample I-2 and control sample in example 7 of the present application;

FIG. 15 is a graph showing the comparison of grape downy mildew inhibition activity of test sample I-3 and control sample in example 7 of the present application;

FIG. 16 is a graph showing comparison of wheat leaf rust inhibitory activities of test sample I-1 and control sample in example 8 of the present application;

FIG. 17 is a graph showing comparison of wheat leaf rust inhibitory activity of test sample I-2 and control sample in example 8 of the present application;

FIG. 18 is a graph showing comparison of wheat leaf rust inhibitory activity of test sample I-3 and control sample in example 8 of the present application.

Detailed Description

The present application is described in detail below with reference to examples, but the present application is not limited to these examples.

All reagents used in the examples of the present application were purchased commercially unless otherwise specified.

The relative opening of the air holes in the embodiment of the application is calculated as follows:

measuring the opening of the air hole by using ImajeJ software, and taking the average value of the measured opening of the air hole as calculation data;

the test solutions of the flavonoid compounds in the embodiment of the application are all obtained by dissolving the flavonoid compounds in a basic buffer solution.

The application discovers that the substances containing the main groups of the flavonoid compounds in the general formulas I-IV or the corresponding salts and adducts of the compounds in the general formulas I-IV have similar effects, i.e. the substances containing the main groups of the compounds in the general formulas I-IV or the groups of the compounds in the general formulas I-IV can generate the air hole induction closing effect. The embodiment of the application mainly takes flavonoid compounds contained in general formulas I-IV as examples for illustration.

Example 1

The relative opening of the pores at a concentration of 50. Mu.M of the known flavonoid is determined by the following method:

(1) Taking 4mm tobacco leaf disc, placing in basic buffer solution, adding 150 μmol ^-1 ·m ^-2 ·s ^-1 Incubating for 2h under the illumination of intensity;

(2) Incubation with 50 μm of a test solution of a known flavonoid is continued for 2h under the same conditions as in step (1);

(3) Observing the opening and closing conditions of air holes of the tobacco leaf disc after incubation by a microscope and photographing;

(4) Pore opening was measured using ImajeJ software and mean and standard deviation calculated. And calculating the relative opening value of the flavonoid compound for inducing the pores to close according to the opening value of the pores at the concentration of 50 mu M of the flavonoid compound.

The base buffer in the test consisted of 10mM MES (Tris, ph=5.6), 5mM potassium chloride and 50 μm calcium chloride.

The test results are shown in the following table.

Table 1: test results of flavonoids of formula I

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Table 2: test results of flavonoids of the general formula II

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Table 3: results of the test for flavonoids of the general formula III

Table 4: results of the test for flavonoids of the general formula IV

Wherein, in the difference significance, 0.05 > P > 0.01; * Represents P < 0.01

As can be seen from tables 1-4, the flavonoid compounds and derivatives thereof all induce closure of the pores.

Example 2

The flavonoid-induced plant stomatal closure activity is typically measured using flavonoid compounds I-1, I-2, and I-3 (i.e., compounds corresponding to general formula I and numbered 1, 2, and 3).

The plant used in the test was nicotiana benthamiana (Nicotiana benthamiana) from the laboratory of crop stomata biology and regulatory technology at the modern agricultural institute of university of Beijing.

The testing method comprises the following steps:

(1) Taking 4mm tobacco leaf disc, placing in basic buffer solution, adding 150 μmol ^-1 ·m ^-2 ·s ^-1 Incubating for 2h under intense light, the composition of the base buffer used was the same as in example 1;

(2) The tobacco leaf tray incubated in the step (1) is respectively added with pure DMSO as a control solution and test solutions of flavonoid compounds I-1, I-2 and I-3 with the concentration of 100 mu M, and the incubation is continued for 2 hours under the same conditions as those in the step (1), and the test solutions are respectively marked as a control sample, a test sample I-1, I-2 and I-3;

(4) After incubation of the different incubation solutions, the pore opening of the tobacco leaf disc was measured and the mean and standard deviation calculated.

FIG. 1 shows the results of the flavonoid I-1, I-2, I-3 activity for inducing closure of stomata in plants. The upper graph shows the measurement and statistical calculation results of the relative opening degree of the air holes, the lower graph shows the photos of the air holes of the tobacco leaf disc after 4 solutions are incubated, and as can be seen from the graph in fig. 1, the air holes of the tobacco leaf disc after the flavonoids compounds I-1, I-2 and I-3 are incubated are completely closed, so that the flavonoids compounds show good activity in inducing the closing of the air holes.

Example 3

The effect of flavonoids on leaf temperature and stomatal conductance was tested and flavonoids I-1, I-2, I-3 are typically exemplified.

Selecting 4 Nicotiana benthamiana with similar growth vigor and plant age, placing at 20deg.C under 180 μmol illumination intensity ^-1 ·m ^-2 ·s ^-1 And (3) culturing in the environment, and collecting leaf temperature by using an infrared camera. After the temperature is stable, the pore conductance is measured by an optical synthesizer. The whole tobacco is sprayed with the test solutions of flavonoid compounds I-1, I-2 and I-3 with the concentration of 50 mu M in the pure DMSO of the control solution until the whole tobacco leaves are soaked completely without dripping, and the test solutions are respectively marked as a control sample, a test sample I-1, I-2 and I-3. Continuous leaf Wen CaiAnd (5) collecting and measuring air hole conductivity.

The experimental results are shown in fig. 2-5, wherein fig. 1 is a pore opening diagram of a control sample and a test sample; fig. 2 is She Wentu of control and test samples; fig. 3 is She Wentu of control and test samples; FIG. 4 is a graph of the leaf temperature variation of the control sample and the test sample, showing that the leaf temperature variation of the test sample is greater than that of the control sample; FIG. 5 is a graph of the air pore conductance of the control sample and the test sample, showing that the air pore conductance of the test sample after 2 hours of treatment is lower than that of the control sample; therefore, the whole plant temperature after the flavonoid compounds I-1, I-2 and I-3 are treated is higher, and the plant stomatal conductance after the flavonoid compounds I-1, I-2 and I-3 are combined to reduce, so that the reduction of the leaf stomatal opening is the reason for the reduction of the transpiration and the rise of the leaf temperature.

Example 4

The physiological changes of plants during the process of closing the stomata of plants induced by flavonoid compounds are typically studied by taking flavonoid compounds I-1, I-2 and I-3 as examples. The specific test method is as follows:

(1) Taking tobacco leaves, crushing, placing in a basic buffer solution, and adding 150 mu mol of ^-1 ·m ^-2 ·s ^-1 Incubating for 2h under the illumination condition;

(2) Adding 2',7' -dichlorofluorescein diacetate (2 ',7' -Dichlorodihydrofluorescein diacetate, H2DCF-DA, targetMol) with the concentration of 50 mu M as an active oxygen fluorescence probe into the mixture in the step (1), wherein the solvent is pure DMSO;

(3) Dividing the mixture obtained in the step (2) into four parts, respectively adding the four parts into pure DMSO of a control solution and flavonoid compounds I-1, I-2 and I-3 with 50 mu M concentration, and incubating for 40min, and respectively marking the four parts as a control sample, a test sample I-1, I-2 and I-3;

(4) Fluorescence change (excitation wavelength 480/30nm; absorption wavelength 510nm; dichroic mirror wavelength 505 nm) was observed using a confocal microscope (Nikon ECLIPSE Ti 2), and fluorescence intensity was measured using Imaje J software.

The experimental results are shown in figure 6, the fluorescence intensity of the plant guard cells after the flavonoid compounds I-1, I-2 and I-3 are obviously higher than that of DMSO, which shows that the reactive oxygen species ROS of the plant guard cells after the flavonoid compounds I-1, I-2 and I-3 are obviously increased, and the reactive oxygen species ROS of the guard cells participate in the process of mediating the closure of the pores induced by the flavonoid compounds.

Example 5

The effect of flavonoids on wheat water retention and drought resistance is studied, and flavonoid compounds I-1, I-2 and I-3 are typically taken as examples. The specific test method is as follows:

(1) Respectively selecting three groups of wheat leaves with basically consistent growth conditions, wherein one part of the wheat leaves in each group is used as a control sample, the other part of the wheat leaves is used as a test sample, the test samples in the three groups are respectively soaked in test solutions of flavonoid compounds I-1, I-2 and I-3 with the concentration of 100 mu M for 10min, and the test solutions are respectively marked as test samples I-1, I-2 and I-3;

(2) The control sample and the test samples I-1, I-2, I-3 were subjected to a temperature of 23℃and a relative humidity of 60% and an illumination intensity of 35. Mu. Mol-1. Mu.m ^-2 ·s ^-1 Standing under the condition of (2) and continuously photographing;

FIGS. 7 to 9 are graphs showing growth conditions of test samples I-1, I-2, I-3 and control samples, respectively, wherein the upper graph shows the initial state of each sample, and the lower graph shows the state of each sample after standing for 30 min. As can be seen from the graph, the wheat leaves in the control sample are wilted after illumination for 30min, which indicates that the serious loss of moisture is insufficient to support the leaves to stand upright, and the wheat leaves in the test sample are kept in an upright state, so that flavonoids compounds have the activity of promoting the water retention and drought resistance of the wheat at the concentration of 100 mu M.

Example 6

The effect of flavonoids on the fresh-keeping activity of rose leaves was studied, typically by taking flavonoids I-1, I-2 and I-3 as examples. The testing method comprises the following steps:

(1) Respectively selecting three groups of rose leaves with basically consistent growth conditions, wherein one part of the rose leaves in each group is used as a control sample, the other part of the rose leaves is used as a test sample, the test samples in the three groups are respectively soaked for 10 minutes by using flavonoid compounds I-1, I-2 and I-3 with the concentration of 100 mu M, and the rose leaves are respectively marked as test samples I-1, I-2 and I-3;

(2) The control sample and the test samples I-1, I-2, I-3 were subjected to a temperature of 23℃and a relative humidity of 60% and an illumination intensity of 35. Mu. Mol ^-1 ·m ^-2 ·s ^-1 Standing under the condition of (2) and continuously photographing;

fig. 10 to 12 are graphs showing growth conditions of a control sample and a test sample, respectively, wherein a graph a shows an initial state of the control sample and the test sample, and a graph B shows a state of each sample after standing for 12 hours. From the graph, after 12h of cultivation, the growth vigor and the freshness of the rose leaves under the test sample are obviously better than those of the control sample, and the flavonoid compound has the effect of promoting the fresh-keeping activity of the rose at the concentration of 100 mu M.

Example 7

The study of the inhibitory activity of flavonoids against downy mildew of grape is typically exemplified by flavonoids I-1, I-2, I-3. The specific test method is as follows:

(1) Respectively selecting three groups of grape leaves with basically consistent growth conditions, wherein one part of grape leaves in each group is used as a control sample, the other part of grape leaves is used as a test sample, all the three groups of grape leaves are picked up into a leaf disc with the thickness of 12mm, and the leaf disc is placed in a basic buffer solution, and the strength of the leaf disc is 130 mu mol ^-1 ·m ^-2 ·s ^-1 Floating for 2h under the light conditions of (2) wherein the composition of the base buffer is the same as in example 1;

(2) Placing grape leaves of a test sample into test solutions of flavonoid compounds I-1, I-2 and I-3 with concentration of 100 mu M respectively, and floating for 2 hours under the same conditions as those of the step (1), and recording the test samples I-1, I-2 and I-3;

(3) The test samples I-1, I-2, I-3 were added with the equal volume of the downy mildew sporangium suspension (2X 10) ⁵ /mL), continuing to float for 6h;

(4) The test specimens I-1, I-2, I-3 were placed in petri dishes with the back side up and transferred to an incubator with parameters of 19℃and 80% humidity and 3500Lx Light (Light: dark=12:12) for 6 days.

The results of the test are shown in FIGS. 13-15, wherein the left graph shows the number of the sporangia of the downy mildew, and the right graph shows the infection status of the downy mildew after the downy mildew is inoculated on the downy mildew for 6 days, and the flavonoid compounds I-1, I-2 and I-3 can be seen from the graph, and the aseptic colony of the downy mildew on the downy mildew is generated and not observed. It can be seen that flavonoids show inhibitory activity against grape downy mildew at a concentration of 100. Mu.M.

Example 8

The studies of the inhibitory activity of flavonoids on wheat leaf rust are typically exemplified by flavonoids I-1, I-2, I-3. The specific test method is as follows:

(1) Respectively selecting three groups of wheat with basically consistent growth conditions in one leaf and one heart period, wherein one part of wheat in each group is used as a control sample, the other part of wheat is used as a test sample, and the test sample is subjected to spraying treatment by using flavonoid compounds I-1, I-2 and I-3 with the concentration of 100 mu M;

(2) Inoculating wheat leaf rust bacteria after 2 hours, and culturing in darkness for 24 hours under the conditions that the temperature is 20 ℃ and the relative humidity is 80%;

(3) Culturing at 23deg.C under an environment with relative humidity of 80% and 5000Lx Light (Light: dark=12:12), and counting antibacterial effect at 14 d.

FIGS. 16-18 are graphs comparing growth conditions of a control sample and a test sample, respectively, and it can be seen from the graphs that rust spots appear on the surface of the leaf of the control sample in a large area, which indicates that the leaf rust of wheat is infected, and the leaf surface of the test sample has a few spots, and the growth conditions are obvious due to the control sample. It can be seen that flavonoids show inhibitory activity against wheat leaf rust at a concentration of 100. Mu.M.

While the application has been described in terms of preferred embodiments, it will be understood by those skilled in the art that various changes and modifications can be made without departing from the scope of the application, and it is intended that the application is not limited to the specific embodiments disclosed.

Claims

1. The application of flavonoid and derivatives thereof is characterized in that the flavonoid and derivatives thereof are used as plant regulators to induce plant stomata to close.

2. The use of flavonoids and derivatives thereof according to claim 1, wherein the general formula of the flavonoids and derivatives thereof is selected from any one of the general formulae (I) to (IV);

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ 、R ₉ 、R ₁₀ Each independently selected from any one of-H, -OH, galactose, oligosaccharide, glucuronic acid, nitro, amino, silicon, halogen, C2-C6 alkenyl, C1-C5 alkoxy, hydroxyalkoxy, acyloxy, substituted or unsubstituted pyrrole groups.

3. The use of the flavonoid and the derivative thereof according to claim 1, characterized in that the flavonoid and the derivative thereof are used as plant disease control regulators.

4. The use of the flavonoid and the derivative thereof according to claim 3, wherein the plant disease is a plant disease caused by pathogenic bacteria using stomata as an invasion path.

5. The use of flavonoids and derivatives thereof according to claim 1, wherein the use of flavonoids and derivatives thereof as drought-resistant water-retention regulators for plants.

6. The use of flavonoids and derivatives thereof according to claim 1, wherein the use of flavonoids and derivatives thereof as plant fresh-keeping agents.

7. The use of flavonoids and derivatives thereof according to claim 1, wherein the plants are selected from any one of angiosperms, gymnosperms, ferns.