Do guard cells have single or multiple defense mechanisms in response to flg22?

Abstract

Bacterial flagellin (flg22) induces rapid and permanent stomatal closure. However, its local and systemic as well as tissue- and cell-specific effects are less understood. Our results show that flg22 induced local and systemic stomatal closure in intact tomato plants, which was regulated by reactive oxygen- and nitrogen species, and also affected the photosynthetic activity of guard cells but not of mesophyll cells. Interestingly, rapid and extensive local expression of Ethylene response factor 1 was observed after exposure to flg22, whereas the relative transcript levels of Defensin increased only after six hours, especially in systemic leaves. Following local and systemic ethylene emission already after one and six hours, jasmonic acid levels increased in the local leaves after six hours of flg22 treatment. Using immunohistochemical methods, significant defensin accumulation was found in the epidermis and stomata of flg22-treated leaves and above them. Immunogold labelling revealed significant levels of defensins in the cell wall of the mesophyll parenchyma and guard cells. Furthermore, single cell qRT-PCR confirmed that guard cells are able to synthesise defensins. It can be concluded that guard cells are not only involved in the first line of plant defense by regulating stomatal pore size, but can also defend themselves and the plant by producing and accumulating antimicrobial defensins where phytopathogens can penetrate.

FIGURE 1

Time‐dependent regulation of stomatal activity. Changes in the stomatal conductance (A) and the size of stomatal apertures (B) were examined in the abaxial epidermal strips of intact tomato plants treated foliar with 5 μM flagellin (flg22) at 8:00 a.m. Measurements were carried out one and six hours after treatments (at 9:00 a.m. and 14:00 p.m.). Means ± SE, n = 3. Means were analyzed by two‐way ANOVA, and significant differences among the data were analyzed by Duncan's test. Mean values significantly different at p < 0.05 were signed with different letters, upper case letters indicate the effects of the treatment at the same daytime, and lower case letters indicate the effects of the daytime under the same treatment. (Control: treatment with sterile distilled water; Control+1: untreated leaves from the distal node from the control; flg22: treatment with 5 μM flagellin dissolved in sterile distilled water; flg22 + 1: untreated leaves from the distal node from the flg22‐treated one).

FIGURE 2

Time‐dependent regulation of the photosynthetic activity of stomata and leaves of intact tomato plants. Changes in the maximum quantum yield of PSII (F_v/F_m), the effective quantum yield of PSII photochemistry (ФPSII), the photochemical quenching coefficient (qP) and the non‐photochemical quenching (NPQ) of stomata and leaves were examined in intact tomato plants treated foliar with 5 μM flagellin (flg22) at 8:00 a.m. Measurements were carried out one and six hours later after treatments (at 9:00 a.m. and 14:00 p.m.). Means ± SE, n = 3. Means were analyzed by two‐way ANOVA, and significant differences among the data were analyzed by Duncan's test. Mean values significantly different at p < 0.05 were signed with different letters, upper case letters indicate the effects of the treatment at the same daytime, and lower case letters indicate the effects of the daytime under the same treatment. (Control: treatment with sterile distilled water; Control+1: untreated leaves from the distal node from the control; flg22: treatment with 5 μM flagellin dissolved in sterile distilled water; flg22 + 1: untreated leaves from the distal node from the flg22‐treated one).

FIGURE 3

Time‐dependent regulation of the metabolism of reactive oxygen species in the stomata. Changes in the superoxide (A), hydrogen peroxide (B) and nitric oxide (C) production were examined in the abaxial epidermal strips of intact tomato plants treated foliar with 5 μM flagellin (flg22) at 8:00 a.m. Measurements were carried out one and six hours later after treatments (at 9:00 a.m. and 14:00 p.m.). Means ± SE, n = 3. Means were analysed by two‐way ANOVA, significant differences among the data were analysed by Duncan's test. Mean values significantly different at p < 0.05 were signed with different letters, upper case letters indicate the effects of the treatment at the same daytime, and lower case letters indicate the effects of the daytime under the same treatment. (Control: treatment with sterile distilled water; Control+1: untreated leaves from the distal node from the control; flg22: treatment with 5 μM flagellin dissolved in sterile distilled water; flg22 + 1: untreated leaves from the distal node from the flg22‐treated one).

FIGURE 4

Time‐dependent regulation of the metabolism of reactive oxygen species in leaves of intact tomato plants. Changes in the superoxide production (A), hydrogen peroxide content (B), and nitric oxide production (C) were examined in leaves of intact tomato plants treated foliar with 5 μM flagellin (flg22) at 8:00 a.m. Measurements were carried out one and six hours later after treatments (at 9:00 a.m. and 14:00 p.m.). Means ± SE, n = 3. Mean values significantly different at p < 0.05 were signed with different letters, upper case letters indicate the effects of the treatment at the same daytime, and lower case letters indicate the effects of the daytime under the same treatment. (Control: treatment with sterile distilled water; Control+1: untreated leaves from the distal node from the control; flg22: treatment with 5 μM flagellin dissolved in sterile distilled water; flg22 + 1: untreated leaves from the distal node from the flg22‐treated one).

FIGURE 5

Time‐dependent regulation of the metabolism of defense‐related hormones. Changes in the ethylene (A) and jasmonic acid (B) contents were examined in leaves of intact tomato plants treated foliar with 5 μM flagellin (flg22) at 8:00 a.m. Measurements were carried out one and six hours later after treatments (at 9:00 a.m. and 14:00 p.m.). Means ± SE, n = 3. Mean values significantly different at p < 0.05 were signed with different letters, upper case letters indicate the effects of the treatment at the same daytime, and lower case letters indicate the effects of the daytime under the same treatment. (Control: treatment with sterile distilled water; Control+1: untreated leaves from the distal node from the control; flg22: treatment with 5 μM flagellin dissolved in sterile distilled water; flg22 + 1: untreated leaves from the distal node from the flg22‐treated one).

FIGURE 6

Time‐dependent regulation of defense‐related gene expression. Changes in the expression of SlERF1 (A) and SlDEF (B) were examined in leaves of intact tomato plants treated foliar with 5 μM flagellin (flg22) at 8:00 a.m. Measurements were carried out one and six hours later after treatments (at 9:00 a.m. and 14:00 p.m.). Means ± SE, n = 3. Means were analysed by two‐way ANOVA, significant differences among the data were analysed by Duncan's test. Mean values significantly different at p < 0.05 were signed with different letters, upper case letters indicate the effects of the treatment at the same daytime, and lower case letters indicate the effects of the daytime under the same treatment. (Control: treatment with sterile distilled water; Control+1: untreated leaves from the distal node from the control; flg22: treatment with 5 μM flagellin dissolved in sterile distilled water; flg22 + 1: untreated leaves from the distal node from the flg22‐treated one).

FIGURE 7

Changes in the defensin (DEF) protein levels. Protein accumulation was examined in the epidermal cells (A) and stomata (B) from the leaves of intact tomato plants treated foliar with 5 μM flagellin (flg22) at 8:00 a.m. (C: representative images). Measurements were carried out six hours later after treatments (at 14:00 p.m.). Means ± SE, n = 3. Means were analysed by one‐way ANOVA, significant differences among the data were analysed by Duncan's test. Mean values significantly different at p < 0.05 were signed with different letters (Control: treatment with sterile distilled water; Control+1: untreated leaves from the distal node from the control; flg22: treatment with 5 μM flagellin dissolved in sterile distilled water; flg22 + 1: untreated leaves from the distal node from the flg22‐treated one).

FIGURE 8

Changes in the defensin (DEF) protein accumulation. DEF protein levels were examined in the cell walls of mesophyll cells (A), epidermal cells (B) and stomata (C) from the leaves of intact tomato plants treated foliar with 5 μM flagellin (flg22) at 8:00 a.m. using immunogold labelling (D: representative images from stomata cell wall). Measurements were carried out six hours later after treatments (at 14:00 p.m.). Means ± SE, n = 3. Means were analysed by one‐way ANOVA, significant differences among the data were analysed by Duncan's test. Mean values significantly different at p < 0.05 were signed with different letters (Control: treatment with sterile distilled water; Control+1: untreated leaves from the distal node from the control; flg22: treatment with 5 μM flagellin dissolved in sterile distilled water; flg22 + 1: untreated leaves from the distal node from the flg22‐treated one).

FIGURE 9

Changes in the expression of tomato defensin (SlDEF) in the stomata. Gene expression was examined in the leaves of intact tomato plants treated foliar with 5 μM flagellin (flg22) at 8:00 a.m. using single cell qRT‐PCR. Measurements were carried out six hours later after treatments (at 14:00 p.m.). Means ± SE, n = 3. Means were analysed by one‐way ANOVA, significant differences among the data were analysed by Duncan's test. Mean values significantly different at p < 0.05 were signed with different letters (Control: treatment with sterile distilled water; Control+1: untreated leaves from the distal node from the control; flg22: treatment with 5 μM flagellin dissolved in sterile distilled water; flg22 + 1: untreated leaves from the distal node from the flg22‐treated one).

FIGURE 10

Schematic model of the induction of defensin (DEF) accumulation during flg22‐induced local and systemic defence responses in leaves and stomata of tomato plants. Flg22 induces rapid local reactive oxygen species (ROS) and nitric oxide (NO) accumulation and ethylene (ET) emission, which contribute to the activation of local leaf defence responses, including stomatal closure. ROS and NO accumulation and reduced photosynthetic activity (ФPSII) in guard cells contribute to this process. Local ROS/NO production and ET emission after flg22 treatment trigger not only local but also systemic defence responses, inducing jasmonic acid (JA) accumulation and stomatal closure. As part of the local and systemic defence responses of plants, the antimicrobial DEFs are accumulated in the cell wall of the mesophyll, epidermis and stomata.