Systemic signaling during abiotic stress combination in plants

Abstract

Extreme environmental conditions, such as heat, salinity, and decreased water availability, can have a devastating impact on plant growth and productivity, potentially resulting in the collapse of entire ecosystems. Stress-induced systemic signaling and systemic acquired acclimation play canonical roles in plant survival during episodes of environmental stress. Recent studies revealed that in response to a single abiotic stress, applied to a single leaf, plants mount a comprehensive stress-specific systemic response that includes the accumulation of many different stress-specific transcripts and metabolites, as well as a coordinated stress-specific whole-plant stomatal response. However, in nature plants are routinely subjected to a combination of two or more different abiotic stresses, each potentially triggering its own stress-specific systemic response, highlighting a new fundamental question in plant biology: are plants capable of integrating two different systemic signals simultaneously generated during conditions of stress combination? Here we show that plants can integrate two different systemic signals simultaneously generated during stress combination, and that the manner in which plants sense the different stresses that trigger these signals (i.e., at the same or different parts of the plant) makes a significant difference in how fast and efficient they induce systemic reactive oxygen species (ROS) signals; transcriptomic, hormonal, and stomatal responses; as well as plant acclimation. Our results shed light on how plants acclimate to their environment and survive a combination of different abiotic stresses. In addition, they highlight a key role for systemic ROS signals in coordinating the response of different leaves to stress.

Keywords: abiotic stress; reactive oxygen species; stress combination; systemic acquired acclimation; systemic signaling.

Fig. 1.

Plants can integrate two different systemic signals generated simultaneously at the same leaf during stress combination. (A) Overlap between the transcriptomic response of plants to a local treatment of HL, HS, or a combination of heat and light stresses applied to the same leaf (HL+HS). Venn diagrams for the overlap between the different responses are shown at the bottom for local leaves and at the top for systemic leaves. Black arrows in HL+HS plants represent the number of HL-specific (solid) or HS-specific (dashed) transcripts common between local and systemic leaves. (B) Venn diagram showing the overlap between stress combination-specific transcripts in local and systemic leaves. (C) Bar graph showing the percent overlap between the systemic HL+HS response and local HL, HS, or HL+HS responses. Local leaves were subjected to HL, HS, or a combination of HL+HS, and local and systemic leaves were sampled at 2 and 8 min following stress application. All experiments were repeated at least three times with 40 plants per biological repeat. All transcripts shown were significantly different from controls at P < 0.05 (negative binomial Wald test followed by Benjamini–Hochberg correction).

Fig. 2.

Plants can integrate two different systemic signals generated simultaneously at two different leaves of the same plant during stress combination. (A) Overlap between the systemic transcriptomic response of plants to a local application of HL, HS, or a combination of heat and light stress simultaneously applied to two different leaves (HL&HS). Venn diagrams for the overlap between the different responses are shown at the bottom for local leaves and at the top for systemic leaves. Black arrows in HL&HS plants represent the number of HL-specific (solid), or HS-specific (dashed) transcripts common between local and systemic leaves. (B) Venn diagrams showing the overlap between stress combination-specific transcripts from local (HL or HS) and systemic leaves. (C) Bar graph showing the percent overlap between the systemic HL&HS response and local HL, HS, HL(HL&HS), or HS(HL&HS) responses. All experiments were repeated at least three times with 40 plants per biological repeat. All transcripts included in the figure were significantly different from controls at P < 0.05 (negative binomial Wald test followed by Benjamini–Hochberg correction). HL(HL&HS) and HS(HL&HS) denote a local HL- or HS- treated leaf of a plant subjected to HS or HL on another local leaf, respectively.

Fig. 3.

The systemic response of plants to two different stresses simultaneously applied to two different local leaves is more extensive than the response to two different stresses simultaneously applied to the same local leaf. (A) Overlap between the systemic transcriptomic responses of plants to a combination of heat and light stress applied to the same (HL+HS) or different (HL&HS) local leaves. Venn diagrams for the overlap between the different responses are shown at the bottom for local leaves and at the top for systemic leaves. Black arrows represent the number of HL-specific (solid) or HS-specific (dashed) transcripts common between local and systemic leaves. (B) Heat maps showing the expression pattern of TFs belonging to the ethylene response (AP2-EREBPs), MYB, and heat shock factor (HSF) families (24) in systemic tissues of plants subjected to a local treatment of HL, HS, HL+HS, or HL&HS. All transcriptomics experiments were repeated at least three times with 40 plants per biological repeat (P < 0.05, negative binomial Wald test followed by Benjamini–Hochberg correction).

Fig. 4.

Overlap between the local transcriptomic responses of two different leaves of the same plant, one subjected to HL and the other to HS simultaneously. (A) Venn diagrams for the overlap between the different local responses of plants subjected to HL, HS, or HL&HS. Yellow arrows represent the number of HL-specific (solid) or HS-specific (dashed) transcripts common between the two different local leaves. (B) Heat maps showing the expression pattern of TFs belonging to the AP2-EREBP, MYB, and HSF families (24) in local tissues of plants subjected to HL, HS, HL+HS, or HL&HS. All transcriptomics experiments were repeated at least three times with 40 plants per biological repeat (P < 0.05, negative binomial Wald test followed by Benjamini–Hochberg correction).

Fig. 5.

Acclimation of systemic leaves and stomatal aperture responses to stress combination. (A, Top) Schematic representation of how experiments were conducted. Local leaves were subjected to a short (15 min) pretreatment of HL, HS, HL+HS, or HL&HS, and plants were allowed to acclimate for 45 min. Following acclimation, systemic leaves were challenged with damaging levels of HL or HS, sampled, photographed and subjected to tissue injury assays (ion leakage for HL and chlorophyll content for HS). (A, Middle) Representative systemic leaf images. (A, Bottom) Measurements of systemic leaf injury: increase in ion leakage for HL (Left) and decrease in chlorophyll content for HS (Right). All acclimation experiments were repeated at least three times with 10 plants per biological repeat. Data are mean ± SD. *P < 0.05, two-way ANOVA followed by Tukey’s post hoc test. CT, control; HL or HS, control plants subjected to a systemic HL or HS stress treatment without pretreatment, respectively; Pretreated, plants in which a local leaf was subjected to HL, HS, HL+HS, or HL&HS treatment before the systemic HL or HS treatment; EL, electrolyte leakage. (B) Stomatal aperture in local and systemic leaves of plants subjected to a local HL, HS, HL+HS, or HL&HS treatment. All experiments were repeated at least three times with 500 stomata per plant and 10 plants per biological repeat. Data are presented as mean ± SD. Different letters denote significance at P < 0.05 (ANOVA followed by a Tukey’s post hoc test).

Fig. 6.

Systemic accumulation of ROS in mature bolting plants simultaneously subjected to two different stresses. (A) Heat maps showing the expression of transcripts associated with systemic ROS accumulation (6) in local leaves of plants subjected to light or heat stress while the other leaf is simultaneously subjected to the other stress [HL(HL&HS) or HS(HL&HS), respectively] or HL and HS applied to the same leaf (HL+HS), and in systemic leaves of plants subjected to a local treatment of HL and HS applied to the same (HL+HS) or two different (HL&HS) leaves (SI Appendix, Fig. S2). Transcripts included in A and in SI Appendix, Fig. S2 are significantly different from controls (P < 0.05; negative binomial Wald test followed by Benjamini–Hochberg correction). (B) Representative images (Left), line graphs showing continuous in vivo ROS measurements (Top Right), and bar graphs showing measurements of ROS at 15 min after stress application (Bottom Right), at the middle of the inflorescence stem (corresponding to white arrows in images on the left). All experiments were repeated at least three times with five plants per biological repeat. Data are presented as mean ± SD, **P < 0.01, two-way ANOVA followed by Tukey’s post hoc test. CT, control; n.s., no significant differences with respect to control. (Scale bars, 1 cm.)

Fig. 7.

Quantitative analysis of JA and SA levels in wild-type plants and systemic accumulation of ROS in bolting aos mutants during stress combination. (A) Quantitative analysis of JA and SA in local and systemic wild-type leaves at 0, 2, and 8 min following the application of HL, HS, HL+HS, and HL&HS to local leaves. Hormone analysis was conducted using the same biological material used from transcriptomics analysis. Data are presented as mean ± SD. *P < 0.05, two-way ANOVA followed by Tukey’s post hoc test. (B) Representative images (Left), line graphs showing continuous in vivo ROS measurements (Top Right), and bar graphs showing measurements of ROS at 15 min after stress application (Bottom Right) at the middle of the inflorescence stem (corresponding to white arrows in images on Left) of aos mutant plants subjected to stress combinations. All experiments were repeated at least three times with five plants per biological repeat. Data are presented as mean ± SD. *P < 0.05, two-way ANOVA followed by Tukey’s post hoc test. CT, control; n.s., no significant differences with respect to control. (Scale bars, 1 cm.)

Fig. 8.

Systemic ROS accumulation and plant acclimation of nonbolting wild-type plants during stress combination. (A) Representative images (Left), line graphs showing continuous in vivo ROS measurements at different local and systemic leaves (Top Right; leaves monitored are indicated with letters on the left images), and bar graphs showing measurements of ROS at the indicated leaves at 10 min after stress application (Bottom Right). All experiments were repeated at least three times with five plants per biological repeat. Data are presented as mean ± SD. *P < 0.05, two-way ANOVA analysis followed by Tukey’s post hoc test. (B) Representative systemic leaf images (Top) and measurements of systemic leaf injury (increase in ion leakage for HL [Left] and decrease in chlorophyll content for HS [Right]) (Bottom) of nonbolting plants subjected to stress combination. All acclimation experiments were repeated at least three times with 10 plants per biological repeat. Data are presented as mean ± SD. *P < 0.05, two-way ANOVA followed by Tukey’s post hoc test. CT, control; HL or HS, control plants subjected to a systemic HL or HS stress treatments without pretreatment, respectively; Pretreated, plants in which a local leaf was subjected to a HL, HS, HL+HS or HL&HS treatment before the systemic HL or HS treatment; EL, electrolyte leakage; n.s., no significant differences with respect to control; S, systemic tissue. (Scale bars, 1 cm.)

Fig. 9.

Model showing that the manner in which plants sense the different stresses that trigger systemic signals during stress combination (i.e., at the same or different leaves) makes a significant difference in how fast and efficient systemic ROS signals and transcriptomic, hormonal, stomatal, and acclimation responses are triggered. When two different stresses are simultaneously applied to the same leaf (HL+HS), the systemic response is suppressed. This is reflected in the expression of systemic HL- or HS-response and photosynthetic-associated transcripts (boxed heat maps; SI Appendix, Fig. S3 and Table 1), rates of SA and JA accumulation (Fig. 7A), accumulation of systemic ROS (dashed orange arrow; Figs. 6 and 8A), and the lack of SAA of systemic leaves (Figs. 5A and 8B). In contrast, when the two different stresses are applied to two different leaves of the same plant, the rate of systemic ROS accumulation is faster (dashed black and red arrows; Figs. 6 and 8A); stomata display a rapid response (Fig. 5B); HL-, HS-, and photosynthetic-associated transcripts accumulate in systemic leaves (boxed heat maps; SI Appendix, Fig. S3 and Table 1); and SAA is induced (Figs. 5A and 8B). Plants are depicted as being able to integrate different systemic signals: however, the manner in which plants sense the different stresses that trigger these signals makes a significant difference in how fast and efficient they are able to acclimate.