Identification and characterization of a core set of ROS wave-associated transcripts involved in the systemic acquired acclimation response of Arabidopsis to excess light

Abstract

Systemic acquired acclimation (SAA) plays a key role in optimizing growth and preventing damage associated with fluctuating or abrupt changes in the plant environment. To be effective, SAA has to occur at a rapid rate and depend on rapid signaling pathways that transmit signals from affected tissues to all parts of the plant. Although recent studies have identified several different rapid systemic signaling pathways that could mediate SAA, very little information is known about the extent of their involvement in mediating transcriptomic responses. Here we reveal that the systemic transcriptomic response of plants to excess light stress is extensive in its context and involves an early (2 min) and transient stage of transcript expression that includes thousands of genes. This early response is dependent on the respiratory burst oxidase homolog D protein, and the function of the reactive oxygen species (ROS) wave. We further identify a core set of transcripts associated with the ROS wave and suggest that some of these transcripts are involved in linking ROS with calcium signaling. Priming of a systemic leaf to become acclimated to a particular stress during SAA involves thousands of transcripts that display a rapid and transient expression pattern driven by the ROS wave.

Keywords: Arabidopsis thaliana; WRKY; H2O2 signaling; MYB30; light stress; reactive oxygen species (ROS) wave; systemic acquired acclimation (SAA); systemic signaling; transcriptomics.

Figure 1

Transcriptomic responses of local (L) and systemic (S) leaves of Arabidopsis plants to local application of excess light stress. (a) The experimental design used (top) and a Venn diagram showing the overlap between local and systemic responses to light stress (bottom). (b) Distinct clusters of transcript expression in local and systemic leaves in response to local application of light stress. (c) Venn diagrams showing the overlap between the different groups of clusters in local and systemic leaves. All Venn diagrams had a hypergeometric testing significance of P < 0.001. L, local; S, systemic.

Figure 2

Gene ontology classification and expression pattern of transcripts that accumulate in both local and systemic leaves of Arabidopsis in response to local application of light stress. (a) Gene Ontology annotation of transcripts that accumulate in local and systemic leaves of Arabidopsis in response to light stress. See Table S4 for full description. The P‐value for enrichment compared with the genome distribution from Fisher's Exact with FDR multiple test correction is provided for each GO term. (b) Expression pattern of selected gene ontology groups in local and systemic leaves. See Figure S4 for additional classification groups. ABA, abscisic acid; SAR, systemic acquired resistance; SA, salicylic acid; JA, jasmonic acid; ROS, reactive oxygen species.

Figure 3

Expression pattern of selected transcription factor families that accumulate in both local and systemic leaves of Arabidopsis in response to local application of light stress. HSF, heat shock factor; DREB, dehydration responsive element binding; AP‐2, activating protein‐2; ERF, ethylene response factor, GATA, (T/A)GATA(A/G)‐binding.

Figure 4

Transcriptomic responses of local (L) and systemic (S) leaves of rbohD plants to local application of excess light stress. (a) The experimental design used (top) and a Venn diagram showing the overlap between local and systemic responses to light stress (bottom). (b) Distinct clusters of transcript expression in local and systemic leaves of rbohD plants in response to local application of light stress. (c) Venn diagrams showing the overlap between the different groups of clusters in local and systemic leaves. All Venn diagrams had a hypergeometric testing significance of P < 0.001. L, local; S, systemic; RBOHD, respiratory burst oxidase homolog D.

Figure 5

Gene ontology classification and expression pattern of systemic rbohD‐dependent transcripts in wild type plants. (a) Gene Ontology annotation of systemic rbohD‐dependent transcripts that accumulate in wild type plants in response to local application of light stress. See Table S9 for full description. The P‐value for enrichment compared with the genome distribution from Fisher's Exact with FDR multiple test correction is provided for each GO term. (b) Distinct clusters of expression of rbohD‐dependent transcripts in systemic leaves of wild type plants in response to local application of light stress. Venn diagram in (a) had a hypergeometric testing significance of P < 0.001. ABA, abscisic acid; RBOHD, respiratory burst oxidase homolog D.

Figure 6

Identification of rbohD‐dependent systemic transcripts significantly enhanced in their expression in response to H₂O₂. (a) Experimental design of the H₂O₂ treatment experiment (top) and distinct clusters of expression of H₂O₂–response transcripts. (b) Venn diagrams showing the overlap between transcripts enhanced in their expression in local leaves of wild type plants and seedlings treated with 1 mm H₂O₂ (top); Venn diagrams showing the overlap between transcripts enhanced in their expression in systemic leaves of wild type plants and seedlings treated with 1 mm H₂O₂ (middle); and Venn diagrams showing the overlap between transcripts significantly accumulated in systemic leaves of wild type plants, systemic leaves of rbohD plants and seedlings treated with 1 mm H₂O₂ (bottom). (c) Heat map showing the response of the 82 rbohD‐dependent and H₂O₂‐response systemic transcripts to different stress conditions and signals. All Venn diagrams had a hypergeometric testing significance of P < 0.001. L, local; S, systemic; eATP, external ATP.

Figure 7

Functional analysis of selected rbohD‐dependent systemic transcripts in Arabidopsis. Light stress‐induced cell injury in two independent knockout mutants for seven different genes encoding rbohD‐dependent systemic transcripts. Cell injury was measured following application of light stress to a local leaf (local), and following pretreatment of local leaves and the application of light stress to systemic leaves (SAA). Two independent alleles for each gene were subjected to light stress and cell injury was measured by electrolyte leakage. *P < 0.05. AT1G69890, Actin cross‐linking protein; AT3G13600, Calmodulin‐binding family protein; AT3G54810, GATA8 type zinc finger protein; AT1G56520 and AT5G46270, Disease resistance TIR‐NBS‐LRR family protein; AT5G49520, WRKY48 transcription factor; AT1G29670, GDSL‐motif esterase/acyltransferase/lipase. SAA, systemic acquired acclimation.

Figure 8

Suppression of light‐induced systemic transcripts by diphenyleneiodonium (DPI). (a) Venn diagram showing the overlap between local and systemic responses to light stress in wild type and rbohD plants. (b) Venn diagrams and images showing the position of water or DPI application (in agar) and the overlap between local and systemic responses to light stress in water or DPI‐treated plants. (c) Venn diagrams showing the overlap between the DPI‐suppressed systemic transcripts (2811) and rbohD‐dependent systemic transcripts (3447; Top), the DPI‐suppressed systemic transcripts (2811) and the 82 rbohD‐dependent and H₂O₂‐response systemic transcripts (middle), and the 82 rbohD‐dependent systemic transcripts that are also H₂O₂‐response transcripts, HL systemic‐response transcripts (3914) and HL systemic‐response transcripts that are not inhibited by DPI (1228; Bottom). Venn diagrams in (b) and (c) had a hypergeometric testing significance of P < 0.001. DPI, diphenyleneiodonium; HL, high lights; L, local; S, systemic.

Figure 9

Short list of ROS wave‐associated transcripts, expression pattern of rbohD‐dependent systemic transcripts with WRKY, MYB and GATA binding elements in their promoters and a model for the putative function of MYB30. (a) A short list of ROS‐wave‐associated transcripts each confirmed by two independent methods. All transcripts are rbohD‐dependent systemic transcripts that are either required for systemic plant acclimation to light stress (top), or suppressed in their expression by DPI (bottom). (b) Distinct clusters of expression of systemic transcripts with WRKY, MYB and GATA binding elements in their promoters. (c) A putative model for the function of MYB30 in mediating or amplifying the ROS wave. See text for more details. CPKs, calcium‐dependent kinases; cytCa²⁺, cytosolic Ca²⁺; BROHD, respiratory burst oxidase homolog D; ROS, reactive oxygen species.