Transcriptome profiling of Arabidopsis slac1-3 mutant reveals compensatory alterations in gene expression underlying defective stomatal closure

Abstract

Plants adjust their stomatal aperture for regulating CO₂ uptake and transpiration. S-type anion channel SLAC1 (slow anion channel-associated 1) is required for stomatal closure in response to various stimuli such as abscisic acid, CO₂, and light/dark transitions etc. Arabidopsis slac1 mutants exhibited defects in stimulus-induced stomatal closure, reduced sensitivity to darkness, and faster water loss from detached leaves. The global transcriptomic response of a plant with defective stimuli-induced stomatal closure (particularly because of defects in SLAC1) remains to be explored. In the current research we attempted to address the same biological question by comparing the global transcriptomic changes in Arabidopsis slac1-3 mutant and wild-type (WT) under dark, and dehydration stress, using RNA-sequencing. Abscisic acid (ABA)- and dark-induced stomatal closure was defective in Arabidopsis slac1-3 mutants, consequently the mutants had cooler leaf temperature than WT. Next, we determined the transcriptomic response of the slac1-3 mutant and WT under dark and dehydration stress. Under dehydration stress, the molecular response of slac1-3 mutant was clearly distinct from WT; the number of differentially expressed genes (DEGs) was significantly higher in mutant than WT. Dehydration induced DEGs in mutant were related to hormone signaling pathways, and biotic and abiotic stress response. Although, overall number of DEGs in both genotypes was not different under dark, however, the expression pattern was very much distinct; whereas majority of DEGs in WT were found to be downregulated, in slac1-3 majority were upregulated under dark. Further, a set 262 DEGs was identified with opposite expression pattern between WT and mutant under light-darkness transition. Amongst these, DEGs belonging to stress hormone pathways, and biotic and abiotic stress response were over-represented. To sum up, we have reported gene expression reprogramming underlying slac1-3 mutation and resultantly defective stomatal closure in Arabidopsis. Moreover, the induction of biotic and abiotic response in mutant under dehydration and darkness could be suggestive of the role of stomata as a switch in triggering these responses. To summarize, the data presented here provides useful insights into the gene expression reprogramming underlying slac1-3 mutation and resultant defects in stomatal closure.

Keywords: abscisic acid; anion channel; drought stress; stomata; transcriptome.

Figure 1

Arabidopsis slac1-3 mutants are insensitive to abscisic acid (ABA) and darkness. (A) Graphical presentation of stomatal aperture under ABA, dark treatment, or light. (B) Representative images of stomatal aperture in WT and slac1-3 mutants under ABA, dark, or light. Three replicates were included for each treatment, and at least 30 stomata were measured in each group. Bars represent mean ± S.E. *** Denotes statistically significant difference (t-test p < 0.01).

Figure 2

Determination of plant leaf temperature in Arabidopsis WT and slac1-3 mutants in light and dark conditions. Multiple images of both genotypes under light/dark conditions were captured with thermal Imager. The representative images are shown here.

Figure 3

Differentially expressed gene (DEG) integration of each data set. DEGs of each data set are overlapped and presented as a Venn plot, including total, upregulated, and downregulated genes. DEGs, differently expressed genes. (A) Venn diagram of gene expression and gene number of up- or downregulated in dehydration stress; (B) up- or downregulated in dark conditions; and (C) up- or downregulated between WT and slac1-3 mutant.

Figure 4

RNA-seq analysis of dehydration stress induced upregulated genes in slac1-3 mutant. (A) Heat map of DEGs (891 in Figure 3A) in slac1-3 mutants between control and dehydration stress (B) GO enrichment analysis of DEGs in slac1-3 mutants in control and dehydration stress.

Figure 5

RNA-seq analysis of DEGs (314 in Figure 3B) at daytime or night. (A) Heat map of DEGs in slac1-3 mutants in dark and light conditions (B) GO enrichment analysis of DEGs in slac1-3 mutants in dark and light conditions.

Figure 6

RNA-seq analysis of DEGs (262 in Figure 3C) between day and night. (A) Heat map of DEGs from day to night between slac1-3 mutant and WT. (B) GO enrichment analysis of DEGs from day to night between slac1-3 mutant and WT.

Figure 7

A model representing transcriptomic changes undertaken by Arabidopsis slac1-3 mutant in response to dehydration stress and dark treatment. Under these stimuli, slac1-3 mutant shows defective stomatal closure. Resultantly, plants lose more water and exhibit cooler leaf temperature. Mutation in Arabidopsis SLAC1 is also linked with higher resting calcium level in mutant. These factors could be among the possible links between slac1-3 mutation and differential transcriptomic response in mutant. To compensate for defective stomatal closure and its consequences, mutant undergo biotic and abiotic stress responses. The former involves induction of bacterial and fungal infection pathways, and systemic acquired resistance while the latter involves dehydration, salt, wounding, and cold stress responses, and induction of stress hormone pathways. However, it should be noted that few of these hormones may also be simultaneously associated with biotic stress response.