Different strategies of strigolactone and karrikin signals in regulating the resistance of Arabidopsis thaliana to water-deficit stress

ABSTRACT

Strigolactone and karrikin receptors, DWARF14 (D14) and KARRIKIN INSENSITIVE 2 (KAI2), respectively, have been shown to positively regulate drought resistance in Arabidopsis thaliana by modulating abscisic acid responsiveness, anthocyanin accumulation, stomatal closure, cell membrane integrity and cuticle formation. Here, we aim to identify genes specifically or commonly regulated by D14 and KAI2 under water scarcity, using comparative analysis of the transcriptome data of the A. thaliana d14-1 and kai2-2 mutants under dehydration conditions. In comparison with wild-type, under dehydration conditions, the expression levels of genes related to photosynthesis and the metabolism of glucosinolates and trehalose were significantly changed in both d14-1 and kai2-2 mutant plants, whereas the transcript levels of genes related to the metabolism of cytokinins and brassinosteroids were significantly altered in the d14-1 mutant plants only. These results suggest that cytokinin and brassinosteroid metabolism might be specifically regulated by the D14 pathway, whereas photosynthesis and metabolism of glucosinolates and trehalose are potentially regulated by both D14 and KAI2 pathways in plant response to water scarcity.

With increasing global warming, drought is becoming one of the most detrimental environmental stresses, which limits the productivity of many crops.^1,2 To avoid and tolerate the water-deficit conditions, plants have evolved a series of strategies, such as the closure of stomata and cuticle thickening to reduce water loss from the leaf surface, biosyntheses of secondary metabolites [e.g. anthocyanins, glucosinolates (GSLs) and trehalose (Tre)] to increase acclimatization, and tradeoff with growth and developmental rates.^3,4 Phytohormones like cytokinins (CKs), brassinosteroids (BRs) and abscisic acid (ABA) play important roles in the regulation of such specific responses, enabling plants to adjust to such adverse conditions.^5–8 Recently, a relatively new class of phytohormone, strigolactones (SLs), and their structural analogs karrikins (KARs) found in smoke water, have also been reported to be involved in the regulation of plant growth and response to drought.^9–13

SLs were first identified to be required for parasite seed germination and involved in inhibition of shoot branching, while KARs are reported to play roles in seed germination and many other aspects of plant development.^14–17 Studies on the positive roles of SLs in plant drought responses initiated with analyzing the drought-responsive phenotypes of the Arabidopsis thaliana (Arabidopsis) SL-signaling more axillary growth (max2) mutant, and SL-deficient max3 and max4 mutant plants.^13,18 Later on, further studies using Arabidopsis mutants defective in SL-specific genes like SL-deficient max1 and SL-receptor dwarf14 (d14), as well as Lotus japonicus and tomato (Solanum lycopersicum) SL-deficient transgenic plants silenced for a gene homologous to MAX3 (e.g. the CAROTENOID CLEAVAGE DIOXYGENASE 7) strengthened the positive regulatory roles of SLs in drought resistance.^10,12,19,20 Recently, comparative studies of d14 and KAR-receptor karrikin insensitive 2 (kai2) mutant plants revealed promoting contributions of SL and KAR/KL (unknown endogenous KAI2 ligand) pathways to Arabidopsis drought resistance, with KAR/KL pathway playing a major role.^10,11 Detailed examinations suggested a complex interaction between D14 and KAI2 signaling pathways as these two pathways differentially contribute to various mechanisms related to anthocyanin accumulation, cuticle formation, cell membrane integrity and leaf senescence in plant response to drought.¹¹ In this study, by comparing the available transcriptome data of the Arabidopsis d14, kai2 and max2 mutants with that of wild-type (WT) plants under dehydrated conditions, we provide evidence that the mechanisms underlying photosynthetic processes and the metabolism of GSLs, Tre, CKs and BRs are mediated either commonly or specifically by SL and KAR/KL pathways in plant responses to water-deficit conditions.

To analyze transcriptome profiles of d14-1 and kai2-2 mutants in comparison with WT plants under 2-h- and/or 4-h-dehydration conditions, first we obtained transcriptome data from Gene Expression Omnibus (GEO) database (accession no. GSE128424 for d14-1 mutant versus WT plants,¹¹ and accession no. GSE90622 for kai2-2 mutant versus WT plants;¹⁰ www.ncbi.nlm.nih.gov/geo). Second, we filtered the differentially expressed genes (DEGs) by the cutoff of two-fold change [corrected P-value (i.e., q-value) < 0.05] for studying gene expression changes in different mutants, e.g. d14-1 and kai2-2, versus WT plants under 2-h- and/or 4-h-dehydration conditions. Thus, the four DEG sets derived from ‘d14-1 vs. WT dehydration 2 h (d14-1-D2/WT-D2)’ and ‘d14-1 vs. WT dehydration 4 h (d14-1-D4/WT-D4)’ comparisons, and from ‘kai2-2 vs. WT dehydration 2 h (kai2-2-D2/WT-D2)’ and ‘kai2-2 vs. WT dehydration 4 h (kai2-2-D4/WT-D4)’ comparisons were analyzed to combine them into the two DEG lists for ‘d14-1-D/WT-D’ and ‘kai2-2-D/WT-D’ comparisons, respectively. A total of 1,363 (Supplementary Table S1a) and 1,454 (Supplementary Table S1b) DEGs were found for ‘d14-1-D/WT-D’ and ‘kai2-2-D/WT-D’ comparisons, respectively.^10,11 Subsequently, enrichment analysis of the DEGs in various functional categories and pathways was carried out by conducting both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses with the aid of Metascape (http://metascape.org) (Supplementary Table S2).²¹ We selected the top fifteen enriched terms for further analysis based on the P-values (Figure 1(a,b)).

Figure 1.

Top 15 enriched terms of differentially expressed genes (DEGs) obtained from comparisons of the transcriptome data of d14-1 and kai2-2 mutants with wild-type (WT) plants under dehydration. Classification of DEGs obtained from ‘d14-1-D/W-D’ (a) and ‘kai2-2-D/W-D’ (b) comparisons through enrichment analysis using Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The x-axis is the accumulative hypergeometric P-values of genes mapped by the terms, representing the abundance of the GO or KEGG terms. The y-axis is the top 15 enrichment GO and KEGG terms. ‘d14-1-D/W-D’, ‘d14-1 dehydrated 2 h vs. WT dehydrated 2 h and/or d14-1 dehydrated 4 h vs. WT dehydrated 4 h’; ‘kai2-2-D/W-D’, ‘kai2-2 dehydrated 2 h vs. WT dehydrated 2 h and/or kai2-2 dehydrated 4 h vs. WT dehydrated 4 h’.

In order to discuss easily, the enriched terms were further categorized into four groups: (i) drought resistance-related processes, (ii) other abiotic stress-related processes, (iii) hormone-related processes, and (iv) development-, metabolism- and biotic stress-related processes (Figure 1(a,b)). The drought resistance related-processes have been previously discussed and supported by phenotypic analyses, such as ‘response to hydrogen peroxide’ (GO:0042542), ‘flavonoid biosynthesis process’ (GO: 0009813), ‘cutin, suberin and wax biosynthesis’ (ath00073 pathway by KEGG), ‘lipid catabolic process’ (GO:0016042), ‘aging’ (GO:0007568, which is related to senescence), which were confirmed by the cuticle deficiency, lower anthocyanin levels and delayed senescence in mutant kai2-2, as well as lower anthocyanin levels and delayed senescence in mutant d14-2, when compared with WT.^10,11 The observation of the terms related to other abiotic stresses, such as ‘response to iron ion starvation’ (GO:1990641), suggested that KAI2 might also be involved in the regulation of plant response to iron deficiency. Furthermore, the abiotic stress-related term ‘response to radiation’ (GO:0009314) was enriched in ‘kai2-2-D/WT-D’ transcriptome (Figure 1(b)), which might explain the insensitivity of kai2 mutant plants to light.²² The relevance of KAI2 in other GO terms, such as ‘response to wounding’ (GO:0009611) and ‘response to iron ion starvation’ (GO:1990641) under dehydration (Figure 1(b)), are still needed to be proved in the future. Additionally, the classification of DEGs also suggests that secondary metabolism, hormonal signals, developmental and photosynthesis processes are regulated by D14 and KAI2, which might be related to the drought responses. As MAX2 is the checkpoint of both D14- and KAI2-mediated signaling pathways, we also analyzed the two DEG sets of ‘max2-3 vs. WT dehydration 2 h (max2-3-D2/WT-D2)’ and ‘max2-3 vs. WT dehydration 4 h (max2-3-D4/WT-D4)’ (accession no. GSE48949) to combine a DEG list of 5,455 members for the ‘max2-3-D/WT-D’ comparison (Supplementary Table S1 c).¹³ Analysis of the list of MAX2-mediated genes might allow us to verify the genes regulated by D14 and/or KAI2. Such comparative analysis of the three DEG lists derived from ‘d14-1-D/WT-D’, ‘kai2-2-D/WT-D’ and ‘max2-3-D/WT-D’ comparisons (Supplementary Table S1) revealed some interesting results. To our interest, in the next lines we focused on GSL-, Tre-, hormone- and photosynthesis-related genes.

GSLs are sulfur-containing plant secondary metabolites and are particularly abundant in members of the Brassicaceae family, including Arabidopsis.^23–25 Previous investigations suggested the roles of GSLs in regulating plant defense, innate immunity, and growth.^23,25–29 Recently, more lines of evidence indicated that the biosynthesis of GSLs is induced by drought, and GSLs and their degradation products are involved in enhancing plant drought resistance through promoting stomatal closure.^29–35 Our transcriptome data (accession no. GSE128424) also revealed that dehydration-induced the expression of some GSL biosynthesis-related genes (Figure 2(a); Supplementary Table S3), such as CYTOCHROME P450 79F1 (CYP79F1), CYP79F2, CYP83A1, ALKENYL HYDROXALKYL PRODUCING 2 (AOP2), BILE ACID TRANSPORTER 5 (BAT5), UDP-GLUCOSYL TRANSFERASE 74B1 (UGT74B1), SULFOTRANSFERASE 16 (SOT16), SOT17, SOT18, and FLAVIN-MONOOXYGENASE GLUCOSINOLATE S-OXYGENASE 1 (FMOGS-OX1).¹¹ Importantly, the expression of several GSL biosynthesis-related genes was down-regulated in ‘d14-1-D/WT-D’ (e.g. UGT74B1, and SOT16), ‘kai2-2-D/WT-D’ (e.g. CYP79F1 and AOP2), and ‘max2-3-D/WT-D’ (e.g. UGT74B1 and SOT16) comparisons (Figure 2(a); Supplementary Table S3), which implied that D14, KAI2 and MAX2 might enhance drought resistance by promoting GSL biosynthesis. Further genetic experiments are required to support D14-, KAI2- and MAX2-mediated regulation of GSL metabolism, and the responses of d14-1, kai2-2 and max2-3 mutants to exogenous GSLs. Additionally, the observed down-regulation of GSL biosynthesis-related genes suggests lower stress-induced accumulation of GSLs in d14, kai2, and max2 mutants, compared with WT plants. Since GSLs have long been implicated in plant defense and immunity, the role of D14, KAI2 and MAX2 in plant defense and innate immunity responses is an important subject of future investigations.

Figure 2.

Expression levels of genes related to metabolism of glucosinolates, trehalose, cytokinins and brassinosteroids, and photosynthesis in wild-type (WT), d14-1, kai2-2, max2-3, and d14-2 kai2-2 mutant plants under non-dehydrated and dehydrated conditions. (a,b) Fold-changes of genes related to glucosinolate and trehalose biosyntheses (a), and cytokinin catabolism, brassinosteroid biosynthesis and photosynthesis (b) obtained from different transcriptome data as indicated by the heatmap presentation. Relative expression levels are shown by intensities of colors expressed in linear fold-change with saturation at 6. Red and blue colors show up-regulation and down-regulation, respectively. Asterisks indicate the data points that pass the q-value < 0.05. ‘WT-D2/WT-C’, ‘WT dehydrated 2 h vs. WT well-watered control’; ‘WT-D4/WT-C’, ‘WT dehydrated 4 h vs. WT well-watered control’; ‘d14-1-D2/d14-1-C’, ‘d14-1 dehydrated 2 h vs. d14-1 well-watered control’; ‘d14-1-D4/WT-C’, ‘d14-1 dehydrated 4 h vs. d14-1 well-watered control’; The‘kai2-2-D2/W-D2ʹ, ‘kai2-2 dehydrated 2 h vs. WT dehydrated 2 h’; ‘kai2-2-D4/W-D4ʹ, ‘kai2-2 dehydrated 4 h vs. WT dehydrated 4 h’; The‘max2-3-D2/W-D2ʹ, ‘max2-3 dehydrated 2 h vs. WT dehydrated 2 h’; ‘max2-3-D4/W-D4ʹ, ‘max2-3 dehydrated 4 h vs. WT dehydrated 4 h’. (c) Expression patterns of photosynthesis marker genes in WT, d14-2, kai2-2, and d14-2 kai2-2 plants under non-dehydrated and dehydrated conditions. Aerial parts (without inflorescence) of 24-day-old soil-grown plants were used for expression analysis using qRT-PCR. Relative expression levels were normalized to a value of 1 in the non-dehydrated WT. Data are means ± SEs (n = 5 biological replicates). Different letters show significant differences in all combinations by a Tukey’s honest significant difference test (P < .05). Asterisks indicate significant differences in comparison of relative transcript levels between different genotypes under the same treatment (*P < .05 and **P < .01; Student’s t-test).

Tre is an important intermediate product in sugar metabolism in plants, which is synthesized from trehalose-6-phosphate by trehalose-6-phosphate phosphatase (TPP). The TPP family consists of 11 members (TPPA-TPPJ) in Arabidopsis with different gene expression patterns.³⁶ In addition to serving as a carbon source, Tre also functions as an osmolyte and signaling molecule under adverse environmental conditions, such as dehydration, high salinity and high temperatures.^36–40 Many studies also suggested that Tre enhances drought resistance through inducing stomatal closure and activating ABA signaling.^36,38,41 For example, overexpression of TPPF gene improves the drought resistance in Arabidopsis transgenic plants, and tppf mutant plants are drought-sensitive compared with WT plants.⁴² Additionally, a recent investigation suggested that TPPE gene is regulated by ABA and involved in the stomatal movement by inducing accumulation of reactive oxygen species (ROS) under drought.⁴¹ Therefore, we were interested in deciphering the functions of D14, KAI2 and MAX2 in Tre biosynthesis. The microarray data from WT plants under dehydration revealed up-regulated expression of TPPB, TPPD and TPPF genes (Figure 2(a); Supplementary Table S3), suggesting that Tre biosynthesis is promoted by dehydration. Importantly, the expression levels of these Tre biosynthesis-related genes were reduced in d14-1 (TPPB, TPPD and TPPF), kai2-2 (TPPD) and max2-3 (TPPB and TPPD) mutants versus WT plants under 2 h and/or 4 h dehydration (Figure 2(a); Supplementary Table S3), which implied that D14, KAI2, and MAX2 might enhance drought resistance through promoting Tre biosynthesis. It is now interesting to carry out detailed experiments to provide genetic evidence validating the D14-, KAI2- and MAX2-mediated regulation of Tre metabolism.

As CK and BR signals negatively regulate plant drought resistance,^5–7,43,44 we were curious about the potential crosstalk between SL and CK signaling,⁴⁵ as well as between SL and BR signaling under water-deficit conditions. Under the well-watered conditions, a crosstalk between SL and CK signaling in rice (Oryza sativa) in regulating plant development was reported, where SL promoted CK degradation through transcriptional activation of CK OXIDASE 9 (CKX9) gene.⁴⁶ The crosstalk between SL and BR signals is supported by the fact that SL induces degradation of a bHLH transcription factor, BRASSINAZOLE-RESISTANT 2 (BZR2)/BRASSINOSTEROID INSENSITIVE 1-EMS-SUPPRESSOR (BES1), which is a BR transcriptional activator involved in regulating shoot branching.⁴⁷ However, the involvement of the SL-CK and SL-BR interactions in drought resistance remains to be clarified. Through microarray data analyses, we found that many CKX genes are significantly down-regulated in mutants d14-1 (e.g. CKX1, CKX5 and CKX7) and max2-3 (e.g. CKX1, CKX2, CKX3, CKX5 and CKX7), but not in mutant kai2-2, when compared with WT under dehydration (Figure 2(b); Supplementary Table S3). These results suggest that SL (but not KAR) signal might promote CK catabolism under dehydration. With respect to BRs, the BR biosynthesis-related genes CYP90A1 and CYP90D1 are repressed by dehydration, suggesting that plants are acclimatized to drought by decreasing BR levels. Additionally, CYP90A1 and CYP90D1 were significantly up-regulated in mutants d14-1 and max2-3, but not in mutant kai2-2 under dehydration (Figure 2(b); Supplementary Table S3), implying that SL (but not KAR) signal enhances drought resistance through inhibiting BR biosynthesis, at least in part.

The expression profile of photosynthesis-related genes displayed a trend similar to the BR biosynthesis-related genes, i.e. they showed down-regulation in WT plants in response to dehydration, and up-regulation in d14-1, kai2-2 and max2-3 mutants versus WT plant under dehydration (Figure 2(b); Supplementary Table S3). These results suggest a negative regulation of the photosynthetic processes by D14, KAI2 and MAX2. To confirm the expression patterns of these genes as a means to show the regulatory involvement of D14 and KAI2 in photosynthesis under normal and dehydration conditions, we examined the transcript levels of several key photosynthesis-related genes in non-dehydrated and dehydrated WT, d14-2 and kai2-2 plants using quantitative reverse transcriptase-PCR (qRT-PCR). The primers used for qRT-PCR are listed in Supplementary Table S4. Furthermore, in order to observe any possible interaction between D14 and KAI2 in photosynthesis, we included the d14-2 kai2-2 double mutant in the qRT-PCR analysis. Under non-dehydrated conditions, the expression levels of several photosynthesis-related genes (e.g. LIGHT HARVESTING COMPLEX II CHLOROPHYLL B BINDING PROTEIN 4.2, LHCB4.2; PHOTOSYSTEM I SUBUNIT D-1, PSAD-1; and RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL CHAIN 1A, RBCS1A) were higher in mutants d14-2, kai2-2 and d14-2 kai2-2 than WT (Figure 2(c)). These data suggest that D14 and KAI2 receptors might act as negative regulators of photosynthesis under non-stressed conditions, which is supported by the higher shoot biomass of mutants d14-2, kai2-2 and d14-2 kai2-2 than WT under well-watered conditions.¹⁰ Under dehydration, the transcript levels of the RBCS1B gene in the d14-2 and WT plants were comparable, whereas its transcript levels were significantly higher in kai2-2 and d14-2 kai2-2 than WT plants (Figure 2(c)), implying the contribution of KAI2 in photosynthesis under dehydration. Since the expression levels of the RBCS1B gene were higher in the dehydrated d14-2 kai2-2 double mutant than dehydrated kai2-2 single mutant plants, D14 might also play a negative role in photosynthesis, through RBCS1B gene, under dehydration conditions, at least in the kai2-2 mutant background. Furthermore, the observed higher expression levels of PSAD-1 gene in d14-2 mutant and of RBCS1A genes in kai2-2 mutant than WT plants after 4 h of dehydration implied that both D14 and KAI2 receptors may be involved in the growth adjustment by regulating the photosynthesis through different genes (Figure 2(c)), to reallocate the energy from growth to protect plants against adverse conditions.^48–50 Additionally, the observation of higher expression levels of LHCB4.2, PSAD-1 and RBCS1A genes in d14-2 kai2-2 double mutant than in d14-2 and kai2-2 single mutant plants under non-stressed conditions, and higher expression levels of RBCS1B gene in d14-2 kai2-2 double mutant than in d14-2 and kai2-2 single mutant plants under dehydration conditions suggests the existence of a crosstalk between D14 and KAI2 in photosynthesis under both normal and dehydration conditions.

In summary, further comparative analyses of the DEGs of ‘d14-1-D/WT-D’, ‘kai2-2-D/WT-D’ and ‘max2-3-D/WT-D’ comparisons, provided several more novel mechanisms of SL and KAR/KL-mediated plant responses to water-deficit conditions. SL signal might enhance drought resistance by promoting GSL and Tre biosyntheses, inducing CK degradation, and inhibiting BR biosynthesis and photosynthesis (Figure 3). On the other hand, the KAR/KL signal might enhance drought resistance by promoting GSL and Tre biosyntheses and inhibiting photosynthesis, but not by affecting CK and BR metabolism (Figure 3). Detailed genetic studies involving physiological, hormonal and metabolic analyses are required to prove the hypothetical model shown in Figure 3.

Figure 3.

Hypothetical model for the differences between SL-mediated D14 and KAR/KL-mediated KAI2 signaling pathways in enhancing drought resistance. D14 pathway promotes glucosinolate and trehalose biosyntheses, while decreasing the levels of cytokinins and brassinosteroids and the capacity of photosynthesis. KAI2 pathway promotes glucosinolate and trehalose biosyntheses, while decreasing the capacity of photosynthesis. Glucosinolates and trehalose are positively, while cytokinins, brassinosteroids and photosynthesis are negatively associated with plant adaptation to drought. Arrows indicate promotion, and blunt bars indicate inhibition.

Funding Statement

This research was funded by a Japan Society for the Promotion of Science [#17K07459 and #20K05871]; National Key R&D Programme (NKP) from Ministry of Science and Technology of the People’s Republic of China [#2018YFE0194000].

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.