Nitric oxide is associated with strigolactone and karrikin signal transduction in Arabidopsis roots

Nitric oxide (NO), strigolactones (SLs) and karrikins (KARs) are bioactive signal molecules controlling root morphology. Our knowledge regarding the signal interplay of NO with SLs or KARs is limited. Our previous results pointed out that there is signal interplay between SL and S-nitrosoglutathione reductase (GSNOR)-mediated NO/S-nitrosothiol (SNO) levels in Arabidopsis. In this addendum, we further prove that the pharmacological increment of SL levels by the application of rac-GR24 decreases GSNOR abundance, while reducing SL synthesis by TIS108 intensifies GSNOR protein level and possibly promotes NO signaling in Arabidopsis. Additionally, we observed that the endogenous NO level in the roots of htl-3 (KAR receptor) and d14 (SL receptor) mutants is significantly higher compared to the wild-type. These results, together with the previous ones, confirm the interplay of GSNOR-regulated NO/SNO signaling with SL- and KAR-dependent signal transduction in Arabidopsis.

SLs are recognized as terpenoid lactons consisting of triciyclic lacton (ABC ring) and butenolide group (D ring).^1–4 SLs are synthesized from all-trans β-carotene by β-carotene isomerase DWARF 27 (D27) and two carotenoid cleavage dioxygenases CCD7 and CCD8.⁵ These enzymes produce carlactone (CL) in two subsequent steps from 9-cis-β-carotene. CL is a bioactive precursor of SLs which is oxidized by MORE AXILLARY GROWTH 1 (MAX1)⁵ yielding canonical and non-canonical SLs. The perception and signal transduction of SL is catalyzed by DWARF14 (D14) and MORE AXILLARY GROWTH 2 (MAX2), respectively.⁶ D14 is an α/β hydrolase hormone receptor, consisting of a conserved Ser-His-Asp triad.⁷ D14 binds the hormone and after that cleaves the enol ether bound between the ABC and D rings. The second component of the SL response pathway is MAX2, a member of the F-BOX protein family. MAX2 contains leucine-rich repeats and it is the part of SCF-type ubiquitin ligase complex.⁸ Karrikins are important smoke components which stimulate seed germination after fire.⁹ KARs are produced by combusting carbohydrates, for example, cellulose in the plant. Interestingly, KARs and SLs share common response pathway element MAX2; furthermore, they have similar structure (butenolide group). KARRIKIN-INSENSITIVE2 (KAI2) is required for the perception of KARs.^10,11 Both SLs and KARs represent new classes of phytohormones, and regulate shoot branching, secondary growth, leaf senescence and control many aspects of root development like primary root length, adventitious root initiation, lateral root formation and root hair growth.^8,12–16

The above root growth parameters are regulated also by nitric oxide (NO) signal molecule. NO is a hydrophobic, small diatomic gaseous molecule which has various effects on plant physiology and development including stomatal closure, stimulating primary root length, promoting lateral root formation and triggering biotic and abiotic stress responses.^17,18 As a free radical, NO can rapidly react with reactive oxygen species and can modify proteins. One of the most important signaling pathway is S-nitrosation. In this process, NO reacts with cysteine residue of glutathione and forms S-nitrosoglutathione (GSNO). GSNO is a stable molecule; it can be transported via xylem; therefore, it is a mobile reservoir of NO signal.¹⁹ GSNO is enzymatically degraded by a GSNO reductase (GSNOR) which is a highly conserved cysteine-rich homodimer enzyme, and contains two catalytic zinc ions in each subunit.²⁰ Our previous results indicated that SL deficiency in max1-1 and max2-1 seedlings caused elevated NO/S-nitrosothiol (SNO) levels due to decreased abundance and activity of GSNOR enzyme.²¹ Since GSNOR is the key enzyme regulating NO-related signal transduction, we further examined the putative SL-induced alterations in its level.

We applied SL mimic rac-GR24 (2 µM) and the inhibitor of SL synthesis TIS108 (5 µM) in order to modify SL level and signaling in wild-type Arabidopsis thaliana (Col-0). Four-d-old seedlings were exposed to the treatments and GSNOR protein abundance was analyzed in 7-d-old seedlings by Western blot according to Oláh et al.²¹ Compared to control, rac-GR24 resulted in reduced GSNOR protein abundance, but the application of SL inhibitor TIS108 increased the amount of GSNOR protein (Figure 1). When rac-GR24 and TIS108 were applied together, the level of GSNOR protein increased compared to untreated samples. These results seemingly contradict the previous results obtained using max1-1 and max2-1, which may be due to the fact that exogenous applications represent different conditions compared to untreated mutants. Another possible explanation may be that the effect of the MAX1 and MAX2 mutations on GSNOR is not SL-specific, but there is some indirect relationship between gene defect and GSNOR protein levels. Moreover, due to the poor detectability of SLs in plant tissues, the effect of rac-GR24 and/or TIS108 on endogenous SLs levels of the seedlings is not known. These methodological issues should be considered.

Figure 1.

(a) Abundance of GSNOR protein in 7-d-old Arabidopsis thaliana seedlings grown without (mock) or with rac-GR24 (2 µM), TIS108 (5 µM) or rac-GR24 plus TIS108. Relevant bands showing GSNOR signals were quantified by Gelquant software (provided by biochemlabsolutions.com) and the values of pixel densities are presented in panel B. Different letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05)

Since MAX2 mediates both SL and KAR signaling, max2-1 mutant alone is not sufficient to determine whether NO is associated with SL or KAR signals or with both of them in the root system. Therefore, the endogenous NO levels of KAR receptor (htl-3) and SL receptor (d14) mutants were compared with that of the wild-type max1-1 and max2-1. The detection of endogenous NO levels was performed according to Oláh et al.²¹ Interestingly, the htl-3 mutation resulted in ~four times higher NO levels in the root compared to the wild-type, and also the deficiency of D14 receptor led to elevated NO levels (Figure 2). In order to support the hypothesis that KAR and NO signal pathways interfere, we evaluated possible S-nitrosation of KAR-specific signal proteins KAI2 and SUPRESSOR OF MAX1 (SMAX1). Peptide sequences were extracted from UNIPROT (www.uniprot.org) and submitted to in silico prediction software GPS-SNO 1.0,²² freely available at http://sno.biocuckoo.org/, iSNO-PseAAC,²³ freely available at http://app.aporc.org and DeepNitro,²⁴ freely available at http://deepnitro.renlab.org. Interestingly, only one of the software tools (iSNO-PseAAC) predicted S-nitrosation of KAI2 protein, while in case of SMAX1, several cysteine (Cys) residues were predicted to be S-nitrosated by all three programs (Table 1). These suggest that these KAR-specific signal proteins may be modified/regulated by NO-dependent S-nitrosation; however, this hypothesis should be experimentally proved in the future.

Figure 2.

Level of nitric oxide (pixel intensity, (a)) and representative microscopic images taken from root tips labeled with DAF-FM DA (b) in 7-d-old wild type (Col-0), KAR receptor mutant (htl-3), SL receptor mutant (d14), SL biosynthesis mutant (max1-1) and SL/KAR signaling mutant (max2-1) Arabidopsis thaliana seedlings grown under stress-free conditions. Different letters indicate significant differences according to Duncan’s test (n = 3, P ≤ 0.05). Scale bar = 110.6 µm

Table 1.

In silico prediction of S-nitrosation of proteins involved in KAR perception and signaling using GSP-SNO 1.0, iSNO-PseAAC and DeepNitro software tools. Predictions were carried out using medium threshold. Amino acid positions and peptides predicted by more than one computational tools are in italic

GPS-SNO 1.0			iSNO-PseAAC		Deep-Nitro
Protein name	Position	Peptide	Position	Peptide	Position	Peptide
KAR perception
KAI (A. thaliana)			154	EAIRSNYKAWCLGFAPLAVPP
			209	RQILPFVTVPCHILQSVKDLA
KAR signaling
SMAX1 (A. thaliana)	637	VAATVSQCKLGNGKR	115	KRAQAHQRRGCPEQQQQPLLA	115	KRAQAHQRRGCPEQQQQPLLA
	962	SSGTYGDCTVARLEL	417	SFVPANRTLKCCPQCLQSYER	479	QKKWNDACVRLHPSF
			580	GDVQVRDFLGCISSESVQNNN	809	KFGKRRASWLCSDEERLTKPK
			637	AAAVAATVSQCKLGNGKRRGV
			809	KFGKRRASWLCSDEERLTKPK
			865	QGFSGKLSLQCVPFAFHDMVS
			962	RVSSSGTYGDCTVARLELDED

Collectively, these results further support the hypothesis regarding the crosstalk between NO, SL and KAR in regulating Arabidopsis root development. Further experiments should clarify the detailed effect of KAR on NO metabolism and signaling and vice versa.

Funding Statement

This work was supported by the Emberi Eroforrások Minisztériuma [ÚNKP-20-3-SZTE-478]; National Research, Development and Innovation [128644]; National Research, Development and Innovation [120383]; National Research, Development and Innovation [135303].