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Endogenous salicylic acid suppresses de novo root regeneration from leaf explants [1]

['Sorrel Tran', 'Department Of Plant Pathology', 'College Of Agricultural', 'Environmental Sciences', 'University Of Georgia', 'Athens', 'Georgia', 'United States Of America', 'Madalene Ison', 'Nathália Cássia Ferreira Dias']

Date: 2023-03

Plants can regenerate new organs from damaged or detached tissues. In the process of de novo root regeneration (DNRR), adventitious roots are frequently formed from the wound site on a detached leaf. Salicylic acid (SA) is a key phytohormone regulating plant defenses and stress responses. The role of SA and its acting mechanisms during de novo organogenesis is still unclear. Here, we found that endogenous SA inhibited the adventitious root formation after cutting. Free SA rapidly accumulated at the wound site, which was accompanied by an activation of SA response. SA receptors NPR3 and NPR4, but not NPR1, were required for DNRR. Wounding-elevated SA compromised the expression of AUX1, and subsequent transport of auxin to the wound site. A mutation in AUX1 abolished the enhanced DNRR in low SA mutants. Our work elucidates a role of SA in regulating DNRR and suggests a potential link between biotic stress and tissue regeneration.

Tissue regeneration is a core technology for modern agriculture and horticulture. It is widely used for crop improvement, propagation of valuable varieties and generation of chimeric plants. Plants must integrate physiological and environmental cues to complete this dramatic and sophisticated reprogramming process. Difficulties in regenerating adventitious roots from cuttings, such as the age-dependent decline of rooting, is still a bottleneck in propagating economically and ecologically important plants. We discovered that Salicylic acid (SA), a key hormone for plant defense, suppresses root regeneration from cuttings. Depleting endogenous SA or disrupting SA signaling enhances plants’ regeneration ability. Our study provides new knowledge for overcoming challenges in vegetative propagation by manipulating the SA response.

Funding: This project is supported by National Science Foundation under Grant NO. IOS-2039313 to L.Y. and C.J. T. Research at the Teixeira lab is funded by the São Paulo State Research Foundation (Fapesp; 2018/24432-0) and by the Serrapilheira Institute (G-1811-25705). NCFD received a fellowship from Capes (00188887.483949/2020-00). Funding for the Agilent UPLC-QTOF was provided by the U.S. Department of Agriculture, National Institute of Food and Agriculture, Equipment Grant Program award no. 2021-70410-35297 (to C.J.T and L.Y.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here we report that endogenous SA suppresses DNRR from leaf explants. SA response is activated rapidly after cutting accompanied by an accumulation of free SA. NPR4 serves as a key receptor of SA in regulating the suppression of DNRR, and distinct signaling components are recruited for SA-mediated defense and regeneration. SA inhibited the transport of auxin to the cutting site. AUX1 is transcriptionally suppressed by SA after cutting and its mutation rescues the enhanced rooting in an SA-deficient mutant. Taken together, our results revealed key signaling components required for the SA-mediated suppression of wound-induced DNRR.

Salicylic acid (SA) is essential to launch a robust defense against various biotrophic and hemi-biotrophic pathogens such as Pseudomonas syringae pv. tomato DC3000 [ 12 ]. In Arabidopsis, mutants defective in SA biosynthesis or signaling show enhanced susceptibility to viral, bacterial, oomycete, and fungal pathogens [ 13 ]. SA is also involved in responses to abiotic stresses, such as drought, and in the regulation of development, including flowering time and root patterning [ 14 – 16 ]. The current understanding of the SA signaling pathway is largely gained from studies of plant immunity. SA is perceived by paralogs of the NONEXPRESSER OF PATHOGENESIS RELATED 1 (NPR1) gene. Six Arabidopsis NPR1 paralogs (NPR1, NPR2, NPR3, NPR4, NPR5, and NPR6) share a BTB/POZ (Broad-complex, Tramtrack, and Bric-a-brac/Poxvirus and Zinc-finger) domain, and an ankyrin repeat domain [ 17 ]. NPR1, NPR3 and NPR4 have been demonstrated to bind SA and to transduce SA-induced immune signaling in Arabidopsis [ 18 – 21 ]. NPR1 contains a transcription co-activation domain at its C-terminus and can activate the expression of genes required for Systemic Acquired Resistance (SAR) upon SA perception [ 22 ]. On the other hand, NPR3 and NPR4 act redundantly to repress SA-mediated defense responses in the absence of SA [ 23 , 24 ]. NPR3 and NPR4 negatively regulate defenses by independently regulating NPR1-controlled genes [ 20 ] or through degrading NPR1 [ 19 ].

The process of DNRR solely relies on the dynamic interactions among endogenous hormones, since no exogenous hormones (e.g., auxin or cytokinin) are added to induce cell differentiation [ 7 ]. In the current model of DNRR, jasmonic acids (JAs) serve as a wound-induced early signal to activate a group of transcription factors, including the ETHYLENE RESPONSE FACTOR 109 (ERF109) shortly after cutting [ 8 , 9 ]. ERF109 serves as a link between early JA signals and subsequent auxin biosynthesis because it directly activates the expression of ANTHRANILATE SYNTHASE α1 (ASA1)—a tryptophan biosynthesis gene in the auxin production pathway [ 8 ]. In addition to ASA1, other genes for auxin biosynthesis (e.g., YUCCA1, YUCCA4, and YUCCA6) are activated in distal tissues, leading to a synthesis of auxin in the leaf mesophyll cells [ 8 ]. Newly synthesized auxin is transported to the wound site where it activates members of the auxin response transcription factors (ARFs). The activation of auxin response is evident one day after cutting (DAC) at the wound site [ 5 ]. ARF7 and ARF19 can directly activate WUSCHEL RELATED HOMEOBOX 11 and 12 (WOX11 and 12) to initiate cell fate transition [ 5 ]. Auxin-induced expression of WOX11 in cambium cells is considered as a first step for cell fate transition during DNRR [ 10 , 11 ], which occurs approximately at two DAC. WOX11/12 subsequently activates a root quiescent center marker, WOX5, initiating adventitious root formation [ 10 ].

Plants have a remarkable ability to regenerate after wounding [ 1 , 2 ]. Regeneration of adventitious roots from leaf explants or stem cuts lays a foundation to propagate valuable crops and fruits for agriculture and horticulture [ 3 ]. Regeneration requires a signaling cascade from the perception of wound signals, gain of reprogramming competence, conversion of cell fate, and eventually, patterning of the new organ [ 4 , 5 ]. Arabidopsis leaf explants detached from a stem can develop adventitious roots from the wound site without exogenous supplementation of phytohormones [ 5 , 6 ]. This process is referred as de novo root regeneration (hereafter DNRR) [ 4 , 5 ].

Results

Endogenous SA suppressed DNRR from Arabidopsis leaf explants To investigate the role of endogenous SA in DNRR, we compared the rooting ability of leaf explants from the wild type, Columbia-0 (Col-0), and transgenic plants overexpressing NahG, a salicylate hydroxylase derived from the bacterium Pseudomonas putida, under a constitutive (cauliflower mosaic virus 35S, hereafter 35S) promoter (35S::NahG, hereafter NahG). Overexpression of NahG reduces the free SA level by converting SA to catechol [25]. The ratio of leaf explants forming adventitious roots were significantly higher in NahG than those in wild type, Col-0 (Fig 1A and 1B). NahG explants also generated an increased number of adventitious roots on each explant (Fig 1C). We also observed a reduced rooting ability in older Col-0 leaf explants, which is consistent with a previous work that showed an age-dependent decline in regeneration [26,27] (Fig 1D). The age-dependent decline of rooting was completely abolished in NahG explants (Fig 1D). Conversely, in Arabidopsis mutants with a high level of SA, such as snc1 (SUPPRESSOR OF NPR1-1) and cpr1 (CONSTITUTIVE EXPRESSER OF PR GENES 1), the formation of adventitious roots from leaf explants was significantly suppressed (Fig 1A–1C). We acknowledge that SNC1 is involved in a microRNA pathway and immune signaling [28–30], whereas CPR1 regulates SNC1 stability, with SA elevation as an indirect effect [31]. We therefore sprayed SA onto leaves one hour before cutting to investigate an SA-specific response on DNRR. We observed that exogenous SA at a concentration beyond 5 μM inhibited adventitious root formation like the phenotypes seen in the snc1 and cpr1 mutants (Figs 1E and S1). Thus, we conclude that endogenous SA inhibits adventitious root formation on leaf explants. PPT PowerPoint slide

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TIFF original image Download: Fig 1. SA repressed the formation of adventitious roots from leaf explants. (A) Representative images of leaf explants at 11 DAC from wild type and mutants. Explants were cut from the first two rosette leaves. (B) The rooting ratio in wild type Col-0 and mutants. * indicates p<0.01 when compared to Col-0 using student t-test. Long and short bars represent means and standard errors, respectively. (C) The proportion of leaf explants with indicated number of adventitious roots from wild type and mutants. * indicates significant difference using a Mann–Whitney U test when compared to Col-0. (D) The rooting ratio of explants from plants with different age. Different letters indicate significant difference using one way ANOVA. (E) Rooting ratio of Col-0 explants with SA treatment. Each dot in B, D, and E represents an independent experiment with 40–60 explants. Error bars indicate standard error. The rooting data were collected at 11 DAC. https://doi.org/10.1371/journal.pgen.1010636.g001

The SA pathway was activated after leaf excision To monitor the dynamics of the SA response during DNRR at the transcriptome level, we further investigated the expression pattern of SA-responsive genes in the first 12 hours after leaf excision [8]. SA-responsive genes were defined in Yang et al [32]. Similar to the present study, Yang et al. also used two-week-old seedlings. In total, 6410 genes were differentially expressed in at least one time point after wounding compared to the control condition (time 0). Of the 2357 SA-activated genes [32], 1101 were differentially expressed in at least one time point after cutting. Similarly, 878 of the 1593 genes that are repressed by SA were also differentially expressed in at least one time point after cutting (Fig 2A). Thus, SA-responsive genes account for 31% (1979 of 6410) of the total genes that respond to wounding within the first 12 hours. PPT PowerPoint slide

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TIFF original image Download: Fig 2. SA response was activated after wounding. (A) Venn diagram showing the overlap of genes that were differentially expressed upon wounding in the first 12 hours after cutting [8] and markers of the SA response [32]. The SA-induced genes are shown in the light pink circle, SA-repressed genes are shown in the light blue circle, and in the green circle are genes that showed altered expression upon wounding in at least one time-point of the experiment. A total of 1101 genes are activated by SA and differentially expressed upon wounding. Furthermore, 878 of the SA-repressed genes are differentially expressed in wounded leaves. (B) Expression profile of the 1101 SA-activated genes that were differentially expressed in leaves during DNRR Marker genes of the SA response were defined by the treatment of Arabidopsis seedlings with exogenous SA [32]. Genes activated by SA and differentially expressed in our experiment were submitted to hierarchical clustering based on their expression profile in wounded leaves. Differential expression at each time point is indicated with a color code (induced: purple; repressed: green). NPR4 and NPR3 are part of cluster 3 (C3) and are highlighted next to the heatmap. A representative profile of each cluster is shown on the right (red line: average behavior; grey line: individual genes). (C) Expression profile of the 878 SA-repressed genes that were differentially expressed in leaves during DNRR. Marker genes of the SA response were defined by the treatment of Arabidopsis seedlings with exogenous SA [32]. Genes repressed by SA and differentially expressed in our experiment were submitted to hierarchical clustering based on their expression profile in wounded leaves. Differential expression at each time point is indicated with a color code (induced: purple; repressed: green). AUX1 is part of cluster 1 (C1) and is highlighted next to the heatmap. A representative profile of each cluster is shown on the right (red line: average behavior; grey line: individual genes). (D) Accumulation of free SA in explants. Each dot represents an independent SA-measurement from two leaf explants by LC-MS (n = 8). The Y axis is peak area normalized for loading based on internal standards. https://doi.org/10.1371/journal.pgen.1010636.g002 A hierarchical clustering analysis of the SA-responsive genes showed that a subset of them were either activated or repressed within as early as 10 minutes after leaf excision, indicating a rapid SA response after cutting (Fig 2B and 2C and S1 Table). Despite the activation of SA pathway as a whole, key genes involved in NPR-mediated defense signaling were downregulated in this early wound response, implying negative feedback or a specific repression of SA-mediated defense signaling (S2A Fig). A subset of SA-repressed genes (cluster 4 and 5 in Fig 2C) was activated upon wounding, indicating potential cross-regulation by other stimuli. Indeed, 50% of all genes included in either clusters 4 and 5 can also be activated by jasmonic acid (JA) [32] (S2B Fig), a known antagonist of the SA-mediated defense gene expression [33]. Given the activation of the JA pathway after leaf excision [8], these genes in clusters 4 and 5 (Fig 2C) may represent the sector of JA-SA crosstalk in the early stage of DNRR. We also examined the levels of free SA in wounded and non-wounded leaf explants from 12-day-old seedlings grown on plates (Fig 2D). Consistent with the observed activation of the SA response, accumulation of free SA was observed 30 min after cutting (Fig 2D). Collectively, this data shows that leaf excision triggers SA accumulation and an associated transcriptional response that is highly reminiscent of the response observed after exogenous SA treatment.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010636

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