Stress-Triggered Long-Distance Communication Leads to Phenotypic Plasticity: The Case of the Early Root Protoxylem Maturation Induced by Leaf Wounding in Arabidopsis

Root architecture and xylem phenotypic plasticity influence crop productivity by affecting water and nutrient uptake, especially under those environmental stress, which limit water supply or imply excessive water losses. Xylem maturation depends on coordinated events of cell wall lignification and developmental programmed cell death (PCD), which could both be triggered by developmental- and/or stress-driven hydrogen peroxide (H2O2) production. Here, the effect of wounding of the cotyledonary leaf on root protoxylem maturation was explored in Arabidopsis thaliana by analysis under Laser Scanning Confocal Microscope (LSCM). Leaf wounding induced early root protoxylem maturation within 3 days from the injury, as after this time protoxylem position was found closer to the tip. The effect of leaf wounding on protoxylem maturation was independent from root growth or meristem size, that did not change after wounding. A strong H2O2 accumulation was detected in root protoxylem 6 h after leaf wounding. Furthermore, the H2O2 trap N,N1-dimethylthiourea (DMTU) reversed wound-induced early protoxylem maturation, confirming the need for H2O2 production in this signaling pathway.


Introduction
Plant adaptive capacity and acclimatization resources play a pivotal role in increasing plant fitness and survival, especially in fast-changing environmental conditions. Thus, the unravelling of variation in phenotypic plasticity in traits of agronomic interest could provide us with beneficial tools for the development of crops more efficiently adaptable to a changing environment. Phenotypic plasticity integrates genetically determined developmental processes and environmental influences [1], and because of this, identifying phenotypic traits showing favourable adaptive plasticity will provide the basis for further studies focused on assessing the underlying genetic basis.
Root systems play a prominent role in crop health and productivity, especially under resource-limited environmental conditions, and plasticity of root traits, such as root growth and architecture, confers functional adaptivity to soils that are poor in water and nutrients [2]. In this regard, root development and differentiation follow different dynamics and may respond to different signalling pathways under physiological or stress conditions, allowing adaptive plasticity in sub-optimal growth conditions.

Leaf Wounding Promotes Alteration of Protoxylem Maturation in Root without Affecting Root Length and Meristem Size
To explore the effect of leaf wounding on root xylem phenotypic plasticity, 7-day-old Arabidopsis seedlings were injured by cutting a cotyledonary leaf, and then roots were observed under Laser Scanning Confocal Microscope (LSCM) 3 days after the injury, for the investigation of the distance from the root apical meristem of the first protoxylem cell with fully developed secondary wall thickenings (whose location is here referred as "protoxylem position") and meristem size. Figure 1 shows images acquired under LSCM after PI staining and relative bright-field images of root apexes from unwounded control and leaf-wounded seedlings. Plantlets in which a cotyledonary leaf was cut present an anticipation of the maturation of protoxylem, as shown by the earlier presence of cells with fully developed secondary wall thickenings that appear closer to the apical meristem as compared to unwounded control plants. Figure 2 demonstrates that these qualitative data were confirmed by statistically significant quantitative analysis. In fact, the mean distance of the first protoxylem cell with fully developed secondary wall thickenings from the root apical meristem was approximately 1620 µm in leaf-wounded plants as compared to unwounded control plants, showing a distance of approximately 2060 µm. The effect of leaf wounding on protoxylem position was specific and not dependent upon variation in root growth or meristem size, which were unchanged in leaf-wounded plants compared to unwounded control plants ( Figure 3; Table 1). present an anticipation of the maturation of protoxylem, as shown by the earlier presence of cells with fully developed secondary wall thickenings that appear closer to the apical meristem as compared to unwounded control plants. Figure 2 demonstrates that these qualitative data were confirmed by statistically significant quantitative analysis. In fact, the mean distance of the first protoxylem cell with fully developed secondary wall thickenings from the root apical meristem was approximately 1620 µm in leaf-wounded plants as compared to unwounded control plants, showing a distance of approximately 2060 µm. The effect of leaf wounding on protoxylem position was specific and not dependent upon variation in root growth or meristem size, which were unchanged in leafwounded plants compared to unwounded control plants ( Figure 3; Table 1).     . Analysis under LSCM after PI staining of the leaf wounding effect on the length of the meristematic zone, determined by measuring the distance between the quiescent centre and the first elongating cell in the cortex cell file. The images presented show roots from 10-day-old unwounded control and leaf-wounded seedlings, injured at the age of 7 days by cutting the cotyledonary leaf with scissors and analysed 3 days after the injury; roots presented are representative of experiments repeated at least five times with ten seedlings analysed each time. Shown images were obtained aligning serial overlapping micrographs of the same root by Photoshop Software (Adobe). Bar: 50 µm. Table 1. Analysis of differences in root growth and meristem size in leaf-wounded seedlings grown in medium with or without the H 2 O 2 -scavenger DMTU. The effect of leaf wounding on root growth was evaluated as the difference between the length measured at the onset of the wounding and that measured after 3 days. The length of the meristematic zone was determined by measuring the distance between the quiescent centre and the first elongating cell in the cortex cell file. These experiments were repeated at least five times with ten seedlings analysed each time (mean values ± SD; n = 50). The statistical significance levels between unwounded control and wounded plants were evaluated by p levels as follows: ns, not significant.  Figure 2 also shows that the H 2 O 2 -scavenger N,N 1 -dimethylthiourea (DMTU), provided at the working concentration of 100 µM, according to a previous report [25], opposes the effect of leaf wounding on early protoxylem maturation consistently with what was previously demonstrated for the MeJA-mediated induction of protoxylem differentiation [20]. To confirm that the effect of the wound-induced signalling on the early maturation of protoxylem cells require H 2 O 2 , this compound was detected in situ in Arabidopsis roots following leaf wounding by exploiting the fluorogenic peroxidase substrate Amplex Ultra Red (AUR). Figure 4 shows that 6 h after leaf wounding, a strong AUR signal was revealed in the root zone where the first protoxylem cell with fully developed secondary cell wall thickenings is found, which was not detectable in unwounded control roots, which is suggestive of a tissue-specific H 2 O 2 production triggered by a long-distance leaf-to-root communication and leading to early protoxylem differentiation.

Discussion
Leaf-to-root long-distance communication is crucial in coordinating biochemical and physiological events between aerial and underground organs, especially in response to changes in environmental conditions [26][27][28][29]. Leaf damage is a frequent injury during the plant lifespan, and may be caused by both herbivores, such as chewing insects, and atmospheric conditions. The wound site is an easy passage for both pathogen entry and water loss, and the presence of leaf mechanical damage triggers several local responses devoted to healing the wound [21,[30][31][32]. Furthermore, complex signalling networks propagate information from the wound site through the whole plant body, allowing systemic responses [29], among which, xylem root remodelling could represent a strategy for enhancing water uptake and counteracting the excessive water loss caused by the wound.
The analysis of root growth, protoxylem position and meristem size in plants in which the cotyledonary leaf has been cut shows a DMTU-reversible early protoxylem differentiation occurring 3 days after injury (Figures 1 and 2), which is independent from variation in meristem size and root growth, which were unchanged (Table 1; Figure 3). A root protoxylem-specific accumulation of H 2 O 2 was detectable 6 h after the injury, supporting its involvement in the variation of protoxylem position (Figure 4). This response is consistent with previous data, where roots of MeJA-treated plants showed a H 2 O 2 -dependent remodelling of the protoxylem, which appeared to be closer to the root tip, independent of root growth or meristem size [20]. Based on the effects of MeJA treatment on protoxylem differentiation, it has been hypothesized that under stress conditions, extracellular H 2 O 2 production may drive early xylem differentiation independently from the auxin/cytokinin/T-Spm loop [17]. In particular, in differentiating protoxylem elements, the H 2 O 2 production driven by cell wall-localized oxidation of PAs was suggested to be involved in both developmental programmed cell death (PCD) and peroxidase-mediated lignin polymerization [17,19,20], which represent key steps in the terminal phase of the xylem differentiation process. PAs are oxidized to aminoaldehydes by AOs, which include copper-containing amine oxidases (CuAOs) and flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs), with the production of a corresponding amine moiety and the biologically active compound H 2 O 2 [33]. Among the cell-wall sources of ROS, it has been known for a long time that AOs are involved in wound-healing responses [30,33] and root xylem differentiation [19][20][21]. Our results suggest the occurrence of a systemic signalling linking an abiotic stress such as leaf wounding with distal root phenotypic plasticity such as variation in protoxylem position, and open the question of unravelling the responsible ROS source.  . In situ H2O2 detection by analysis under LSCM after AUR staining of roots from 7-day-old unwounded control and leaf-wounded seedlings 6 h after injury. The corresponding bright-field and overlay images are shown. Micrographs show the root zone corresponding to the site of appearance of the first protoxylem cell with fully developed secondary cell wall thickenings (arrows) and have been taken at the level of the central root section. Images are representative of those obtained from ten seedlings from five independent experiments. In the red degrading scale, the average values of fluorescence intensity, measured as the sum of the pixels of each 65 µm 2 rectangle, are indicated for unwounded control and leaf-wounded plants, and these were 60 × 10 3 ± 19 × 10 3 and 220 × 10 3 ± 38 × 10 3 , respectively (mean values ± SD; n = 25). The maximum pixel sum for a completely saturated square was approximately 1800 × 10 3 . Bar: 10 µm.
The analysis of root growth, protoxylem position and meristem size in plants in which the cotyledonary leaf has been cut shows a DMTU-reversible early protoxylem differentiation occurring 3 days after injury (Figures 1 and 2), which is independent from variation in meristem size and root growth, which were unchanged (Table 1; Figure 3). A root protoxylem-specific accumulation of H2O2 was detectable 6 h after the injury, supporting its involvement in the variation of protoxylem position In situ H 2 O 2 detection by analysis under LSCM after AUR staining of roots from 7-day-old unwounded control and leaf-wounded seedlings 6 h after injury. The corresponding bright-field and overlay images are shown. Micrographs show the root zone corresponding to the site of appearance of the first protoxylem cell with fully developed secondary cell wall thickenings (arrows) and have been taken at the level of the central root section. Images are representative of those obtained from ten seedlings from five independent experiments. In the red degrading scale, the average values of fluorescence intensity, measured as the sum of the pixels of each 65 µm 2 rectangle, are indicated for unwounded control and leaf-wounded plants, and these were 60 × 10 3 ± 19 × 10 3 and 220 × 10 3 ± 38 × 10 3 , respectively (mean values ± SD; n = 25). The maximum pixel sum for a completely saturated square was approximately 1800 × 10 3 . Bar: 10 µm.

Plant Materials, Treatments and Root Growth Analysis
Arabidopsis seedlings (Columbia-0 ecotype) were grown in vitro in a growth chamber at 23 • C and 55% relative humidity under a photoperiod of 16 h light and 8 h dark. Sterilization of seeds was carried out according to Valvekens et al. [34]. After cold stratification at 4 • C, seeds were grown in one-half-strength Murashige and Skoog salt mixture added with 0.5% (w/v) sucrose and 0.8% (w/v) agar. Plates were kept in vertical position to allow root growth on the solid medium surface. For analysis under LSCM of root protoxylem position and meristem size, 7-day-old seedlings were selected for homogeneity in root length and then transferred onto fresh medium with or without 100 µM DMTU. After the transfer, seedlings were injured by cutting the cotyledonary leaf with scissors, and after 6 h (AUR staining) or 3 days (PI staining), they were collected for analysis under LSCM. The effect of leaf wounding on root growth was evaluated as the difference between the length measured at the onset of the wounding and that measured after 3 days.

Protoxylem Position and Meristem Size Analysis under LSCM by Cell Wall PI Staining and Bright-Field Examination of Root Tissues
Root apices from 10-day-old unwounded control and leaf-wounded seedlings treated or not with 100 µM DMTU for the last 3 days, were incubated for 5/10 min in PI (10 µg mL −1 ) to highlight cell wall and protoxylem [35] and then observed under LSCM using a 488 nm argon laser, with a 600-680 nm band-pass filter and a 40× oil immersion objective. The PI staining was allowed to proceed until protoxylem was completely highlighted. Roots were concurrently analysed by bright-field microscopy, using the same laser beam as described above. To analyse protoxylem maturation, the distance from the root apical meristem of the first protoxylem cell with fully developed secondary wall thickenings was measured following the method described by Ghuge et al. [20] considering the point where a sharp intensification of protoxylem PI staining was detectable as indicative of fully differentiated secondary cell wall thickenings (this point is referred to here as the protoxylem position). Analysis of protoxylem position was validated by the correspondence between the site where the sharp increase in the PI-induced fluorescence was revealed under LSCM and that of protoxylem appearance under bright-field microscope [20]. The length of the meristematic zone was determined by measuring the distance between the quiescent centre and the first elongating cell in the cortex cell file [13,36,37]. The images shown were obtained by aligning serial overlapping micrographs of the same root using Photoshop Software (Adobe, San Jose, CA, USA). Protoxylem position and meristem size were estimated exploiting the Leica Application Suite Advanced Fluorescence software, and then used for statistical analysis.

Hydrogen Peroxide In Situ Detection
To reveal the in situ extracellular H 2 O 2 accumulation, the fluorogenic peroxidase substrate AUR (Molecular Probes, Invitrogen, Carlsbad, CA, USA) was exploited [38], and the fluorescence of the peroxidase reaction product was detected under LSCM in root apices from 7-day-old unwounded control and leaf-wounded seedlings 6 h after injury, as hereafter described. Root apices were stained by incubation in 100 µM AUR for 5/10 min and then observed under LSCM using a 543 nm helium-neon laser with a 550-700 nm band-pass filter. For the measurement of the AUR fluorescence intensity in roots of unwounded control and leaf-wounded plants, five rectangles of approximate 65 µm 2 for each analysed root were drawn over the protoxylem maturation zone and the sum of the pixels corresponding to the fluorescence present in each rectangle was measured exploiting the quantitative analysis of the LAS-AF software used to acquire the confocal images.

Statistics
The analyses under LSCM of protoxylem position, meristem size and H 2 O 2 accumulation after PI and AUR staining, as well as root growth analysis, were performed on five independent experiments on a minimum of ten plants per treatment, yielding reproducible results. Images from single representative experiments are shown. Statistical tests of protoxylem position, meristem size and root growth were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) with one-way ANOVA. The statistical significance of differences was evaluated by p levels as follows: ns, not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; and ****, p ≤ 0.0001. The average values of fluorescence intensity for unwounded control and leaf-wounded plants were obtained by analysing five roots for treatment, and five rectangles of approximately 65 µm 2 for each analysed root.