FLIM-based detection of early wound-response signatures suggesting rooting hotspots in Argania spinosa

Abstract

Root regeneration from stems requires cells to reprogram their developmental trajectories. This reprogramming becomes possible when multiple factors converge at specific sites, ultimately giving rise to adventitious roots in what often appears to be a random pattern. Here, we asked whether such rooting `hotspots' could be detected by fluorescence-lifetime imaging microscopy. Using stem cuttings from a difficult-to-root Argan tree (Argania spinosa) clone, we examined wounded tissues by microscopy. Upon 440 nm illumination, discrete and asymmetric regions emerged in which fluorescence events with lifetimes below 2 ns increased within 3 h. Further, we show that similar lifetime signal appeared both around newly initiating root primordia and along what seemed to be their outward pathway through the stem tissues. These observations suggest that early, localized modifications in cell wall composition occur prior to visible root formation and may mark sites with the competence to initiate adventitious roots.

Graphical Abstract

Graphical Abstract.

Introduction

Adventitious roots are post-embryonic roots that originate from non-root tissues, either as part of a natural developmental program or in response to diverse stress signals (Steffens and Rasmussen 2016). The tissues capable of giving rise to adventitious roots differ among species and organs and may include the cambium between vascular bundles, the pericycle, phloem parenchyma, inner cortical parenchyma or non-differentiated secondary phloem (Fahn 1990, Bellini et al. 2014, Wan et al. 2023). These sites are consistently located near the xylem and phloem of the originating organ, enabling successful vascular reconnection with the newly formed root.

In contrast to lateral roots—which initiate following a defined spatiotemporal pattern regulated by auxin signaling in the root apex (De Smet et al. 2007, Moreno-Risueno et al. 2010)—adventitious roots tend to arise at seemingly random positions (Bellini et al. 2014, Verstraeten et al. 2014). Identifying the early events that mark tissues competent to form adventitious roots therefore remains a major challenge and represents the focus of this study.

A key limitation in vegetative propagation of elite tree genotypes is the progressive decline in rooting capacity during maturation (Bellini et al. 2014, Verstraeten et al. 2014), a longstanding challenge for both growers and researchers (Bellini 2024, Roth et al. 2024). Alterations in cell wall composition have been associated with rooting ability (Duman et al. 2020, Pizarro and Díaz-Sala 2021), raising the question of whether detectable cell wall changes may mark sites of adventitious root initiation.

Fluorescent molecules provide a powerful means to explore this question, as their abundance, distribution and synthesis dynamics can be monitored using confocal microscopy and fluorescence lifetime imaging microscopy (FLIM) (Donaldson 2020, Maceda and Terrazas 2022). FLIM quantifies fluorescence lifetime—the duration a fluorophore remains in an excited state before photon emission—which reflects specific molecular structures and is influenced by subcellular conditions. Accordingly, spatial and temporal shifts in fluorescence lifetime may signal early events that precede cellular differentiation.

In this study, we used stem cuttings of argan tree, a rooting-recalcitrant species (Mazri et al. 2022, Tzeela et al. 2022) to examine whether autofluorescence signatures correlate with early events leading to adventitious root formation.

Materials and methods

Plant material and growth conditions

Argania spinosa clones, ARS14 which is difficult to root and ARS7 which is very difficult to root (Tzeela et al. 2022), were cultivated in pots containing coconut fiber/pit/slow released fertilizer mixture (RAM158 (TUF Merom Golan)/granulated polystyrene 9:1 (v/v)) under greenhouse conditions. The greenhouse climate control provided 25–28 °C and 70% shadow. Stem cuttings were collected from the current year’s growth, 15–20 cm in length and bearing the apical meristem. Transverse sections (~0.3–0.5 mm) were manually prepared from basal internodes (1–4 cm above the base) using a razor blade. The experiment was repeated eight times for ARS14 and three times for ARS7.

For analysis of adventitious-root primordium induction, seeds of ARS14 were mechanically cracked and germinated in a soil mixture (4:1 (v/v) vermiculite/RAM158). After 1 month, seedlings having stems exceeding 10 cm in height were cut at the level of the hypocotyl or epicotyl. The bases of the cuttings were immersed for 1 min in a solution of indole-3-butyric acid potassium salt (K-IBA, 1 g L⁻¹, dissolved in ddH₂O) and planted in a soil mixture (3:2:1 (v/v) granulated polystyrene/vermiculite/RAM158) on rooting tables. The tables were heated to 25 °C in the winter, covered with nonwoven fabric and humidity was kept at 90% by fog sprayers operated for 10 s every 10 min from 6 a.m. to 6 p.m. This experiment was repeated three times (18 epicotyl and 22 hypocotyl cuttings).

Slide preparation and microscopy

Untreated, freshly collected cuttings were first imaged immediately (0 min) after immersion in distilled water. The water was then removed with filter paper, and 0.1 M 2-(N-morpholino) ethane sulfonic acid (MES) buffer (pH 5.6) was added. Samples were subsequently imaged at 180 min. Sections prepared from auxin induced juvenile cuttings were immersed in distilled water on glass slides.

Image acquisition was done using a Stellaris 8 FALCON (Fast Lifetime Contrast) FLIM laser scanning microscope (Leica, Wetzlar, Germany), equipped with a White Light Laser (WLL) with pulse frequency 80 MHz (12.5 ns), HC PL FLUOTAR 5×/0.15 objective (Leica, Wetzlar, Germany) and Leica Application Suite X software (LASX, Leica, Wetzlar, Germany). Autofluorescence life time of stem sections was initially excited using 440, 492, 575, 600 nm and detected within the range of 480–700 nm depending on excitation wavelength. Eventually 440 nm was used. Image acquisition parameters were as follows; resolution 512 × 512, 75% laser intensity for 440 and 492 nm excitation light, 25% for 575 and 600 nm excitation light, line accumulation X10.

Results

Changes in fluorescence lifetime of stem tissues after wounding in relation to adventitious root formation

To determine whether specific fluorescence lifetime changes occur after wounding, we used the A. spinosa line ARS14 and first tested several excitation wavelengths. Excitation at 440 nm (the detection range was 480–590 nm) consistently revealed a light-blue domain in the heat map color coded image of fluorescence life time, indicating reduced fluorescence lifetime that appeared in the peripheral young xylem adjacent to the cambial region as early as 1 h after wounding and increased 3 h after wounding. This effect was asymmetric and did not involve all cells within the affected layers (Figure 1A–E). Among 33 analyzed cuttings of ARS14, 16 cuttings demonstrated the fluorescence increase while in 7 cuttings it was not detected. In ARS7, a highly recalcitrant-to-root clone of A. spinosa, no comparable changes in fluorescence lifetime were observed under similar conditions (Figure 1F–H). To further characterize the fluorescence lifetime changes observed in ARS14 at time 0 and after 180 min, phasor plot analysis was performed (Figure 1I–L), comparing an affected region of interest (ROI; inset in Figure 1M) with an unaffected ROI from the same section (inset in Figure 1N). The fluorescence signal displayed multi-exponential behavior, indicating contributions from multiple fluorophores. Quantitative analysis of the fluorescence lifetime distribution revealed a pronounced increase in events with lifetimes in the range of 0.029–2 ns in the affected ROI (Figure 1M). Although short-lifetime fluorescence also increased in the unaffected ROI, the magnitude of this increase was substantially lower (Figure 1N). Such localized, asymmetric reductions in fluorescence lifetime near the cambium may reflect early cellular events associated with changes in cell wall composition. To link this observation more directly with root formation, we needed a system in which multiple primordia develop simultaneously. For this purpose, we used highly juvenile tissues—hypocotyl and epicotyl segments from young seedlings germinated from seeds. After auxin treatment, sections were collected as soon as the cutting bases began to swell and were examined for both root primordia and fluorescence lifetime patterns. Application of 1 g L⁻¹ K-IBA was sufficient to trigger the initiation of multiple roots (9 out of 22 hypocotyls and 6 out of 18 epicotyls contained root primordia). Adventitious root primordia were observed in 7–8 days at the level of hypocotyls (Figure 2C) and in 14–16 days at the level of epicotyls (Figure 2A and B).

Figure 1.

Autofluorescence lifetime in A. spinosa stem cuttings following wounding. (A) ARS14 cutting imaged in this experiment. (B and C) Fluorescence intensity and fluorescence lifetime at time 0. (D and E) Fluorescence intensity of the same section 180 min after wounding. (F) Fluorescence lifetime of ARS7 at time 0. (G) Fluorescence lifetime of the same ARS7 section 180 min after wounding. (H) ARS7 cutting imaged. (I–L) Phasor plots of affected and unaffected regions of interest (insets in M and N) at time 0 and 180 min after wounding. (M and N) Fluorescence lifetime distributions within the same region of interest. The affected area corresponds to the region displaying the bluish lifetime signal (arrow in E).

Figure 2.

Short-lifetime fluorescence is likely associated with adventitious root formation. (A) An asymmetrical pre-accumulation of short-lifetime fluorescence is observed in the young xylem (arrow 1). The corresponding transmitted-light image (middle) and the epicotyl (right) are shown. (B) An early root primordium is surrounded by the same short-lifetime fluorescence (arrows 2 and 3). In the same section, a more developed primordium appears without this surrounding signal (arrow 6). This section corresponds to the epicotyl shown. (C) At an intermediate developmental stage, the primordium remains encased in an `envelope' of short-lifetime fluorescence that extends outward, delineating a potential path for further growth (arrows 4 and 5). This section corresponds to the hypocotyl shown on the right. Color scales indicate fluorescence lifetimes (ns).

The sections of both hypocotyls and epicotyls displayed a range of developmental stages of adventitious root primordia (Figure 2A–C). In Figure 2A-1, an asymmetric accumulation of bluish signal—with lifetimes of 1 ns or less—is visible. Figure 2B-2 and B-3 show two early root primordia surrounded by this reduced-fluorescence lifetime pattern. In the more advanced primordia shown in Figure 2C-4 and C-5, the bluish signal outlines the primordia and extends into the overlying tissues, marking what appears to be the emerging pathway through which the young root will grow. By contrast, in a mature primordium (Figure 2B-6), the bluish lifetime signal is no longer detectable.

Discussion

Here, we show that changes in cell wall fluorescence lifetime may mark early events in adventitious root formation. Cell wall autofluorescence mainly originates from lignin-associated phenolics in secondary cell walls. Lignin exhibits excitation peaks between 360 and 465 nm, emits in the visible range (400–700 nm) and displays fluorescence lifetimes from 0.07 to 12.7 ns (Radotić et al. 2006). Importantly, lignin fluorescence lifetime varies with its compositional profile; for example, the Arabidopsis thaliana c4h-3 mutant, with an increased syringyl/guaiacyl (S/G) ratio, shows reduced lifetimes compared with wild type plants (Van Acker et al. 2013, Escamez et al. 2021).

Lignin also interacts extensively with hemicellulose and pectin (Delmer et al. 2024), and these associations influence fluorescence lifetime. Increased lignin–carbohydrate interactions are linked to more loosely packed lignin and longer lifetimes, whereas densely packed lignin domains exhibit shorter lifetimes (Zeng et al. 2015). Consistently, Arabidopsis gux1gux2 mutant plants lacking xylan side chains required for lignin attachment show increased lignin fluorescence lifetimes (Escamez et al. 2021). Together, these observations suggest that the increased occurrence of short lifetimes (0.029–2 ns) in our study reflects changes in lignin composition and/or structural organization.

These changes may arise from modification of pre-existing lignin or rapid deposition of stress lignin, which is enriched in p-hydroxyphenyl units and more condensed (Cesarino 2019) and can be deposited within hours (Xu et al. 2024). Lignin remodeling near the cambial region may alter cell wall properties, providing mechanical or biochemical cues that trigger cambial cell reprogramming toward adventitious root formation. At later stages, reinforced cell walls may also help guide and protect emerging root primordia.

Consistent with these observations, previous studies have demonstrated that cell wall properties play a critical role in adventitious root formation (Abu-Abied et al. 2015, Duman et al. 2020, Pizarro and Díaz-Sala 2021). In particular, dynamic changes in lignin composition and distribution have been reported during this process (Syros et al. 2004, Li et al. 2009, Wamhoff et al. 2024), and such changes were shown to distinguish between easy- and difficult-to-root subspecies (Wamhoff et al. 2024). In addition, we cannot exclude the possibility that local bursts of reactive oxygen species triggered by wounding (Vilasboa et al. 2020) contributed to the observed fluorescence changes.

Our findings provide a foundation for future investigations in difficult-to-root tree species, combining correlative fluorescence and mass spectrometry imaging approaches (Liu et al. 2025) to uncover in situ chemical changes associated with root regeneration.

Author contributions

A.K. performed all experiments and participated in writing the manuscript; S.Y. cultivated the plant material; E.B. assisted with microscopy; and E.S. designed the experiments and wrote the paper.

Conflict of interest

There are no conflicts of interest to declare.

Funding

The research was funded by grant number 20-01-0072 from the Ministry of Agriculture of Israel and grant number IS-5598-23 from the Binational Agricultural Research and Development Fund (BARD) for E.S. The authors thank the Ministry of Aliyah and Integration for partially funding the fellowship of A.K.

Data availability

All data are available.