Phytochrome Mediates Light Signal for Cortical Microtubule Randomization that Enables
Root Hair Formation in Lettuce Seedlings
Wakana Harigaya and Hidenori Takahashi*
Department of Biology, Faculty of Science, Toho University, 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan
Received September 26, 2018; accepted October 26, 2018
Summary When hydroponically cultured lettuce seedlings are transferred from a medium at pH 6.0 to another one at pH 4.0, the formation of root hairs takes place. Previous studies have revealed that this low-pH-induced root hair formation requires a phytochrome-mediated light signal and randomization of the existing transverse cortical microtubule (CMT) arrays in the future hair-forming cells, which occurs before the bulge formation. Here, we investigated the relationship between these two requisites, i.e., whether phytochrome is involved in CMT randomization. To test this, seedlings were cultured throughout in the dark except the various periods and timings of red (R) or far-red (FR) light irradiation. In seedlings that had already been transferred to pH 4.0 me-dium, R light irradiation induced CMT randomization, whereas neither FR light irradiation nor continuous dark culturing instead of R light irradiation did. R light irradiation during the pre-culture at pH 6.0 also caused CMT randomization later when the seedlings were transferred to pH 4.0 medium. Irradiation of FR light immediately after the R light irradiation suppressed the R light-induced CMT randomization, regardless of whether R/FR ir-radiation was carried out before or after the transfer of seedlings to pH 4.0 medium. The efficacy of R light and R/FR photo-reversibility in the induction of CMT randomization proves that the light signal needed for CMT randomization was indeed mediated by phytochrome, during lettuce root hair formation.
Key words Cortical microtubule, Lettuce, Photo-reversibility, Phytochrome, Root hair.
Root is an indispensable organ for the growth and development of plants. It not only acquires water and nutrients but also serves to anchor the plants to the soil and participate in the environmental interactions around the root (Bibikova and Gilroy 2003, Schmit and Gaudin 2017). Root hairs are specialized cylindrical structures that are projected from root epidermal cells (Dolan et al. 1993). The presence of root hairs helps increase the sur-face area of a root, which allows the root to effectively uptake water and nutrients from the surroundings (Gri-erson et al. 2014). The root hairs are formed through two distinctive stages, i.e., root hair initiation, when a small bulge appears in the outer cell wall and root hair elongation from the bulge, which is not accompanied by cell division.
It is well-known that plant hormones ethylene and auxin promote the formation of root hairs (Tanimoto et al. 1995, Leyser et al. 1996, Masucci and Schiefel-bein 1996, Pitts et al. 1998, Zhu et al. 2006, Feng et al. 2017). Besides these internal regulators of the plants, an external light condition also affects the formation of root hairs. Cutter (1969) found that the aquatic plant Elodea formed root hairs only in the dark. Phenotypic analyses of Arabidopsis mutants for phytochromes A and B sug-
gest involvement of phytochromes in root hair formation (Reed et al. 1993, De Simone et al. 2000a, Shin et al. 2010).
Another key regulator of root hair formation is rep-resented by the cortical microtubule (CMT) arrays in root epidermal cells. Initiation of root hair is associated with the disruption or rearrangement of CMTs in the cells (Emons and Derksen 1986, Bibikova et al. 1999, Baluska et al. 2000, Bao et al. 2001, Van Bruaene et al. 2004). Importance of the appropriate CMT organization for root hair initiation is supported by the study of Bao et al. (2001), in which, reduced α-tubulin gene expres-sion caused ectopic or multiple root hairs in Arabidopsis.
Using lettuce seedlings, Inoue and Hirota (2000) found a useful “all-or-none” phenomenon concerning root hair induction, which made it suitable for investi-gation of the mechanism of root hair formation. When hydroponically cultured lettuce seedlings on pH 6.0 medium were transferred to pH 4.0 medium, the forma-tion of root hairs was observed, whereas no root hairs formed when they were transferred to pH 6.0 medium. In the seedlings on pH 4.0 medium, root epidermal cells respond to low pH and small bulges appear 4 h after the medium change (Inoue and Hirota 2000). Prior to the bulge formation, CMT arrays in future hair-forming cells show a remarkable change with low pH stimulus as a trigger. The arrays are transverse to the longitudinal
axis of the cell (i.e., the root axis) during the pre-culture at pH 6.0. They begin reorienting 5 min after the transfer to pH 4.0 medium, and the distribution becomes random within 30 min. However, CMTs in the control seedlings, which were transferred to pH 6.0 medium, maintain the transverse arrays (Takahashi et al. 2003a).
As in other plants, light is also an influential factor in lettuce root hair formation, because no root hairs were formed in the dark, even if the seedlings were transferred to pH 4.0 medium (De Simone et al. 2000c, Takahashi and Inoue 2008). De Simone et al. (2000b) found that R light is effective for root hair initiation, whereas FR light is not. Furthermore, this induction by R light was canceled when it was followed by FR light irradiation. These results suggest an involvement of phy-tochrome in the root hair initiation in lettuce. Takahashi and Inoue (2008) revealed that the CMT randomization needs light as well as low pH stimulus and that it does not occur in the dark. However, it is still unclear whether phytochrome participates in CMT randomization, an es-sential factor for root hair initiation in lettuce.
Therefore, in this study, we investigated whether phytochrome mediates the light signal for CMT ran-domization during lettuce root hair formation. We used dark-grown seedlings and examined the effects of R and FR light irradiation on CMT organization in their root epidermal cells.
Materials and methods
Plant material and culture conditionsLettuce (Lactuca sativa L. ‘Grand Rapids’) seeds were
immersed in distilled water for 3 h under white light (7.5 W m-2) to induce germination. After the incubation at 4°C in the dark for 24 h to synchronize the germina-tion, they were pre-cultured at 25°C for 24 h on a nylon mesh stretched on culture medium (Inoue et al. 2000) at pH 6.0. Germinated seedlings were transferred to pH 4.0 medium together with the nylon mesh, followed by further culturing. The seedlings were irradiated with R or FR light for an appropriate period at appropriate timing based on the experiment or kept in the dark as a control. Handling of seeds and seedlings was performed under a dim green safety light (De Simone et al. 2000c). Except for the periods of handling and light irradiation, they were cultured in the dark. For details of the timing and period of R and FR light irradiation, refer to the cor-responding figure legends.
Light sourcesR light (6.6 W m-2) was obtained by using combination
of white fluorescent lamps (FL20SS-BRN/18, Toshiba Lighting and Technology Corp., Tokyo) and a 3-mm thick red acrylic resin filter (Acrylite no. 102, Mitsubishi Rayon Co. Ltd., Tokyo). FR light (6.7 W m-2) was ob-tained by using a combination of FR fluorescent lamps
(FL20S FR-74, Toshiba Lighting and Technology Corp., Tokyo) and a 3-mm thick black acrylic resin filter (Dela-glas A900, Asahi Chemicals Co. Ltd., Tokyo).
Microscopic observationFor examination of root hair initiation, the seedlings
were harvested 12 h after the change of the medium such that the initiation, if observed, had saturated, and were observed with an inverted microscope (CKX41, Olym-pus Corp., Tokyo).
For CMT observation, seedlings were harvested at appropriate time points in each experiment (for details of the timing, refer to the corresponding figure legends). Their roots were subjected to indirect immunofluores-cent staining with monoclonal anti-α-tubulin antibody (Sigma-Aldrich Co. LLC., St. Louis, MO), as described by Takahashi et al. (2003a). The CMT images were observed using a confocal laser microscope (Fluoview FV500, Olympus Corp., Tokyo) with PlanApo 60× ob-jective lens (Olympus Corp., Tokyo). Angles between the long axis of each root epidermal cell and each CMT array in the corresponding cell were measured using Im-ageJ software (U.S. National Institutes of Health, Mary-land). Prior to the measurement, CMT images were ro-tated on a computer monitor so that the long axis of the corresponding cell became horizontal. All CMTs in the cell were manually traced and labeled every time they were traced in order to avoid duplication of measure-ment. The angles were then automatically measured by a function of the software and they were classified into nine types, from 0 to 180° at intervals of 20°. Percent-ages of each CMT type were determined in each cell.
Results
Effect of R or FR light irradiation on root hair initiationAfter the seedlings were pre-cultured on pH 6.0 me-
dium for 24 h in the dark, the medium was replaced with a fresh one at pH 4.0. Subsequent to the medium change, the seedlings were kept in the dark for a period of 3 h, at the end of which, the seedling sensitivity to light reaches the maximum level (De Simone et al. 2000c). They were then irradiated with R or FR light for 1 h and kept in the dark until 8 h after the end of the irradiation (Fig. 1), when root hair initiation if observed, saturates (Inoue and Hirota 2000). In the case of the seedlings irradiated with R light, root hair bulges were observed on the root surface. In contrast, no or few bulges were observed on the seedlings irradiated with FR light or on the control seedlings kept throughout in the dark without light ir-radiation.
CMT organization in the R light or FR light irradiated seedlings
In all the culture conditions we had examined previ-ously, transverse CMT arrays, which were observed
2019 Phytochrome and CMT Randomization in Lettuce 55
during the pre-culture at pH 6.0, were destroyed when-ever root hairs formed, whereas they were maintained when root hairs did not form (Takahashi et al. 2003a, c, Takahashi and Inoue 2008). To examine the CMT organization in the seedlings irradiated with R or FR light, seedlings cultured in the same condition as in Fig. 1 were subjected to indirect immunofluorescent micros-copy (Fig. 2). Just before the irradiation, about half of the total CMT arrays were transverse (80–100°) to the longitudinal axis of the root epidermal cell (Fig. 2a, b). After the R light irradiation, the percentage of the trans-verse arrays decreased to 32.2% (Fig. 2e, f). In contrast, FR light irradiation did not cause a significant change in CMT organization: about half the arrays were still trans-verse even after the irradiation (Fig. 2g, h). In the control seedlings that were kept in the dark without irradiations, about half the CMT arrays were transverse again (Fig. 2c, d), similar to the FR light-irradiated seedlings (Fig. 2g, h). These results indicated that CMT randomiza-tion was induced by R light but not by FR light or in the dark, suggesting a probable involvement of phytochrome in CMT randomization.
The effect of FR light irradiation following R light irra-diation on CMT organization
To obtain evidence for the involvement of phyto-chrome in CMT randomization, the R light-irradiated dark-grown seedlings were immediately subjected to FR light irradiation. If phytochrome mediates a light signal for CMT randomization, the FR light would cancel the induction of CMT randomization that was initiated by R light. As preparation for CMT randomization progresses moment by moment in the cell during the R light ir-radiation, and we could no longer stop the progress if it reaches to a certain stage, the R light irradiation period should be short while examining R/FR photo-revers-ibility. Thus, we shortened the period of R light irradia-tion to 1 min and investigated whether it was enough to cause the CMT randomization. Transverse CMT arrays decreased to 33.8% by 1 min R light irradiation (Fig. 3a, b). This value was comparable to that of the seedlings subjected to 1 h R light irradiation (Fig. 2e, f), indicating that 1 min R light irradiation was enough to change the CMT organization. Therefore, R light irradiation period was set for 1 min in the following photo-reversibility experiments.
Fig. 1. Effects of R and FR light irradiation after the medium change on root hair initiation. After the pre-culture at pH 6.0 in the dark, seedlings were transferred to pH 4.0 medium and were kept in the dark for 3 h to maximize their sensitivity to light (De Simone et al. 2000c). The seedlings were then irradiated with R or FR light for 1 h, and further cultured for 8 h in the dark. As a control, seedlings were kept in the dark (D) for 1 h instead of light irradiation and were further cultured for 8 h in the dark. (A) Schematic representation of the culture condition. Black boxes indicate culturing in the dark, whereas white and gray boxes indicate R and FR light irradiation, respectively. On the time axis, the time point of seed-ling transfer to pH 4.0 medium is represented as 0 h. Note that the width of the boxes does not proportionally reflect the time length. (B) Root images. Arrowhead points to the root hair bulge. Scale bar=100 µm.
56 W. Harigaya and H. Takahashi Cytologia 84(1)
When the 1 min R light irradiation was followed by 59 min FR light irradiation (i.e., total 1 h irradiation), the percentage of transverse CMT arrays was 37.2% (Fig. 3c, d). This value was higher than that of the seedlings irradiated only with 1 min R light (Fig. 3a, b). However, it was a little lower compared with the control seedlings that were continuously maintained in the dark (Fig. 2c, d). This could be because the induction of the CMT randomization progressed so quickly with the onset of R light irradiation that FR light irradiation as early as 1 min after the beginning of R light irradiation could not suppress the progress enough.
The effect of R light irradiation before the pH 4.0 treat-ment
The unsatisfactory result of the photo-reversibility
Fig. 2. CMT organization in root epidermal cells of the seedlings irradiated with R or FR light during pH 4.0 treatment. After the pre-culture at pH 6.0 in the dark, seedlings were trans-ferred to pH 4.0 medium and were kept in the dark for 3 h. The seedlings were then irradiated with R or FR light for 1 h and were subjected to indirect immunofluorescence micros-copy using anti-α-tubulin antibodies. As controls, seedlings just before the light irradiation (0 h) and those kept in the dark (D) for 1 h instead of light irradiation were observed. (A) Schematic representation of the culture condition. Black boxes indicate culturing in the dark, whereas white and gray boxes indicate R and FR light irradiation, respectively. On the time axis, the time point of seedling transfer to pH 4.0 medium is represented as 0 h. Note that the width of the boxes does not proportionally reflect the time length. (B) a, c, e, g: CMT images. Scale bar=20 µm. b, d, f, h: The angles between the CMTs and the long axes of the root epidermal cells of at least 5 seedlings. The angles were classified into nine types from 0 to 180°, with an interval of 20°. The num-ber of microtubules (%) shown are mean values±standard error. An asterisk indicates a significant difference from 0 h sample (b) at p<0.05 based on Dunnett’s test.
Fig. 3. CMT organization in root epidermal cells of the seedlings irradiated with R/FR light during pH 4.0 treatment. After the pre-culture at pH 6.0 in the dark, seedlings were trans-ferred to pH 4.0 medium and were kept in the dark for 3 h. The seedlings were then irradiated with R light for 1 min and kept in the dark (D) for 59 min. Alternatively, the seed-lings were irradiated with R light for 1 min and subsequently with FR light for 59 min. They were then subjected to indi-rect immunofluorescence microscopy using anti-α-tubulin antibodies. (A) Schematic representation of the culture con-dition. Black boxes indicate culturing in the dark, whereas white and gray boxes indicate R and FR light irradiation, respectively. On the time axis, the time point of seedling transfer to pH 4.0 medium is represented as 0 h. Note that the width of the boxes does not proportionally reflect the time length. (B) a, c: CMT images. Scale bar=20 µm. b, d: The angles between the CMTs and the long axes of the root epidermal cells of at least 18 seedlings. The angles were classified into nine types from 0 to 180°, with an interval of 20°. The number of microtubules (%) shown are mean values±standard error. An asterisk indicates a significant difference from R1→D59 sample (b) at p<0.05 based on Student’s t-test.
2019 Phytochrome and CMT Randomization in Lettuce 57
experiment in Fig. 3 suggested that triggering of CMT randomization should be completely suppressed during R light irradiation to achieve a clearer photo-reversibil-ity of its induction by R/FR irradiation. We, therefore, changed the irradiations timing from the one during the pH 4.0 treatment to the one before it. Because the root hair formation of lettuce seedlings needs both low pH and a light signal (Takahashi et al. 2003a, Takahashi and Inoue 2008), R light irradiation during the pre-culture at pH 6.0 is not expected to induce CMT randomization.
First, we examined whether R light irradiation during the pre-culture at pH 6.0 in the dark could induce CMT randomization later when the seedlings were transferred to pH 4.0 medium. At the same time, we searched for an effective timing of the R light irradiation. Dark-grown seedlings were irradiated with R light for 1 h at 4, 2, or 1 h before the end of the pre-culture at pH 6.0 for 24 h, and CMT arrays were observed at 30 min after the trans-fer to pH 4.0 medium. The transverse CMT arrays de-creased to about 30% in all these conditions, irrespective of the irradiation timing (Fig. 4c–h). This percentage was comparable with that in the case of R light irradia-tion at 3 h after the medium change (Fig. 2e, f). On the contrary, about half of the CMTs remained in a trans-verse position in the control seedlings kept in the dark (Fig. 4a, b). These results suggest that R light irradiation before the pH 4.0 treatment can induce CMT randomiza-tion later when the pH of the medium is lowered.
Prior to the examination of R/FR photo-reversibility during the pre-culture, we further examined the effect of R light irradiation with shorter periods (1 or 2.5 min), both of which started 1 h before the end of the pre-cul-ture. The decrease in the pre-existing transverse CMT array was observed in both conditions at 30 min after the transfer to the pH 4.0 medium (Fig. 5), the degree of which was comparable to each other irrespective of the irradiation period. Furthermore, it was also comparable with that of the seedlings irradiated for 1 h (Fig. 4h), suggesting that 1 min R light irradiation is enough to cause CMT randomization. Therefore, R light irradiation period was set as 1 min in the following R/FR photo-reversibility experiments.
The effect of FR light irradiation following R light irra-diation before the pH 4.0 treatment
Dark-grown seedlings were irradiated with R light for 1 min at 1 h before the transfer to pH 4.0 medium and were immediately subjected to FR light irradiation for different time periods (1, 2, or 59 min). Alternatively, the R light irradiated seedlings were kept in the dark for 59 min without FR irradiation. At 30 min after the transfer to pH 4.0 medium, CMT organization in these seedlings was observed (Fig. 6). In case of the 1 min FR light irradiation (Fig. 6c, d), the percentage of transverse CMT arrays was as low as in the control seedlings ir-radiated only with R light (Fig. 6a, b), indicating that
Fig. 4. CMT organization in root epidermal cells of the seedlings irradiated with R light at various timings before pH 4.0 treatment. During the pre-culture at pH 6.0 for 24 h in the dark, seedlings were irradiated with R light for 1 h at 4, 2, or 1 h before the transfer to pH 4.0 medium. As a control, seedlings were pre-cultured throughout in the dark. After the transfer to pH 4.0 medium, these seedlings were kept in the dark for 30 min and were subjected to indirect immuno-fluorescence microscopy using anti-α-tubulin antibodies. (A) Schematic representation of the culture condition. Black and white boxes indicate culturing in the dark and R light irradi-ation, respectively. On the time axis, the time point of seed-ling transfer to pH 4.0 medium is represented as 0 h. Note that the width of the boxes does not proportionally reflect the time length. (B) a, c, e, g: CMT images. Scale bar=20 µm. b, d, f, h: The angles between the CMTs and the long axes of the root epidermal cells of at least 7 seedlings. The angles were classified into nine types from 0 to 180°, with an inter-val of 20°. The number of microtubules (%) shown are mean values±standard error. Asterisks indicate significant differ-ences from the dark sample (b) at p<0.05 based on Dunnett’s test.
58 W. Harigaya and H. Takahashi Cytologia 84(1)
1 min FR irradiation was insufficient to cancel the effect of R light. On the other hand, FR light irradiation for 2 min resulted in a larger percentage of transverse CMT arrays compared with the control seedlings (Fig. 6c, d). When the period of FR light irradiation was prolonged to 59 min, the decrease in transverse CMT arrays was suppressed even more, and its percentage was as high as 40.8% (Fig. 6g, h). This value was larger than that of the seedlings subjected to R/FR irradiation after the transfer to pH 4.0 medium (Fig. 3c, d).
Discussion
Involvement of phytochrome in CMT randomizationPreviously, we had reported that the CMT random-
ization, which is needed for lettuce root hair forma-
tion, requires light (Takahashi and Inoue 2008). In this study, we examined whether the light signal for CMT randomization is mediated by phytochrome. When the
Fig. 5. CMT organization in root epidermal cells of the seedlings irradiated with R light of various periods before the pH 4.0 treatment. During the pre-culture at pH 6.0 for 24 h in the dark, seedlings were irradiated with R light for 1 or 2.5 min, at 1 h before the medium change, and were then kept in the dark until the pre-culture period reached totally 24 h. After the transfer to pH 4.0 medium, these seedlings were kept in the dark for 30 min and were subjected to indirect immuno-fluorescence microscopy using anti-α-tubulin antibodies. (A) Schematic representation of the culture condition. Black and white boxes indicate culturing in the dark and R light irradi-ation, respectively. On the time axis, the time point of seed-ling transfer to pH 4.0 medium is represented as 0 h. Note that the width of the boxes does not proportionally reflect the time length. (B) a, c: CMT images. Scale bar=20 µm. b, d: The angles between the CMTs and the long axes of the root epidermal cells of at least 9 seedlings. The angles were clas-sified into nine types from 0 to 180°, with an interval of 20°. The number of microtubules (%) shown are mean values± standard error. A significant difference was not observed between R1 (b) and R2.5 (d) samples at p<0.05 based on Student’s t-test.
Fig. 6. CMT organization in root epidermal cells of the seedlings irradiated with R/FR light before the pH 4.0 treatment. Dur-ing the pre-culture at pH 6.0 for 24 h in the dark, seedlings were irradiated with R light for 1 min, then with FR light for 0, 1, 2, or 59 min at 1 h before the medium change and were kept in the dark until the pre-culture period, for a total of 24 h. After the transfer to pH 4.0 medium, these seedlings were kept in the dark for 30 min and were subjected to indi-rect immunofluorescence microscopy using anti-α-tubulin antibodies. (A) Schematic representation of the culture con-dition. Black boxes indicate culturing in the dark, whereas white and gray boxes indicate R and FR light irradiation, respectively. On the time axis, the time point of seedling transfer to pH 4.0 medium is represented as 0 h. Note that the width of the boxes does not proportionally reflect the time length. (B) a, c, e, g: CMT images. Scale bar=20 µm. b, d, f, h: The angles between the CMTs and the long axes of the root epidermal cells of at least 9 seedlings. The angles were classified into nine types from 0 to 180°, with an inter-val of 20°. The number of microtubules (%) shown are mean values±standard error. An asterisk indicates a significant difference from R1 sample (b) at p<0.05 based on Dunnett’s test.
2019 Phytochrome and CMT Randomization in Lettuce 59
dark-grown seedlings were irradiated with R or FR light after their transfer to pH 4.0 medium, CMT randomiza-tion was induced by R light but not by FR light (Fig. 2). This induction by R light was canceled by the follow-ing irradiation of FR light (Fig. 3). The similar effect was observed for each of R and FR irradiations, when they were carried out before lowering the pH of the medium (Figs. 4–6). De Simone et al. (2000c) reported that phytochrome plays an essential role in lettuce root hair formation, as in Arabidopsis. However, how phyto-chrome participates in the process had not been clarified. Furthermore, although reorganization of CMT arrays during root hair formation and the effect of light condi-tion on root hair formation have been reported in other plants (Emons and Derksen 1986, Bibikova et al. 1999, Baluska et al. 2000, De Simone et al. 2000a, Bao et al. 2001, Van Bruaene et al. 2004, Shin et al. 2010), the re-lationship between the two had remained unknown. Our finding that phytochrome controls CMT reorganization prior to bulge formation leads to the hypothesis that the increase in Arabidopsis root hair numbers in light (De Simone et al. 2000a) could have resulted from enhanced CMT reorganization through phytochrome signaling, similar to the case of lettuce.
Realignment of plant's CMT arrays is induced by vari-ous environmental and developmental factors including light (Cyr 1994, Le et al. 2005, Lian et al. 2017, Ma et al. 2018). Although there are some reports about the involvement of phytochrome in the reorganization of CMT arrays (Zandomeni and Schopfer 1993, Fischer and Schopfer 1997, Ma et al. 2018), these studies were conducted on the aerial parts of the plants. Our results provide a novel finding which shows involvement of phytochrome in the regulation of CMT arrays in roots.
Interestingly, it is a root apical portion among the plant body of lettuce seedlings that perceives light for root hair formation (De Simone et al. 2000b). Recent studies on Arabidopsis have revealed that not only shoots but also roots undergo photomorphogenesis, and that root itself can perceive light (Usami et al. 2004, Correll and Kiss 2005, Dyachok et al. 2011, Lee et al. 2017). A significant level of phytochrome gene expres-sion was observed in roots (Johnson et al. 1991, Somers and Quail 1995, Goosey et al. 1997, Tóth et al. 2001, Salisbury et al. 2007, Lee et al. 2016). Furthermore, it is not the mesophyll-localized phytochrome but the root-localized one that is needed for the formation of root hairs in Arabidopsis (Oh et al. 2014). These findings suggest that phytochrome exists in lettuce root apex as well. Since root apex is a place where CMT randomiza-tion occurs in lettuce seedling root hair formation (Inoue and Hirota 2000, Takahashi et al. 2003a), the existence of phytochrome in the same portion is convenient for the lettuce seedlings to promptly control the CMT organiza-tion in response to the light condition. CMT randomiza-tion and the following root hair formation could be just
one of the events concerning photomorphogenesis in lettuce root.
Effect of the irradiation timing on CMT randomizationIn this study, light irradiations were performed in
either of the following two timings: after or before low-ering the pH of the medium. Transverse CMT arrays decreased by R light irradiation and their resultant per-centages were comparable, irrespective of the irradiation timing [“after”: 33.8% (Fig. 3a, b) and “before”: 33.4% (Fig. 6a, b)]. However, the canceling effect against this decrease by the following FR light irradiation showed a minor difference between the two irradiation timings. FR light had larger canceling effect in case of “before” irradiation (Fig. 6g, h) than “after” irradiation (Fig. 3c, d).
This difference could be explained by a viewpoint whether the two necessary conditions for root hair induction, i.e., R light and low pH, were satisfied or not. When the seedlings were irradiated after medium change, R light would immediately begin to induce CMT randomization because low pH condition had al-ready been satisfied. In such a situation, inactivation of phytochrome by the following FR light may not be in time to fully cancel the CMT randomization. In contrast, FR light irradiation may be in time in case of the R/FR irradiation before the medium change, because “low pH” had not yet been satisfied during the preculture, and thus, CMT randomization could not be prepared imme-diately by R light irradiation.
Until the irradiation experiments before the medium change were carried out, we had hypothesized that the CMT randomization needs simultaneous satisfaction of R light and low pH. However, unexpectedly, it became clear that these two conditions need not be satisfied si-multaneously. When R light irradiation was placed at as early as 4 h before lowering the medium pH (Fig. 4c, d), the percentage of the transverse CMT arrays was com-parable with those in irradiation experiments just before the medium change (Fig. 4g, h) or after the medium change (Fig. 2e, f). Although phytochrome changes from Pr to Pfr form by R light irradiation and from Pfr to Pr form by FR light irradiation, Pfr form is also converted back to Pr form with time in the dark (Mancinelli 1994, Rockwell et al. 2006). Judging from the fact that 1 h R light irradiation at 4 h before lowering the medium pH succeeded to cause CMT randomization later when the seedlings were treated with pH 4.0 medium, the effect of R light was maintained at least for 3 h as far as lettuce root hair formation is concerned.
Plausible mediator of the output signal from phyto-chrome
In this study, we revealed that the light signal needed for lettuce root hair formation is mediated by phyto-chrome and is transmitted to cause CMT randomization.
60 W. Harigaya and H. Takahashi Cytologia 84(1)
This raises the question as to how the output signal from phytochrome is mediated. The most plausible candidate is the plant hormone ethylene. In lettuce seedlings, eth-ylene production is higher in the light than in the dark, as observed in Arabidopsis (Cao et al. 1999, Takahashi et al. 2003b). This may be due to the increased expres-sion of lettuce 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene (Ls-ACS1) in the light, which gene product catalyzes the synthesis of ethylene precursor (Takahashi et al. 2003b). It is well known that ethylene reorganizes the CMT arrays in plant cells (Steen and Chadwick 1981, Lang et al. 1982, Roberts et al. 1985). Furthermore, the effect of light on the induction of CMT randomization is reproducible in the dark by application of ACC to the seedlings (Takahashi and Inoue 2008). All these findings agree with the idea that phytochrome signaling directs CMT randomization with the aid of ethylene signaling.
The recent study by Ma et al. (2018) proposed cross-talk between light- and ethylene-signaling, both of which lead to microtubule reorganization. In this path-way, microtubule organization is altered by its binding with microtubule-destabilizing protein 60 (MDP60), the gene expression of which is regulated by phytochrome-interacting factor 3 (PIF3). Since microtubule-associated proteins (MAPs) that play a role in lettuce CMT ran-domization have not yet been identified, future studies on MAPs could reveal a direct mechanism of CMT ran-domization.
Another candidate for the mediator of phytochrome signaling is Ca2+, a well-known intracellular second messenger integrated in the regulation of various physi-ological processes (Gilroy et al. 1990, DeFalco et al. 2010, Dodd et al. 2010, Steinhorst and Kudla 2013). Besides, Ca2+ also regulates microtubule organization in a cell (Soga et al. 2006, Wang and Nick 2017). Re-cent studies have revealed that the IQ67 DOMAIN (IQD) family of calmodulin-binding proteins links Ca2+ signaling to microtubules (Bürstenbinder et al. 2013, 2017a, b). The increase of cytosolic Ca2+ in response to R light irradiation has been observed in various plant species (Chae et al. 1990, Kim et al. 1990, Shacklock et al. 1992, Fallon et al. 1993, Molchan et al. 2001). Collectively, the prospect arises that the photosignaling for CMT randomization from phytochrome is mediated by Ca2+. The importance of Ca2+ in elongating root hair is well known (Jones et al. 1995, Bibikova et al. 1997, Wymer et al. 1997, Monshausen et al. 2008, Cárdenas 2009, Candeo et al. 2017). However, deficiency of root hair formation in lettuce seedlings cultured on Ca2+-depleted medium (Konno et al. 2003) proposes another role of Ca2+ in the earlier stage of root hair formation. Considering that Ca2+ regulates microtubule organiza-tion (Soga et al. 2006, Wang and Nick 2017), CMT ran-domization is probably prevented and thus, no root hairs are formed on Ca2+-depleted medium.
We report that light signaling, which is needed for CMT randomization in lettuce seedlings, is mediated by phytochrome. Since CMT randomization occurs prior to the formation of root hair bulges, this finding indicates that phytochrome participates in the process of root hair initiation through the rearrangement of CMT organiza-tion. In Arabidopsis, phytochrome negatively controls the length of root hairs (Reed et al. 1993, Schiefelbein 2000, Oyama et al., 2002). An examination of whether and how phytochrome plays a role in the elongation pro-cess of lettuce root hairs would be an interesting aspect of future research. At the same time, identification of MAPs that regulate CMT randomization would con-tribute towards the elucidation of a direct mechanism of CMT randomization.
Acknowledgements
We are grateful to Prof. Y. Inoue in Tokyo Universi-ty of Science for the generous provision of lettuce seeds. This work was supported by JSPS KAKENHI Grant No. 15770032 and Sasakawa Scientific Research Grant No. 22-420.
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