Physiological roles of Arabidopsis MCA1 and MCA2 based on their dynamic expression patterns

Determining the mechanisms by which plants sense and respond to mechanical stimuli is crucial for unraveling the detailed processes by which plants grow and develop. Mechanosensitive (MS) channels, including MCA1 and its paralog MCA2 in Arabidopsis thaliana, may be essential for these processes. Although significant progress has been made in elucidating the physiological roles of MS channels, comprehensive insights into their expression dynamics remain elusive. Here, we summarize recent advancements and new data on the spatiotemporal expression patterns of the MCA1 and MCA2 genes, revealing their involvement in various developmental processes. Then, we describe findings from our study, in which the expression profiles of MCA1 and MCA2 were characterized in different plant organs at various developmental stages through histochemical analyses and semiquantitative RT‒PCR. Our findings revealed that MCA1 and MCA2 are preferentially expressed in young tissues, suggesting their pivotal roles in processes such as cell division, expansion, and mechanosensing. Lastly, we discuss the differential expression patterns observed in reproductive organs and trichomes, hinting at their specialized functions in response to mechanical cues. Overall, this review provides valuable insights into the dynamic expression patterns of MCA1 and MCA2, paving the way for future research on the precise roles of these genes in planta. Supplementary Information The online version contains supplementary material available at 10.1007/s10265-024-01575-8.


Introduction
Mechanical forces profoundly influence plant growth and development, necessitating sophisticated mechanisms for sensing and responding to stimuli; these stimuli are produced by environmental forces and intrinsic force-generation events, such as cell division and expansion.(Hamant et al. 2008;Louveaux et al. 2016).Among these mechanisms, mechanosensitive (MS) channels have emerged as crucial components that help transduce mechanical signals into biochemical responses.In Arabidopsis thaliana (L.) Heynh.(Arabidopsis), MCA1 (mid1-complementing activity (1) and its paralog MCA2, which belong to the MS channel family, have garnered significant attention due to their roles in mediating various physiological processes (Hamilton et al. 2015;Kurusu et al. 2013).While previous studies have provided valuable insights into their functions, comprehensive knowledge on their expression dynamics is still needed to decipher their precise roles in planta.In this review, we explore the potential functions of MCA1 and MCA2 in Arabidopsis, emphasizing recent progress in elucidating the spatiotemporal expression profiles of MCA1p::GUS, the MCA1 promoter fused to the reporter gene β-glucuronidase (GUS), and MCA2p::GUS.Through this review, we aim to illuminate the possible physiological roles of MCA1 and MCA2 in various developmental processes.

Overview of MCA channels
The MCA1 and MCA2 proteins in Arabidopsis consist of 521 and 516 amino acid residues, respectively, and share 73% amino acid sequence identity.Both proteins exhibit identical structural features, including a single transmembrane segment near the N-terminus, an EF-hand-like motif slightly beyond the middle region on the N-terminal side, a coiled-coil motif in the middle, and a PLAC8 motif in the C-terminal half (Nakagawa et al. 2007).These proteins also form a homotetramer to produce Ca 2+ -permeable channels in yeast (Nakano et al. 2011), insect cells (Shigematsu et al. 2014) and in vitro (Yoshimura et al. 2021).
Although Arabidopsis MCA1 and MCA2 are not always studied comparatively, certain research has shown that the proteins share some physiological roles and also have differences.The growth of the mca1-null mca2-null double mutant, but not that of single mutants, is sensitive to excess external Mg 2+ ; therefore, the physiological roles of MCA1 and MCA2 for plant growth may overlap under excess Mg 2+ conditions (Yamanaka et al. 2010).MCA1 and MCA2 are involved in the response to hypergravity in hypocotyls (Hattori et al. 2020).Both mediate a coldinduced increase in the cytosolic free calcium concentration, [Ca 2+ ] cyt .(Mori et al. 2018).MCA1, but not MCA2, is needed for primary roots to enter hard agar media from soft agar media (Nakagawa et al. 2007;Yamanaka et al. 2010).Moreover, MCA2, but not MCA1, plays a role in Ca 2+ uptake in roots (Nakayama et al. 2010).Researchers have shown that MCA1 is involved in the hypoosmotic shock-induced increase in [Ca 2+ ] cyt in seedlings (Nakagawa et al. 2007) and in the gravity-induced increase in [Ca 2+ ] cyt (Nakano et al. 2021).An electrophysiological study with a Xenopus oocyte system suggested that MCA1 and MCA 2 are MS channels (Furuichi et al. 2012).A recent in vitro electrophysiological study clearly demonstrated that MCA1 and MCA2 are MS channels that are inherently sensitive to membrane stretching, a type of mechanical force (Yoshimura et al. 2021).
In some cases, MCA1 and MCA2 perform different functions because the location of their expression is different.For example, as mentioned above, MCA1, but not MCA2, is necessary for primary roots to enter hard agar medium from soft agar medium (Nakagawa et al. 2007;Yamanaka et al. 2010).This difference may be explained by the differential expression of MCA1 and MCA2 in the root tip (Yamanaka et al. 2010).Unlike MCA2p::GUS, MCA1p::GUS was shown to be expressed at the Arabidopsis primary root tip (Yamanaka et al. 2010), suggesting that MCA1 is responsible for detecting the hardness of agar.This is a good example of how the physiological roles of MS channels can be well linked with their localization.
Such known examples are as follows: The Arabidopis pollen-specific aquaporins, NIP4;1 and NIP4;2, are required for pollen development and pollinations through water transport (Giorgio et al. 2016), while one of the highest expressed PIP1 members in leaves, AtPIP1;2, acts as a CO 2 transport facilitator (Heckwelf et al. 2011;Weig et al. 1997).The root localization of the Arabidopsis nitrate transporter NRT2.1 is involved in nitrate uptake from the soil, while the seed localization of NRT2.7 is responsible for nitrate storage into the seed (Xu et al. 2024).

Expression patterns in leaves
The expression patterns of MCA1 and MCA2 in leaves are intriguing, suggesting that these proteins play crucial roles in leaf development.Our histochemical analyses with standard methods (see Table S1) revealed that both genes were preferentially expressed in younger cotyledons and leaves.

Cotyledons
Previous studies have reported that both MCA1p::GUS and MCA2p::GUS are expressed in the cotyledons of 10-day-old Arabidopsis plants (Yamanaka et al. 2010).However, the analysis is limited to 10-day-old plants, and detailed studies, including those on spatiotemporal expression, have not been conducted at all.Therefore, we conducted the analysis of their spatiotemporal expression patterns.Figure 1a shows that MCA1p::GUS is stably expressed in the veins of cotyledons up to 15 days after sowing (DAS), but its expression decreases at 20 DAS and becomes undetectable at 30 DAS. Figure 1b shows that MCA2p::GUS was expressed throughout the veins and mesophyll cells of the cotyledons up to 15 DAS, with scattered points indicating particularly high expression; however, at 20 and 30 DAS, the expression in both the veins and mesophyll cells was greatly reduced (Fig. 1b).The above results suggest that the expression of both MCA1 and MCA2 is high in young cotyledons and gradually decreases as cotyledons age.

True leaves
Leaf development starts at the shoot apical meristem (SAM), from which the leaf primordium emerges (Barton 2010).In an investigation on the expression of MCA1 and MCA2 in leaf primordia, histochemical analysis of the leaf primordia was performed at 3, 4, and 5 DAS.As shown in Fig. 2a, histochemical analysis revealed that the expression of MCA1p::GUS was weak and restricted to the tip of the leaf primordium.At 5 DAS, patchy expression and tip expression were detected.In contrast, the expression of MCA2p::GUS was observed throughout the entire leaf primordium, although it was more pronounced at the tips (Fig. 2b).At 5 DAS, patchy expression was also observed.These results suggest that MCA1 and MCA2 are involved in early development of the SAM, albeit to varying degrees.
Researchers have shown that for the first set of true leaves, MCA1p::GUS is clearly expressed in the veins of these leaves at 10 DAS (Yamanaka et al. 2010), but its expression gradually decreases by 15 DAS (Fig. 3a).In this case, the expression tended to decrease mainly from the center of the first set of true leaves.At 20 and 30 DAS, very little MCA1p::GUS expression was observed.
MCA2p::GUS is expressed in veins and mesophyll cells at 10 through 30 DAS (Yamanaka et al. 2010;Fig. 3b), but its expression level is greater at 10 and 15 DAS than at 20 and 30 DAS (Fig. 3b).Interestingly, the expression levels tended to remain high at the tip of the first set of true leaves and decreased in the remainder of the leaves.This tendency is similar to that of MCA1p::GUS.
Young leaves emerge as the plant body ages.It is interesting to examine whether MCAs expression is greatly influenced by the leaf age or the age of the plant body relative to the leaf.To test this possibility, we performed histochemical analysis on four leaves of different ages from plants at 30 DAS (Fig. 4).MCA1p::GUS expression was reduced in the veins of older (rosette) leaves (Fig. 4a, leaf No. 1-3), whereas it was clearly expressed in the veins of younger (cauline) leaves (Fig. 4a, leaf No. 4).In addition, MCA2p::GUS expression was lower in the veins and mesophyll cells of the central and surrounding areas of older (rosette) leaves(Fig.4b, leaf No. 1-3), whereas MCA2p::GUS was widely and strongly expressed in the veins and mesophyll cells of younger (cauline) leaves (Fig. 4b, leaf No. 4).Therefore, MCA1 and MCA2 were found to be highly expressed in new leaves, suggesting that both gene products are functional in areas with active cell division and expansion during Arabidopsis development.
Similarly, a rice homolog and a maize homolog of Arabidopsis MCAs reportedly participate in cell division and expansion, which may contribute to the growth of plant organs and bodies (Kurusu et al. 2012;Liang et al. 2020;Liu et al. 2015;Rosa et al. 2017).These results suggest that MCA1 and MCA2 are involved in early development of the leaf, although it is likely that MCA1 is less involved in this event than MCA2 and spatially limited because the expression of MCA1 is less than that of MCA2 and limited to the tip of the young leaf.Since the expression of MCA1 and MCA2 is influenced by the leaf age, not the age of the plant body, the MCA family might be involved in regulating cell division and expansion in young leaves across species.
Cell division and expansion occur actively in young leaves, putting pressure on neighboring cells and tissues and leading to mechanical stress within the leaf structure.In addition, Ca 2+ regulates cell division and expansion by modulating the activity of enzymes involved in cell wall synthesis and rigidity during leaf growth (Hepler 2005).Since MCA1 and MCA2 are expressed at the site where mechanical stress is added, it is likely that both gene products are involved in the leaf development because of their ability to a. MCA1 p::GUS

Spatiotemporal expression patterns in primary roots
We found that the spatiotemporal expression patterns of MCA1p::GUS and MCA2p::GUS in primary roots remained unchanged for at least 15 DAS (Fig. 5).During the developmental period, MCA1p::GUS was stably expressed in the root tip areas, including the meristematic zone and the stele, pericycle, and endodermis of the transition and elongation zones; however, expression was not observed in the root cap, which included columellar cells (Yamanaka et al.2010).In contrast, MCA2p::GUS was not detected in the root tip.We cannot definitively state whether MCA1p::GUS is expressed in the cortex that generates faint GUS staining because the histochemical GUS assay accompanies the intercellular diffusion of reaction products (Guivarc'h et al. 1996).A cross-sectional experiment of primary roots has revealed that MCA1p::GUS is not expressed in the cortex (Yamanaka et al. 2010).
Although little is known about the sensitivity of the root meristematic zone to mechanical forces, this zone is involved in the response to external forces (Chiatante et al. 2021;Potocka et al. 2011).In addition, the cell division rate of the stele, in which MCA1p::GUS staining was the highest, was reported to be greater than that of surrounding tissues, such as the cortex and epidermis (Rahni and Birnbaum 2019).While the stele of the root apical meristem is exposed to mechanical stresses generated by both external and internal forces, it is important to consider that other regions of the root may also experience significant mechanical stresses.Additionally, the role of MCA1 in sensing mechanical stresses in the meristematic zone is not yet fully understood, and there may be other factors or mechanisms involved in this process.Therefore, It is plausible to assume that MCA1 could play a role in sensing mechanical stresses in the meristematic zone because of the expression of MCA1p::GUS (and not that of MCA2p::GUS), and the inability of the mca1-null mutant to penetrate harder agar medium.(Nakagawa et al. 2007;Yamanaka et al. 2010).

Semiquantitative analysis of organ-specific expression
The histochemical analysis revealed the developmental patterns of MCA1p::GUS and MCA2p::GUS in plant organs.To confirm the above results using a different method, we employed semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).For these experiments, we prepared total RNA from cotyledons, the first set of true leaves, the second set of true leaves, and primary roots of plants at 10, 15, and 20 DAS.Then, we performed a semiquantitative RT-PCR analysis with the prepared total RNAs as templates to examine the organspecific expression of MCA1 and MCA2.mRNA encoding Arabidopsis tubulin β-1 (TUB1 gene product) was used as an internal control to normalize the gene expression levels of the different samples.
In cotyledons, the MCA1 and MCA2 levels were the highest in plants at 10 DAS and decreased at 15 DAS and further at 20 DAS (Fig. 6).In the first set of true leaves, the MCA1 and MCA2 levels were high in the plants at 10 and 15 DAS but decreased at 20 DAS.These results suggest that MCA1 and MCA2 are highly expressed when cotyledons and leaves are young.This finding was confirmed by the observation of the second set of true leaves that were the youngest and presented the highest expression levels.In primary roots, high expression levels of MCA1 appeared to be maintained from 10 to 20 To summarize the expression data described above, the results obtained by semiquantitative RT-PCR were consistent with those obtained by histochemical analyses; therefore, these data clearly show that the expression of MCA1 and MCA2 is the highest in newly produced organs.

Expression patterns in the vasculature and lateral roots
We histochemically investigated the expression patterns of MCA1p::GUS and MCA2p::GUS in primary roots and leaves, although basic histochemical analysis has already Primary roots, including the meristematic zone, of MCA1p::GUS and MCA2p::GUS plants at 5, 10 and 15 DAS were GUS-stained and photographed using a phase-contrast microscope.Naming of the meristematic zone was based on Verbelen et al. (2006), who defined that the meristem stretched up to 200 μm away from the root cap junction (RCJ).Note that only roots with clearly identifiable RCJs are marked.
This figure shows a typical example from three or more experiments.Similar results were seen in the MCA1p::GUS lines 1, 3, and 5, and the MCA2p::GUS lines 3, 4, and 5. Therefore, we examined at least nine samples independently.Scale bar = 50 μm.The drawing of the root tip was hand-drawn with reference to the figures presented in three papers (Dolan et al. 1993;Rahni and Birnbaum 2019;Schres et al. 2002) been performed (Yamanaka et al. 2010).The vasculature, particularly the phloem, transmits mechanical stressinduced, long-distance Ca 2+ signals in Arabidopsis (Toyota et al. 2018).Vascular tissues in plants play a role in the transportation of water, minerals, and nutrients, and Ca 2+ has crucial roles in cell wall stability, signaling, and the regulation of enzyme activity (Thor 2019).Therefore, MCA1 and MCA2 expressed in this tissue may be involved in regulating the above events.Indeed, as previously shown, MCA1p::GUS was expressed in the vascular tissues of the cotyledons, true leaves, and primary roots (Fig. 7a-g).In addition, its expression in the primary root was undetectable in other specific cells or regions, including columellar cells, the lateral root cap, the epidermis, and the cortex (Fig. 5a).MCA1p::GUS was also expressed in the lateral root primordium (Fig. 7b), the stele of the hypocotyl (Fig. 7c), the vascular bundle of the petiole and vein of the cotyledon (Fig. 7d, e), and the petiole and vein of the first leaf (Fig. 7f, g).No expression of MCA1p::GUS was detected in stomatal guard cells (Fig. 7h) whose opening is controlled by turgor pressure, a mechanical force (Yi et al. 2022).are not simply a portion of panels a and i, respectively, but also a portion of other plant samples The expression patterns of MCA2p::GUS were different from those of MCA1p::GUS, although both were expressed in the stele and vascular bundles in addition to the surrounding tissues (Fig. 7k-p).MCA2p::GUS expression was observed in stomatal guard cells (Fig. 7q) but not the root tip (Fig. 5b).MCA2p::GUS was expressed at the base of the lateral root primordium but not at the distal end (Fig. 7j).
Additionally, MCA1 and MCA2 were strongly expressed in young lateral roots (Fig. 7b, j).The development of lateral root primordium encounters mechanical stress caused by the overlaying tissues (Pond 1908) and external factors, including soil compaction.Thus, it is plausible to assume that MCA1 and MCA2 are ivolved in the development of lateral roots.

Expression patterns in reproductive organs
Reproductive organs are specialized structures with unique mechanosensory functions.Flower organs, such as stamen filaments and pistils, are sensitive to touch (Braam 2005).Although MCA1p::GUS expression was scarcely detected in reproductive organs (Fig. 8a, b), MCA2p::GUS was robustly expressed in various flower tissues.A closer look at the flowers under magnification before anthesis revealed the following interesting findings in relation to mechanical stress (Fig. 8b, e).The stamen consists of the anther and filament, both of which should receive mechanical forces during flower development due to contact with other organs and an active uptake of water (Ishiguro et al. 2001;Scott et al. 2004).Figure 8e shows that the intensity of GUS staining was high at the anther and the upper portion of the filament where water uptake is active (Ishiguro et al. 2001).Weak staining was observed in MCA1p::GUS flowers (Fig. 8b).In addition, the pistil also receives mechanical forces during flower development (Smyth 2005).It consists of the stigma, style, and ovary.Figure 8e shows that the intensity of GUS staining was high at the stigma and style, which supposedly encounter mechanical forces due to the elongation of the pistil during flower development (Bull-Hereñu et al. 2022).Thus, MCA2 may be involved in mechanosensing processes associated with pistil elongation.

Spatiotemporal expression patterns in trichomes
Trichomes also exhibit specialized structures with unique mechanosensory functions (Fig. 9a).As shown in Fig. 9b  and c trichomes in a spatiotemporal manner with common and unique features, as both genes were strongly expressed in the skirt cells that support trichome cells; however, the expression of MCA1p::GUS became undetectable on the 1st true leaves at 10 DAS but was detectable at 7 DAS.In the newly formed true leaves, MCA1p::GUS was expressed even at 10 DAS.Therefore, age-dependent expression of MCA1 may occur in both trichomes and skirt cells.
Trichomes are mechanosensory cells that are activated by mechanical stresses, such as brushing; help protect plants from herbivore attack; and participate in the plant immune response (Matsumura et al. 2022;Zhou et al. 2017).Zhou et al. (2017) showed that when trichomes were mechanically stimulated by compression or brushing, Ca 2+ oscillations were induced in skirt cells.In addition, it is shown that when the neck of a trichome is flicked with a wire, Ca 2+ influx is induced in the trichome base, and Ca 2+ waves spread out to surrounding cells (Matsumura et al. 2017).Notably, these are the cells and locations at which MCA1 and MCA2 are highly expressed.Therefore, it is plausible that the two Ca 2+ -permeable mechanosensitive channels are involved in these physiological processes in trichomes and skirt cells.

Conclusion
The dynamic expression patterns of MCA1 and MCA2 offer valuable insights into the roles of these proteins in plant growth and development.Based on their preferential expression in young tissues and specialized functions in reproductive organs and trichomes, MCA1 and MCA2 are likely key components that sense and respond to mechanical cues at an early stage of development.As the location and timing of the expression of MCA1 and MCA2 genes have been determined, we can focus on a specific organ at a limited timeframe and analyze it at the molecular level.Therefore, our knowledge of plant mechanosensing will undoubtedly be advanced by future studies aimed at dissecting the molecular mechanisms that underly the differential expression patterns of MCA1 and MCA2 genes and functional divergence.This knowledge should help researchers regulate plant growth under stress conditions and harness the potential of MCA1 and MCA2 in agricultural and biotechnological applications.

Fig. 1
Fig. 1 Developmental changes over time in the expression patterns of MCA1p::GUS and MCA2p::GUS in cotyledons.Cotyledons of MCA1p::GUS plants and MCA2p::GUS plants at 3, 5, 10, 15, 20 and 30 DAS were GUS-stained and photographed using a binocular stereomicroscope.This figure shows a typical example from three or more experiments.Similar results were seen in the MCA1p::GUS lines 1, 3, and 5, and the MCA2p::GUS lines 3, 4, and 5. Therefore, we examined at least nine samples independently.Scale bar = 0.1 cm

Fig. 2
Fig. 2 Expression patterns of MCA1p::GUS and MCA2p::GUS in the leaf primordia.Seedlings including the shoot apical meristem (SAM) of MCA1p::GUS plants and MCA2p::GUS plants at 3, 4, and 5 DAS were GUS-stained and photographed using a binocular stereomicroscope.This figure shows a typical example from three or more exper-

Fig. 3
Fig. 3 Developmental changes in the expression patterns of MCA1 and MCA2 in the first true leaves.The first true leaves of MCA1p::GUS plants and MCA2p::GUS plants at 10, 15, 20 and 30 DAS were GUS-stained and photographed using a binocular stere-

Fig. 4
Fig. 4 Expression patterns of MCA1 and MCA2 in the true leaves of plants at 30 DAS.True leaves of MCA1p::GUS and MCA2p::GUS plants at 30 DAS were GUS-stained and photographed using a binocular stereomicroscope.Note that the numbers 1 through 4 are the order of appearance of the youngest leaves; the lower the number, the older the leaf.This figure shows a typical example from three or more

Fig. 5
Fig. 5 There were no detectable spatiotemporal changes in MCA1 and MCA2 expression patterns in the root meristematic zone over time.Primary roots, including the meristematic zone, of MCA1p::GUS and MCA2p::GUS plants at 5, 10 and 15 DAS were GUS-stained and photographed using a phase-contrast microscope.Naming of the meristematic zone was based onVerbelen et al. (2006), who defined that the meristem stretched up to 200 μm away from the root cap junction (RCJ).Note that only roots with clearly identifiable RCJs are marked.

Fig. 6
Fig. 6 Developmental changes in transcript levels of and MCA2 in plants at 10, 15 and 20 DAS.Cotyledons, a 1st set of true leaves, a 2nd set of true leaves, and primary roots of approximately 10 wild-type plants at 10, 15, and 20 DAS were collected separately and frozen in liquid nitrogen.Total RNA was extracted from each plant, and cDNA was synthesized using the RNA as a template.The amount of MCA1 and MCA2 in the template mRNA was then

Fig. 7
Fig. 7 Expression patterns of MCA1p::GUS and MCA2::GUS in various plant parts.Tissue-specific expression of MCA1p::GUS and MCA2::GUS was investigated by differential interference microscopy.a and i, Full-body photo of a seedling at 7 DAS; b and j, lateral root primordia at 10 DAS; c and k, hypocotyl at 7 DAS; d and l, petiole of a cotyledon at 7 DAS; e and m, vein of a cotyledon at 7 DAS; f and o, petiole of the 1st true leaf at 10 DAS; g and p, vein of the 1st true

Fig. 8
Fig. 8 Tissue-specific expression patterns of MCA1p::GUS and MCA2::GUS in reproductive organs.GUS staining was performed on flowers at 45 DAS. a and d, flowers; b and e, enlarged photographs of one of the flowers shown in a and d, respectively.The stigma (sg), style (st), and ovule (ov) of the gynoecium were pointed out by red bars.The anther (an) and filament (fi) of the stamen were pointed out by black bars。The naming of tissues was based on Moubayidin and Østergaard (2017).; c and f, enlarged anther.The arrow in c indicates the position of the tip of the filament (Aloni et al. 2006).This figure shows a typical example from five experiments.Similar results were seen in the MCA1p::GUS lines 1, 3, 5, and 9, and the MCA2p::GUS lines 3, 4, and 5. Therefore, we examined at least 15 samples independently.Scale bar = 0.2 cm (a, c), 100 μm (b, d)

Fig. 9
Fig. 9 Expression patterns of MCA1p::GUS and MCA2::GUS in trichomes during true leaf development.a. Scanning electron micrograph of a trichome cell from A. thaliana with surrounding skirt cells and epidermal cells.The scanning electron micrograph taken by Stefan Eberhard was obtained online (https:// wellc omeco llect ion.org/ works/ pvacp mdu).A portion of the micrograph is presented here.Naming of the positions of a trichome cell and the surrounding