Starch-dependent sodium accumulation in the leaves of Vigna riukiuensis.

doi:10.21203/rs.3.rs-2572700/v1

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Starch-dependent sodium accumulation in the leaves of Vigna riukiuensis.

https://doi.org/10.21203/rs.3.rs-2572700/v1

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Journal Publication

published 26 May, 2023

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This research provides insight into a unique salt tolerance mechanism of Vigna riukiuensis. V. riukiuensis is one of the great genetic resources of salt tolerance. We have previously reported that V. riukiuensis accumulates a higher amount of sodium in the leaves, whereas V. nakashimae, a close relative of V. riukiuensis, suppresses sodium allocation to the leaves. We first suspected that V. riukiuensis would have developed vacuoles for sodium sequestration, but there were no differences compared to a salt-sensitive accession. However, many starch granules were observed in the chloroplasts of V. riukiuensis. In addition, forced degradation of leaf starch by shading treatment resulted in no radio-Na (²²Na) accumulation in the leaves. We performed SEM-EDX to locate Na in leaf sections and detected Na in chloroplasts of V. riukiuensis, especially around the starch granules but not in the middle of those. Our results could provide the second evidence of Na-trapping system by starch granules, following the case of common reed that accumulates starch granule at the shoot base for binding Na.

genus Vigna

wild crop relatives

starch granules

autoradiography

salt tolerance

Salt tolerance has been one of the most important issues in plant science as soil salinity is a major constraint of crop production. In addition, given a rapid depletion of ground water plus a rapid growth of global population, there is a concern that demands for drinking water may compete with those for food production (Wada et al., 2010). Thus, there is a growing demand for salt-tolerant crops, which can be grown with salinized water (Panta et al., 2014).

To this goal, we have screened genetic resources of wild Vigna species and identified several species that are highly tolerant to salt stress (Iseki et al., 2016, Yoshida et al., 2016). The genus Vigna is a reservoir of diversity and many species are adapted to harsh environments, such as marine beach, desert, limestone karsts and marshes (Tomooka et al., 2014, van Zonneveld et al., 2020). As expected, the species collected from coastal areas presented high tolerance to salt (Iseki et al., 2016)

The following studies on sodium allocation have revealed that V. riukiuensis accumulates relatively higher amount of sodium in the leaves, whereas V. nakashimae, a close relative of V. riukiuensis, suppresses sodium accumulation to the leaves (Yoshida et al., 2016, Noda et al., 2022). Thus, we have hypothesized that V. riukiuensis has a mechanism of salt-includer, which isolates sodium into vacuoles to lower sodium concentration in cytoplasm.

To confirm this hypothesis, in this study, we first observed vacuoles of the leaf cells to see whether there are any structural differences between V. riukiuensis and other species. However, we could not find any characteristic features in the vacuoles. Instead, we found lots of starch granules in the chloroplast of V. riukiuensis leaves. Since there has been a report that common reed accumulates starch granules that bind Na at the shoot base (Kanai et al., 2007), we were intrigued to test whether the starch granules in V. riukiuensis also has ability to bind Na. To do so, we performed further experiments including element mapping.

Plant material and growth conditions

All plant seeds were provided by the NARO Genebank in Tsukuba, Japan (https://www.gene.affrc.go.jp/index_en.php). Seeds were germinated on Seramis clay (Westland Deutschland GmbH, Mogendorf, Germany) for 1 week and then transferred to hydroponic solution in a growth chamber (Light: 28°C for 14 h and Dark: 25°C for 10 h. Light intensity 500 µM^− 1 s^− 1 m-²) until autoradiographic imaging. Hydroponic solution contained diluted nutrient solution of a 1:1 ratio of OAT House No.1 (1.5 g L^− 1) : OAT House No.2 (1 g L^− 1) (Otsuka Chemical Co, Japan), which contained 18.6 mEq L^− 1 N, 5.1 mE1 L^− 1 P, 8.6 mEq L^− 1 K, 8.2 mEq L^− 1 Ca and 3.0 mEq L^− 1 Mg.

Visualization of ²²Na

Pre-cultured plant was transplanted to new hydroponic solution containing 5 kBq ²²Na (PerkinElmer, USA) with non-radioactive 100 mM ²³NaCl. After adding the radio-isotope, plants were incubated again in a long-day condition (Light: 28 ℃ for 14 h and Dark: 25 ℃ for 10 h. Light intensity 200 µmol s^− 1 m^− 2) for 3 days. After incubation, we carefully washed the roots and then enclosed the whole plant body into a plastic bag, and exposed it to a Storage Phosper Screen (BAS-IP-MS-2025E, GE Healthcare, UK) in Amersham exposure cassettes (GE Healthcare, UK) for 24 h. We then scanned the exposed screen with a laser imaging scanner Typhoon FLA-9500 (GE Healthcare, UK). To arrange radioactive intensity equally at each image, photo-stimulated luminescence and contrast were equalized by Multi Gauge version 3.0 (Fujifilm, Japan). All the experiments were independently done on more than three biological replicates.

ICP-MS

We germinated the seeds on Seramis clay, cultivated for 1 week and then transferred 4 plants of each species to hydroponic solution (as described above) in a growth chamber (Light: 28℃ for 14 h and Dark: 24℃ for10 h). When the 3rd leaves have fully expanded, we transferred the plants to hydroponic culture with 100 mM NaCl for 2 days. After incubation, we separately collected the 1st and the 2nd leaves and dried at 50°C for 3 days. The leaves were digested with 200 µL 69% HNO₃ at 90ºC for 0.5 h. The digestate was diluted 1-in-140 with Milli-Q water and inductively coupled plasma-mass spectrometry (ICP-MS, NexION 350S, PerkinElmer, Waltham, MA, USA) determined the contents of Na. The Tukey HSD of statistical analysis was used to compare differences in the measured variables of leaf Na and K concentration, respectively. Differences were significant when p < 0.05.

Starch staining and correlation between starch and Na accumulation with shading in V. riukiuensis

Iodine staining

At the beginning of the treatment, we grew V. angularis, V. nakashimae and V. riukiuensis in hydroponic solution for 7 days, 14 days and 21 days, respectively. Plants were grown in 100 mM NaCl hydroponic solution or non-NaCl solution for 3 days. After incubation, we harvested fresh leaves, immediately immersed in hot water for 10 min and decolorized in 99.5% ethanol for 5 minutes. Finally, samples were immersed 1/50 iodine solution (20 g l^− 1 KI, 10 g Iodine) for 10 minutes.

Shading experiment

V. riukiuensis plants were grown in hydroponic solution for 21 days. The middle leaflets of the 1st, 2nd and 3rd leaves were masked with aluminum foil (shaded) or cling film (non-shaded control). Plants were transferred to 100 mM ²²NaCl solution (5 kBq) for 3 days under long-day condition. After ²²Na treatment, ²²Na localization was visualized using the method described above.

Electron microscopy and SEM-EDX

We treated V. angularis, V. nakashimae and V. riukiuensis with 100 mM NaCl for 3 days. We cut the leaves into 5x5 mm² pieces, and the samples were fixed with 4% (w/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 50 mM cacodylate buffer for 2.5 h at room temperature. The samples were washed six times by the same buffer and postfixed with 1% osmium tetroxide in 50 mM cacodylate buffer for 2 h. After washing by the double distilled water, the samples were dehydrated in methanol series (25, 50, 75, 90, 100%) and substituted to methanol:propylene oxide (1:1) to 100% propylene oxide. Next, the samples were substituted in propylene oxide:Epon812 series (3:1, 1:1, 1:3) and finally embedded in 100% Epon812 resin (TAAB, UK). Semi-thin sections (1 or 2 µm) were cut with a diamond knife on an ultramicrotome (EM UC7, Leica Microsystems, Germany). To test whether there are any structural differences between each species, 1 µm thickness sections were observed by a field-emission scanning electron microscope (FE-SEM). Sections were dried on glass slides and stained with 0.4% uranyl acetate and lead citrate solution. Sections were coated with osmium tetroxide with an osmium coater (HPC-1SW, Vacuum device, Japan), then observed by FE-SEM (SU8220, Hitachi High-Tech, Japan) at accelerating voltage 5 kV with an yttrium aluminum garnet backscattered electron detector. For scanning electron microscope-energy dispersive x-ray spectrometry (SEM-EDX), 2 µm thickness sections were dried on carbon tape at 60°C and set on scanning electron microscope (SEM) stub. The Na imaging condition was high vacuum and acceleration voltage mode as follows: Acceleration voltage, 15 kV; Spot intensity, 80; Beam exposure time, 1 hour. As beam irradiation causes a slight elongation of the sections, electron microscopy images were acquired after beam irradiation in low vacuum and acceleration voltages. Spectral acquisition of Na and electron microscope images were acquired by EMAX Evolution (version EMAX 2.2 SP2, HORIBA, Japan) and SU3500 (Hitachi High-Tech, Japan), respectively. Spectral data was converted into Na mapping image by ImageJ version 1.51j8 (National Institutes of Health, USA). We checked intensity of Na signal in energy dispersive X-ray spectroscopy (EDX) images by quantitative analysis provided in the EMAX Evolution. Sodium mapping and intensity of signal in these species were tested by more than three biological replicates.

Sodium allocation in V. riukiuensis and its relatives

To confirm that V. riukiuensis accumulates higher amount of Na⁺ in the leaves, we evaluate Na allocation in V. angularis, V. nakashimae and V. riukiuensis by autoradiograph and mass spectrometry. The results reproduced our previous observations (Yoshida et al., 2016, Noda et al., 2022), where Na allocation to the leaves were low in V. nakashimae but high in V. riukiuensis (Fig. 1). V. angularis, which is sensitive to salt stress, allocated more Na in the lower leaves than in the higher leaves.

Electron microscope image of leaf cells

Because V. riukiuensis accumulated higher amount of Na⁺ in the leaves, we considered that it isolated Na⁺ to the vacuoles. To test whether any specific features are observable in the vacuoles of V. riukiuensis, we obtained electron microscope images on the leaf cells of V. angularis and V. riukiuensis.

However, we could not find any specific features in vacuoles of V. riukiuensis. Regardless of salt stress, the vacuoles were fully developed and occupied most of the cell compartments in both the species (Fig. 2). The only difference we found between the two species was in that V. riukiuensis contained well-developed starch granules in the chloroplasts whereas V. angularis did not (Fig. 2).

Iodine-starch staining

To confirm that V. riukiuensis accumulates starch granules in the leaf cells, we did iodine-starch staining on the leaves of V. angularis, V. nakashimae and V. riukiuensis (Fig. 3). As a result, we observed strong staining in the leaves of V. riukiuensis but not in those of V. angularis, confirming high starch content in V. riukiuensis but low in V. angularis. However, we also observed strong staining in the leaves of V. nakashimae. Thus, it did not seem that the Na⁺ allocation to the leaves in V. riukiuensis was because of the higher content of starch.

Suppressed Na ⁺ allocation to shaded leaves of V. riukiuensis

However, we still suspected that starch granules in V. riukiuensis positively affected Na⁺ allocation to the leaves. To test the hypothesis, we shaded some leaflets of V. riukiuensis plants for 24h to have all the starch granules degraded (Fig. 4). We then fed the plants with ²²Na and took autoradiograph to visualize Na allocation. As a control, we wrapped the leaflets of the same positions with cling film to exclude a possibility that “covering” had negatively affected Na accumulation by decreasing transpiration.

The results were remarkable. The autoradiography after ²²Na treatment clearly showed that the shaded leaflets did not accumulate ²²Na, while all other leaves (including “wrapped” leaflets in control) exhibited strong indication of ²²Na allocation (Fig. 5).

Scanning electron microscope-energy dispersive x-ray spectrometry (SEM-EDX)

As the results motivated us to test whether the starch granules have capability of binding Na⁺ in V. riukiuensis. To do so, we performed SEM-EDX to locate Na in leaf sections (Fig. 6). We should note that the process of preparing leaf sections (including fixation) washes free Na⁺ ions away from vacuoles, cytoplasm and apoplastic spaces. Thus, SEM-EDX can detect only Na bound to cellular components.

As expected from the results of iodine-starch staining experiments (Fig. 3), the SEM image showed that there were few starch granules in the leaf sections of V. angularis whereas there were a lot in those of V. nakashimae and V. riukiuensis (Fig. 6).

The following EDX analysis detected few Na in the sections of V. angularis and V. nakashimae but detected Na in the sections of V. riukiuensis, especially in chloroplasts (Fig. 6). Moreover, Na was not in the middle of starch granules but was enriched around them (Fig. 6). As such, in the leaves of V. riukiuensis, Na was isolated in chloroplasts, especially around starch granules.

In this study, we demonstrated that V.riukiuensis accumulates starch granules that bind Na, which could be the second evidence of Na-binding starch granules in plants. The only report before this study was of common reed, which forms Na-binding starch granules in the shoot base in response to salt stress (Kanai et al., 2007). Kanai et al. have demonstrated co-localization of Na with starch granules and the isolated starch granules contained higher amounts of Na than other parts of the shoot base did (Kanai et al., 2007). Although V. riukiuensis forms Na-binding starch granules in the leaves not in the shoot base (Figs. 1–6), these facts suggest that the Na-trapping system by starch granules had independently evolved in multiple salt-tolerant species.

Since we previously revealed that V. riukiuensis allocate relatively higher amount of Na to the leaves, we have considered it has an includer-type mechanism, which sequesters excess Na⁺ into vacuoles (Yoshida et al., 2016, Noda et al., 2022). However, recent studies argue that vacuole is not an ideal organelle for Na⁺ sequestration, as tonoplast is permeable to Na⁺ and thus allows back-leak to cytosol (Shabala et al., 2019). Thus, for halophytic species including V. riukiuensis, it would be better to have a mechanism other than, or in addition to, the one using vacuole.

What is complicating in our results is in that V. nakashimae also accumulates lots of starch granules in the chloroplasts but does not allocate Na to the leaves. One possibility is that V. nakashimae have supreme ability to exclude Na⁺ out of the leaves. If Na⁺ does not enter leaf cells, it can never reach chloroplasts where starch granules are formed. The other possibility is that the starch granules in V. nakashimae lacks Na-binding ability. Although pure amylose chains do not have any ability to bind cations including Na⁺, modification of hydroxyl groups such as phosphorylation turns them into cation exchangers (Matsumoto et al., 1998). Given amylose chains are extended from the surface of starch granules (Goren et al., 2018), V. riukiuensis and common reed may have higher enzymatic activity in modifying those chains.

It should be noted that V. riukiuensis may also have ability to exclude Na out of the leaves, at least when the leaves have run out of starch granules (Fig. 5). One may argue that the shading leads to stomatal closure and reduces xylem flow that transports Na to the leaves. However, the results of the control plants do not support such arguments. We also covered the leaflets of the control plants with transparent film to minimize the effect of transpiration, but sodium allocation did not seem to be reduced compared to other leaves or leaflets (Fig. 5). In addition, as observed in Fig. 5, ²²Na have entered into veins of the shaded leaves but not into the mesophylls. Thus, there should be a mechanism to suppress Na⁺ transport from xylem to mesophyll cells.

One limitation in this study is that we do not know how much the starch granules contribute to salt tolerance in V. riukiuensis. However, this is a testable issue because V. riukiuensis can be crossed with V. angularis. We have already crossed them and will perform genetic analyses to see if there is any correlation between starch content in leaves and salt tolerance.

To conclude, we have demonstrated common reed is not the only species that have evolved Na-binding starch granules. As it is effective in multiple plant taxa, we are intrigued to apply this system for developing salt-tolerant crops. By combining other mechanisms of salt tolerance, it would bring synergistic effects on salt tolerance.

Funding

This study was financially supported by JSPS KAKENHI Grant Number 18H02182, 19KK0148, 20H02885, JST PRESTO Grant Number 11103610, Moonshot R&D Program for Agriculture, Forestry and Fisheries by Cabinet Office, Government of Japan (JPJ009237), Environmental Radioactivity Research Network Center (Y-19-05) and Interdisciplinary Project on Environmental Transfer of Radionuclides (No. Y-1).

Conflict of interest: The authors have no conflicts of interest to declare that are relevant to the content of this article.

Author contributions

YN, AH, KT, NK, JF, KT and KN planned the research.

YN, AH, MA, MS, MW and KT performed experiments.

YN, AH, MS and KN analyzed data.

AH, MS, KT and JF tested the results.

YN and KN wrote the paper.

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Download PDF

Journal Publication

published 26 May, 2023

Read the published version in Journal of Plant Research →

Reviewers agreed at journal
22 Feb, 2023
Editor assigned by journal
15 Feb, 2023
First submitted to journal
10 Feb, 2023

You are reading this latest preprint version

Starch-dependent sodium accumulation in the leaves of Vigna riukiuensis.

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Plant material and growth conditions

Visualization of ²²Na

ICP-MS

Starch staining and correlation between starch and Na accumulation with shading in V. riukiuensis

Electron microscopy and SEM-EDX

Results

Electron microscope image of leaf cells

Iodine-starch staining

Scanning electron microscope-energy dispersive x-ray spectrometry (SEM-EDX)

Discussion

Declarations

References

Status:

Journal Publication

Version 1

Starch-dependent sodium accumulation in the leaves of Vigna riukiuensis.

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Plant material and growth conditions

Visualization of 22Na

ICP-MS

Starch staining and correlation between starch and Na accumulation with shading in V. riukiuensis

Electron microscopy and SEM-EDX

Results

Electron microscope image of leaf cells

Iodine-starch staining

Scanning electron microscope-energy dispersive x-ray spectrometry (SEM-EDX)

Discussion

Declarations

References

Status:

Journal Publication

Version 1

Visualization of ²²Na