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Article

Investigation of Water Distribution and Mobility Dynamics in Recalcitrant Quercus acutissima Seeds during Desiccation Using Magnetic Resonance Methods

by
Haiyan Chen
and
Yongbao Shen
*
Collaborative Innovation Centre of Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Forests 2023, 14(4), 738; https://doi.org/10.3390/f14040738
Submission received: 27 February 2023 / Revised: 25 March 2023 / Accepted: 2 April 2023 / Published: 4 April 2023
(This article belongs to the Special Issue Advances in Plant Science Research: Celebrating Nanjing Forestry University’s 120th Anniversary)
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Abstract

:
Recalcitrant seed vigor is closely related to seed moisture, so how do the water distribution and status change during seed drying? In this study, we investigated the association between water content (WC) and germination of Quercus acutissima seeds and used nuclear magnetic resonance (NMR) to monitor the water dynamics during seed drying. Results showed that freshly dispersed seeds had 38.8% WC, but drying to 14.8% WC resulted in a complete loss of vigor. Magnetic resonance images (MRI) reveal that the embryonic axis had the highest WC and the fastest rate of water loss, and seeds lost water from the embryonic axis to the apex and from the center to the end of cotyledons during desiccation. According to low-field NMR results, the proportion of free water in fresh seeds was the highest at 55%, followed by bound water at 10% and immobile water at 35%. During drying, the bound water and free water of seeds were lost simultaneously, and free water was lost most when the seeds died. Our results revealed that Q. acutissima seeds are highly sensitive to desiccation and that the water loss sites of the seeds were at the micropyle and scar. During desiccation, the bound water could not be retained, and the water balance in the seeds was broken, eventually leading to seed death.

1. Introduction

According to the water content of the seed when harvested and the response of seed vigor to desiccation, seeds can be categorized into orthodox, recalcitrant, and intermediate seeds [1,2]. The water content and water status of seeds are significant determinants of seed longevity and are closely related to the desiccation tolerance of the seeds [3]. Orthodox seeds can be dried to low WC (3%–7%) without loss of viability, and their longevity is prolonged with the decrease in WC and storage temperatures [4]. It has been reported that 8% of the world’s seed plants produce desiccation-sensitive seeds [5]. Unlike orthodox seeds, recalcitrant seeds remain sensitive to desiccation both during development and after they are shed from the parent plant [6]. They do not undergo maturation drying, are typically shed at a relatively high WC (30%–65%), and suffer lethal damage when they lose only a small proportion of water [7]. Water is the mediator of seed physiological metabolism, and water status is essential for biological activity [8]. It is generally believed that seeds have two states of water: free water and bound water. In living organisms, bound and free water exist in different proportions yet maintain a dynamic balance. It has been shown that the survival and effectiveness of orthodox seeds depend on the ability of the cells to retain bound water [9]. In addition, it was speculated that desiccation-sensitive seeds might lack water tightly bound to macromolecules (or cannot be maintained after drying), but there was not enough evidence [10]. High sensitivity to water loss is a significant factor affecting the storage and long-term preservation of recalcitrant seeds, but little is known at present regarding the changes in their water distribution and status in recalcitrant seeds during drying.
Recalcitrant seeds are typically characterized by larger size and thinner seed coats [11]. In addition, it was found that some specific structures of the seed coat of recalcitrant seeds can reduce the risk of seed death after dispersal. Xia [12] discovered that the morphological structure of the seed coat of Quercus species affects the pace of water entrance and exit, with a smaller scar drying more slowly. Depending on the environment in which the seeds are dispersed, Aesculus chinensis seeds may have a thick waxy layer covering the seed coat to minimize water loss [13]. However, in the classical sense, these characteristics help to avoid desiccation rather than tolerate desiccation [14]. Recalcitrant seeds are still at risk of water loss and death after prolonged drying. Therefore, it is ecologically vital to investigate the sites of seed water loss during drying.
MRI and LF-NMR provide a non-destructive, real-time visualization technology to monitor the water content, status, movement, and spatial distribution of seeds [15,16]. MRI is the most advanced imaging technique to investigate the internal anatomy of organisms [17]. However, only a few studies have been applied to the changing rules of water migration during seed drying. Ishida [18] used MRI to visualize the drying process of Oryza sativa seeds at different temperatures and investigated the water reduction in different parts of the seeds [18]. Song [19] explored the water migration of chestnut seeds during storage based on MRI and discovered that water was lost from the cotyledons faster than from the embryonic axis [19]. LF-NMR employs the spin relaxation properties of hydrogen nuclei in a magnetic field to explain, from a microscopic standpoint, the status and migration rule of water in a sample via the variation in relaxation time [20]. As a novel nondestructive testing technology, LF-NMR provides an excellent opportunity to visualize the changes in water status within the seed tissue during drying and has been widely used. Jiang [20] found differences in water status changes during the dehydration of orthodox and recalcitrant seeds using NMR [21]. Sun [21] found that there were three water statuses in Ginkgo biloba seeds, namely bound, immobile, and free water, and the change of free water during drying was more significant [22]. Jin [22] found that the drying treatment removed almost all of the free water and most of the immobile water during the drying of walnuts and had no significant effect on the content bound water and oil [23].
Quercus acutissima Carruth is a temperate deciduous broad-leaved forest tree native to East Asia, widespread in southeastern China. It is one of the most economically and ecologically valuable tree species in China [24,25]. Therefore, the protection, cultivation, and utilization of Q. acutissima are essential. Q. acutissima seeds are typical temperate recalcitrant seeds that are difficult to store for more than one year after they mature and disperse [26]. Presently, most research focuses on the physiological and biochemical changes of recalcitrant seeds during desiccation [27,28,29]. In order to reduce the risk of death from water loss once recalcitrant seeds mature and disperse, it is vital to figure out the sites of seed water loss and the relationship between changes in water status during drying and desiccation sensitivity.
In this study, we used nuclear magnetic resonance and scanning electron microscopy (SEM) to investigate the water loss of Q. acutissima seeds by drying. In this study, we used nuclear magnetic resonance and scanning electron microscopy (SEM) to study the water loss of Q. acutissima seeds by drying. The objectives were (1) to establish the relationship between WC and germination of Q. acutissima seeds; (2) to understand the water distribution and the water loss sites of Q. acutissima seeds by SEM of the seed coat and MRI; (3) to utilize LF-NMR to comprehend the changing rules of water status and content in the drying process of recalcitrant seeds, combined with the changes in seed germination rate, to study their relationship with the desiccation sensitivity of seeds.

2. Materials and Methods

2.1. Seed Collection and Conditioning

In mid-November 2021, Quercus acutissima seeds were harvested from the practice forest farm of Nanjing Forestry University (119°12′58′′ E, 32°7′26′′ N). Water selection was performed to remove insect-infested grain after the seeds were collected, and seeds without mechanical damage were chosen for testing.

2.2. Seed Drying

Three replicates of 100 similar-sized seeds (the longitudinal and transverse diameters were approximately 2.2 cm and 2.1 cm, respectively) were selected and placed in a sealed bag containing silica gel (seed to silica gel mass ratio of approximately 1:3, with all seeds buried in the silica gel). The silica gel was replaced when the color changed from dark blue to pink. During the first 24 h of drying, the seeds were weighed every 2 h; after drying for 24 h, the seeds were weighed every 12 h to monitor the change in seed water content in real-time. Samples were taken whenever seed WC dropped by 3%, using the initial WC as a control (CK); each sample was repeated three times.

2.2.1. Determination of Initial Water Content

The initial water content of the seeds was measured using the constant low-temperature oven drying method, following the regulations of the International Seed Testing Association [30]. We weighed the aluminium case with the lid (M1), then cut ten whole Q. acutissima seeds into 3–5 mm slices and evenly placed 5–8 g of these slices in a dried and weighed aluminium box with the lid (M2). After drying the sample for 17 h at 103 °C in a constant temperature oven, we removed it and placed it in a desiccator for 30 min to cool before weighing it with the box (M3). The seed WC was calculated using the following formula:
W C ( % ) = ( M 2 − M 3 ) / ( M 2 − M 1 ) × 100 %

2.2.2. Determination of Relative Water Content

According to the equation described by Feng [31], the current relative WC (R) of the seeds was calculated based on the initial WC (G), the weight of seed before (W1) and after (W2) desiccation:
R ( % ) = ( W 2 − W 1 × ( 1 − G ) ) / W 2 × 100 %

2.3. Germination Test

After desiccation, the seed germination was tested, and there were three replicates in each treatment, with 50 seeds in each replicate. The germination boxes (13 × 19 × 12 cm) were disinfected with 75% alcohol, after removing the seed coat [32], the de-coated seeds were placed on cotton beds, and subjected to germination tests in a constant temperature light box at 25 °C. Furthermore, we watered the seeds at 12:00 p.m. every day. The germination period was 28 days [33]. The germination rate was counted at the end of the experiment, and an embryonic axis protruding seed coat length of 2 mm was used as the criterion to determine whether the seed had germinated [34].

2.4. Observation of Seed Coat Structure

We used SEM (ThermoFisher Scientific, Waltham, MA, USA) to observe the microstructure of the seed coat. Five fresh seeds were randomly selected for assessment. The seed coat surface, apex, and scar sections of the seeds were prepared by cutting them longitudinally into 1 cm2 pieces and mounting them on a sample table with double-sided tape. The samples were first fixed with formaldehyde–acetic acid–alcohol solution and then dried by critical-point drying. The samples were observed under SEM operated at 15 and 20 kV at 0.45–0.68 Torr.

2.5. Changes in Water Distribution and Water Status during Seed Drying

2.5.1. Changes in Water Distribution during Drying

A 7.0 T MRI scanner (Pharma Scan, BioSpin GmbH, Bruker Co., Billerica, MA, USA) was used to track the moisture spatial distribution during Q. acutissima seed desiccation. Three seeds were selected and dried with silica gel; the MRI scans were performed when the WC of the seeds was 38.8%, 35.8%, 32.8%, 29.8%, 26.8%, 23.8%, 20.8%, 17.8%, 14.8%. The direction of the MRI slice was parallel to the cross-section of the seed, and its thickness was 1 mm. The parameters set for MRI measurement were a TR of 2000 ms and a TE of 13 ms, with averages based on 20 repetitions. The spatial distribution of water in the seed was visualized using false colors (relative scales from zero (blue) to maximum (red)) [35].

2.5.2. Seed Measurement Region of Interest Signal-to-Noise Ratio Determination

The Measurement Region of Interest was delineated at the seed embryonic axis, cotyledon center, and cotyledon end (Figure 1c). The proton density-weighted images of seeds at each stage of water loss scanned by a high-field NMR instrument were used to obtain the image signal S of the region of interest and to determine the image noise. The signal-to-noise ratio (SNR) was calculated using the following formula:
s i g n a l − t o − n o i s e   r a t i o = S i m a g e   n o i s e × 100 %

2.5.3. Changes in Water Status and Content during Seed Drying

The LF-NMR measurements were made using a 21 MHz NMR Analyzer (MesoMR23-060H-Ⅰ, 0.5T; Suzhou Niumei Instrument Co., Ltd., Suzhou, China). Three replicates of 10 seeds were selected and dried with silica gel; the LF-NMR measurements were performed when the WC of the seeds was 38.8%, 35.8%, 32.8%, 29.8%, 26.8%, 23.8%, 20.8%, 17.8%, 14.8%. The samples were placed in an NMR tube with an outer diameter of 25 mm, and the temperature of the LF-NMR instrument was maintained at 32 °C during the transverse relaxation time measurements. The parameters for NMR measurement were set as follows: an echo time (TE) of 0.05 ms and a waiting time (TW) of 4000 ms, with data from 18,000 echoes acquired using eight repeated scans. The simultaneous iterative reconstruction technique (SIRT) was used to invert the CPMG data, which has the advantage of fast and straightforward iterative convergence and stable results [36].

2.5.4. Establishment of NMR Detection Method for Seed Water

The sum of the peak areas (total amplitude S) of the T2 (transverse) relaxation curves is proportional to the number of hydrogen atoms in the sample which is mainly from water molecules. The WC of Q. acutissima seeds can be calculated using the sum of the areas in the transverse relaxation curve of the NMR signal. The regression equation between the water quality in Q. acutissima seeds and the total amplitude S of NMR signals can be obtained using regression analysis based on the quality loss and the decrease in total signal amplitude at different drying stages.

2.6. Data Analysis

Excel (Version 2013; Microsoft Corp., Redmond, WA, USA) processed the experimental data. SPSS (Version 26.0; IBM Corp., Armonk, NY, USA) was used to conduct variance analysis on the measurement indicators. The MRI images were examined using Bruker ParaVision 5.1 software (Bruker, Germany). The Origin 2022 (OriginLab Corp, Northampton, MA, USA) and ImageJ (Rawak Software Inc., Stuttgart, Germany) were used to process the images. Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). A correlation analysis was performed to estimate Pearson correlation coefficients. All reported values in this paper were mean ± SD for three replicates in each group. p-values < 0.05 were considered significant.

3. Results

3.1. Changes in Water Content and Germination Rate of Quercus acutissima Seeds during Desiccation

Q. acutissima is a dicotyledon; its seeds are nuts, the testa is thick and hard, the endotesta is thin film-like when fresh, and tightly faces the cotyledons (Figure 1). As desiccation time increased, Figure 2 shows a general declining trend in the WC and germination rate of Q. acutissima seeds. The initial WC of freshly harvested seeds was 38.8%, and the germination rate was 98.67%. The WC of the seeds decreased dramatically at the beginning of drying, but the rate of decrease slowed after 51 h. When the WC reduced from 38.8% to 35.8%, the germination rate declined from 98.67% to 94.67% without any significant difference. However, the germination rate dropped precipitously as dehydration intensified after 51 h of drying. With further desiccation to 26.8% WC (209 h), the germination rate was 52.0%, and nearly half of the seeds lost vigor. The seed germination rate dropped to 0 when the WC decreased to 14.8%. The Pearson correlation analysis (Table S2) revealed a highly significant positive correlation (p < 0.01, R = 0.982) between the germination rate and WC of Q. acutissima seeds, demonstrating that they were typical recalcitrant seeds that were extremely sensitive to water loss.

3.2. Ultrastructure of the Seed Coat

Q. acutissima seed coat (Figure 3A) is composed mainly of the cuticle, palisade layer, and parenchyma, as revealed by the SEM image. There is a thick waxy deposit on the surface of the cuticle (Figure 3B). The palisade layer, which is tightly packed and consists of a monolayer of palisade cells without-gaps, is immediately adjacent to the cuticle. These two layers are the main structures that form a barrier to water flow (water uptake and water loss).
However, we found two water gaps in the seed coat. The first one is located at the micropyle of the seed (Figure 3D,E), which is known from the cross-section of the micropyle (Figure 3E), and consists of parenchyma tissue with many tiny pores in the apex of the endotesta (Figure 3F). The rest of the micropyle is a closely arranged perianth remnant structure (Figure 3C). The scar (Figure 3G), in addition to the micropyle, is another water gap in the seed coat. There are many vascular bundles in the scar (Figure 3H), and its cross-section is loose parenchyma (Figure 3I), which is more efficient to transport water than the dense, waxy layer of the seed coat. It is inferred that the scar is the primary water entry and exit point for Q. acutissima seeds.

3.3. MRI Analysis during Seed Desiccation

Figure 4a demonstrates the MRI of a Q. acutissima seed cross-section during drying. We can observe that the fresh seed was full and had high WC, the red regions were the most distributed, and the testa, with a certain WC, was plainly visible from the MRI. Nevertheless, we can observe that water distribution in the seeds was uneven. The SNR can digitize the results of MRI, and a higher SNR means a stronger 1H signal intensity in the region, indicating a higher WC. Figure 4b reveals that the embryonic axis of fresh seeds had the highest SNR at 8.56, followed by the cotyledon center at 3.82 and the cotyledon end at 1.86, with the red water signal primarily localized in the embryonic axis and central cotyledon. It demonstrated that the embryonic axis and cotyledon centers have a high WC, and that there is a considerable difference between the WC of the embryonic axis and the cotyledon margins.
During seed dehydration, the SNR of the embryonic axis and the cotyledon center constantly declined as seed WC decreased. After 7 h of drying (the WC reduced from 38.8% to 35.8%), the water signal of the testa disappeared, and the SNR of the embryonic axis and the cotyledon fell to 8.13 and 3.8, which were both minor drops of 5% and 7%, respectively. At this time, the testa played a protective role; the seeds were not significantly injured by dehydration, and the germination rate remained high.
After drying for 120 h (the WC was reduced to 29.8%), the SNR of the embryonic axis and cotyledon center decreased significantly by 34% and 24%, respectively. The MRI images show that the red water signal began accumulating continuously at the end of the cotyledons, and the SNR of the cotyledon end increased to 2.53. This indicated that during drying, the embryonic axis and the center of the cotyledons, where the seeds had higher WC, lost water first after drying. Water was lost from the embryonic axis to the cotyledon periphery and the center to the end of the cotyledons.
After drying for 209–358 h (the WC reduced from 26.8% to 20.8%), the SNR of the embryonic axis showed a significant decrease and the area of the red water signal of the embryonic axis was reduced, indicating that the embryonic axis lost significant amounts of water and suffered dehydration injury. Simultaneously, the SNR of the end of the cotyledons kept increasing from 1.55 to 2.17, indicating that the seed water was progressively lost from the inside out. However, at this time, there was no significant difference in decreasing the cotyledon center’s SNR, which indicated that with the increase in drying, the rate of water loss in the embryonic axis part was higher than in other parts.
Drying for 444–545 h (the WC lowered to 14.8%), the embryonic axis water signal eventually disappeared with the SNR decreased by 83% to 1.45, and the embryonic axis only had a little water at this time. The cotyledon water signal area was also reduced gradually, with SNR decreased by 52% to 1.85. In addition, the cotyledon periphery water signal faded away, and the seeds shrank (Table S1). When the seeds died, the SNR of the cotyledon center was the highest, and there was only a tiny amount of red water signal in the cotyledons of the seed.

3.4. Division of T2 Relaxation Time and Water Status during Desiccation

The duration of the transverse relaxation time reflects the water-binding forces and the degree of free hydrogen protons; therefore, the water status of a sample can be determined by comparing the peak positions of the poly-exponential simulation of CPMG relaxation curves. The shorter the relaxation time, the more tightly the hydrogen nucleus is attached to the substance, the more difficult it is to remove the hydrogen nucleus, and the lower is the water freedom [37]. Figure S1 demonstrates that the relaxation curve has three prominent peaks, and the T2 relaxation times in the interval where each peak is placed can be identified as T21, T22, and T23, respectively. The shortest relaxation time of T21 (0.1–1 ms) corresponds to bound water, primarily hydrogen-bonded to macromolecules in the cell, and has poor mobility [38]. The intermediate relaxation time of T22 (1–10 ms) is immobile water, mainly intercellular water with restricted but slightly higher mobile than bound water. T23, with the longest relaxation time (10–1000 ms), is free water, which flows freely within the cells and is a good solvent that can participate in the material metabolism of the cells [39].

3.5. Changes in T2 Inversion Spectrum during Dehydration of Q. acutissima Seeds

The relaxation range and peak time shifted due to the change in freedom degrees of the water during drying. Table 1 reveals that the bound water relaxation range remained relatively the same at first (39.8%–29.8%) and decreased from 0.34 ms to 0.17 ms when the WC dropped from 29.8% to 14.8% (p < 0.05). The change in the peak time of bound water was small, but it also showed a slow decreasing trend, and the peak point time decreased significantly from 0.13 ms to 0.10 ms when the seed WC dropped to 14.8%.
The free water relaxation time and peak time consistently decreased during dehydration. When the seed WC declined to 14.8%, the relaxation range decreased from 534.60 ms to 247.58 ms, a reduction of 53.68% relative to the initial value. The peak time was significantly reduced from 30.03 ms at the beginning to 26.75 ms when the WC dropped to 23.8%; in addition, when the WC reduced to 14.8%, the peak time further decreased to 18.04 ms, a reduction of 39.93%.
The relaxation range changes of the immobile water were more complex than that of the bound water and free water. When the seed WC dropped from 38.8% to 35.8%, the relaxation range reduced from 6.15 ms to 5.58 ms (p < 0.05). The immobile water relaxation range then increased significantly to 6.78 ms (35.8–29.8%). As the degree of desiccation intensified, with the WC decreased to 14.8%, the relaxation range decreased significantly to 4.6 ms, a fall of 24.7% from the initial. The peak time of immobile water had been decreasing during desiccation. In the early drying stages, the peak time decreased slowly from 1.91 ms to 1.83 ms (38.8%–29.8%) but fell drastically from 1.83 ms to 0.78 ms in the later stages (26.8%–14.8%). Overall, the relaxation ranges and peak time of the three water states were significantly diminished during drying, indicating that the hydrogen proton degree of freedom gradually decreased, and the water binding capacity became stronger during drying.

3.6. Dynamic Changes of Peak Area and Its Proportion during Desiccation of Q. acutissima Seeds

We found that there is a significant linear relationship between Q. acutissima seed WC and peak area (Figure S2). Therefore, the peak areas S21, S22, and S23 can be used to quantify the relative contents of bound, immobile, and free water in the seeds of Q. acutissima. Figure 5 depicts the dynamic changes in the peak areas and proportion of the three water statuses during the desiccation of Q. acutissima seeds. Throughout the drying process, the total water content of Q. acutissima seeds declined consistently. In contrast, there was increase or decrease in the content of each water status; the changes in the trend of the proportion of water in each status also varied, indicating that the various water statuses in the seed cells were mutually transformed.
Bound, immobile, and free water accounted for 10%, 35%, and 55% of the total water in the fresh Q. acutissima seeds respectively, with the highest content of free water, which ensures vigorous metabolic activity and makes the seeds easier to germinate after they have dispersed. During desiccation, the peak area and proportion of free water declined with intensified drying due to partial conversion and drying loss. During the decrease of seed WC from 38.8% to 14.8%, the peak area decreased markedly from 21,087.02 to 4060.41, a decrease of 80.74%.
A different trend from free water, the peak area of immobile water showed a fluctuating trend. When the WC decreased from 35.8% to 32.8%, the peak area of immobile water increased markedly from 12,200.29 to 12,993.20. The increase in immobile water content may be because as the seed WC decreased, the cell structure began to contract, and some free water combined with the macromolecular groups to transform into immobile water. When the WC decreased from 32.8% to 14.8%, the peak area of the immobile water dropped to 9461.87, which was a 31.03% decrease, and the decline rate was slow. The change in the proportion of peaks of the immobile water shows a decreasing (38.8%–35.8%) and then an increasing (35.8%–14.8%) trend. As the seed WC decreased from 35.8% to 14.8%, the rate of free water loss within Q. acutissima seeds was higher than that of immobile water, causing the proportion of immobile water to continue to increase.
During drying, the peak area of bound water showed a tendency to rise first and then fall, and the peak proportion showed a slowly increasing trend. As seed WC dropped from 38.8% to 32.8%, the peak area of bound water increased slightly from 3748.30 to 3833.09 with no significant difference change. The peak area of bound water gradually decreased when the WC dropped below 29.8%. When the water content decreased to 14.8%, the bound water peak area reduced to 2067.71, a drop of 44.83%. When seeds died, the proportion of immobile water was the maximum; during drying, the free water lost the most.

4. Discussion

4.1. Effect of Desiccation on Seed Germination of Q. acutissima

The desiccation sensitivity of the seeds of Quercus species is widespread, and their seeds do not undergo maturation drying, so they shed at relatively high-water content and metabolic activity [40,41]. Ganatsas [42] found that the viability of Q. coccifera and Q. pubescens acorns was highly dependent on WC and started to decline when the WC was lower than 28% and 26%, respectively. Leon-Lobos [43] also observed that the WC and germination rate of Q.ilex seeds and Q.cerris seeds decreased significantly with desiccation. In this study, freshly matured and shed Q. acutissima seeds were found to have high WC (38.9%) and germination rate (98.67%). Our study shows that the Q. acutissima seeds continuously lost germination upon moisture loss. The semi-lethal water content of Q. acutissima seeds was 26.8%; when the water content further decreased to 14.8% (545 h), Q. acutissima seeds completely lost their germination ability. The study results confirm the typical recalcitrant character of the seeds of Q. acutissima that are sensitive to desiccation. Therefore, the seeds must be maintained at a high moisture content during ex situ storage and transportation.

4.2. Water Loss Site of Q. acutissima Seed

MRI, as a non-destructive detection tool, can visualize the spatial distribution of seed water and water migration paths during water loss [44,45]. This study observed that the order of the water loss was first the embryonic axis and the cotyledons around the embryonic axis, then the center of the cotyledons, and finally, the end of the cotyledons. Sobrino-Vesperinas [46] discovered that the scar (the point of attachment of the pericarp to the parent plant), which contains vascular bundles, is the primary point of water uptake/loss in the Q. suber seed coat. In addition, an investigation of water absorption in Quercus revealed that the scar was the main route for water uptake in Q. nuttallii and Q. palustris [47]. In this study, we observed that the water loss paths were from the embryonic axis to the periphery of the cotyledons and from the center to the end of the cotyledons by MRI images during the desiccation of Q. acutissima seeds. Combined with the seed coat SEM observation, it was found that there are many tiny pores for water entry and loss in the endotesta at the micropyle, as well as water-transporting vascular bundles at the scar. These two water gaps are consistent with the water loss paths shown in MRI, so we inferred that the scar and the micropyle are the main sites of water loss in Q. acutissima seeds.
By dissecting the seed coat structure and measuring the water uptake rate of the seeds of Quercus species, Xia [12] discovered that both the micropyle and the scar had high water uptake rates, but the scar was thought to be the primary water loss site due to the area of the micropyle being small. Nevertheless, our study showed that the SNR of the embryonic axis declined at a significantly higher rate than that of the cotyledon center, indicating that the embryonic axis lost water more quickly. Combined with LF-NMR results, we deduced that the embryonic axis is composed primarily of free water, whereas the cotyledon center is composed of mainly bound and immobile water. This suggests that the cells of the embryonic axis were more vacuolated, which predisposed the cells to greater sensitivity to water loss. This also provides a plausible explanation for why in the MRI, the water signal of the embryonic axis disappeared, and only the water signal of the center of the cotyledons remained when the seed died. When seeds dry, the apex becomes the crucial point for water loss due to the high free water content and rapid loss of free water in the embryonic axis. Correlation analysis showed that the embryonic axis SNR was highly significantly and positively correlated with seed germination, suggesting that water loss from the embryonic axis was an essential factor in seed vigor loss. This is congruent with the findings of Han [48] in the drying of chestnut seeds, where massive water loss and atrophy of the embryonic axis was the leading cause of seed death. The embryonic axis of Q. acutissima seed is located at the micropyle (the gap where water can pass easily), making it more vulnerable to desiccation injury. Therefore, the micropyle is as equally an important water loss site as the scar. Further research on effectively stopping water loss at the water loss site of Q. acutissima seeds will provide a reference for the transportation and preservation of recalcitrant seeds of the Quercus species.

4.3. The Changes in Water Status during Desiccation

During drying, we found that the relaxation range and peak time of all three water statuses decreased, indicating that water freedom was reduced. The reason may be because of the continual loss of water, the seeds underwent significant crumpling, the internal cell gaps became smaller, the internal cell structure more compact, the mobility of hydrogen protons decreased, and thus, the freedom of each water state decreased. Song [19] reported that the relaxation ranges of free, immobile, and bound water in chestnut seeds significantly reduced as storage duration increased. Sun [49] found, in a study of water changes during room temperature storage of Ziziphus jujube, that the mobility of bound water, immobile water, and free water inside the jujube increased as storage duration increased, and the peaks migrated to the right overall. The patterns of changes in the water status of the experimental materials during water loss were different, and we inferred that the differences in nutrients, storage conditions, and drying methods of various experimental materials made them respond differently to water loss.

4.4. Changes in the Water Content of Each Status during Drying

Water is vital for the survival of living organisms, and bound, and free water exist in various proportions yet maintain a dynamic balance [50]. When the seeds of Q. acutissima reached maturity and had been shed, free water content was highest and dropped greatest (80.74%) during the drying. Intracellular water was insufficient to sustain the active metabolism of recalcitrant seeds due to the extensive loss of free water. As a result, cellular activity declined, and seed vigor was lost. This result agrees with research on changes in the water status of olecranon peach during storage; the loss of excessive free water led the fruit to senescence [51].
Vertucci [10] speculated that desiccation-sensitive seeds might lack water tightly bound to macromolecules (or cannot be maintained after drying). Jiang [21] discovered that, after maturation and drying of orthodox seeds, free water can be lost entirely, whereas bound water is retained. On the contrary, it is worth pointing out that, in this study, bound water and free water loss occurred simultaneously while the drying of Q. acutissima seeds, and bound water decreased by 44.83%. Analysis of correlation revealed that the drop in seed germination rate had a highly significant positive correlation with both the massive loss of free water in the seed and the continual decline in bound water, demonstrating that Q. acutissima seed vigor is extremely sensitive to the loss of bound and free water. Bound water plays an important role in the stability of the cellular macromolecular structure and is directly related to the water retention capacity and degree of dehydration of the cell [52]. Due to the destruction of the internal structure and membrane of Q. acutissima seed tissues during drying and the degradation of other substances, the bound water became less restricted and was gradually lost [53]. Khan [54] suggested that the decrease in bound water may be attributed to the rupture of the cell membrane, hence the decrease in bound water would have a considerable impact on seed vigor. Using low-field NMR to identify the vigor of rice seeds, Song [55] similarly concluded that the bound water content of rice seeds with less vigor was lower. This implied that the loss of bound water causes the desiccation injury to the seeds to become eventually irreversible, resulting in the death of the seeds. Notably, the content of bound water did not change significantly from 0 to 51 h of drying (38.8% to 32.8%), and the germination rate of the seeds remained high (>80%). Consequently, brief, sporadic desiccation events do not affect the survival of seeds, while continued desiccation leads to mortality [41]. Hence, to keep the seed vigor of Q. acutissima at a high level, it is vital to avoid drying the seeds for a long time and to keep the seed moisture content above 32.8% during storage and transport.
Kuroki [56] discovered that Haberlea rhodopensis could maintain a constant ratio of all water statuses during rapid drying. The finding implied that resurrection plants have a dynamic mechanism to maintain the water molecular structure in a constant status, which may be favorable for desiccation tolerance. Nevertheless, the equilibrium of the water status ratio was upset during Q. acutissima seed desiccation. In fresh Q. acutissima seeds, free water > immobile water > bound water; when the seeds died, immobile water > free water > bound water in the seeds. The proportion of the water status shifted significantly toward a sharp reduction in free water. It is challenging to keep the water status ratio of Q. acutissima in equilibrium during drying, which may be one of the reasons why recalcitrant seeds are sensitive to desiccation.

5. Conclusions

Our study shows that Q. acutissima seeds are extremely sensitive to water loss, with vigor being lost at a WC lowered to 14.8%. As a non-destructive, real-time visualization method, NMR provides new insights into water distribution and status changes during recalcitrant seed desiccation. Water in seeds can be divided into free water, immobile water, and bound water. As the seeds crumpled and hardened during drying, the free degree of all three statuses of water gradually decreased. We discovered that seed scar and micropyle were water loss sites during seed drying. The embryonic axis of Q. acutissima seeds has a higher WC than other parts of the seeds, and we inferred that most of the water in the embryonic axis is free water. Hence the water loss rate from the embryonic axis is faster. Fresh seeds have the highest proportion of free water which is lost the most during drying. Notably, bound water is also continuously lost during drying, and the seeds eventually die because the water balance is broken. Therefore, we recommend maintaining the seed moisture content above 32.8% to avoid the loss of bound water causing irreversible damage during seed storage. Further study on how to effectively prevent water flow from the water loss sites of Q. acutissima seeds is important for the maintenance of seed vigor.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14040738/s1, Figure S1: Transverse relaxation time (T2) inversion spectrum of fresh Q. acutissima seed; Figure S2: Correlation of seed water and NMR signal amplitude in Q. acutissima; Table S1: Changes in the transverse and longitudinal diameter of seeds in MRI during drying; Table S2: Pearson correlation analysis of various indexes of Q. acutissima seeds during desiccation.

Author Contributions

Y.S. conceived the original research plans; H.C. designed and performed the experiments; H.C. analyzed the data; H.C. wrote the article with the contributions of all the authors. Y.S. modified the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by The Subject of Key R & D Plan of Innovation and Popularization of Forestry Technology in Jiangsu Province Project (LYKJ [2021]03) and Shandong Province (Major Scientific and Technological Innovation Project) (2021LZGC023).

Data Availability Statement

Not applicable.

Acknowledgments

We would like to thank the College of Forestry, Nanjing Forestry University, and Co-innovation Center for Sustainable Forestry in Southern China, Southern Tree Inspection Center National Forestry Administration.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Morphological structure of Quercus acutissima seeds. (a) Front view of an intact seed; (b) seed with half of the seed coat removed; (c) transverse section of the seed; (d) scar at the bottom of seed; (e) longitudinal section of the seed; (f) micropyle of seed.
Figure 1. Morphological structure of Quercus acutissima seeds. (a) Front view of an intact seed; (b) seed with half of the seed coat removed; (c) transverse section of the seed; (d) scar at the bottom of seed; (e) longitudinal section of the seed; (f) micropyle of seed.
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Figure 2. Changes in water content and germination rate of Q. acutissima seeds during desiccation.
Figure 2. Changes in water content and germination rate of Q. acutissima seeds during desiccation.
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Figure 3. SEM images of the seed coat. (A) Testa cross-section (160×); (B) testa surface (600×); (C) perianth remains on the surface of the testa (300×); (D) micropyle (35×); (E) cross-section of the micropyle (80×); (F) endotesta in the middle of the micropyle (600×); (G) scar; (H) surface of scar (1000×); (I) cross-section of scar (500×). CU, cuticle; PI, palisade layer; P, parenchyma layer; WD, waxy deposits; VBS, vascular bundles; PE, perianth remains; S, stylar; WE, point of water entry.
Figure 3. SEM images of the seed coat. (A) Testa cross-section (160×); (B) testa surface (600×); (C) perianth remains on the surface of the testa (300×); (D) micropyle (35×); (E) cross-section of the micropyle (80×); (F) endotesta in the middle of the micropyle (600×); (G) scar; (H) surface of scar (1000×); (I) cross-section of scar (500×). CU, cuticle; PI, palisade layer; P, parenchyma layer; WD, waxy deposits; VBS, vascular bundles; PE, perianth remains; S, stylar; WE, point of water entry.
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Figure 4. The changes in water signal (a) and SNR (b) of MR images during desiccation of Q. acutissima seeds. The spatial distribution of protons within the seed tissues is visualized by false colors (relative scales from zero (blue) to maximum (red)), red and blue represent hydrated, and water-free regions, respectively.
Figure 4. The changes in water signal (a) and SNR (b) of MR images during desiccation of Q. acutissima seeds. The spatial distribution of protons within the seed tissues is visualized by false colors (relative scales from zero (blue) to maximum (red)), red and blue represent hydrated, and water-free regions, respectively.
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Figure 5. Changes in the peak area and proportion of free water, immobile water, and bound water during desiccation of Q. acutissima seeds. Different lower-case letters after the values refer to a significant difference at the p < 0.05 level.
Figure 5. Changes in the peak area and proportion of free water, immobile water, and bound water during desiccation of Q. acutissima seeds. Different lower-case letters after the values refer to a significant difference at the p < 0.05 level.
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Table
Table 1. LF-NMR parameters of Q. acutissima seeds during desiccation.
Table 1. LF-NMR parameters of Q. acutissima seeds during desiccation.
Water Content (%)T21T22T23
Relaxation Range/msPeak Time/msRelaxation Range/msPeak Time/msRelaxation Range/msPeak Time/ms
38.80.34 ± 0.02 a0.13 ± 0.02 a6.15 ± 0.24 abc1.91 ± 0.00 a534.60 ± 0.48 a29.33 ± 1.22 a
35.80.35 ± 0.00 a0.12 ± 0.01 ab5.58 ± 0.71 cd1.91 ± 0.00 a487.57 ± 20.36 b28.02 ± 1.14 ab
32.80.35 ± 0.01 a0.11 ± 0.01 bc6.43 ± 0.27 ab1.87 ± 0.08 a475.34 ± 19.85 bc29.38 ± 2.04 a
29.80.34 ± 0.00 a0.11 ± 0.01 bc6.78 ± 0.26 a1.83 ± 0.00 a444.10 ± 48.35 cd28.02 ± 1.14 ab
26.80.30 ± 0.01 b0.11 ± 0.01 bc6.65 ± 0.50 ab1.59 ± 0.11 b412.61 ± 0.28 de28.02 ± 1.14 ab
23.80.23 ± 0.00 c0.11 ± 0.01 bc6.41 ± 0.27 ab1.48 ± 0.00 c403.11 ± 0.28 e26.75 ± 1.06 b
20.80.19 ± 0.00 d0.11 ± 0.01 bc6.00 ± 0.23 bcd1.29 ± 0.00 d376.07 ± 0.26 e24.39 ± 0.99 c
17.80.19 ± 0.01 d0.11 ± 0.01 bc5.45 ± 0.22 d1.10 ± 0.04 e290.81 ± 11.79 f18.47 ± 0.75 d
14.80.17 ± 0.02 d0.10 ± 0.00 d4.63 ± 0.31 e0.78 ± 0.03 f247.58 ± 17.18 g18.04 ± 0.00 d
Note: Different lower-case letters after the values refer to a significant difference at the p < 0.05 level.
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Chen, H.; Shen, Y. Investigation of Water Distribution and Mobility Dynamics in Recalcitrant Quercus acutissima Seeds during Desiccation Using Magnetic Resonance Methods. Forests 2023, 14, 738. https://doi.org/10.3390/f14040738

AMA Style

Chen H, Shen Y. Investigation of Water Distribution and Mobility Dynamics in Recalcitrant Quercus acutissima Seeds during Desiccation Using Magnetic Resonance Methods. Forests. 2023; 14(4):738. https://doi.org/10.3390/f14040738

Chicago/Turabian Style

Chen, Haiyan, and Yongbao Shen. 2023. "Investigation of Water Distribution and Mobility Dynamics in Recalcitrant Quercus acutissima Seeds during Desiccation Using Magnetic Resonance Methods" Forests 14, no. 4: 738. https://doi.org/10.3390/f14040738

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