Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts

Abstract

This study aims to develop a method for isolating and purifying protoplasts/vacuoles from fresh leaves of the Cd hyperaccumulator plant species, Sedum alfredii. The results revealed that preheating cellulase and macerozyme at 50 °C for 5 min significantly accelerated the cell wall degradation. For the most optimal conditions for mesophyll protoplast isolation, the mixture of fresh leaves and cell lysates was followed by a 2-h–long vibration. The protoplast lysate for vacuole isolation was diluted, and 0.675 mmol/L was identified as the most appropriate 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) level, in which S. alfredii large vacuoles are characterized by a high metal and malic acid content. For the best vacuole purification results, we established that 0.8 mol/L was the most optimal mannitol level in the vacuole buffer in terms of vacuole protection during centrifugation, whereas a Ficoll concentration of 0.10 g/ml was adopted in the density-gradient centrifugation

This study aims to develop a method for isolating and purifying protoplasts/vacuoles from fresh leaves of the Cd hyperaccumulator plant species, Sedum alfredii. The results revealed that preheating cellulase and macerozyme at 50 °C for 5 min significantly accelerated the cell wall degradation. For the most optimal conditions for mesophyll protoplast isolation, the mixture of fresh leaves and cell lysates was followed by a 2-h–long vibration. The protoplast lysate for vacuole isolation was diluted, and 0.675 mmol/L was identified as the most appropriate 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) level, in which S. alfredii large vacuoles are characterized by a high metal and malic acid content. For the best vacuole purification results, we established that 0.8 mol/L was the most optimal mannitol level in the vacuole buffer in terms of vacuole protection during centrifugation, whereas a Ficoll concentration of 0.10 g/ml was adopted in the density-gradient centrifugation.

Sedum alfredii (Crassulaceae) is a rare plant species that hyperaccumulates cadmium (Cd), which is native to China (Yang et al., 2004). A better understanding of mechanisms involved in Cd hyperaccumulation by S. aflredii may facilitate its usage in phytoremediation of Cd-polluted soils. Previous studies on S. alfredii revealed that Cd chelates with malic acid and mainly accumulates in the parenchyma cells consisting of large vacuoles (Tian et al., 2011). This strategy of Cd sequestration in S. alfredii differs from that employed by most other hyperaccumulators (Leitenmaier and Küpper, 2013), suggesting that vacuolar sequestration of Cd in parenchyma cells could be of great importance for Cd detoxification in shoots of S. alfredii. A better understanding of the processes involved in vacuolar sequestration of Cd would contribute to the knowledge on Cd hyperaccumulation mechanisms operating at cellular and subcellular levels in this plant species. Vacuole and protoplast isolation from plant tissues, however, is quite challenging in most cases. To date, purified isolation of vacuoles was successfully achieved in only a few plant species, including barley (Hordeum vulgare L. cv. Baraka), Thlaspi caerulescens and Arabidopsis (Shimaoka et al., 2004; Ma et al., 2005; Robert et al., 2007; Huang et al., 2012; Song et al., 2014). While these results are certainly beneficial, they cannot be generalized, as properties of cells from different plant species are not identical. As most available cases of vacuole isolation pertain to brassica family plants, the methods used are not applicable to S. alfredii, as its properties are distinct from those of brassica plants. Accordingly, we explored several improvements in this study, resulting in highly optimized methods for protoplast and vacuole isolation from the hyperaccumulator S. alfredii.

First, protoplast activities were improved by accelerating the cell hydrolysis processes. The results indicated that preheating cellulase and macerozyme in the cell lysate at 50 °C for 5 min significantly accelerated the cell wall degradation rate. In order to facilitate protoplast isolation, we also ripped apart the epidermis of leaves and sliced leaf tissues to increase the tissue surface area exposed to the cell lysate. Moreover, vibrating the plant tissue and cell lysate mixed solution can improve the contact between tissues and the enzyme solution, thus accelerating the release of the protoplasts from the tissues. Treatments of three different durations (1, 2, and 3 h) were applied when vibrating the plant tissue and cell lysate mixed solutions in the incubator. The results showed that the 2-h vibration was the most optimal for isolating protoplasts from the plant tissue and cell lysate mixed solution (Fig. 1). After being centrifuged twice at 80g and 10 °C, suspensions containing a high concentration of protoplasts with the best quality can be obtained (Fig. 2).

Fig. 1.

Microscopic images of resulted protoplasts of S. alfredii after vibration

The leaves were mixed with the preheated cell lysate, and then vibrated for 1 (a), 2 (b), and 3 h (c), respectively

Fig. 2.

Microscopic image of resulted protoplasts from young leaves of S. alfredii after centrifugation

The S. alfredii leaves were mixed with the preheated cell lysate, and then vibrated for 2 h. To get rid of the residues in the mixed solution, the mixed solution was centrifuged twice at 80g and 10 °C

To accelerate the vacuole isolation, we optimized the CHAPS concentration, a substance used to break down cell membrane structures (Schuck et al., 2003). Three different CHAPS levels (0.450, 0.675, and 0.900 mmol/L) in protoplast lysate were applied to release the vacuoles from the protoplast. The results revealed that the CHAPS concentration of 0.675 mmol/L was the best for obtaining a sufficient number of intact vacuoles from protoplasts. After being isolated by protoplast lysate, the vacuoles should be suspended in a specific buffer to remove CHAPS for their membrane protection. An appropriate concentration of mannitol in vacuole buffer is important to avoid damage during centrifugation. We tried a range of mannitol concentrations (0.5 to 1.0 mol/L), and indicated 0.8 mol/L mannitol as the optimal choice for vacuole purification of S. alfredii. In order to purify the vacuoles effectively, another major change was conducted as compared to the previous method (Ma et al., 2005). During vacuole purification, Ficoll was used as the medium of density-gradient centrifugation. We investigated the effects of three Ficoll concentration treatments (0.05, 0.10, and 0.15 g/ml) on vacuole isolation during centrifugation. The findings revealed that many intact protoplasts (green) and the cell residues were aggregated in the 0.05 g/ml Ficoll concentration treatment group (Fig. 3a). In the 0.15 g/ml Ficoll group (Fig. 3c), some protoplasts could still be observed and the vacuoles were not purified completely. However, purified vacuoles (red) were clearly observed under the microscope in the 0.10 g/ml Ficoll group (Fig. 3b). Therefore, a Ficoll concentration of 0.10 g/ml is the most optimal choice and should be adopted for vacuole purification in the density-gradient centrifugation.

Fig. 3.

Microscopic images of vacuoles after being centrifuged at 1500g and 10 °C for 5 min

To purify the vacuole solution, 0.05 (a), 0.10 (b), and 0.15 g/ml (c) Ficoll were added to the bottom of the vacuole solution. The purified vacuoles were collected from the Ficoll layer after centrifugation. The vacuoles were marked using a neutral red (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article)

In conclusion, the present study provides an optimized method of isolating protoplasts and vacuoles from S. alfredii leaves. The isolated protoplasts and vacuoles can be used for physiological and biological investigations of metal sequestration in the hyperaccumulator S. alfredii at the cellular or subcellular level.

Materials and methods

Plant culture

S. alfredii seeds were collected from an old Pb/Zn mine area in Zhejiang, China, and were germinated on a mixture of perlite and vermiculite moistened with deionized water. Four weeks after germination, plants were subjected to 4 d exposure to 1/4, 1/2, or full strength nutrient solutions according to Tian et al. (2011).

Protoplast isolation

After a 60-d preculture period, fresh leaves were cut from the plants of S. alfredii, resulting in 5.0 g of leaf samples. The epidermis of the fresh leaves was ripped apart. In order to determine the effect of preheating on cell hydrolysis, the leaf slices were mixed with a cell lysate containing 15 g/L cellulase, 4 g/L macerozyme, 0.4 mol/L mannitol, 20 mmol/L KCl, 20 mmol/L 2-(N-morpholino)ethanesulfonic acid (MES), 10 mmol/L CaCl₂, and 5 mmol/L β-mercaptoethanol. They were separated into two groups, one of which was preheated at 50 °C for 5 min. Protoplast concentration was obtained. The isolated protoplasts were observed using a microscope (NIKON-ECLIPSE-E600).

Vacuole isolation

For this stage of our study, we used the protoplast lysate containing 0.5 mol/L mannitol, 0.5 mmol/L ethylene glycol bis(2-aminoethyl ether)-N,N,N',N' tetraacetic acid (EGTA), 0.5 mmol/L CHAPS, 20 mmol/L MES, 125 mmol/L CaCl₂, 5 mmol/L KCl, and pH 8.0, as reported previously (Yang et al., 2004; Ma et al., 2005; Robert et al., 2007). To determine the concentration of the protoplast lysate that yields the vacuoles with the most optimal characteristics, protoplast lysate was diluted with deionized water at different ratios (protoplast lysate/water, 9:1 and 4:1 (v/v)). Next, 15 ml of the diluted protoplast lysates were added to three protoplast suspensions, together with a 75 μl neutral red solution (3.46 mmol/L neutral red, 0.33 μmol/L acetic acid, 0.05% chloroform) used to dye the vacuoles. The protoplast lysis processes and the released vacuoles were observed under a microscope. After vacuole isolation for 30 min, the above solution was centrifuged at 600g and 10 °C for 3 min, whereby the supernatant was gently removed. Next, 15 ml of vacuole buffer containing 20 mmol/L MES, 113.63 mmol/L CaCl₂, 4.55 mmol/L KCl, pH 8.0, and different mannitol levels (0.5, 0.6, 0.8, and 1.0 mmol/L) was added to the solution in order to resuspend the isolated vacuoles. The solution was once again centrifuged at 600g and 10 °C for 3 min to completely remove EGTA and CHAPS. In order to purify the vacuole solution, 15 ml of 0.05, 0.10, and 0.15 g/ml Ficoll solutions, containing 20 mmol/L MES, 0.8 mol/L mannitol, 113.63 mmol/L CaCl₂, 4.55 mmol/L KCl, and pH 8.0, were added to the bottom part of the vacuole solutions derived from the previous centrifugations, respectively. The vacuole solutions were centrifuged at 1500g and 10 °C for 5 min (Thermo-S00264).