Highly efficient endosperm and pericarp protoplast preparation system for transient transformation of endosperm-related genes in wheat

Plant protoplasts constitute a versatile system for transient gene expression and have frequently been used in high-throughput to screen and identify functional characterization of plant genes. Wheat (Triticum aestivum L.) is one of the most important crops for our daily life. Endosperm-trait related genes are associated with grain yield or quality in wheat. However, very few studies have explored on the use of protoplasts isolated from endosperm and pericarp tissue of developing grain. In this study, endosperm tissues of developing wheat grains at 8 DPA (days post-anthesis) were collected. It was shown that, after being digested with the enzymolysis solution containing 0.714 M mannitol for 2 h, total 1.1 × 105 of intact protoplasts containing 80% vital individuals were isolated from 0.6 g samples. Pericarp protoplasts were successfully purified from wheat grains at 4 DPA using the optimized method. Transcription factor TaABI5 and amyloplast protein TaSSIIIa were transfected to the prepared protoplasts, and they were successfully localized in the nucleus and the surface of starch granule, respectively. It is an effective and reproductive method for endosperm and pericarp protoplast isolation and of great importance to further investigate gene’s functions and regulations related to endosperm development and differentiation in plants. Endosperm and pericarp protoplast preparation system for endosperm-related gene transformation in wheat.


Introduction
Generating stable transgenic lines is a powerful tool, but long transformation cycle and low transformation efficiency are still major problems in characterize plant gene function (Chen et al. 2006).Protoplasts are plant and bacterial cells that have completely removed their cell walls using either mechanical or enzymatic methods.Using electroporation and PEG induced transformation (Salmenkallio-Marttila et storage proteins to protein bodies from 5 to 8 DPA.During the whole process of development, the number and volume of amyloplast increased continuously accompanying the increase of starch granules in size and number.At the later stage of development, the protein matrix fills the endosperm cell due to the expansion and extrusion of amyloplast and protein bodies (Jing et al., 2013).The walls of endosperm cells are usually only consisted of primary walls, which determine cell fusion process when cells are filled with storage starches and proteins (Wang et al. 1998).
Pericarp is the mainly component of caryopsis and can accumulate a large number of starch granules, being account for more than 70% of the caryopsis dry matter content at anthesis (Schnyder et al. 1993).With the development of grain, starch granules are gradually degraded to provide nutrients for grain development and storage synthesis, and at last, the pericarp is degenerated into a protecting tissue of endosperm (Zhuo et al. 2023).Pericarp can be divided into exocarp, mesocarp, and endocarp.Exocarp cells form thick walls and generally accumulate a few starch granules at 3 DPA (Xiong et al. 2013).The mesocarp contains more than 10 layers of parenchyma cells, being responsible for the main amyloplast starch synthesis of the pericarp at the early developmental stage (Xiong et al. 2013;Yu et al. 2015a, b).Endocarp is consisted of two layers of cells, cross cells and tube cells, and the former contains chlorophyll (Xiong et al. 2013).Power and Chapman (1985) were the first to explore the isolation of endosperm protoplasts from barley grains by treating with the CPW (cell protoplast washing) salt solution first and then purifying under density gradient centrifugation with Percoll.The fusion expression vector containing GUS was introduced into the barley endosperm protoplast for transient expression (Diaz and Carbonero 1992;Diaz et al. 1993).Later on, Keeling et al. (1989) reported their works on preparation of endosperm protoplasts from 12-DPA wheat grains.The starch granules, released due to cell breakage during the process of fragmentation, were removed by using Percoll or Nycodenz solution for density gradient centrifugation.However, it is still difficult to eliminate the interference of starch granules on protoplast acquisition.This situation is also presented in maize.Since there existed large starch granules and high starch content in maize grains, protoplasts isolated from maize grains are fragile.Therefore, it is necessary to establish an effective and reproductive method for endosperm protoplast isolation in plants, especially to eliminate the interference of starch granules.
As far as today, there is only one report on pericarp protoplast isolation in tomato, but not in Gramineae (Fieuw and Willenbrink 1991).Here, we proposed and optimized the isolation and transient transformation system suit for both endosperm and pericarp protoplast preparation of wheat grain, which laid the foundation for the subsequent study on the function analysis of grain specific expression genes in plants.

Plant materials
Common wheat Fielder and Chinese Spring (CS) were planted in an artificial climate chamber and grown in an environment of 16 h of light/8 h of darkness and 55-60% relative humidity.Developing grains were harvested from the central spike of Fielder at 5, 8, 11, and 14 DPA.

Plasmid construction
Using the mRNA of CS grains as templet, the full-length coding sequences of TaABI5 and TaSSIIIa were cloned and subsequently inserted into the 16318hGFP vector (donated by Dr. Tang, Beijing Academy of Agriculture and Forestry Sciences).The primer sequences used in this study were shown in Table S1.

Endosperm and pericarp protoplast isolation
Wheat endosperm protoplast preparation was performed following the Diaz and Carbonero's method (Diaz and Carbonero 1992) with some changes in cellular osmotic potential maintaining and protoplast purification.Grains collected from different stages were cut along the abdominal seam.Endosperm tissues were obtained by crushing halfgrain.Hand-cut endosperm pieces (0.5-1.0 mm, 0.4 g) were incubated in 2.0 mL of solution buffer I (CPW salt solution: 0.659 M D-Mannitol, 0.2 mM dipotassium hydrogen phosphate, 1.0 mM potassium nitrate, 10.0 mM calcium chloride dihydrate, 1.0 mM magnesium sulfate heptahydrate, 1.0 µM potassium iodide, 0.1 µM copper sulfate pentahydrate, and 2.0 mM MES, pH 5.7) for 30 min in the dark at 24℃.To optimize the conditions of cellular osmotic potential maintaining, the concentration gradients of D-Mannitol in the buffer I solution was 0.604 M (CPW11), 0.659 M (CPW12), 0.714 M (CPW13), and 0.769 M (CPW14).Removal of the supernatant, 2 mL solution buffer I was added to wash endosperm tissues 2-3 times.Endosperm tissues were socked in 2.0-mL solution II (solution I containing 1.5% fibrin enzyme, 0.3% pectinase, and 0.1% BSA) and vacuumed at 0.6 kPa for 30 min.Endosperm tissues were digested for 1.5-2.0h on a shaker incubator at 40 rpm in darkness, 22 ± 2℃.Equal volume of solution I was added to the enzymatic hydrolysate, and the protoplasts were filtered through a 100-mm nylon mesh and collected with a 10 mL round bottom tube.In the following steps, the time-gradient natural sinking method instead of density gradient centrifugation (Diaz and Carbonero 1992) was used to purify protoplasts.Specifically, protoplasts were stood on ice water bath for 10 min.Removal of the supernatant, an equal volume of solution I was added slowly to the samples along the side tube wall.Samples were bathed in ice water for 8 min.The washing steps were repeated again except for treatment of 6 min on ice bath.Removal of the supernatant, the protoplasts were diluted with the solution III (0.769 M D-Mannitol, 15.0 mM magnesium chloride hexahydrate, and 4.0 mM MES, pH 5.7) into a concentration of 1.0 × 10 6 /mL for the protoplast transformation.
Wheat pericarp protoplast preparation was performed according to the method in barley stem and leaf protoplast isolation (Bai et al. 2014) with some modifications.Pericarp of 4 DPA grains was used for the preparation.Removal of the solution buffer I (containing 0.604 M mannitol), the sample was treated with the enzyme solution (0.6 M mannitol, 10 mM MES, 1.5% fibrin enzyme, 0.3% pectinase, and 0.1% BSA, pH 5.7) in the 0.6 kPa environment for 30 min, and followed a 2-hr digestion under the condition of 40 rpm, dark, and 22 ± 2℃.
The size of protoplast was measured by ImageJ software (https://imagej.en.softonic.com/).Fluorescein diacetate (FDA) staining was used to detect the activity of protoplasts (Bai et al. 2014).Cells were counted under a 200 magnified visual field with a Zeiss LSM Laser confocal microscope (Zeiss, Oberkochen, Germany).The number of cells marked by FDA to the total of cells from three visual fields was used as ratio of vital protoplasts.

Protoplast transformation
Ten µg of plasmid was mixed with 100 µL protoplasts.Equal volume of PEG-Ca 2+ conversion solution (40% PEG3350 (w/v), 0.6 M mannitol, and 10 mM CaCl 2 , pH5.7) was added and then mixed softly.After standing in the dark at 22 ± 2℃ for 15 min, 1 mL of solution I was repeatedly added and mixed softly following a centrifuge at 150 g for 3 min by a fixed angle centrifuge (Eppendorf 5424R, Hamburg, Germany).The sample mixed with 200 µL solution I was incubated in the dark at 22 ± 2℃ for 14-18 h.Fluorescent signals were observed using the Zeiss LSM 510 confocal laser microscope at the wavelength of 450-510 nm.

Data analysis
The one-way ANOVA analysis was performed with the SPSS Statistics 19.0 software (SPSS, Inc., Chicago, IL, USA).Duncan multiple comparisons was used to compare mean values at the 0.05 probability level.All experiments were independently designed for three repetitions.

Protoplast preparation
For endosperm protoplast preparation, each sample of 0.4 g from wheat grains at 5 to 14 DPA was collected and treated with the solution I (CPW salt solution containing 12% (w/v) mannitol) for 30 min.It is indicated that endosperm protoplasts exhibited comprehensive variation in diameter, from 13.74 to 82.28 μm (Fig. 1a-d).While barley stem and leaf protoplasts were 24-26 and 26-34 μm by ImageJ software, respectively (Bai et al. 2014).The average diameter was about 45 μm.Especially, the number of vital protoplasts was lower than 20% (Fig. 1e, g).
In the enzymolysis solution, CPW concentration was varied from CWP11 to CPW14 (11-14% mannitol, respectively).A significantly high yield of protoplasts was obtained at CPW13, reaching to 4.12 × 10 4 per mL (Fig. 2ab, S1a).FDA staining further showed that the proportion of green fluorescent cells in CPW13 treatment was 76.67%, highest among the four kinds of CPW concentration.At 5 DPA, Fielder grains had produced a large number of starch granules, which were gradually increased in size and number along with the endosperm development (Fig. S2).In order to identify the time-point for protoplast preparation, endosperms were collected at 5, 8, 11, and 14 DPA.The 8-DPA grains contributed the highest number and proportion of protoplasts, reaching to 5.13 × 10 4 per mL and 60.12%, respectively (Fig. 2cd, S1b).In order to determine the most suitable dosage of endosperm tissue, endosperm tissues were divided into 0.2, 0.4, 0.6, and 0.8 g for enzymolysis, respectively.As shown in Fig. 3 and Fig. S1, 0.6 g endosperm contributed the most at 5.95 × 10 4 per mL and 70.37% vital protoplasts among different dosages (Fig. 3ab,  S1c).In addition, enzymolysis time affects the quantity and quality of endosperm protoplasts in the enzymolysis buffer.Of the time gradient of 0.5, 1.0, 2.0, and 3.0 h enzymolysis, the highest number of protoplasts and proportion of vital protoplasts could be obtained at 2.0 h, being 8.97 × 10 4 and 62.67% (Fig. 3cd, Fig. S1d), respectively.
Wheat endosperm tissues were treated with CPW11, CPW12, CPW13, and CPW14 solutions.It was shown that the number of vital protoplasts obtained from CPW13 solution were the largest among the four CPW solutions, reaching to 76.67%.Under the condition of 2 h treatment with CPW12, the 0.4 g endosperms collected the range of 8-14 DPA released the 8.97 × 10 4 intact protoplasts.While the vital protoplasts only accounted for 62.67% intact endosperm protoplasts (Fig. 4).TaABI5-GFP fluorescence signal was enriched in the nuclei of wheat endosperm protoplasts but absent from amyloplasts or cytoplasm.In contrast, the TaSSIIIa was specifically localized in the amyloplast (Fig. 4d).Notably, the starch granules were surrounded by fluorescence signals, indicating that the surface of starch granules was the key place for starch synthesis of TaSSIIIa in endosperms.According to the method, it was easy to perform subcellular localization of endosperm proteins.

Isolation of pericarp protoplasts and transient transformation
The pericarp striped from 4-DPA grains was incubated in lysate for 2 h to prepare protoplasts (Fig. 5a, b).The size of pericarp protoplast was in the range of 12.98-50.87μm, with the corresponding average size 31.47μm (Fig. 5ce).It also can be seen that the diameter of wheat pericarp protoplasts was generally smaller than that of endosperm protoplasts.The average transformation efficiency of protoplast.Compared with the optimized development stage (8-DPA, 60.12%), material dosage (0.6 g, 70.37%), and enzymolysis time (2 h, 62.67%), the concentration of mannitol at 76.67% (CPW13,) contributed better on the vital protoplast preparation.
Shortly, the optimal conditions for the separation of endosperm protoplasts were as follows: 0.6 g of 8-DPA endosperms was treated with a standard volume of CPW13 for 2 h.Under the situation, 1.10 × 10 5 of intact protoplasts could be isolated from endosperm tissues, and the vital cells reached to 80% (Fig. 1f, g).

Subcellular localization
Transformation efficiency of endosperm protoplast was measured by transient expression of the GFP reporter gene.The average transformation efficiency reached 62.5% when protoplasts were transformed by the 16318hGFP vector (Fig. S3).Two endosperm-expressed genes fused with the reporter GFP were individually conducted into the

Discussion
The 8-DPA endosperm was suitable for protoplast preparation In barley, stem and leaf protoplasts were 24-26 and 26-34 μm, respectively, and the vital protoplasts are about 97% of total protoplasts isolated from stems or leaves (Bai et al. 2014).However, the vital protoplasts obtained from endosperms were far below 90% (Figs. 2 and 3), resulting from damaging effect of starch granules upon the delicate membrane of the protoplasts (Diaz and Carbonero 1992) and larger cell size.Starch granules formed at the 5 DPA, and grew increasingly larger in size and number with the development of endosperms.At 17 DPA, endosperm cells had been filled with starch granules (Fig. S2), which was pericarp protoplasts was 36.2% under the conduction of the 16318hGFP vector (Fig. S3).When TaABI5 and TaSSIIIa were transient expressed in pericarp protoplasts according to the above optimized method, fluorescence could be clearly detected in the nucleus and amyloplast, respectively (Fig. 5f).The consensus results with endosperm protoplasts indicated that pericarp protoplasts could be applied to the subcellular localization of wheat endosperm-expressing genes.The proposed system could be used for subcellular localization of grain-specific proteins New protocols were developed to understand the distribution of cereal proteins in mesophyll cell protoplasts of Arabidopsis, tobacco, maize, rice, and wheat (Cai et al. 2022;Yang et al. 2021;Wu et al. 2022;Gu et al. 2021).However, neither these methods can still meet the specific expression of genes in endosperm, especially the starch synthesis enzymes located in the amyloplast.Based on the endosperm protoplast preparation (Keeling et al. 1989;Diaz and Carbonero 1992), we optimized a method for transient subcellular transformation in endosperm protoplasts (See Materials and methods).The transcription factor TaABI5 is a member of bZIP family, and its orthologues in rice is clearly localized in the nuclei (Yoshida et al. 2022).TaSSIIIa encodes a starch consistent with the previous reports (Wang et al. 1998).Diaz and Carbonero (1992) failed to isolate vital protoplasts from endosperm at late developmental stages.In contrast, endosperm tissues remain free nuclear phase at the early developmental stages.At about 3 DPA, cellularized endosperm starts the division process, and the number of its cells reaches the half of peak at 8 DPA (Wang et al. 1998).Subsequently, cell expansion instead of cell division becomes the main developmental mode of endosperm, accompanying the storage starches and proteins dramatic accumulation.Therefore, the 8-DPA endosperm that contained the more cells and less starch granules than other stage were the optimal donor for protoplast preparation.

Pericarp protoplasts were alternative materials for subcellular localization and function analysis of endosperm proteins
The pericarp, consisting of exocarp, mesocarp, and endocarp, is the main component of wheat caryopsis at the early development stage and ultimately converts into a protecting tissue that covers the mature caryopsis (Zhou et al. 2001(Zhou et al. , 2009)).It is now clear that exocarp and mesocarp contain amyloplasts, in which a large amount of starch is synthesized at the early development stage (Yu et al. 2015a, b).The presence of amyloplast in the two kinds of tissues synthase, which were transferred into chloroplast of wheat leaf protoplasts indicating that it is involved in the synthesis of amylopectin in endosperm amyloplasts (Gu et al. 2021).In this study, proper localization of TaABI5 and TaSSIIIa indicated that the system was suitable for exploring subcellular distribution of grain proteins.Moreover, attachment of TaSSIIIa to granules suggested that endosperm protoplast could provide more details for subcellular localization of amyloplast proteins in contrast to mesophyll protoplasts.increase in number and size accompanying pericarp cell division and expansion, reaching a peak at 5 DPA (Zhuo et al. 2023), and gradually decrease due to decomposing pericarp cells (Yu et al. 2015a).This study proposed that the 4-DPA pericarp tissue might be selected for the separation of pericarp protoplasts (Fig. 5a, b).Starch granules in the pericarp protoplasts were smaller than those in endosperm cells (Figs. 4d and 5f), which avoided protoplast damage to some extent during a series of handling and centrifugations.Additionally, the transcription factor TaABI5 and amyloplast protein TaSSIIIa were properly localized in pericarpderived protoplasts as in endosperm-derived protoplasts.These results indicated that pericarp protoplasts prepared from 4-DPA grains were alternative to the endosperm protoplasts for subcellular localization and function analysis of endosperm proteins.

Conclusions
In conclusion, we established a simple and highly efficient transient expression system, in which starch granules released by broken protoplasts were removed using gravity sedimentation instead of Percoll or Nycodenz solution coupled with density gradient centrifugation.Based on the system, two endosperm proteins, especially the amyloplast protein TaSSIIIa, were properly localized on the surface of starch granules.The proposed system can be used for instantaneous expression of grain-specific expression genes, protein localization, and protein-protein interaction, serving prediction and plant functional gene verification.

Fig. 1
Fig. 1 Isolation of endosperm protoplasts from premature wheat grains.(a) Wheat plants at 8-14 DPA.Scale bar represents 10.0 cm.(b) Developing wheat grains used for the separation of endosperm protoplasts.Scale bar = 2.0 mm.(c) different magnifications of freshly isolated endosperm protoplasts.Arrows indicate the starch granules.Scale bars represent 40 μm.(d) Diameter distribution of endosperm protoplasts.(e) and (f) Activity of endosperm protoplasts before (e) and after (f) optimization representing that 0.6 g of 8-DPA endosperms was treated with CPW13 for 2 h.Scale bars represent 40 μm.(g) Ratio of vital endosperm protoplasts before and after optimization.Bars represent SD from at least three independent replicates.P < 0.05

Fig. 2
Fig. 2 Effect of CPW concentration and developing stage on the activity of isolated endosperm protoplasts.(a) and (c), fluorescein diacetate (FDA) staining of different CPW concentration (11-14% mannitol respectively) and developing stage, respectively.(b) and (d), ratio of vital protoplasts derived from different CPW concentration and developing stage, respectively.Bars represent SD from at least three independent replicates.Different lower case letters were significantly different at the 0.05 level.Scale bar = 50 μm

Fig. 3
Fig. 3 Effects of premature grain usage and digestion time on the activity of isolated endosperm protoplasts.(a) and (c), fluorescein diacetate (FDA) staining of different premature grain usage and digestion time, respectively.(b) and (d), ratio of vital protoplasts derived from different premature grain usage and digestion time, respectively.Bars represent SD from at least three independent replicates.Different lower case letters were significantly different at the 0.05 probability level.Scale bar = 50 μm

Fig. 4
Fig. 4 Subcellular localization in developing endosperm protoplasts.(a) and (b) Tissue expression analysis of TaABI5 and TaSSIIIa in wheat.Data were collected from Wheat eFP Browser.(c) The schematic illustration of the plasmids 16318hGFP, TaABI5, and TaSSIIIa.(d)TaABI5 and TaSSIIIa were transiently expressed in protoplasts, respectively.Scale bar = 20 μm