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A simple and highly efficient protocol for 13C-labeling of plant cell wall for structural and quantitative analyses via solid-state nuclear magnetic resonance

Abstract

Background

Plant cell walls are made of a complex network of interacting polymers that play a critical role in plant development and responses to environmental changes. Thus, improving plant biomass and fitness requires the elucidation of the structural organization of plant cell walls in their native environment. The 13C-based multi-dimensional solid-state nuclear magnetic resonance (ssNMR) has been instrumental in revealing the structural information of plant cell walls through 2D and 3D correlation spectral analyses. However, the requirement of enriching plants with 13C limits the applicability of this method. To our knowledge, there is only a very limited set of methods currently available that achieve high levels of 13C-labeling of plant materials using 13CO2, and most of them require large amounts of 13CO2 in larger growth chambers.

Results

In this study, a simplified protocol for 13C-labeling of plant materials is introduced that allows ca 60% labeling of the cell walls, as quantified by comparison with commercially labeled samples. This level of 13C-enrichment is sufficient for all conventional 2D and 3D correlation ssNMR experiments for detailed analysis of plant cell wall structure. The protocol is based on a convenient and easy setup to supply both 13C-labeled glucose and 13CO2 using a vacuum-desiccator. The protocol does not require large amounts of 13CO2.

Conclusion

This study shows that our 13C-labeling of plant materials can make the accessibility to ssNMR technique easy and affordable. The derived high-resolution 2D and 3D correlation spectra are used to extract structural information of plant cell walls. This helps to better understand the influence of polysaccharide-polysaccharide interaction on plant performance and allows for a more precise parametrization of plant cell wall models.

Introduction

The plant cell walls are made of a complex network of interacting polymers including polysaccharides and lignin, which is a complex polyphenolic network. They can be classified into primary and secondary cell walls depending on their composition and physical characteristics [1]. Secondary cell walls are the main constituents of plant lignocellulosic biomass from major feedstocks used in biofuels industry [2, 3]. Cellulose in these cell walls alone represents the most abundant renewable source available worldwide (about 1.5 × 1012 tons of the total annual biomass) produced via photosynthesis. However, due to lignin, the accessibility to this renewable source is reduced, which negatively impacts the biofuels industry [4, 5].

Despite technological advances allowing the isolation and structural analysis of individual polysaccharides from cell walls of various plant tissues, our understanding of how these polysaccharides are organized into specific molecular three-dimensional (3D) architectures is very limited [6, 7]. Elucidating this 3D organization of plant cell walls is a prerequisite for the full understanding of how plants can adapt to environmental and growth conditions specific to cell types. For structural analysis, the individual polysaccharides are first extracted from cell walls by treatments with various chemicals. However, the 3D structure that these polymers adopt within the cell wall is lost and can only be predicted through molecular computer modeling. X-ray diffraction and magic-angle spinning solid-state nuclear magnetic resonance (ssNMR), along with better computer modeling, allowed the determination of isolated semi-crystalline structures of cellulose microfibrils [8,9,10,11,12]. These studies showed that the β(1–4)-d-glucan chains of cellulose microfibrils can fold in a two-fold helical screw conformation (one 360° twist per two glycosidic bonds) forming stiffened sheets in parallel arrangement via inter and intra chains hydrogen bonds and sheets are held together through weak van der Waals forces to form microfibrils [13]. However, the dynamic of the interactions of these cellulose microfibrils with other cell wall polymers (in particular hemicellulose and lignin) is poorly characterized, and we are still lacking a clear model for the 3D network formed by these interactions [14,15,16]. Multidimensional ssNMR, which relies on the resolution of 13C chemical shifts of cell wall polysaccharides in 2D and 3D 13C-13C correlation NMR spectra, has been instrumental in probing these interactions in native cell walls from never-dried plant tissues [17, 18]. For example, originally, it was thought that cellulose microfibrils are cross-linked by xyloglucan and heteroxylans (both represent major hemicellulose polymers within the cell walls of dicots and monocots, respectively). However, recent studies using ssNMR have shown that while only a minor portion of xyloglucan interacts with cellulose microfibrils [19], the majority of heteroxylans binds to cellulose microfibrils in a flattened two-fold helical screw fold, which has similar rigidity to the cellulose microfibrils [20]. Previous investigations of primary cell wall structure using ssNMR were conducted on Arabidopsis (Arabidopsis thaliana), Brachypodium (Brachypodium distachyon) seedlings, and maize (Zea mays) coleoptiles that were grown in darkness using 13C-glucose-containing liquid media to achieve near-complete labeling [19, 21, 22]. Cosgrove and colleagues employed a custom growth chamber to label Arabidopsis inflorescence stems [23].

The major limitation of ssNMR is the low natural abundance of 13C isotope in plant material (1.1%), which results in limited NMR sensitivity and makes it challenging to apply multidimensional (2D/3D) correlation experimental schemes to achieve high resolution [15]. In addition, the cost of 13CO2 needed to grow plants in standard growth chambers further limited the accessibility of researchers to ssNMR analysis [19]. The 13C-labeling in more mature plants has been challenging, and most of the samples studied so far were from commercial sources. To overcome this, Gao and Mortimer developed a specialized growth chamber designed to achieve higher labeling efficiency in mature plants [24]. However, it requires large amounts of 13CO2 to fill the growth chamber, which is costly.

In this work, we describe a simple and cost-effective protocol for 13C-labelling of plant materials using 13C-labeled precursors (e.g., glucose, sucrose, CO2) to facilitate accessibility to multidimensional ssNMR analysis. This protocol allows more than 60% 13C-labelling of plant cell walls of rice seedlings and can be adapted to any plant species. This important enabling protocol allows high-resolution ssNMR analysis to be more accessible to our research community, helping to address many unsolved questions related to biomass structure.

Methods

Plant materials and chemicals

Rice seeds (Oryza sativa) were from Kitaake cultivar. MS media was purchased from Sigma Aldrich (Burlington, MA). 13C-labeled glucose was purchased from Cambridge Isotope Laboratories (cat# CLM-1396-PK) and 1L 13CO2 (99.0 atom % 13C, < 3 atom % 18O) was from Sigma Aldrich (cat# 364592-1L, Burlington, MA). The stems of wild type (WT) rice are uniformly 13C-labeled and commercially available from IsoLife, Netherlands.

Uniform 13C-labeling of cell walls of rice seedlings

De-husked fresh immature rice mutant seeds (Oryza sativa -Kitaake) were placed in a sterile 50 mL conical tube with 70% ethanol for 5 min. The ethanol solution was replaced with 40% Clorox solution (containing 7.5% sodium hypochlorite) and incubated for 15 min at room temperature with occasional shaking. The solution was discarded, and the seeds were washed with sterile ddH2O five to six times in a laminar flow hood and dried on an autoclaved paper towel. Using sterile forceps, the seeds were placed on autoclaved jars containing half-strength Murashige and Skoog media supplemented with 13C-labeled glucose (1% w/v of the media) and incubated at 22 °C in a tissue culture room with continuous light (~ 30 μmol/m2/s) until the seeds germinated (4–5 days). After germination, the jars were transferred to a dry-seal vacuum-desiccator and supplemented with 1L 13CO2. The seedlings were then grown for 2 weeks under continuous light (~ 30 μmol/m2/s) to allow the shoot and root to develop. The supplementation system of 13CO2 is described below.

Set up of 13CO2 supplementation system

After germination, the jars were placed in a dry-seal vacuum-desiccator (diameter 100 mm, ~ 2.2 L volume, 27ʺ Hg, 914 hPa) that can be connected to a vacuum pump (or gas sources) via sleeve valve with a vacuum hose (Fig. 1A). To supplement 13CO2 to the vacuum-desiccator without affecting the ambient atmospheric pressure within the vacuum-desiccator, a vacuum was applied (via the pump) to the desiccator containing the jars through the side arm sleeve valve. The cover of the desiccator has a sleeve valve on the top with a molded arrow for alignment to the open position that allows to turn the sleeve valve to close the desiccator. The time needed to vacuum the equivalent of 1 L of air from the desiccator was determined arbitrarily (in our case, the time was ~ 2 min of vacuuming the vacuum-desiccator). The 13CO2 is first collected from the low-pressure cylinder (Sigma Aldrich) in a balloon. To release 13CO2 from the balloon into the desiccator, the balloon was tightly connected to the sleeve valve, and the cover aligned with the open position (Fig. 1).

Fig. 1
figure 1

Setup of the 13CO2 supply system. A After seed germination, the jars, are placed in a dry-seal vacuum-desiccator that can be connected to a vacuum pump via sleeve valve with a vacuum hose. To supplement 13CO2 to the vacuum-desiccator without affecting ambient atmospheric pressure, a vacuum was applied to the vacuum-desiccator via the side arm sleeve valve, which has a molded arrow for alignment to the open position that allows to turn the sleeve valve to close the desiccator. The 13CO2 is collected from the low-pressure cylinder in a balloon (B). The balloon is tightly connected to the sleeve valve of the desiccator (C), and the cover is aligned with the open position to release 13CO2 into vacuum-desiccator (D)

Solid-state NMR analysis

All newly labeled samples used in this study maintained their native hydration without disruption. Native 13C plant tissue samples were directly packed into 3.2 mm magic-angle spinning (MAS) rotors for ssNMR measurements (30–50 mg for each 3.2 mm MAS rotor). The commercial materials used for comparison are stems of rice WT that are uniformly 13C-labeled. All experiments were collected on a 400 MHz (9.4 Tesla) Bruker Neo spectrometer using a 3.2 mm HCN MAS probe under 15 kHz MAS at 298 K. 13C chemical shifts were referenced to the tetramethylsilane (TMS) scale by externally referencing to the Met Cδ of a model peptide N-formyl-Met-Leu-Phe-OH (MLF) at 14.0 ppm. The radiofrequency (rf) field strengths were 71.4 kHz for 1H decoupling, 50 kHz for 1H cross polarization (CP) contact pulse, and 41.67 kHz for 13C hard pulses.

1D 13C CP spectrum was measured using 1,024 scans and recycle delays of 2 s, which allows to complete the experiment within 34 min. CP was measured with ramped (70–100%) 1H rf amplitude for 1ms contact time and 18 ms acquisition time, a typical condition for characterizing plant materials [19, 25, 26]. Quantitative 1D 13C direct polarization (DP) spectrum was finished within 5 h using 512 scans and a long recycle delay of 35 s. The use of sufficiently long recycle delays allow for recovery of equilibrium of magnetization between scans, thus ensuring quantitative, unbiased detection of all molecules and carbons. 2D 13C-13C correlation experiment was conducted using 33 ms CORD mixing [25]. The spectra were collected using 32 scans, recycle delays of 2 s, and 256 indirect points (TD1), allowing the completion of each experiment within 4.5 h. To estimate the labeling percentage, the 2D experiments were measured on both our custom-labeled samples and a commercially labeled rice sample with known (97%) 13C-labeling percentage.

Statistical analysis

Further % of standard error during 13C labeling is determined by the following equation;

\(SE= \frac{SD}{\sqrt{n}}\), where SD is the standard deviation of percentage of 13C labeling determined from the 1D cross-sections of 2D spectrum and n is the number of cross-sections taken.

$$SD=\sqrt{\frac{\sum {(\% \,of \,labeling \,determined \,from \,one \,crosssection-average \,\% \,of \,labeling)}^{2}}{Number \,of \,crosssections}}$$

Four cross-sections were used for these calculations and the chemical shifts used are at 105, 76, 73 and 63 ppm from each 1D cross sections.

Results and discussion

Our custom-designed system does not affect rice seedlings growth

The 13C-based multi-dimensional ssNMR approaches have been instrumental in revealing information on plant cell walls in their native environment. However, the requirement of enriching plants with 13C to obtain 2D and 3D correlation spectra needed for adequate analysis limits the applicability of these approaches. Here, we present a simple protocol for 13C labeling of rice seedlings grown under standard light and temperature conditions of tissue culture growth chambers. In this study, a rice mutant generated through CRISPR/Cas9 technology in a glycosyltransferase gene from GT43 family (CAZy database) and wild type (WT) plants were used.

To determine whether the growth of rice seedlings is affected under our custom-based protocol, we compared the growth of WT rice seedlings in vacuum-desiccator with atmospheric CO2 (without 13CO2 and 13C-glucose) with the growth of seedlings grown under our 13C labeling experiment (under the same light and temperature conditions used). As indicated in Fig. 2, two-week-old rice seedlings from both experiments are undistinguishable despite that the two experiments were carried out on different dates. There were no statistically significant differences in the fresh weight (p-Value = 0.3911, N = 3) and the height (p-Value = 0.3574, N = 3) of the seedlings, and both have an average fresh weight of 0.33 g per seedling and an average height of 25 cm.

Fig. 2
figure 2

Comparison of 2-week-old rice WT plants grown in a vacuum-desiccator under either 13CO2 (A) or atmospheric CO2 (B). There were no statistically significant differences in height (p-Value = 0.3911, N = 3) and weight (p-Value = 0.3574, N = 3)

Our isotopic labeling protocol produced high 13C labeling percentage

To estimate 13C incorporation in rice tissues (in mutant and WT plants), we compared 2D 13C-13C correlation spectra of commercially 13C-labeled (97%) rice stems with those of rice plants grown under our custom-designed system (Fig. 3). Key 1D cross sections were extracted from the 2D spectrum at 105, 76, 73, and 63 ppm and compared between the two samples after normalization to the diagonal peaks and off-diagonal peaks of each cross section. Since both samples were measured under identical conditions with the same mixing times, the intensity ratios of the off-diagonal peaks reflect the isotopic enrichment ratio between the samples. The 13C-labeling percentages were determined to be ~ 62% for rice mutant and ~ 66% for WT using the following equation:

Fig. 3
figure 3

13C-labeling of rice mutant (A) and WT (B) seedlings grown under our custom-designed protocol. 2D 13C-13C correlation spectra collected on our custom-labeled rice samples (orange) showed good spectral quality compared to commercial samples. Highlighted regions and dashed lines show the positions where 1D cross sections were extracted and compared in our rice seedlings and commercial samples. After normalization by the diagonal peaks (asterisks), the off-diagonal peaks in our samples showed 50–70% intensity compared to those from the commercial sample having 97% 13C labeling. The experiments were conducted within 4.5 h and with only 32 scans

$$13C\%=\frac{Normalized \,intensity \,of \,off-diagonal \,peaks \,of \,custom-labeled \,sample}{Normalized \,intensity \,of \,off-diagonal \,peaks \,of \,commercial \,sample} \times 97\%$$

If a different model sample was used for comparison, this value in the equation should be replaced by the 13C percentage specific to the model sample being used. It should be noted that the efficiency of 13C incorporation can be further increased if the amount of 13CO2 used can be increased.

Not only our results demonstrate the efficient enrichment of 13C in our samples under our protocol but showed also a good reproducibility, as the 2D 13C-13C correlation spectra collected from rice mutant and WT seedlings were similar. To determine the variability in 13C-labeling of mutant and WT samples, standard deviation (SD) and standard error (SE) were calculated. As indicated in Table 1, there was less variability of data compared to the mean (small SD values) and less discrepancy in a sample's mean compared with the population mean (small SE values).

Table 1 Average percentage of 13C-labeling of rice mutant and WT tissues grown under our protocol. Standard deviation (SD) and standard error (SE) of labeling were calculated

In previous works, it has been shown that when plants were supplied with 100% 13CO2 (in designed sealed growth chamber), the typical enrichments were 90–95% [20, 24]. Under our custom-designed protocol, 1L of 99% 13CO2 was diluted in ~ 2.2 L (volume of the vacuum-desiccator), which corresponds to ~ 45% 13CO2 inside the desiccator. At this level of supplied 13CO2, our protocol resulted in 13C enrichments of over 60%. Thus, it can be concluded that a positive correlation exists between percentage of labeling and the amount of 13CO2 used in the experiment.

Our isotopic labeling protocol required less analysis time of plant materials

High-resolution ssNMR analyses of native cell walls include 1D polarization experiments and 2D 13C/1H-13C correlated experiments. The 1D spectra, which require experimental times ranging from a few minutes to several hours, quickly provide information on the composition and dynamics of molecules within plant biomass [19, 25]. Using our custom-designed protocol a sufficient 13C enrichment for 1D 13C CP spectra to be completed in half an hour, revealing the structure of rigid molecules (Fig. 4). In this analysis, we compared a rice mutant in one of the genes from GT43 family to rice WT. Quantitative detection through a 13C DP experiment with long recycle delays was achieved within only 4.5 h (instead of several hrs) and provided comprehensive molecular composition data. In addition, experimental time can be significantly reduced with advanced techniques like the Multi-CP scheme for quantitative detection [27].

Fig. 4
figure 4

1D 13C solid-state NMR spectra of rice samples prepared using our custom-designed protocol. Top panel shows 1D 13C CP spectra of select rigid molecules measured within 34 min. The bottom panel shows 1D quantitative 13C DP spectra that unbiasedly detect all molecules, which were completed within 4.5 h. Comparison between mutant and wild type (WT) of rice plants grown using our custom-designed protocol. The key spectral regions for carbohydrates, protein/lipid, carbonyl, and aromatic sites are marked. Asterisks mark the interior cellulose carbon 4 peak (iC4) at 89 ppm, which was used to normalize the intensity of the spectra. This is because it represents a distinctive peak that is consistently well-resolved across nearly all plant samples, making it broadly applicable to other studies. Moreover, using cellulose content for normalization is practical, as most NMR studies prioritize quantifying the relative abundances of different carbohydrates rather than absolute quantities (e.g., mg/g). This approach allows for straightforward comparisons with other biomolecules present in the sample

The isotopic enrichment demonstrated through the 2D CORD spectrum (Fig. 4) is sufficient for other 2D correlation experiments, such as the 2D double-quantum (DQ) and single-quantum (SQ) 13C correlation experiment, known as refocused J-INADEQUATE [28]. This method provides information about atoms directly correlated through bonds, making it particularly useful for resonance assignment. The pulse sequence can be easily coupled with either CP or DP for initial polarization, allowing detection of both rigid and mobile molecules. For example, the variations observed in the CP spectra of carbohydrate versus protein/lipid (Fig. 4) arise from differences in molecular dynamics, where lipids and proteins were more rigid in the mutant sample, thereby enhancing their signals in the CP spectrum. However, the total quantity of carbohydrate versus protein/lipid remains comparable between the two samples, as evidenced by the similar spectral patterns in the quantitative DP spectra, which detect all molecules. Utilizing this protocol has enabled the 13C chemical shift assignments of many major cell wall polysaccharides, including cellulose, heteroxylans, and pectin with various morphologies, substructures, substituents, or conformations (e.g., twofold and threefold screw conformation of heteroxylans), as well as lignin [15, 20, 25, 26, 29, 30].

Interactions between the major components of native plant cell walls can be determined by probing the proximities of different moieties using the pulse sequence as shown in Fig. 3 and many analogs available in the NMR toolbox. By extending the mixing time, it is possible to detect further spatial correlations between different cell wall components, thereby revealing their interactions. Such an approach has been applied to various plant samples to understand the interactions happening between cellulose and pectin, cellulose and hemicellulose such as heteroxylans, carbohydrates, and lignin [20, 21, 29, 30]. The rapid and cost-efficient 13C-enrichment protocol established here is a crucial step for expanding the applications of solid-state NMR in understanding biomass structure and dynamics.

Conclusions

In this work, we describe a detailed and easy protocol to achieve high efficiency 13C-labeling of plant materials for multi-dimensional ssNMR analysis of native plant cell walls. The rational was that high cost and limited availability of 13C-labeled plant material has limited ssNMR adoption by plant cell wall scientists. The setup described here can generate 13C-labeled plants at low cost and increased efficiency under light. Currently, 13C enrichment requires growing plants either in liquid culture with 13C-labeled glucose in dark or in growth chambers with 13CO2, which is time consuming and expensive. For comparison, the cost of acquiring commercially available 13C-labeled plant samples typically ranges from €700 to €3,000 per gram (prices for only materials already in stock). However, if a specific plant species or mutant strain needs to be cultivated, the cost increases substantially. The total cost of our 13C labeling is ~ $59 per gram fresh weight. Thus, our protocol represents an excellent alternative where plants can grow under light and at low costs. Furthermore, using a larger vacuum-desiccator (e.g., 20 L or 65 L volume, such as Techni-DomeTM360 vacuum-desiccator or cabinet style desiccator for more height), which can allow for plant growth to up to 3 weeks for sufficient formation of secondary cell walls in certain tissues. Not only does this protocol make the 13C-labeling of plant tissues more affordable, but it can be adapted to researchers’ needs and budget.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

ssNMR:

Solid-state nuclear magnetic resonance

MS:

Murashige and Skoog

2D:

Two dimensional

3D:

Three dimensional

CO2 :

Carbon dioxide

CP:

Cross polarization

DP:

Direct polarization

DQ:

Double quantum

SQ:

Single quantum

MAS:

Magic-angle spinning

rf:

Radiofrequency

TMS:

Tetramethylsilane

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Acknowledgements

The solid-state NMR part of this work was supported by U.S. Department of Energy, Office of Science, Basic Energy Sciences under award number DE-SC0023702.

Funding

The work performed at OU was funded primarily by Baker/Ohio University Research Committee (Baker/OURC) Award. The solid-state NMR part of this work was supported by U.S. Department of Energy, Office of Science, Basic Energy Sciences under award number DE-SC0023702.

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M.B., A.V, T.W., and A.F. designed the research; A.V., M.B., T.J., W.Z., and D.D. performed the research, T.W. and A.F. analyzed the data, and A.F wrote the paper with the help from the other co-authors. All authors reviewed the manuscript.

Corresponding author

Correspondence to Ahmed Faik.

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Javaid, T., Venkataraghavan, A., Bhattarai, M. et al. A simple and highly efficient protocol for 13C-labeling of plant cell wall for structural and quantitative analyses via solid-state nuclear magnetic resonance. Plant Methods 21, 5 (2025). https://doi.org/10.1186/s13007-024-01310-3

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  • DOI: https://doi.org/10.1186/s13007-024-01310-3

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