Calcium lignosulfonate modulates physiological and biochemical responses to enhance shoot multiplication in Vanilla planifolia Andrews

Abstract

Utilisation of calcium lignosulfonate (CaLS) in Vanilla planifolia has been reported to improve shoot multiplication. However, mechanisms responsible for such observation remain unknown. Here, we elucidated the underlying mechanisms of CaLS in promoting shoot multiplication of V. planifolia via comparative proteomics, biochemical assays, and nutrient analysis. The proteome profile of CaLS-treated plants showed enhancement of several important cellular metabolisms such as photosynthesis, protein synthesis, Krebs cycle, glycolysis, gluconeogenesis, and carbohydrate synthesis. Further biochemical analysis recorded that CaLS increased Rubisco activity, hexokinase activity, isocitrate dehydrogenase activity, total carbohydrate content, glutamate synthase activity and total protein content in plant shoot, suggesting the role of CaLS in enhancing shoot growth via upregulation of cellular metabolism. Subsequent nutrient analysis showed that CaLS treatment elevated the contents of several nutrient ions especially calcium and sodium ions. In addition, our study also revealed that CaLS successfully maintained the cellular homeostasis level through the regulation of signalling molecules such as reactive oxygen species and calcium ions. These results demonstrated that the CaLS treatment can enhance shoot multiplication in V. planifolia Andrews by stimulating nutrient uptake, inducing cell metabolism, and regulating cell homeostasis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-023-01293-w.

Introduction

Lignosulfonate (LS) is a natural low-cost complex polymer obtained from waste products of the paper industry (Low et al. 2019). The usage of LS as a chelating agent has become apparent in fertilization production and amendment of soil properties for years (Cieschi et al. 2016). In the agriculture sector, ion-chelated LS has successfully promoted plant growth in Oryza sativa (Low et al. 2019), Zea mays (Ertani et al. 2011), Populus tremula × P. tremuloïdes, Saintpaulia ionantha and Sequoiadendron sempervirens (Docquier et al. 2007). Several studies have also proposed that the addition of ion-chelated LS in fertilizer was able to accelerate the movement of metal ions to the plant and reduced trace elements pollution in ground water (Rodella and Chiou 2009; Cieschi et al. 2016). In addition, ion-chelated LS could also act as an auxin protector and enhance the production of endogenous auxin (Kevers et al. 1999), producing more roots to improve micronutrients uptake by the plant and promote plant growth (Carrasco et al. 2012). As an environmental-friendly and low-cost chelating agent, various ion-chelated LS had been widely adopted in the formulation of fertilizer, which benefited plant growth and yield.

Vanilla planifolia is a tropical vine plant that belongs to the family Orchidaceae. Commercialization of V. planifolia has been an economically lucrative industry due to its flavouring pods which are widely used in food and beverages, pharmaceuticals and fragrance. Conventionally, V. planifolia is cultivated through stem cuttings. However, this method has several shortcomings, which include labour-intensive, time-consuming and prone to disease infection (Janarthanam and Seshadri 2008; Mengesha et al. 2012). Hence, the micropropagation technique was preferred for the mass production of V. planifolia (Izzati et al. 2013). Our recent study showed that supplementation of CaLS was able to enhance the shoot multiplication of V. planifolia Andrews (Wan Abdullah et al. 2020). Such growth enhancement may be due to the increment of photosynthesis activity, protein synthesis, sugar biosynthesis, and accumulation of phenolic compounds (Wan Abdullah et al. 2020).

One of the key factors in modulating plant development and growth is via their environmental cues. Supplementation of nutritional components in the medium has a major impact on the effectiveness of plant tissue culture in optimising plant growth (Zahara et al. 2017). As a sessile organism, plants have evolved complex mechanisms to take advantage of various metabolisms, which are beneficial for their growth. Such complex mechanisms include metabolism processes in plants, especially during differentiation processes, which determined the fate of the shoot apical meristem (SAM) cells (Fleming 2006). For instance, during the initiation of axillary shoot from meristematic cells, the cells undergo changes in the acquisition process of carbon source from heterotrophic to autotrophic (Fleming 2006). This requires the plant cells to develop an efficient photosynthesis system to synthesize carbohydrate molecules that play crucial roles in facilitating plant growth and differentiation (Pien et al. 2001). Moreover, initiation of axillary shoot from SAM also requires continuous extensive cell division and differentiation of the meristem cells. Therefore, an increased rate of protein metabolism in the developing cells is required to sustain the needs of the highly demanding energy processes (Ghosh and Pal 2013). Increased turnover of storage proteins to new proteins had also been reported previously during organogenesis (Das et al. 2006).

Reactive oxygen species (ROS) on the other hand, have been regarded as the key signalling molecules in plants, which are known for their roles in modulating plant immune response. However, recent studies have shown that they also participated in numerous plant development processes throughout the plant life cycle (Mhamdi and Van Breusegem 2018; Chapman et al. 2019). Regulation of ROS metabolism is governed by the sophisticated antioxidant system in the plant cell (Foyer and Noctor 2003). In brief, an interplay of metabolism processes in a complex network system of a plant is crucial for regulation to ensure proper plant growth and development.

Previously, we have demonstrated that the addition of CaLS in V. planifolia Andrews culture successfully improved shoot multiplication (Wan Abdullah et al. 2020). However, the precise underlying mechanisms of CaLS in promoting shoot multiplication remain largely unknown. Hence, the present study aimed to investigate the physiological changes in V. planifolia and to elucidate the possible mode of action of CaLS on its shoot multiplication. Such information is crucial to further refine the usage efficiency of CaLS as a plant growth additive and enhance its potential applications in agricultural practices.

Materials and methods

Plant materials

Young plants of V. planifolia Andrews were obtained from Lum Chin Nursery, Selangor, Malaysia (GPS coordinates: 3.086359, 101.679054). The intercalary meristems were obtained from excised nodal segments (approximately 1.0 cm) of the plants and surface sterilized according to the protocol described by Lim and Lai (2017).

Lignosulfonate preparation

The CaLS used in this study was purchased from Sigma-Aldrich (CAS number: 8061–52-7). The stock solution was prepared by dissolving 500 mg of CaLS in 10 mL distilled water. The stock solution was then filtered sterilized with a 0.22 µm cellulose acetate membrane.

Culture condition and sample preparation

Shoot induction medium containing Murashige and Skoog medium (Murashige and Skoog 1962) supplemented with 1.0 mg L⁻¹ BAP, 30 g L⁻¹ sucrose and 3 g L⁻¹ gelrite, pH 5.8 was prepared and autoclaved at 1.2 kg cm⁻² for 15 min at 121 °C. For CaLS-treated V. planifolia (CaLS), 150 mg L⁻¹ sterile CaLS was added after the medium was cooled (approximately 50 °C). The nodal segments of V. planifolia were excised and cultured on the shoot induction medium. The shoot induction medium without CaLS was used as a control treatment. All cultures were maintained at 25 ± 2 °C with 16 h photoperiod of 40 µmol m⁻² s⁻¹ white fluorescent light. After four weeks of culture, the shoot was harvested and ground to a fine powder in the presence of liquid nitrogen. The sample was then stored at − 80 °C until further analysis.

Protein extraction and peptide digestion

Protein extraction and peptide digestion were performed according to Wan Abdullah et al. (2021) with minor modifications. Briefly, 100 mg of ground plant sample was dissolved in 500 μL ice-cold protein extraction buffer containing 50 mM ammonium bicarbonate and 10 mM phenylmethylsulfonyl fluoride (PMSF). Then, the mixture was subjected to sonication and centrifugation according to Yang et al. (2020) to obtain the total soluble protein. The protein concentration was then determined via Bradford assay (Bradford 1976). Next, 50 μg of the protein was resuspended in 100 μL of 50 mM ammonium bicarbonate (pH 8.0) in the presence of 0.05% RapiGest (Waters Corporation, Milford, MA) and the mixture was incubated at 80 °C for 15 min. Next, 5 mM dithiothreitol (DTT) was added to the protein mixture and incubated for 30 min at 60 °C. The protein mixture was then subjected to an alkylation process whereby 10 mM iodoacetamide was added to the mixture and incubated in the dark for 45 min at room temperature. Trypsin gold was added to the mixture at the concentration of 100:0.25 (Protein:Trypsin) to start the proteolytic digestion. The digestion was conducted overnight at 37 °C. Subsequently, 1 μL of 99% trifluoroacetic acid (Sigma-Aldrich, USA) was added to the mixture followed by incubation at 37 °C for 20 min to stop the enzymatic digestion process. The tryptic peptide solution of each sample was centrifuged at 18,000 × g for 20 min and the resulting supernatants were collected and kept at − 80 °C until subsequent analysis.

Peptide separation and MS analysis

NanoLC-MS/MS analysis was performed on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, USA) according to the previous study (Wan Abdullah et al. 2021). Briefly, 2 μL of 1 μg μL⁻¹ peptides from each sample were injected and separated on an EASY-nLC 1000 (Dionex, Thermo Scientific, USA) equipped with an Easy-Spray Column Acclaim PepMap C18 100 Å (2 μm, 50 μm × 15 cm) (Thermo Scientific, USA). Separation of the sample was achieved by using an acetonitrile (ACN) gradient (5% to 50%) in 0.1% formic acid (FA) for over 45 min, at a flow rate of 250 nL min⁻¹. Next, each sample was further subjected to a gradient of 85% ACN in 0.1% FA for 2 min. Before injecting the subsequent sample, the column was equilibrated back to 5% ACN with 0.1% FA over 1 min. Mass spectrometry was conducted in a positive ion mode with a source temperature of 250 °C and a nanospray voltage of 1.5 kV. The instrument was operated in data-dependent acquisition (DDA) mode with an Orbitrap MS (OTMS) survey scan using the following parameters as set by Wan Abdullah et al. (2021). All precursors were filtered using a 20 s dynamic exclusion window and intensity threshold of 5000. The MS/MS spectra were analyzed using OTMS with the exact parameters as described by Wan Abdullah et al. (2021). Precursors were then fragmented by collision-induced dissociation (CID) and high-energy collision dissociation (HCD) at a normalized collision energy of 30% to 28%.

The raw data were then processed using the Thermo Scientific Proteome Discoverer Software v2.1. The SEQUEST HT was used as the database searching algorithm. The MS ion intensities were calculated based on the accurate mass and time tag strategy (Ramdas et al. 2019). The accurate alignment of the detected LC retention time and m/z value across different analyses, together with the area under chromatographic elution profiles of the identified peptides could be compared between different samples. For protein identification, the data were searched against the UniprotKB database restricted to plants (2021_03: 8,121,425 sequences) with a 1% strict FDR and 5% relax FDR criteria using Percolator®. Search parameters were set up according to Wan Abdullah et al. (2021). Identified protein with either one of the following criteria implies greater confidence of protein: (i) have at least one unique peptide or (ii) SEQUEST HT score more than 200 or (iii) coverage score above 25%.

Protein quantification and data analysis

Protein quantification and data analysis were performed based on three biological replicates and every biological replicate consists of three technical replicates. A protein file with three technical replicates in txt.format from Proteome Discoverer™ was first uploaded to Perseus for subsequent comparative analysis between control and CaLS-treated samples. Further analysis in Perseus was performed according to Ramdas et al. (2019). First, the average protein intensity from three biological replicates of the same treatment was grouped into one matrix. The data was then log₂-transformed to scale normalized and stabilize the variance to the same mean intensity. Filtration based on the valid values of at least two samples was then performed for each sample group in order to eliminate the proteins that were only found in a single biological replicate. Next, the missing values in the data were imputed with the random numbers that were drawn from a normal distribution (Ramdas et al. 2019). Subsequently, the histogram was plotted in order to ensure the samples are normally distributed. Differentially abundant proteins between control and treatment were detected using a t-test. The p-value was adjusted for multiple-testing using the Benjamin–Hochberg false discovery rate. The Benjamin–Hochberg test was used as it is one of the powerful procedures that decreases the false discovery rate. Proteins were considered significant and differentially abundant between the two conditions, with an adjusted p-value < 0.05 and a t-test difference ≤  − 1 or ≥ 1 (Ramdas et al. 2019).

Gene expression analysis by real-time PCR

The RNA isolation and cDNA synthesis were performed according to Kok et al. (2020). Briefly, the total RNA was isolated from 100 mg powdered sample using RNeasy Plant Mini Kit (Qiagen, Germany). Subsequently, 1 μg of isolated total RNA was converted to the first strand cDNA using QuantiNova Reverse Transcription Kit (Qiagen, Germany). Real-time PCR analysis was conducted using Bio-Rad CFX96 system with QuantiNova SYBR Green PCR (Qiagen, Germany) according to Kamarudin et al. (2017). The PCR reaction conditions used were as follows: 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 5 s. The experiment was performed on three technical replicates with three biological replicates for each sample. The data were analyzed using Bio-rad CFX Manager 3.1 software. The relative expression levels (2^−ΔΔCT) were calculated according to Livak’s method (Livak and Schmittgen 2001). Actin and ubiquitin genes were used as reference genes to normalize the gene expression between the samples. The genes specific and reference genes primers used were listed in Supplementary Table S1.

Inductively coupled plasma optical emission spectroscopy (ICP-OES)

Shoot samples for each treatment group were harvested and dried at 65 °C for 72 h. The dried samples were ground to a fine powder and added to hydrochloric acid (HCl) and nitric acid (4:1). The samples were then digested in microwave oven (CEM Mathews, NC, USA). The digested samples were left to cool and diluted with distilled water. Determination of mineral ions (Ca, Na, K, Mg, Fe, Mn, Zn and Cu) was carried out using PerkinElmer Avio 500 ICP-OES system as outlined in USEPA Method 6010D (U.S. Environmental Protection Agency 2014).

Determination of rubisco activity

The activity of rubisco was determined spectrophotometrically according to the protocol reported by Khan et al. (2015). Approximately 1 g of shoot sample was homogenized with an extraction buffer containing 0.25 M Tris–HCl (pH 7.8), 0.05 M MgCl₂, 0.0025 M EDTA and 37.5 mg DTT. The homogenate was centrifuged at 10,000 × g for 10 min at 4 °C. The corresponding supernatant was mixed with a reaction mixture containing 100 mM Tris–HCl (pH 8.0), 40 mM NaHCO₃, 10 mM MgCl₂, 0.2 mM NaDH, 4 mM adenosine triphosphate (ATP), 0.2 mM EDTA, 5 mM DTT, 1 U glyceraldehyde-3-phosphodehydrogenase, 1 U 3-phosphoglyceratekinase and 0.2 mM ribulose-1,5-bisphosphate. The absorbance was measured at a wavelength of 340 nm.

Assay of hexokinase activity

The hexokinase (HK) activity assay was performed as previously described by Kumar et al. (2016). Shoot sample (1 g) was homogenized with 5 mL of 0.1 M Tris–Cl extraction buffer (pH 7.5) containing 50 mM β-mercaptoethanol, 5 mM MgCl₂, 50 mM sucrose, 1% Triton X-100 and 1% insoluble polyvinylpyrrolidone. The mixture was centrifuged at 10,000 × g for 30 min. The standard reaction mixture for HK comprised of 50 mM Tris–Cl buffer (pH 7.5), 5 mM MgCl₂, 1 mM NADP + , 5 mM glucose, 2.5 mM ATP, 2.25 U G-6-P dehydrogenase and 200 μL plant extract, in a total volume of 3 mL. The sample was measured at 340 nm using a spectrophotometer reader and expressed as μmol NADP + reduced min⁻¹.

Assay of isocitrate dehydrogenase activity

The isocitrate dehydrogenase (ICDH) activity was determined according to Kumar et al. (2016). The shoot sample (1 g) was homogenized with 5 mL of 50 mM Na-phosphate extraction buffer (pH 7.5) containing 5 mM MgCl₂ and 1% insoluble polyvinylpyrrolidone. The mixture was centrifuged at 10,000 × g for 30 min. The plant extract (100 μL) was added to a 3 mL assay mixture containing 50 mM Tris–Cl buffer (pH 7.5), 5 mM MgCl₂, 1 mM NADP + and 10 mM DL-isocitrate. The determination of ICDH activity, expressed as μmol NADP + reduced min⁻¹, was monitored spectrophotometrically at 340 nm.

Determination of carbohydrate concentration

Carbohydrate concentration was determined via anthrone colorimetry (Clegg 1956). Briefly, 0.5 g of shoot sample was heated for 30 min in 10 mL of distilled water and centrifuged at 15,000 × g for 15 min. The plant extract (0.5 mL) was mixed with 1 mL anthrone reagent and cooled on ice. Then the sample was transferred to a cuvette and measured at 630 nm using a spectrophotometer reader. Quantification of carbohydrate concentration was obtained by interpolation of the absorbance against the calibration curve of glucose.

Assay of glutamate synthase activity

The assay for glutamate synthase (GOGAT) activity was carried out according to the protocol previously described by Ertani et al. (2011) with minor modifications. Shoot sample (1 g) was homogenized in a mortar with 10 mL of 100 mM HepesNaOH solution (pH 7.5), 5 mM MgCl₂ and 1 mM DTT. The extract was then centrifuged at 20,000 × g for 15 min at 4 °C. The standard assay mixture contained 25 mM HepesNaOH (pH 7.5), 2 mM L-glutamine, 1 mM α-ketoglutaric acid, 0.1 mM NADH, 1 mM Na₂EDTA and 200 μL of supernatant, in a total volume of 2 mL. The GOCAT activity was determined by monitoring spectrophotometrically the oxidation of NADH at 340 nm and expressed as μmol g⁻¹ FW.

Detection of total protein content

Total protein content was determined according to Bradford (1976). Shoot sample (150 mg) was ground into a fine powder and homogenized with 10 mM potassium phosphate buffer (pH 7.0) containing 4% (w/v) polyvinylpyrrolidone. The extract was then centrifuged at 12,000 × g for 30 min at 4 °C. The resulting supernatant was added to the Bradford reagent and incubated for 5 min. Absorbance was read at 595 nm and expressed as mg g⁻¹ FW.

Assay of peroxidase activity

The peroxidase activity assay was conducted by monitoring the consumption of hydrogen peroxide in a spectrophotometer at 420 nm over 3 min (Fortunato et al. 2012). In brief, 250 mg shoot samples were homogenized with 2 mL of 100 mM potassium phosphate buffer (pH 6.8) containing 1 mM EDTA, 1 mM PMSF and 300 mg polyvinylpolypyrrolidone. Then, the plant extract was centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant (100 μL) was added to a 3 mL reaction mixture consisting of 100 mM potassium phosphate buffer (pH 6.8), 100 mM pyrogallol and 100 mM H₂O₂. The peroxidase activity was expressed as U mg⁻¹ protein.

Determination of hydrogen peroxidase content

Determination of hydrogen peroxide (H₂O₂) content was performed using the protocol reported by Velikova et al. (2000). Shoot sample (200 mg) was homogenized with 2 mL of 0.1% trichloroacetic acid in an ice bath and centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant (0.5 mL) was then added to 0.5 mL of 10 mM potassium phosphate buffer (pH 7) and 1 mL of 1 M potassium iodide. The sample was measured at 390 nm using a spectrophotometer.

Statistical analysis

All data presented were the average ± standard deviation (SD) values of three biological replicates. The Student’s t-test was applied in evaluating the level of significant differences at p < 0.05 between the different treatments using the SPSS v.20 software (IBM Corp., Armonk, USA).

Results

Application of CaLS on V. planifolia Andrews cultures

The application of CaLS significantly improved the growth of V. planifolia shoot as shown in Fig. 1. This result was coherent with the previous study on CaLS in shoot multiplication (Wan Abdullah et al. 2020).

Fig. 1.

Representative images of Vanilla planifolia Andrews shoot under a control and b calcium lignosulfonate treatment (CaLS). Scale bar represents 1 cm

Comparative proteomic analysis of V. planifolia treated with CaLS

Comparative proteome analysis was carried out between the control and CaLS-treated V. planifolia using Perseus Software v1.6.0.7 (Max Planck Institute of Biochemistry). The analysis detected 247 proteins in the control treatment and 223 proteins in CaLS treatment, whereas both groups shared 93 proteins in common (Fig. 2a). The volcanic plot showed a total of 26 proteins with significantly different abundance in response to CaLS treatment (Fig. 2b). Furthermore, a total of 130 proteins was exclusive to CaLS-treated V. planifolia; while a total of 154 proteins was exclusive to the control group (Fig. 2c). From the KEGG pathway analysis, the differentially expressed proteins were mainly involved in the pathways related to stress response, photosynthesis, Krebs cycle, glycolysis and gluconeogenesis, protein synthesis, carbohydrate synthesis, lipid synthesis, and calcium signalling (Fig. 2d). Upon CaLS treatment, the identified proteins with the greatest increase in abundance were oxygen-evolving enhancer protein 2–1 (+ 6.223), oxygen-evolving enhancer protein 1 (+ 5.264), and CBS domain-containing protein CBSX3 (+ 5.084) (Table 1). Meanwhile, the proteins with the greatest decrease in abundance were ferredoxin-NADP reductase, leaf isozyme (− 6.253), ferredoxin-NADP reductase (− 5.177), and 40S ribosomal protein S4 (− 4.684) (Table 2). Four key genes that encoded for the upregulated proteins were selected, including 4-coumarate: CoA ligase (4CL) and Caffeoyl-CoA O-methyltransferase (CCOAMT) which were involved in lignin biosynthesis and stress response; Photosystem 2D (PS2D) in photosynthesis; and Rubisco large subunit-binding protein subunit beta (RBCL) in carbohydrate metabolism. Two key genes that encoded the downregulated proteins were also chosen, namely ATP synthase subunit alpha (ASA) which is involved in energy synthesis and oxidative phosphorylation; and ribosomal protein S4 (RPS4) associated with protein synthesis. The proteomics data were validated via RT-qPCR, where the relative transcript abundance of selected genes was found to be consistent with their proteomics profiles (Supplementary Fig. S1).

Fig. 2.

Comparative proteomic analysis in Vanilla planifolia Andrews shoot under control and calcium lignosulfonate treatment (CaLS). a Venn diagram of the total protein obtained; b volcanic plot analysis showing differences of protein abundance; c total of differentially abundant protein identified; d KEGG-pathway analysis of upregulated and downregulated expressed proteins

Table 1.

Top 15 proteins showing significant increase in abundance of protein in Vanilla planifolia Andrews shoot under control and calcium lignosulfonate treatment (CaLS)

No	Accession no	Protein description	Pathway	Difference in protein abundance
1	Q9XF97	60S ribosomal protein L4	Protein synthesis	6.223
2	Q9FSF0	Malate dehydrogenase	Krebs cycle	5.264
3	Q2QXR8	Pyruvate kinase 2	Krebs cycle	5.084
4	P54609	Cell division control protein 48 homolog A	Cellular process	4.549
5	Q40977	Monodehydroascorbate reductase	Stress response	4.158
6	Q93WJ8	Monodehydroascorbate reductase 2	Stress response	4.152
7	P08927	Rubisco large subunit-binding protein subunit beta	Photosynthesis	4.135
8	Q08480	Adenylate kinase 4	DNA synthesis	3.890
9	O23755	Elongation factor 2	Protein synthesis	3.885
10	P51824	ADP-ribosylation factor 1	Cellular process	3.715
11	Q06GR5	Photosystem II D2 protein	Photosynthesis	3.708
12	P50346	60S acidic ribosomal protein P0	Protein synthesis	3.670
13	Q9STX5	Endoplasmin homolog	Protein modification	3.346
14	Q6ER94	2-Cys peroxiredoxin BAS1	Stress response	3.315
15	Q9SF16	T-complex protein 1 subunit eta	Protein synthesis	3.238

Table 2.

Top 15 proteins showing significant decrease in abundance of protein between Vanilla planifolia Andrews shoot under control and calcium lignosulfonate treatment (CaLS)

No	Accession no	Protein description	Pathway	Difference in protein abundance
1	P41344	Ferredoxin–NADP reductase, leaf isozyme	Photosynthesis	− 6.253
2	P41346	Ferredoxin–NADP reductase	Photosynthesis	− 5.177
3	P46299	40S ribosomal protein S4	Protein synthesis	− 4.684
4	P49211	60S ribosomal protein L32-1	Protein synthesis	− 4.460
5	Q9AT35	60S ribosomal protein L23a	Protein synthesis	− 4.453
6	P00761	Trypsin	Protein modification	− 4.432
7	Q02028	Stromal 70 kDa heat shock-related protein	Stress response	− 4.175
8	Q9M5L0	60S ribosomal protein L35	Protein synthesis	− 4.092
9	P22953	Probable mediator of RNA polymerase II transcription subunit 37e O	Protein synthesis	− 4.088
10	Q9SRX2	60S ribosomal protein L19-1	Protein synthesis	− 3.980
11	P22738	60S ribosomal protein L3-2	Protein synthesis	− 3.645
12	P49688	40S ribosomal protein S2-3	Protein synthesis	− 3.495
13	P83291	NADH-cytochrome b5 reductase-like protein	Krebs cycle	− 3.481
14	Q03685	Luminal-binding protein 5	Stress response	− 3.437
15	P62786	Histone H4 variant TH091	DNA modification	− 3.384

The Gene Ontology (GO) analysis showed all identified proteins were categorised into functional classes based on their biological process, molecular function, and cellular localization (Supplementary Fig. S2). According to the biological process, the identified proteins were classified into the following nine groups, namely cellular process, metabolic process, biological regulation, stimulus response, signalling, multicellular organisms, localization, cellular component organisation, and developmental process. However, most of the identified proteins were involved in cellular and metabolic processes (34.24% and 33.15%). In terms of molecular function, the major identified proteins were grouped in catalytic activity and binding (44.07% and 40.68%). While, for the cellular localization perspective, the identified proteins were mainly localised in the nucleus (20%), cytoplasm (18.82%), and chloroplast (17.65%).

Nutrient ion contents in V. planifolia under CaLS treatment

Nutrient ion analysis was performed via ICP-OES to determine the effect of CaLS on the nutrient content in V. planifolia. Our result showed that the CaLS-treated plants contained higher concentrations of calcium, sodium, magnesium and zinc as compared to the control (Table 3). With CaLS treatment, the calcium ion was increased by twofold, while sodium ion showed fourfold increment. However, only a slight decrease of potassium, iron and manganese ions was observed in CaLS-treated V. planifolia (Table 3).

Table 3.

Nutrient ions contents of Vanilla planifolia Andrews shoot under control and calcium lignosulfonate treatment (CaLS). Data shown are from a pooled sample of n = 5

	Nutrient Content (mg/kg)
	Na	K	Ca	Mg	Fe	Mn	Zn	Cu
CTRL	86.5 ± 8.07	3320 ± 133.21	388 ± 19.62	128 ± 9.28	4.14 ± 0.31	19.5 ± 3.34	3.39 ± 0.43	ND
CaLS	346 ± 20.18*	3080 ± 82.84	849 ± 33.29*	133 ± 10.21	3.81 ± 0.45	14.2 ± 3.91	3.64 ± 0.55	ND

ND, not detected. Means ± standard deviation followed by * in a row which indicates statistical significance between control and CaLS−treated samples (p < 0.05)

Changes in growth-related compounds in CaLS-treated V. planifolia

To evaluate the shoot induction effects of CaLS in V. planifolia, several biochemical and enzymatic assays were performed to determine the changes in growth-related compounds upon CaLS treatment. This included Rubisco activity, hexokinase activity, isocitrate dehydrogenase activity, total soluble carbohydrate, glutamate synthase activity, and total protein content. The enzymatic assays showed CaLS treatment significantly increased Rubisco activity, hexokinase activity and isocitrate dehydrogenase activity. An increment in the Rubisco activity was recorded in CaLS-treated plants (0.122 μmol min⁻¹), compared to the control (0.091 μmol min⁻¹) (Fig. 3a). Similar trends were also detected in hexokinase activity and isocitrate dehydrogenase activity for CaLS treatments (0.15 μmol min⁻¹ and 0.20 μmol min⁻¹, respectively) as compared to the control plants (0.11 μmol min⁻¹ and 0.08 μmol min⁻¹, respectively) (Fig. 3b, c). In total soluble carbohydrate, a higher concentration was detected in CaLS treatment (1.01 mg g⁻¹ FW) as compared to the control (0.76 mg g⁻¹ FW) (Fig. 3d). Glutamate synthase activity and total protein contents showed significant increment in CaLS-treated plants. CaLS-treated plants showed a higher glutamate synthase activity (1.89 μmol g⁻¹ FW) as compared to the control (1.44 μmol g⁻¹ FW) (Fig. 3e). Similarly, an increment of total protein content was observed in plants treated with CaLS (4.02 mg g⁻¹ FW), compared to the control (3.03 mg g⁻¹ FW) (Fig. 3f).

Fig. 3.

Biochemical analyses on growth-related enzyme activities and contents in Vanilla planifolia Andrews shoot under control and calcium lignosulfonate treatment (CaLS). a Rubisco activity; b hexokinase activity; c isocitrate dehydrogenase activity; d total soluble carbohydrate; e glutamate synthase activity; f total protein content. Data are presented as means ± standard deviations of n = 3 biological replicates. Asterisk (*) above bars indicate significant differences at p < 0.05

Changes in stress-related compounds in CaLS-treated V. planifolia

The levels of peroxidase and H₂O₂ were determined to evaluate the changes in ROS accumulation and ROS scavenging system in V. planifolia under CaLS treatment. Compared to the control plants (2.82 U mg⁻¹ protein), plants treated with CaLS treatment exhibited higher peroxidase content (3.54 U mg⁻¹ protein) (Fig. 4a). However, the concentration of H₂O₂ was much lower in CaLS treatment (1.80 nM g⁻¹ FW) than control (3.05 nM g⁻¹ FW) (Fig. 4b).

Fig. 4.

Biochemical analyses on stress-related compounds activity in Vanilla planifolia Andrews shoot under control and calcium lignosulfonate treatment (CaLS). a Peroxidase content; b hydrogen peroxide content. Data are presented as means ± standard deviations of n = 3 biological replicates. Asterisk (*) above bars indicate significant differences at p < 0.05

Discussion

CaLS improved nutrient ion uptake in V. planifolia

Nutrients are vital for plant growth and development. Enhancement of nutrient uptake in plants could be linked with an increase in nutrient availability in the growth media (Wan Abdullah et al. 2021). Our results showed that calcium was significantly increased in CaLS-treated plants. As reported previously, CaLS could be dissociated into calcium ions and lignosulfonate which in turn increased the bioavailability of calcium ions in the media (Cieschi et al. 2016). Subsequently, calcium ions could be taken up by the roots and transported into plant cells via the calcium ion channel. According to Wan Abdullah et al. (2020), CaLS improved the root length and root percentage in V. planifolia. The improvement of the root structure could lead to better nutrient absorption in V. planifolia. Therefore, the increment of calcium concentration in V. planifolia was believed to be due to the exogenous calcium supply from CaLS and also the improvement of root structure. Calcium ion is essential for cell wall and membrane stability, as well as signalling transduction in physiological and developmental processes (López‐Lefebre et al. 2001). Changes in calcium ion levels might activate the downstream signalling molecules, triggering gene transcription and protein translation to support plant growth and development. Previous studies reported that calcium ion signalling induced numerous responses in the regulation of plant growth and development such as nutrient sensing (Liu et al. 2017; Kudla et al. 2018), and phytohormone regulation (Li et al. 2019). Besides, calcium facilitates the regulation of nutrient uptake and homeostasis (Aranda-Peres et al. 2009; Wan Abdullah et al. 2021). Calcium activates the Ca²⁺ decoding complexes such as CBL interacting protein kinases (CIPKs) and Ca²⁺-dependent protein kinases (CDPKs), in which these complexes regulate the transport proteins responsible for nutrient uptake (Behera et al. 2017; Dubeaux et al. 2018). For instance, CIPK23 regulates the expression of ferrous iron transporter which controls the uptake of iron and non-iron essential heavy metals such as manganese, zinc, cobalt and cadmium (Barberon et al. 2011; Thor 2019). This study also revealed that CaLS-treated plants had higher contents of sodium, magnesium and zinc as compared to the control. Sodium was the major increased nutrient in CaLS-treated plants. Sodium ion has been postulated to stimulate plant growth and maintain osmotic potential in plants. Sodium ions were found to improve growth performance and maintain osmotic potential in Zygophyllum xanthoxylum and sugar beet (Ma et al. 2012; Wu et al. 2015). Ma et al. (2012) also suggested that the growth stimulating effect of sodium ions could be associated with the improvement in stomatal conductance and leaf development. Thus, these results suggested that CaLS enhances nutrients absorption in V. planifolia, which may lead to improved cellular metabolism and improved shoot multiplication.

CaLS enhanced photosynthesis capacity in V. planifolia

The proteomic analysis identified 49 differentially expressed proteins associated with photosynthesis. The major upregulated proteins related to photosynthesis in CaLS treatment were Rubisco large subunit-binding protein subunit beta and Photosystem II D2 protein. Photosystems are the energy-transforming complexes in chloroplast which absorb light and excite electron in thylakoid membrane to generate ATP and NADPH. Photosystem II (PSII) is the first light-capturing complex and participated in splitting of water molecules into electrons and hydrogen ions (Gao et al. 2018). The generated ATP and NADPH then enter the Calvin cycle to produce sugar and other carbohydrate molecules. Rubisco is the first enzyme used in the Calvin cycle. Rubisco catalyses the process of carbon fixation and takes part in photorespiration, which maintains the level of ribulose-1,5-bisphosphate and prevents photo damage in plant cells (Huang et al. 2015). As reported previously, the lack of Rubisco large subunit-binding protein and downregulation in PSII photochemistry had resulted in decreased photosynthesis rate and reduced plant growth (Wu et al. 2013; Liu et al. 2019). Besides, the increasing levels of calcium ion in V. planifolia was believed to activate the downstream signalling molecules to induce transcriptional and metabolic responses crucial for photosynthesis. For instance, the application of calcium was found to improve photosynthesis in cucumber (Liang et al. 2009) and spring wheat (Dolatabadian et al. 2013). Hochmal et al. (2015) also reported the important role of calcium ions in chloroplast to regulate the photosynthetic pathway. In light-dependent reaction, calcium ion acts as a cofactor in forming an active site for PSII and facilitates water oxidation to produce ATP (Popelkova et al. 2011; Wang et al. 2019). In addition, calcium ion has been postulated to increase photosynthetic capacity by regulating Calvin cycle enzymes. Previous studies reported that reduction of the Calvin cycle enzymes decreased photosynthetic capacity and starch biosynthesis, thus affecting plant growth (Koßmann et al. 1994; Harrison et al. 1997). Besides, it was also shown that calcium ions could improve photosynthesis by regulating stomata opening and closure (Young et al. 2006; Schulze et al. 2021). Young et al. (2006) suggested that repression of calcium transients in Arabidopsis plants caused impairment of stomatal opening and closure. In this study, our results also revealed that CaLS increased Rubisco activity and carbohydrate content in V. planifolia shoot. Therefore, CaLS treatment may enhance the photosynthesis capacity and carbohydrate synthesis in V. planifolia, thereby promoting plant growth and development.

CaLS increased carbohydrate and protein metabolism in V. planifolia

Carbohydrate is an important energy source in plant cells. The carbohydrates produced from photosynthesis are broken down into simple sugar and transported to other plant parts for cellular metabolism (Slewinski and Braun 2010). Sugar metabolism begins with glycolysis to form pyruvate molecules, which are further used in the Krebs cycle. Energy was released in the form of ATP during glycolysis and Krebs cycle (Zhong et al. 2016). In this study, a total of 49 differentially expressed proteins involved in carbohydrate and energy metabolism were upregulated in CaLS treatment, whereby malate dehydrogenase and pyruvate kinase were the major upregulated proteins (Table 1). In addition, biochemical analysis recorded that CaLS significantly increased the activity of hexokinase and isocitrate dehydrogenase in V. planifolia shoot. Hexokinase and pyruvate kinase are key enzymes in glycolysis, while isocitrate dehydrogenase and malate dehydrogenase are crucial for the Krebs cycle. In the first step of glycolysis, hexokinase catalyses the conversion of glucose into glucose-6-phosphate. The glucose-6-phosphate then undergoes a series of conversions into phosphoenolpyruvate. The phosphoenolpyruvate is converted to pyruvate by the enzyme pyruvate kinase. Along the process of glycolysis, ATP molecules are generated (Oliver et al. 2008; Salazar-Roa and Malumbres 2017). Upon completion of glycolysis, pyruvate undergoes oxidation and attaches to coenzyme A to form acetyl-CoA. The acetyl-CoA is crucial for the Krebs cycle, where it delivers the acetyl group to the Krebs cycle to be oxidised and combined with oxaloacetate to form citrate (Ciccarone et al. 2017; Wang et al. 2017). Citrate is then converted into isocitrate, and isocitrate is oxidised to form α-ketoglutarate. This reaction is catalysed by isocitrate dehydrogenase (Sienkiewicz-Porzucek et al. 2010; Salazar-Roa and Malumbres 2017). The α-ketoglutarate then undergoes a series of reactions to form oxaloacetate. Oxaloacetate is formed from malate by the catalysis reaction of malate dehydrogenase (Yao et al. 2011). With the presence of oxaloacetate, the Krebs cycle can be repeated to produce more energy. This cycle generates ATP directly, as well as NADH and FADH₂ which are used for ATP production through oxidative phosphorylation (Wang et al. 2017). Previous studies reported that an increase in glucose-6-phoshate, a hexokinase product, raises the glycolytic rate, whereas a reduction in pyruvate kinase decreases the pyruvate level and other organic acids in the Krebs cycle which affects the Krebs cycle efficiency (Claeyssen and Rivoal 2007; Oliver et al. 2008). Sienkiewicz-Porzucek et al. (2010) also reported that the reduction of isocitrate dehydrogenase decreased the flux through the Krebs cycle, thus slowing down the process of the Krebs cycle. However, overexpression of malate dehydrogenase enhanced plant growth via alteration of energy generation (Yao et al. 2011). Hence, our results suggested that CaLS could enhance the rate of glycolysis and Krebs cycle, contributing to increasing ATP production in V. planifolia. The generated ATP could then be used to provide adequate energy for plant growth and development.

Protein metabolism is regulated by the rate of protein turnover, which is crucial to maintain the basic requirement of cellular metabolism and developmental processes (Goff 2011). According to Wullschleger et al. (2006), ribosome biogenesis and protein translation are crucial in controlling cell growth. Our results showed that the glutamate synthase activity and total protein content were significantly increased in CaLS treatment, whereby 25 differentially expressed proteins associated with protein synthesis were increased in abundance. The major upregulated proteins include 60S ribosomal protein L4, elongation factor 2, 60S acidic ribosomal protein P0, and T-complex protein 1 subunit eta. Protein synthesis consists of two stages, namely transcription and translation. DNA acts as a template to produce mRNA in nucleus during transcription and the mRNA is transferred to ribosome. At the same time, ribosome, consisting of a large subunit and a small subunit, is synthesised in the cytoplasm, preparing for protein translation. The 60S ribosomal protein L4 and 60S acidic ribosomal protein P0 participate in production of the large ribosomal subunits in plant cells, facilitating ribosome assembly for protein synthesis (Schmeing 2013). Stelter et al. (2015) also reported that 60S ribosomal protein L4 is associated with assembly chaperone (Acl4) and is released to be incorporated into the pre-60S to prevent cellular degradation and unnecessary interaction, aiding in the efficiency of ribosome assembly. As reported previously, defective ribosomal biogenesis results in a reduction in protein synthesis, which then affects plant development (Cho et al. 2013). During the initiation stage of translation, a small ribosomal subunit and tRNA are attached to the mRNA, and then followed by a large ribosomal subunit. As the ribosome moves along the mRNA, tRNA is released and a new tRNA is attached. This process is repeated whereby the amino acid chain is formed. In this elongation stage, elongation factor 2, a GTPase protein, catalyses the ribosomal translocation process (Guo et al. 2002). Previous reports also postulated that the deactivation of the translational elongation factor inhibits protein translation, making an unfavourable condition for cell growth (Li et al. 2011; Pereira et al. 2015). At the termination stage, the polypeptide chain is released as a stop codon is detected. The polypeptide chain then undergoes protein folding and modifications. Protein folding is important to ensure the structurally folded is functional to modulate cell developmental processes. T-complex protein 1 subunit eta’s role as a cytosolic chaperonin was reported to be involved in protein folding (Hill and Hemmingsen 2001). Therefore, our data revealed that CaLS promotes protein metabolism activity in the shoot of V. planifolia, which regulates various vital metabolic processes to improve shoot multiplication.

CaLS induced ROS scavenging system and maintained cellular homeostasis in V. planifolia

ROS are produced in plant cells during basic cellular metabolism, mainly in photosynthesis, photorespiration, and β-oxidation of fatty acids, (De Gara et al. 2010). Among the ROS, H₂O₂ is known to be cytotoxic, resulting in cellular damage, lipid peroxidation, and protein denaturation (You and Chan 2015). Previous studies revealed that H₂O₂ is involved in signalling for defence mechanisms, hormonal responses, and plant growth and development (Ye et al. 2011; Schippers et al. 2016; Mhamdi and Van Breusegem 2018). However, excessive ROS will cause stress in plants, which adversely affects plant growth and metabolism. To prevent oxidative stress in plant cells, ROS-scavenging enzymes are produced to eliminate excessive ROS and maintain ROS at a low level for signalling events in plant growth, development and stress response (You and Chan 2015; Chapman et al. 2019). The proteomics profiling revealed that a high abundance of stress response-related proteins was detected in CaLS-treated plants. Enzymes involved in the ROS scavenging activity in V. planifolia shoot were upregulated upon CaLS treatment, including monodehydroascorbate reductase (MDAR) and 2-Cys peroxiredoxin. The MDAR plays a vital role in the ascorbate–glutathione cycle for plant protection against ROS. The ROS scavenging enzymes catalyse the reduction of H₂O₂ into water molecules and monodehydroascorbate. The MDAR maintains the scavenging activity by reducing the monodehydroascorbate into ascorbate, where ascorbate is a major antioxidant in plants (Vadassery et al. 2009). The role of 2-Cys peroxiredoxin as peroxidase is also well-recognized in eliminating H₂O₂ and balancing redox homeostasis in plant cells (Awad et al. 2015). Likewise, the biochemical analysis showed that the concentration of peroxidase was increased in V. planifolia treated with CaLS. However, the accumulation of H₂O₂ in the shoot of V. planifolia was significantly reduced in CaLS treatment as compared to the control. This might be due to the high levels of ROS scavenging enzymes induced in the plant cells. The ROS scavenging system was strengthened as monodehydroascorbate reductase, 2-Cys peroxiredoxin, and peroxidase were upregulated upon CaLS treatment. The strengthened ROS scavenging system can effectively eliminate the accumulation of H₂O₂ and maintain a minimal level of H₂O₂ for plant growth and development. Hence, CaLS potentially induces the shoot growth of V. planifolia through the elimination of ROS and maintenance of cellular homeostasis.

Conclusion

In summary, CaLS has a significant impact on the shoot multiplication of V. planifolia. Our study revealed that CaLS treatment increased the concentration of calcium ions in V. planifolia. The increment of calcium concentration induced signalling responses stimulated nutrient absorption and maximized photosynthesis capacity in V. planifolia. Increase in carbohydrate synthesis and energy generation due to an increase in photosynthesis and upregulation of proteins encoded for cellular metabolism had contributed to enhancing shoot growth. CaLS also activated the production of ROS scavenging enzymes in V. planifolia, resulting in reduced ROS accumulation and optimised shoot growth. Taken together, CaLS promoted shoot multiplication in V. planifolia Andrews through enhanced nutrient uptake, upregulated cellular metabolism, and maintenance of cellular homeostasis in plant cells (Fig. 5). In future, a combination of omics technology could be performed to gain a deeper understanding on underlying mechanisms of CaLS in improving V. planifolia shoot multiplication.

Fig. 5.

Proposed mechanism of CaLS on shoot multiplication of Vanilla planifolia Andrews

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

KSL conceived the study conception and design. KLT and WMANWA carried out all the experiments and wrote the manuscript. JOA, DUL, CYW, MHYMY, JYL and WHC edited and reviewed the manuscript. All authors read and approved the manuscript.

Funding

The work was supported by the Fundamental Research Grant Scheme [FRGS/1/2019/STG05/UPM/02/27] from the Ministry of Higher Education, Malaysia.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Declarations

Conflict of interest

The authors have no competing interests to declare that are relevant to the content of this article.