Expression analysis of phosphatase III-B subfamily in common bean reveals two storage proteins highly expressed during seed formation and germination
Lucia O. Pareja, Mercedes Diaz-Baena, Gregorio Galvez-Valdivieso, Pedro Piedras
TL;DR
This study identifies two storage protein genes in common beans that are highly active during seed development and germination, and may help plants respond to stress.
Contribution
The study identifies two new putative vegetative storage protein genes in common bean with distinct expression patterns and potential stress-related roles.
Findings
PvNTD9 and PvNTD10 are highly expressed in developing seeds, flowers, and pods.
PvNTD9 and PvNTD10 show different expression kinetics during post-germinative development.
PvNTD9 lacks phosphatase activity and may have a non-enzymatic function.
Abstract
Germination and seedling development are crucial phases in the plant life cycle with economical and agronomical implications. During these stages, seedlings activate a wide range of strategies to ensure an adequate supply of nutrients and defence against environmental stress. The common bean phosphatase III-B subfamily comprises 11 genes, named PvNTD1 to PvNTD11. Phylogenetic alignment with Arabidopsis thaliana III-B phosphatases identified PvNTD9, PvNTD10 and PvNTD11 as candidates Vegetative Storage Proteins (VSP) homologs. Among these, PvNTD9 and PvNTD10 exhibited high expression in developing seedlings, flowers and developing pods, tissues characterized by an intense mobilization and accumulation of nutrient. These genes showed different expression kinetics during post-germinative development, with PvNTD9 reaching maximum expression earlier than PvNTD10, which suggests that their…
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Taxonomy
TopicsPlant nutrient uptake and metabolism · Plant Stress Responses and Tolerance · Seed Germination and Physiology
Introduction
Legume seeds are characterized by their high nutritional values and protein content (20–45%), representing an ideal source of vegetable proteins (Bera et al. 2023). Among legumes, common bean (Phaseolus vulgaris L.) is t most widely consumed by humans, both as dry seeds and fresh pods (Petri et al., 2015; Ntatsi et al. 2018). Common bean is considered the most important source of protein in many developing world countries, being also an important source of minerals, vitamins, antioxidants, polyphenols, and other phytochemicals (Ntatsi et al. 2018; Castro-Guerrero et al. 2016).
The transition from seed to seedling is a critical step in the plant life cycle, playing a key role in plant development and optimal crop production. Germination sensu stricto begins with the imbibition of the seeds and ends with the emergence of the embryo (Rajjou et al. 2012), after which postgerminative development starts. Both stages require fine-tuned regulation by internal and environmental factors (Farooq et al. 2022). During germination and seedling development, seedlings depend on stored resources such as reserve proteins and nucleic acids, producing a large mobilization of nutrients (Bewley 1997). In legume, seed germination has additional nutritional relevance due to its impact on digestibility (Bera et al. 2023).
Although protein mobilization during germination has been widely studied due to their relative abundance (Bera et al. 2023), the role of nucleic acids as nitrogen and phosphorus reservoirs has received little attention. Nucleic acids are relatively abundant organic molecules in plant cells and represent the largest pool of organic phosphorus (Veneklaas et al. 2012). RNA degradation in common bean cotyledons occurs in parallel to storage protein breakdown, and the resulting compounds can serve as nitrogen and phosphorus sources (Diaz-Baena et al. 2021). Developing seedlings require large amounts of phosphorus to support the synthesis of new nucleic acids, to the extent that nucleotide availability is essential for successful germination (Stasolla et al. 2003). This fact implies the need of a tight balance between synthesis, degradation and salvage of nucleotides during this developmental stage (Mohlmann et al. 2010). These metabolic pathways converge in the formation of nucleoside monophosphate (NMPs), the substrate of phosphatases that hydrolyses the NMPs to nucleosides. The importance of these enzymes is such that the ratio between phosphorylation and dephosphorylation of nucleotides could be critical for the balance between synthesis and degradation of nucleotides (Bogan and Brenner 2010).
A phosphatase with high affinity for NMPs (PvNTD1) has been identified in common bean during seedling development (Cabello-Diaz et al. 2012). This gene encodes a member of the haloacid dehalogenase (HAD) superfamily of phosphatases (Cabello-Diaz et al. 2015), a ubiquitous group of enzymes that catalyses the transfer of phosphate groups (Gohla 2019). Despite their sequence divergence, these enzymes present four conserved domains that form the catalytic site (Du et al. 2021). This includes: Domain I, characterized by the sequence DxDx(T/L)(L/I), that interacts with Mg^2+^ along with domain IV; Domain II, hh(T/S), that maintains a threonine or a serine preceded by a series of hydrophobic amino acids (h); Domain III, characterized by a variable region with a conserved lysine that, together with domain II, helps stabilize the reaction; and Domain IV with a GDx3-4D sequence (Du et al. 2021).
Within the HAD superfamily, PvNTD1 belongs to the phosphatases III-B subfamily, which includes both acid phosphatases and vegetative storage proteins (VSPs) (PF03767) (https://www.ebi.ac.uk/interpro) (accessed on 16/01/2024). In common bean, this subfamily comprised of 11 members, named PvNTD1 to PvNTD11 (Gálvez-Valdivieso et al., 2021). Among them, PvNTD1, encodes a nucleotidase purified from developing bean axes (Cabello-Díaz et al. 2012; 2015), while PvNTD2 encodes a similar enzyme highly expressed in nodules (Gálvez-Valdivieso et al. 2020). In addition, PvNTD9, PvNTD10 and PvNTD11 are induced in radicles treated with methyl jasmonate (Gálvez-Valdivieso et al. 2021).
Some HAD phosphatases are transcriptionally upregulated under phosphate starvation, indicating their potential involvement in phosphate scavenging and remobilization (Pandey et al. 2017; Du et al. 2021). In support of this, nucleotidase activity increases in Catharanthus roseus cell cultures (Shimano and Ashihara 2006) and in soybean (Glycine max) roots under phosphorus deficiency (Ostergaard et al. 1991). Moreover, some members of the HAD family can also be induced by wounding, insect attacks and jasmonates (Berger et al. 2002; Liu et al. 2005) suggesting possible functions in both metabolism and defense pathways. In addition to their putative role as phosphatases, some members of this subfamily may function as VSPs, as described in Arabidopsis (Liu et al. 2005).
In this manuscript, the participation of phosphatase III-B subfamily genes from common bean under different physiological conditions have been analysed, with special emphasis on germinative and post-germinative development. We propose that PvNTD9 and PvNTD10 may act as VSPs in common bean. We show high expression of these genes in seedlings during germination and early seedling development following different temporal patterns of induction. Interestingly gene expression was detected in all tissues analysed, including both source and sink tissues for the genes. To our knowledge, this is the first study reporting the involvement of VSPs in these physiological processes. A molecular characterization, with focus on PvNTD9 expression under conditions of high nutrient mobilization is also presented.
Materials and methods
Plant material and growth conditions
Common bean (Phaseolus vulgaris L. cv. Great Northern) seeds were sterilized and germinated as previously described (Diaz-Baena et al. 2021). Plant material up to 6 days after start of imbibition (DPI) was collected directly from seedlings developed in Petri dishes as previously described (Diaz-Baena et al. 2021). At 6 DPI seedlings were transferred to pots and plants were grown as previously described (Diaz-Baena et al. 2024). Dark induced senescence was applied to leaves by placing aluminun foil at 26 DPI for 5 days as described previously (Lambert et al. 2017). Wounded leaves were obtained by gently marking the leaves with serrated tweezers. In seedlings, treatment with salt (200 mM), methyl jasmonate (50 μM), salicylic acid (0.5 mM) and phosphate (5 mM) were performed in seedlings at 4 DPI and radicles were collected after 24 h. Wounded radicles were obtained as indicated for leaves but in radicles from seedlings at 4.5 DPI and collected after 10 h. In all cases, tissue samples were harvested, immediately frozen in liquid nitrogen, and stored at − 80 °C until further processing.
Nicotiana benthamiana seeds were sown in pots containing a sterilized substrate composed of blonde peat, black peat, and wood fibres (50:45:5, w:w:w) (Gramoflor). Pots were placed in a growth chamber and watered regularly.
Plants were cultured at ambient CO_2_ concentration in a growth chamber with 16/8 h and 26/20 °C of day/night photoperiod and temperature, respectively. Photosynthetic photon flux density was 300 μmol m^−2^ s^−1^ and relative humidity 70%.
RNA extraction, cDNA synthesis and qRT-PCR
RNA isolation and cDNA synthesis were carried out as previously described (Galvez-Valdivieso et al. 2021). qRT-PCR was carried out with an CFX-Connect system (BioRad) using the iQSYBRGreen Supermix (BioRad) and the gene-specific primers indicated in Supplementary Table 1. The PCR program consisted of an initial denaturation of 5 min at 95 ºC, followed by 40 cycles of 15 s at 95 ºC, 30 s at 60 ºC, and 30 s at 72 ºC. Results were normalized using the geometric mean of two reference genes (actin and ubiquitin) and calculated using the 2^−ΔCT^ method (Livak and Schmittgen 2001).The specificity of the pair of primers was verified by real time PCR and sequencing of the products and following the amplicon dissociation curves.
Cloning, transient expression in Nicotiana benthamiana and purification of recombinant protein
The coding sequence of PvNTD9 was amplified from cDNA from 3 DPI seedling axes using the primers CloNTD9 Forward and CloNTD9 Reverse (Supplementary Table 2). The PCR product was cloned into the pSparkII vector (Canvax) and transformed into Escherichia coli DH5αF’ cells. Plasmid integrity was confirmed by double strand sequencing. Using Gateway (R) technology (Invitrogen). PvNTD9 was reamplified with attB flanked primers (attB1-NTD9F and attB2-NTD9R) which also eliminates the stop codon to allow C-terminal tagging) and introduced into the entry vector pDONR207 via BP recombination. Subsequently, LR recombination was performed into the destination vector pGWB502/C-SHTAP, which incorporates two Strep-tag II and 6xHis tag at the C-terminal end of the expressed protein. The final vector was transformed into E. coli DH5α.
Agrobacterium tumefaciens GV3101::pMP90RK (Koncz and Schell 1986) cells were transformed with the expression vector pGWB502/C-SHTAP carrying the PvNTD9 coding sequence. The expected recombinant protein including the tags corresponds to 36 kDa. LB medium supplemented with antibiotics (25 mg/L rifampicin, 50 mg/L kanamycin, 15 mg/L gentamicin and 100 mg/L spectinomycin) was inoculated with an overnight culture and incubated at 28 °C with shaking until an optical density at 600 nm of 0.4–0.5 was reached. At this point, cultures were harvested by centrifugation (1000 g for 15 min), resuspended in infiltration buffer (10 mM MES pH 5.6, 10 mM MgCl_2_ and 200 µM acetosyringone) to an OD_600_ of 0.4 and incubated for 2 h with gentle shaking at room temperature. The same process was performed with A. tumefaciens cells transformed with pBIN61-P19 expressing the P19 RNA silencing suppressor (Qu and Morris 2002). Just before infiltration, equal volumes of both cultures were mixed. The mixture was infiltrated into the abaxial side of 3–4-week-old N. benthamiana leaves. A total of 3 leaves per plant was infiltrated from at least 3 plants per experiment. The same number of leaves were infiltrated with empty vector as negative control. Infiltration was performed with a needleless syringe, applying the solution to the abaxial side of the leaf. Once infiltrated, plants were maintained in the growth chamber until the leaves were collected.
Recombinant protein purification was carried out using the batch method. Frozen N. benthamiana leaves were ground and homogenized in extraction buffer (100 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 10 mM DTT, 0.5% Triton X-100) at a 1:3 (w:v) ratio. The homogenate was incubated on ice for 5 min and then centrifuged at 18,000 g for 10 min at 4 °C. The supernatant was transferred to a new tube, and incubated with 40 µl of Streptactin Sepharose (GE Healthcare) on a rotator for 30 min at 4 °C. The mixture was then centrifuged at 700 g for 1 min at 4 °C and the supernatant (flow-through) was collected. The resin was washed five times with 500 µl with wash buffer (100 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.05% Triton X-100). Finally, protein was eluted with three 60 µl aliquots of elution buffer (100 mM Tris–HCl pH 8, 2 mM DTT, 0.05% Triton X-100, 2.5 mM desthiobiotin) by centrifugation at 700 g for 30 s at 4 °C.
Protein gel immunoblot analysis
Proteins were fractionated by SDS-PAGE in 10% acrylamide gels on a Mini PROTEAN II system (Bio-Rad). For protein determination, gels were stained with Coomassie Brilliant Blue. For immunoblot analysis, gels were transferred to PVDF membrane (Bio-Rad). Membranes were blocked with 5% non-fat dry milk and 0.1% Tween-20 in TBS. The primary antibody was mouse anti-Strep-tag II monoclonal antibody (1:3000, IBA), and the secondary was goat anti-mouse IgG conjugated to alkaline phosphatase (1:10,000, Sigma). The alkaline phosphatase activity was detected with BCIP and NBT.
Preparation of crude extracts
Crude extracts from common bean materials were used for the determination of phosphatase activity. Frozen tissues were ground with a mortar and pestle under liquid nitrogen and stored at −80 °C. Crude extracts were obtained mixing extraction buffer (50 mM TES pH 7 and 3.5 mM sodium deoxycholate) with ground tissue in a ratio 4:1 (v:w). The homogenate was centrifuged at 14,000 g for 15 min at 4 °C, and the supernatant was used as crude extract.
Crude extracts from N. benthamiana samples were used for determination of phosphatase activity and as starting material for purification of recombinant protein. The procedure was the same as indicated above for common bean materials but using as extraction buffer 100 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 10 mM DTT, 5% Triton X-100 and 1 mM PMSF.
Determination of phosphatase activity
In vitro enzymatic activity was determined in crude extracts following the release of inorganic of phosphate in the reaction mixture, as indicated by Cabello-Diaz et al. (2015). The standard activity assay was performed in 50 mM MES buffer (pH 5.5) containing 5 mM of the corresponding substrate and an appropriate amount of crude extract or purified protein. One unit of enzymatic activity is defined as the amount of enzyme catalysing the production of 1 μmol of phosphate per minute.
In gel enzymatic activity was measured by mixing crude extract with loading buffer in the absence of reducing agent at a ratio of 1:3 and loaded onto an SDS-PAGE without heat denaturation and reducing treatment. After electrophoresis, gels were washed six times for 10 min in water with gentle shaking and at room temperature followed by two washes of 15 min each with 50 mM sodium acetate (pH 4.9) with 10 mM MgCl_2_ in the same conditions to renature proteins. Finally, gels were incubated at 37 ºC with Fast Black K (0.5 mg/ml) and p-naphthyl phosphate (0.3 mg/ml) in a 50 mM sodium acetate pH 4.9 with 10 mM MgCl_2_. After colour development, the reaction was stopped by removing the solution, and the gels were photographed.
Statistical analysis
All results represent the mean of at least three independent biological replicates, each with two technical replicates. Statistical analyses were performed using SPSS software (version 25). Tukey’s post-hoc test was applied to determine statistically significant differences among treatments, with the significance threshold set at p < 0.05. For experiments involving comparisons between only two conditions, Student’s t-Test was used. Statistical significance levels were indicated as follows: p < 0.05 (), p < 0.01 (), and p < 0.001 ().
Search for sequences
The Phytozome database v.14.0 (http://phytozome.jgi.doe.gov/pz/portal.html) was used to identify phosphatases III-B genes in Phaseolus vulgaris and Arabidopsis thaliana. Multiple sequence alignments were generated using MUSCLE (Edgar 2004) as implemented in MEGA 11 (Tamura et al. 2021). Phylogenetic relationships were inferred using the Neighbour-Joining method (Saitou and Nei 1987) with the Poisson correction model. The reliability of the branching pattern was assessed by bootstrap analysis based on 1000 replicates.
Results
Molecular analysis of phosphatases III-B family genes in common bean
The 11 genes belonging to the phosphatase IIIB subfamily were used to identify homologous genes in Arabidopsis. A total of 10 homologous were identified, and the resulting phylogenetic tree is shown in Fig. 1. PvNTD9, PvNTD10 and PvNTD11 cluster with AtVSP1 and AtVSP2 genes suggesting a possible functional similarity.Fig. 1. Cladogram of phosphatases III-B subfamilies in common bean and Arabidopsis. The three VSPs previously described in Arabidopsis are indicated. Accession numbers for common bean gene are PvNTD1 (Phvul.004G174200), PvNTD2 (Phvul.011G182400), PvNTD3 (Phvul.001G164000), PvNTD4 (Phvul.001G240100), PvNTD5 (Phvul.007G270800), PvNTD6 (Phvul.008G227000), PvNTD7 (Phvul.010G058800), PvNTD8 (Phvul.010G059000), PvNTD9 (Phvul.010G144200), PvNTD10 (Phvul.010G144300), PvNTD11 (Phvul.010G144600). The phylogenetic tree was constructed using the Neighbor-Joining method with Poisson correction in MEGA11. The tree was tested by bootstrap analysis with 1000 replicates, and bootstrap values (> 50%) are shown next to branches. The evolutionary distances are expressed in substitutions per site, as indicated by the scale bar
The phosphatase functionality of VSPs remains unclear, but it is postulated that a conserved aspartic acid in domain I is responsible for the nucleophilic attack. To explore this, we analysed the amino acids sequence surrounding domains I and IV in the putative bean VSPs (PvNTD9, PvNTD10, and PvNTD11), as well as AtVSP1 and AtVSP2 from Arabidopsis, and the soybean VSP (GmVSPα and GmVSPβ). The two functionally characterized phosphatases PvNTD1 and PvNTD2 from common bean were included as references (Fig. 2). The catalytic aspartate in domain I was present in all sequences except PvNTD10 and the soybean VSPs. The presence of aspartate in the rest of sequences could indicate that these are functional phosphatases. However, a tryptophan residue located three amino acids upstream this aspartate, proposed as important for activity (Leapon et al., 2004) was absent in PvNTD9, PvNTD10 and both soybean VSP, but present in PvNTD1, PvNTD2 and the AtVSPs (Fig. 2). Domain GDxxxD, involved in Mg^2+^ binding, was fully conserved in all sequences. Interestingly, PvNTD1 and PvNTD2 also possess a serine residue upstream of domain IV, absent in other sequences, and predicted to be a phosphorylation site (https://services.healthtech.dtu.dk/services/NetPhos-3.1/), possibly representing a regulatory feature that distinguishes true phosphatases from VSPs.Fig. 2. Amino acid alignment of sequences surrounding domain I and IV in the indicated sequences. The key residues are indicated above. Conserved residues across at least seven sequences are shaded in grey. * indicates the tryptophan residue proposed as relevant for phosphatase activity in domain I. + indicates the serine residue potentially phosphorylated in domain IV
Expression of phosphatases III-B subfamily genes in different tissues
The expression level of the 11 genes belonging to the phosphatases III-B family from common bean was analysed in seedlings, leaves, flowers and developing pods (Fig. 3). PvNTD4, PvNTD5 and PvNTD7 showed no detectable expression in any tissues. The patterns of expression of PvNTD1, PvNTD2, PvNTD6 and PvNTD8 were expressed at moderate levels across tissues, following a similar pattern. PvNTD1 was the most highly expressed among them. PvNTD3, PvNTD9, PvNTD10 and PvNTD11 showed higher expression in pods, with PvNTD9 and PvNTD10 exhibiting particularly strong expression in pods and seedlings, but minimal expression in leaves. Notably, PvNTD10 had much higher expression in flowers compared to PvNTD9 suggesting tissue specific regulatory differences.Fig. 3. Expression pattern of phosphatases III-B subfamily (PvNTD1 to PvNTD11) in several common bean tissues (seedlings, leaves, flowers and pods). Seedlings correspond to whole seedlings at 6 DPI; leaves correspond to the second trifoliate leaves at 28 DPI; flowers are fully opened flowers; pods correspond to developing pods without seeds. Expression analysis was performed using qRT-PCR. Values represent mean ± SE of three independent biological replicates with two technical replicates
PvNTD expression during germination and early seedling development
The high expression levels of PvNTD9, PvNTD10 and PvNTD1 in whole seedlings, and the lack of in silico data for these gene in this developmental context, prompted us to investigate the expression of the phosphatase III-B subfamily during germination and early post-germination development in seedlings. Radicle emergence occurred at 2 DPI, allowing us to distinguish germination from postgerminative development.
In cotyledons, no expression of any of the genes was detected at 1 DPI, except for a low level of PvNTD6 (Fig. 4A), which progressively declined by 5 DPI. PvNTD1, PvNTD9 and PvNTD10, showed a marked increase in expression at 3 DPI, just after radicle emergence. Expression of PvNTD1 and PvNTD10 continued to increase at 5 DPI, whereas PvNTD9 remained stable between 3 to 5 DPI. PvNTD2 and PvNTD3 increase their expression at 5 DPI, while the remaining genes were not detectable at any of the time points analysed or showed very low expression.Fig. 4. Expression pattern of PvNTD1 to PvNTD11 in cotyledons (A), embryonic axes (B) and testa (C) during germination and early seedling development. Expression analysis was performed using qRT-PCR on total RNA samples extracted at the indicated days after start of imbibition. Values are mean ± SE of four independent biological replicates with two technical replicates per experiment. Different letters indicate significant differences among the developmental stages for each gene using Tukey’s post hoc analysis (p ≤ 0.05)
Expression analysis in developing axes revealed some similarities to that observed in cotyledons (Fig. 4B). On day 1 after the start of imbibition, no significant expression of any of the genes was detected. PvNTD1, PvNTD9 and PvNTD10 were again the most highly expressed. As in cotyledons, the expression of PvNTD1 and PvNTD10 increased over their development, while PvNTD9 peaked at 3 DPI and remained unchanged thereafter (Fig. 4B).
In testas, only 1 and 3 DPI were analysed due to RNA quality limitations. None of the 11 genes showed detectable expression at 1 DPI (Fig. 4C). In contrast, at 3 DPI, PvNTD1, PvNTD9 and PvNTD10 were clearly induced, with PvNTD9 and PvNTD10 being dominant.
Therefore, PvNTD1, PvNTD9 and PvNTD10 showed the most significant increase in expression during postgerminative development in all seedling tissues with strong induction after radicle emergence. Notably, PvNTD9 showed earlier induction than PvNTD10 suggesting distinct regulatory roles.
Heterologous expression and functional analysis of PvNTD9
The expression data indicated above, together with the fact that PvNTD10 was previously identified as a pod storage protein and that PvNTD1 was previously purified and characterized, prompted us to explore the function of PvNTD9. The open reading frame for PvNTD9 was amplified from 3-day embryonic axis cDNA and cloned for transient overexpression in Nicotiana benthamiana. The predicted size for recombinant protein is 36 kDa. The overexpressed protein was purified by affinity chromatography showing a molecular mass of approximately 34 kDa (Fig. 5A, B).Fig. 5SDS-PAGE (A) and Western blot (B) analysis of recombinant PvNTD9 purification. M, molecular mass marker; EC, crude extract of PvNTD9-infiltrated tissues; FT, flow through; L1, wash 1; L5, wash 5; E1–E3: elution fractions; EV: crude extract from leaves infiltrated with the empty vector. Samples were collected from N. benthamiana leaves at 4 days postinfiltration. The molecular mass standards are indicated on the left side**.** Arrows indicate the position of PvNTD9 overexpressed protein
Despite proper expression and purification, the protein lacked phosphatase activity when tested with several phosphorylated substrates (pNPP, phosphoenolpyruvate, ADP, inosine monophosphate, guanosine monophosphate, glucose 6 phosphate, threonine phosphate, naphthyl phosphate, tyrosine phosphate), at neutral and acidic pH values, and in the presence or absence of Mg^2+^ cations. The lack of activity in the purified protein prompted us to perform phosphatase activity assays using crude extracts from N. benthamiana infiltrated leaves to rule out the potential loss of activity during purification. No difference in activity was observed between PvNTD9 expressing leaves, empty vector controls, or non-infiltrated leaves, suggesting that PvNTD9 likely lacks phosphatase activity in vivo under the tested conditions, and that the little activity detected corresponds to endogenous N. benthamiana enzymes.
Total phosphatase activity in common bean tissues
The lack of activity in the overexpressed PvNTD9 prompted us to investigate whether PvNTD9 expression correlates with total phosphatase activity. Crude extracts were prepared from the same tissue used for gene expression analysis (Figs. 3 and 4). Phosphatase activity was comparable across all tissues, regardless of the high expression levels of PvNTD9 or PvNTD10 (Fig. 6A). In cotyledons, axes and testas (Fig. 6B) no increase in phosphatase activity was detected at 3DPI, despite the strong expression of PvNTD9. Interestingly, testas at 1 DPI showed relatively high phosphatase activity, (Fig. 6B) despite the absence of detectable expression of any phosphatase IIIB gene at that time point.Fig. 6. Phosphatase activity in crude extracts from the tissues used in Figs. 3 and 4. Activity was measured using pNPP as substrate. Values represent the mean ± SE of at least three independent biological replicates and two technical replicates. Different letters indicate significant differences among the developmental stages for each gene using Tukey’s post hoc analysis (p ≤ 0.05)
PvNTD9 expression in nutrient remobilization and stress conditions
Given its high induction during seedling development, particularly in stages associated with nutrient redistribution, we investigated whether PvNTD9 might also be involved in other physiological processes that require active nutrient mobilization, such as senescence or stress response. In leaves, no change was observed during dark induced senescence (Fig. 7A). In contrast, in cotyledons, the expression levels decreased progressively from 6 to 10 days DPI (Fig. 7B). Given that roots must respond dynamically to fluctuating environmental conditions, PvNTD9 expression was also analysed in radicles exposed to salt stress, MeJA, SA, mechanical wounding and phosphate supplementation. Among treatments, expression changes were only observed in response to MeJA and wounding (Fig. 7C).Fig. 7. Relative expression of PvNTD9 under different nutrient mobilization or stress related conditions in leaves (A), cotyledons (B) and radicles (C). Expression analysis was determined by qRT- PCR. A Expression was determined in leaves at 31 days after start of imbibition in normal growth conditions (−) or subjected for 5 days to dark-induced senescence by placing aluminium foil to the leaves (senescent). B Expression in cotyledons at 5, 8 and 11 DPI. C Expression in radicles from seedlings at 5 DPI in normal growth conditions (−), treated for 24 h with 200 mM NaCl (salt), 50 μM MeJA (MeJA) for 24 h, 0.5 mM SA for 24 h (SA), 10 h after having been wounded with tweezers (W), and 5 mM phosphate supplementation for 24 h (P). Expression analysis was normalized to the control treatments or to expression in cotyledons at 5 DPI. Different letters indicate significant differences among the developmental stages for each gene using Tukey’s post hoc analysis (p ≤ 0.05)
Wounding induced expression in leaves
The induction of PvNTD9 by mechanical wounding alone with the fact that members of the VSPs are known to be upregulated by this stimulus, prompted us to analyse the expression of all members of the phosphatases III-B subfamily in wounded leaves. Among all the genes analysed, only PvNTD9, PvNTD10 and PvNTD11 showed increased expression after wounding. A time-course expression analysis was performed at 2, 10 and 24 h post-wounding. PvNTD9 showed early induction at 2 h, but its expression did not increase further at late time points (Fig. 8). In contrast, PvNTD10 and PvNTD11 exhibited delayed response, with strong induction at 10 and 24 h (Fig. 8).Fig. 8. Time course expression of PvNTD9, PvNTD10 and PvNTD11 in wounded leaves. Leaves from plants at 31 DPI were wounded using tweezers. Samples were analysed at 2, 10 and 24 h after wounding. The expression analysis was performed by qRT-PCR and normalized to non-wounded leaves. Values represent the mean ± SE of four independent biological replicates and two technical replicates. Student’s t-test was performed comparing the expression of each gene at each time point with the expression of the undamaged leaves. Statistical significance levels were indicated as follows: p < 0.05 (), p < 0.01 (), and p < 0.001 ()
Discussion
Phosphatases are essential enzymatic activities involved in several developmental and metabolic processes in all living organisms. However, functional characterization of these enzymes is complex due to their broad substrate specificity, making it difficult to assign specific physiological roles (Duff et al. 1994). In common bean, the phosphatase III-B subfamily comprises 11 members (Galvez-Valdivieso et al. 2021). Among them, PvNTD1 and PvNTD2 have been shown to encode phosphatases with high affinity for nucleotides (Cabello-Diaz et al. 2015; Galvez-Valdivieso et al. 2020). Nucleotides, phosphorylated molecules composed of a nucleobase and one to three phosphates groups, are critical for plant metabolism and development. Recent studies have also related members of the phosphatase III-B subfamily to the response to methyl jasmonate in common bean seedlings (Galvez-Valdivieso et al. 2021) as well as to the filling of seed during fruit development (Diaz-Baena et al. 2024). Altogether, these findings point to the involvement of some members of this family in important physiological processes.
Alignment and phylogenetic analysis of common bean and Arabidopsis phosphatase III-B subfamilies revealed that PvNTD9, PvNTD10 and PvNTD11 cluster with AtVSP1 and AtVSP2, two well characterized vegetative storage proteins in Arabidopsis. Among the ten Arabidopsis genes classified as VSPs-like based on sequence similarity (Liu et al. 2005), seven are annotated as acid phosphatases and three as bona fide VSPs (AtVSP1, 2 and 3) (Liu et al. 2005). A distinguishing feature among phosphatases III-B subfamily is the conserved DDDD signature, composed of four aspartate residues distributed in two domains separated by a variable spacer (Thaller et al. 1998). However, the functional significance of this motif, particularly the first aspartate in motif I, remains under debate. For instance, this aspartate is absent in soybean VSPs but present in Arabidopsis (Liu et al. 2005) raising questions about the requirement of the complete DDDD motif for enzymatic activity. Supporting this, restoring of the full DDDD signature in soybean VSP led to a 20-fold increase in phosphatase activity (Leelapon et al. 2004).). In addition, all three Arabidopsis VSP containing this motif exhibit phosphatase activity (Chen et al. 2012; Liu et al. 2005; Sun et al. 2018).
Among the proposed PvVSPs in this study, only PvNTD10 lack the first aspartate in motif I (Fig. 2). This gene was previously identified as Pod Storage Protein (PSP, Zhong et al. 1997). Despite the presence of the first aspartate in PvNTD9, the overexpressed protein showed no detectable phosphatase activity suggesting that additional residues beyond the DDDD motif may modulate enzymatic function. For example, a tryptophan residue located three positions upstream of the catalytic aspartate, previously proposed to influence activity (Leapon et al. 2004), is absent in PvNTD9, PvNTD10 and soybean VSPs (Fig. 2). Similarly, a serine residue upstream of the GDxxxD motif, which is predicted to be phosphorylated, might also play a regulatory role. Furthermore, the high Km values (10–20 mM) reported for AtVSPs (Sun et al., 2012; Liu et al. 2005; Chen et al. 2012) raise questions about whether these proteins function primarily as phosphatases in vivo or whether they might instead fulfil alternative, possibly non-enzymatic, roles in the plant. It is also worth considering the possibility that the overexpressed protein was not correctly folded. However, the lack of correlation between the high expression levels in some tissues and the in vitro phosphatase activity determined in those tissues to rule out this possibility. The lack of enzymatic activity for PvNTD9 and PvNTD10, together with the absence of correlation between their transcript levels and total phosphatase activity in the analysed tissues, supports their classification as vegetative storage proteins (VSPs).
Another characteristic of VSPs is their responsiveness to jasmonates and mechanical wounding (Liu et al. 2005). PvNTD9, PvNTD10 and PvNTD11 were all upregulated by wounding (Fig. 8) and by methyl jasmonate treatment (Galvez-Valdivieso et al. 2021). In these two conditions, the only phosphatases III-B genes that induce its expression were PvNTD9, 10 and 11. Therefore supporting their annotation as PvVSPs. In contrast, the two previously purified proteins, PvNTD1 (Cabello-Diaz et al. 2012) and PvNTD2 (Galvez-Valdivieso et al. 2020) did not induce its expression under these two conditions, and these two proteins showed phosphatase activity with several phosphorylated compounds. Our results reveal a coordinated but temporally staggered response of these genes to wounding, with PvNTD9 acting as an early responder and PvNTD10 and PvNTD11 contributing to a sustained response phase, which may reflect functional specialization.
In this study, we report strong postgerminative induction of PvNTD1, 9 and 10 in cotyledons, embryonic axes, and testas after radicle emergence during common bean germination. To our knowledge, this is the first comprehensive analysis of VSP gene expression during germination and early post-germination development, two stages that require extensive mobilization of nutrients. Developing seedlings needs phosphate with nucleotides being essential compounds as building blocks for RNA and DNA synthesis as well as energetic compounds. We previously proposed that nucleic acids present in cotyledons may serve as a phosphate source during seedling establishment (Lambert et. al 2016 Díaz-Baena et al. 2021). Nucleic acid catabolism involves both a phosphatase and a nucleosidase. In this line, we have recently demonstrated the induction of nucleosidase at both molecular and enzymatic levels in cotyledons and embryonic axes during early seedling development (Delgado-García et al. 2021). Using nucleosides monophosphate as substrate, we had purified the main phosphatase in developing embryonic axes in seedlings up to 4 DPI (PvNTD1, Cabello-Diaz et al. 2012). Taken together, these findings suggest that PvNTD1 might be involved in nucleotide degradation as a nucleotidase, while PvNTD9 and PvNTD10 might function primarily as VSPs.
None of these three genes was expressed at 1 DPI, strongly suggesting that their induction occurs after radicle emergence and is not due to residual RNA from seed development in mother plant. Notably, PvNTD9 is induced earlier than PvNTD10, suggesting that coordinated heterodimer formation, such as that reported for soybean VSPα and VSPβ (DeWald et al. 1992; Leelapon et al. 2004), is unlikely. However, homodimerization cannot be ruled out, as faint dimer sized bands were observed in Coomassie stained gels and Western blot from overexpressed proteins. Indeed, PvNTD1 has been previously reported to form homodimers (Cabello-Diaz et al. 2012) needing harsh denaturing conditions to be dissociate into monomers (Cabello-Diaz et al. 2015).
These findings open intriguing questions regarding the physiological significance of phosphatases III-B genes expression after germination. Seedlings are especially vulnerable during this transition and must rapidly develop adaptative strategies for survival and establishment. The function of these two proteins must be different to that of PvNTD1 and PvNTD2, two phosphatases III-B with activity with several compounds and that showed high affinity for nucleotides as substrates (Cabello-Diaz et al. 2012, 2015; Galvez-Valdivieso et al. 2020). The induction of PvNTD9 and PvNTD10 after root emergence, coinciding with the requirement of intense mobilization of nutrient required for the development of the new plant, suggest a critical role during seedling establishment in common beans. Furthermore, the sequential expression pattern for PvNTD9 and PvNTD10 during development and in response to wounding, suggests they may have complementary rather than redundant roles. Their selective induction in specific contexts, rather than a broad activation during all nutrient mobilization processes, highlights their functional specificity. Together, these findings point to specialized contribution of VSPs to nutrient recycling in early developmental stages and provide a basis for future studies aimed to elucidate their precise biochemical roles and regulatory mechanisms.
Supplementary Information
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