Metabolic Responses of the Microalga Neochloris oleoabundans to Extracellular Self- and Nonself-DNA.

Stressed organisms identify intracellular molecules released from damaged cells due to trauma or pathogen infection as components of the innate immune response. These molecules called DAMPs (Damage-Associated Molecular Patterns) are extracellular ATP, sugars, and extracellular DNA, among others. Animals and plants can recognize their own DNA applied externally (self-exDNA) as a DAMP with a high degree of specificity. However, little is known about the microalgae responses to damage when exposed to DAMPs and specifically to self-exDNAs. Here we compared the response of the oilseed microalgae Neochloris oleoabundans to self-exDNA, with the stress responses elicited by nonself-exDNA, methyl jasmonate (MeJA) and sodium bicarbonate (NaHCO3). We analyzed the peroxidase enzyme activity related to the production of reactive oxygen species (ROS), as well as the production of polyphenols, lipids, triacylglycerols, and phytohormones. After 5 min of addition, self-exDNA induced peroxidase enzyme activity higher than the other elicitors. Polyphenols and lipids were increased by self-exDNA at 48 and 24 h, respectively. Triacylglycerols were increased with all elicitors from addition and up to 48 h, except with nonself-exDNA. Regarding phytohormones, self-exDNA and MeJA increased gibberellic acid, isopentenyladenine, and benzylaminopurine at 24 h. Results show that Neochloris oleoabundans have self-exDNA specific responses.

Identification, analysis, and modeling of the YUCCA protein family genome-wide in Coffea canephora.

Auxin is involved in almost every aspect of plant growth and development, from embryogenesis to senescence. Indole-3-acetic acid (IAA) is the main known natural auxin that is synthesized by enzymes tryptophan aminotransferase of arabidopsis (TAA) and YUCCA (YUC) of the flavin-containing monooxygenases family (FMO) from one of the tryptophan-dependent pathways. Genome-wide identification and comprehensive analysis of the YUC-protein family have been conducted in Coffea canephora in the present study. A total of 10 members CcYUC gene family were identified in C. canephora. Phylogenetic analysis revealed that the CcYUC protein family is evolutionarily conserved, and they consist of four groups. In contrast, bioinformatic analysis predicted a hydrophobic transmembrane helix (TMH) for one CcYUC (YUC10) member only. Isoelectric point (pI), molecular mass (Ms), signal peptide, subcellular localization, and phosphorylation sites were predicted for CcYUC proteins. YUC enzymes require the prosthetic group flavin adenine dinucleotide (FAD) and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) for their enzymatic activity. Therefore, we include the molecular docking for CcYUC2-FAD-NADPH-IPyA and yucasin, which is a specific inhibitor for YUC activity. The docking results showed FAD and NADPH binding at the big and small domain sites, respectively, in CcYUC2. IPyA binds very close to FAD along the big domain, and yucasin competes for the same site as IPA, blocking IAA production. Furthermore, in silico point mutations affect the stability of the CcYUC2-4 proteins.

Interaction between fructan metabolism and plant growth regulators.

MAIN CONCLUSION: The relationship of fructan to plant growth regulators is clearly more complicated than it looks and is likely related to differences between fructan molecules in size and structure as well as localization. Fructans are a complex group of carbohydrates composed mainly of fructose units linked to a sucrose molecule. Fructans are present in plants as heterogeneous mixtures with diverse molecular structures and mass, different polymerization degrees, and linkage types between fructosyl residues. Like sucrose, they are frequently stored in leaves and other organs, acting as carbohydrate reserves. Fructans are synthesized in the cell vacuole by fructosyltransferase enzymes and catabolized by fructan exohydrolase enzymes. Several publications have shown that fructan metabolism varies with the stage of plant development and in response to the environment. Recent studies have shown a correlation between plant growth regulators (PGR), fructan metabolism, and tolerance to drought and cold. PGR are compounds that profoundly influence the growth and differentiation of plant cells, tissues, and organs. They play a fundamental role in regulating plant responses to developmental and environmental signals. In this review, we summarize the most up-to-date knowledge on the metabolism of fructans and their crosstalk with PGR signaling pathways. We identify areas that require more research to complete our understanding of the role of fructans in plants.

The source, level, and balance of nitrogen during the somatic embryogenesis process drive cellular differentiation.

Since the discovery of somatic embryogenesis (SE), it has been evident that nitrogen (N) metabolism is essential during morphogenesis and cell differentiation. Usually, N is supplied to cultures in vitro in three forms, ammonium (NH4+), nitrate (NO3-), and amino N from amino acids (AAs). Although most plants prefer NO3- to NH4+, NH4+ is the primary form route to be assimilated. The balance of NO3- and NH4+ determines if the morphological differentiation process will produce embryos. That the N reduction of NO3- is needed for both embryo initiation and maturation is well-established in several models, such as carrot, tobacco, and rose. It is clear that N is indispensable for SE, but the mechanism that triggers the signal for embryo formation remains unknown. Here, we discuss recent studies that suggest an optimal endogenous concentration of auxin and cytokinin is closely related to N supply to plant tissue. From a molecular and biochemical perspective, we explain N's role in embryo formation, hypothesizing possible mechanisms that allow cellular differentiation by changing the nitrogen source.

Current Proteomic and Metabolomic Knowledge of Zygotic and Somatic Embryogenesis in Plants.

Embryogenesis is the primary developmental program in plants. The mechanisms that underlie the regulation of embryogenesis are an essential research subject given its potential contribution to mass in vitro propagation of profitable plant species. Somatic embryogenesis (SE) refers to the use of in vitro techniques to mimic the sexual reproduction program known as zygotic embryogenesis (ZE). In this review, we synthesize the current state of research on proteomic and metabolomic studies of SE and ZE in angiosperms (monocots and dicots) and gymnosperms. The most striking finding was the small number of studies addressing ZE. Meanwhile, the research effort focused on SE has been substantial but disjointed. Together, these research gaps may explain why the embryogenic induction stage and the maturation of the somatic embryo continue to be bottlenecks for efficient and large-scale regeneration of plants. Comprehensive and integrative studies of both SE and ZE are needed to provide the molecular foundation of plant embryogenesis, information which is needed to rationally guide experimental strategies to solve SE drawbacks in each species.

YUCCA-Mediated Biosynthesis of the Auxin IAA Is Required during the Somatic Embryogenic Induction Process in Coffea canephora.

Despite the existence of considerable research on somatic embryogenesis (SE), the molecular mechanism that regulates the biosynthesis of auxins during the SE induction process remains unknown. Indole-3-acetic acid (IAA) is an auxin that is synthesized in plants through five pathways. The biosynthetic pathway most frequently used in this synthesis is the conversion of tryptophan to indol-3-pyruvic acid (IPA) by tryptophan aminotransferase of Arabidopsis (TAA) followed by the conversion of IPA to IAA by enzymes encoded by YUCCA (YUC) genes of the flavin monooxygenase family; however, it is unclear whether YUC-mediated IAA biosynthesis is involved in SE induction. In this study, we report that the increase of IAA observed during SE pre-treatment (plants in MS medium supplemented with 1-naphthaleneacetic acid (NAA) 0.54 µM and kinetin (Kin) 2.32 µM for 14 days) was due to its de novo biosynthesis. By qRT-PCR, we demonstrated that YUC gene expression was consistent with the free IAA signal found in the explants during the induction of SE. In addition, the use of yucasin to inhibit the activity of YUC enzymes reduced the signal of free IAA in the leaf explants and dramatically decreased the induction of SE. The exogenous addition of IAA restored the SE process in explants treated with yucasin. Our findings suggest that the biosynthesis and localization of IAA play an essential role during the induction process of SE in Coffea canephora.

Phenylpropanoids Are Connected to Cell Wall Fortification and Stress Tolerance in Avocado Somatic Embryogenesis.

Somatic embryogenesis (SE) is a valuable model for understanding the mechanism of plant embryogenesis and a tool for the mass production of plants. However, establishing SE in avocado has been complicated due to the very low efficiency of embryo induction and plant regeneration. To understand the molecular foundation of the SE induction and development in avocado, we compared embryogenic (EC) and non-embryogenic (NEC) cultures of two avocado varieties using proteomic and metabolomic approaches. Although Criollo and Hass EC exhibited similarities in the proteome and metabolome profile, in general, we observed a more active phenylpropanoid pathway in EC than NEC. This pathway is associated with the tolerance of stress responses, probably through the reinforcement of the cell wall and flavonoid production. We could corroborate that particular polyphenolics compounds, including p-coumaric acid and t-ferulic acid, stimulated the production of somatic embryos in avocado. Exogen phenolic compounds were associated with the modification of the content of endogenous polyphenolic and the induction of the production of the putative auxin-a, adenosine, cellulose and 1,26-hexacosanediol-diferulate. We suggest that in EC of avocado, there is an enhanced phenylpropanoid metabolism for the production of the building blocks of lignin and flavonoid compounds having a role in cell wall reinforcement for tolerating stress response. Data are available at ProteomeXchange with the identifier PXD019705.

A recent advance in the intracellular and extracellular redox post-translational modification of proteins in plants.

Plants, as sessile organisms, have acquired through evolution sophisticated regulatory signal pathways to overcome external variable factors during each stage of the life cycle. Among these regulatory signals, two pathways in particular, reactive oxygen species and reactive nitrogen species, have become of significant interest in several aspects of plant biology, underpinning these molecules as critical regulators during development, cellular differentiation, and plant-pathogen interaction. Recently, redox posttranslational modifications (PTM), such as S-nitrosylation on cysteine residues and tyrosine nitration, have shed light on multiple protein targets, as they are associated with signal networks/downstream metabolic pathways, capable of transducing the imbalance of redox hemostasis and consequently redirecting the biochemical status under stress conditions. However, most of the redox PTM have been studied only in the intracellular compartment, providing limited information concerning redox PTM in the extracellular matrix of plant cells. Nevertheless, recent studies have indicated the plausibility of redox PTM in extracellular proteins, including cell wall associated proteins. Accordingly, in this review, we endeavor to examine evidence of redox PTM supported by mass spectrometry data in the intracellular and extracellular space in plant cells. As a further example, we focus the last section of this review on illustrating, using molecular dynamics simulation, the effect of S-nitrosylation on the structural conformation of well-known cell wall-associated proteins including pectin methylesterase and xyloglucan endo-transglycosylases.

The cell wall proteome from two strains of Pseudocercospora fijiensis with differences in virulence.

Pseudocercospora fijiensis causes black Sigatoka disease, the most important threat to banana. The cell wall is crucial for fungal biological processes, including pathogenesis. Here, we performed cell wall proteomics analyses of two P. fijiensis strains, the highly virulent Oz2b, and the less virulent C1233 strains. Strains were starved from nitrogen to mimic the host environment. Interestingly, in vitro cultures of the C1233 strain grew faster than Oz2b in PDB medium, suggesting that C1233 survives outside the host better than the highly virulent Oz2b strain. Both strains were submitted to nitrogen starvation and the cell wall proteins were isolated and subjected to nano-HPLC-MS/MS. A total of 2686 proteins were obtained from which only 240 had a known function and thus, bioinformatics analyses were performed on this group. We found that 90 cell wall proteins were shared by both strains, 21 were unique for Oz2b and 39 for C1233. Shared proteins comprised 24 pathogenicity factors, including Avr4 and Ecp6, two effectors from P. fijiensis, while the unique proteins comprised 16 virulence factors in C1233 and 11 in Oz2b. The P. fijiensis cell wall proteome comprised canonical proteins, but thirty percent were atypical, a feature which in other phytopathogens has been interpreted as contamination. However, a comparison with the identities of atypical proteins in other reports suggests that the P. fijiensis proteins we detected were not contaminants. This is the first proteomics analysis of the P. fijiensis cell wall and our results expands the understanding of the fundamental biology of fungal phytopathogens and will help to decipher the molecular mechanisms of pathogenesis and virulence in P. fijiensis.

Biotic interactions in the rhizosphere: a diverse cooperative enterprise for plant productivity.

Microbes and plants have evolved biochemical mechanisms to communicate with each other. The molecules responsible for such communication are secreted during beneficial or harmful interactions. Hundreds of these molecules secreted into the rhizosphere have been identified, and their functions are being studied in order to understand the mechanisms of interaction and communication among the different members of the rhizosphere community. The importance of root and microbe secretion to the underground habitat in improving crop productivity is increasingly recognized, with the discovery and characterization of new secreting compounds found in the rhizosphere. Different omic approaches, such as genomics, transcriptomics, proteomics, and metabolomics, have expanded our understanding of the first signals between microbes and plants. In this review, we highlight the more recent discoveries related to molecules secreted into the rhizosphere and how they affect plant productivity, either negatively or positively. In addition, we include a survey of novel approaches to studying the rhizosphere and emerging opportunities to direct future studies.

Characterization of the PIN Auxin Efflux Carrier Gene Family and Its Expression during Zygotic Embryogenesis in Persea americana.

Auxins are responsible for a large part of the plant development process. To exert their action, they must move throughout the plant and from cell to cell, which is why plants have developed complex transport systems for indole-3-acetic acid (IAA). These transporters involve proteins that transport IAA into cells, transporters that move IAA to or from different organelles, mainly the endoplasmic reticulum, and transporters that move IAA out of the cell. This research determined that Persea americana has 12 PIN transporters in its genome. The twelve transporters are expressed during different stages of development in P. americana zygotic embryos. Using different bioinformatics tools, we determined the type of transporter of each of the P. americana PIN proteins and their structure and possible location in the cell. We also predict the potential phosphorylation sites for each of the twelve-PIN proteins. The data show the presence of highly conserved sites for phosphorylation and those sites involved in the interaction with the IAA.

Protein Profiling of Psittacanthus calyculatus during Mesquite Infection.

Psittacanthus calyculatus is a hemiparasite mistletoe that represents an ecological problem due to the impacts caused to various tree species of ecological and commercial interest. Although the life cycle for the Psittacanthus genus is well established in the literature, the development stages and molecular mechanism implicated in P. calyculatus host infection are poorly understood. In this study, we used a manageable infestation of P. laevigata with P. calyculatus to clearly trace the infection, which allowed us to describe five phenological infective stages of mistletoe on host tree branches: mature seed (T1), holdfast formation (T2), haustorium activation (T3), haustorium penetration (T4), and haustorium connection (T5) with the host tree. Proteomic analyses revealed proteins with a different accumulation and cellular processes in infective stages. Activities of the cell wall-degrading enzymes cellulase and ß-1,4-glucosidase were primarily active in haustorium development (T3), while xylanase, endo-glucanase, and peptidase were highly active in the haustorium penetration (T4) and xylem connection (T5). Patterns of auxins and cytokinin showed spatial concentrations in infective stages and moreover were involved in haustorium development. These results are the first evidence of proteins, cell wall-degrading enzymes, and phytohormones that are involved in early infection for the Psittacanthus genus, and thus represent a general infection mechanism for other mistletoe species. These results could help to understand the molecular dialogue in the establishment of P. calyculatus parasitism.

The Role of Cytokinins during the Development of Strawberry Flowers and Receptacles.

Cytokinins play a relevant role in flower and fruit development and plant yield. Strawberry fruits have a high commercial value, although what is known as the "fruit" is not a "true" botanical fruit because it develops from a non-reproductive organ (receptacle) on which the true botanical fruits (achenes) are found. Given cytokinins' roles in botanical fruits, it is important to understand their participation in the development of a non-botanical or accessory "fruit". Therefore, in this work, the role of cytokinin in strawberry flowers and fruits was investigated by identifying and exploring the expression of homologous genes for different families that participate in the pathway, through publicly available genomic and expression data analyses. Next, trans-zeatin content in developing flowers and receptacles was determined. A high concentration was observed in flower buds and at anthesis and decreased as the fruit approached maturity. Moreover, the spatio-temporal expression pattern of selected CKX genes was evaluated and detected in receptacles at pre-anthesis stages. The results point to an important role and effect of cytokinins in flower and receptacle development, which is valuable both from a biological point of view and to improve yield and the quality of this fruit.

In Vitro Conversion of Coffea spp. Somatic Embryos in SETIS™ Bioreactor System.

Somatic embryogenesis (SE) is an excellent example of mass plant propagation. Due to its genetic variability and low somaclonal variation, coffee SE has become a model for in vitro propagation of woody species, as well as for large-scale production of vigorous plants that are advantageous to modern agriculture. The success of the large-scale propagation of an embryogenic system is dependent on the development, optimization, and transfer of complementary system technologies. In this study, two successful SE systems were combined with a SETIS™ bioreactor immersion system to develop an efficient and cost-effective approach for the in vitro development of somatic embryos of Coffea spp. This study used an efficient protocol for obtaining somatic embryos, utilizing direct and indirect SE for both C. canephora and C. arabica. Embryos in the cotyledonary stage were deposited in a bioreactor to complete their stage of development from embryo to plant with minimal manipulation. Following ten weeks of cultivation in the bioreactor, complete and vigorous plants were obtained. Different parameters such as fresh weight, length, number of leaves, and root length, as well as stomatal index and relative water content, were recorded. In addition, the survival rate and ex vitro development of plantlets during acclimatization was assessed. The best substrate combination was garden soil (GS), peat moss (PM), and agrolite (A) in a 1:1:0.5 ratio, in which the bioreactor-regenerated plants showed an acclimatization rate greater than 90%. This is the first report on the use of SETIS™ bioreactors for the in vitro development of somatic embryos in Coffea spp., providing a technology that could be utilized for the commercial in vitro propagation of coffee plants. A link between research and innovation is necessary to establish means of communication that facilitate technology transfer. This protocol can serve as a basis for the generation and scaling of different species of agroeconomic importance. However, other bottlenecks in the production chains and the field must be addressed.

Auxin-Cytokinin Cross Talk in Somatic Embryogenesis of Coffea canephora.

Cytokinins (CK) are plant growth regulators involved in multiple physiological processes in plants. One less studied aspect is CK homeostasis (HM). The primary genes related to HM are involved in biosynthesis (IPT), degradation (CKX), and signaling (ARR). This paper demonstrates the effect of auxin (Aux) and CK and their cross talk in a Coffea canephora embryogenic system. The transcriptome and RT-qPCR suggest that Aux in pre-treatment represses biosynthesis, degradation, and signal CK genes. However, in the induction, there is an increase of genes implicated in the CK perception/signal, indicating perhaps, as in other species, Aux is repressing CK, and CK are inducing per se genes involved in its HM. This is reflected in the endogenous concentration of CK; pharmacology experiments helped study the effect of each plant growth regulator in our SE system. We conclude that the Aux-CK balance is crucial to directing somatic embryogenesis in C. canephora.

Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism in Catharanthus roseus (L.) G. hairy roots.

Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 µM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.

From genome scissors to molecular scalpel: evolution of CRISPR systems.

From bizarre palindromic repeats to a bacterial defense mechanism, to genome editing tool, and more, Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR has significantly impacted the way we study genome modification in less than a decade. In this review, we would like to highlight some key players over 30 years of research and explain this biotechnological tool's basic mechanisms. We also refer to the evolution of the CRISPR variants and some of the applications derived from them. The understanding and upgrading of this system will be a valuable tool in the years to come to solve some of the challenges in diverse fields from pharmaceuticals to therapeutics, from basic plant genetics to crop improvement, from metabolic engineering to waste management and industrial processing.

Genome-Wide Analysis, Modeling, and Identification of Amino Acid Binding Motifs Suggest the Involvement of GH3 Genes during Somatic Embryogenesis of Coffea canephora.

Auxin plays a central role in growth and plant development. To maintain auxin homeostasis, biological processes such as biosynthesis, transport, degradation, and reversible conjugation are essential. The Gretchen Hagen 3 (GH3) family genes codify for the enzymes that esterify indole-3-acetic acid (IAA) to various amino acids, which is a key process in the induction of somatic embryogenesis (SE). The GH3 family is one of the principal families of early response to auxin genes, exhibiting IAA-amido synthetase activity to maintain optimal levels of free auxin in the cell. In this study, we carried out a systematic identification of the GH3 gene family in the genome of Coffea canephora, determining a total of 18 CcGH3 genes. Analysis of the genetic structures and phylogenetic relationships of CcGH3 genes with GH3 genes from other plant species revealed that they could be clustered in two major categories with groups 1 and 2 of the GH3 family of Arabidopsis. We analyzed the transcriptome expression profiles of the 18 CcGH3 genes using RNA-Seq analysis-based data and qRT-PCR during the different points of somatic embryogenesis induction. Furthermore, the endogenous quantification of free and conjugated indole-3-acetic acid (IAA) suggests that the various members of the CcGH3 genes play a crucial role during the embryogenic process of C. canephora. Three-dimensional modeling of the selected CcGH3 proteins showed that they consist of two domains: an extensive N-terminal domain and a smaller C-terminal domain. All proteins analyzed in the present study shared a unique conserved structural topology. Additionally, we identified conserved regions that could function to bind nucleotides and specific amino acids for the conjugation of IAA during SE in C. canephora. These results provide a better understanding of the C. canephora GH3 gene family for further exploration and possible genetic manipulation.

Tissue-specific proteome characterization of avocado seed during postharvest shelf life.

Avocado is a nutritious and economically important fruit, generating significant income for exporter countries. Recently, by-products of this fruit such as seeds and peels, have raised interest in different industries. However, the biochemical features of the nutraceutical value of these tissues have not been analyzed using molecular approaches during the postharvest shelf life (PSL). We carried out comparative proteomics using tandem mass tagging (TMT) and synchronous-precursor selection (SPS)-MS3. We analyzed testa, cotyledon, and embryo axes from avocado seeds at detachment from the tree (unripe), and after five (breaker) and ten days (ripe) of PSL. We identified 1968 proteins, from which 933 were specific to the testa, 167 to the embryo axis, and 23 to the cotyledon. The testa had a more dynamic proteome than the other tissues, resembling similar stress responses to those observed in peel tissues, such as down-accumulation of translational machinery, cell wall catabolism and synthesis of secondary metabolites. In contrast, the up-accumulation of the biosynthesis of l-glutamine, L-isoleucine, and l-serine was observed in all tissues. Our study provides the basic biochemical and physiological features of avocado seed during PSL and demonstrates that avocado seed tissues could potentially be used as a costless source of high-value compounds. SIGNIFICANCE: Avocado seed as a fruit by-product is a source of different valuable molecules, including those with nutraceutical properties. During PSL, several biochemical and physiological modifications occur in this dispersal unit, which also includes the alteration of several key metabolites' content. However, the proteome profile associated with different metabolic pathways that regulate the inner content of seed metabolites has not been previously studied. Our tissue-specific proteomics TMT-SPS-MS3-based provides the first evidence of molecular and physiological changes in avocado tissues during PSL delivering fundamental knowledge of this organ. In this vein, the modulation of secondary metabolites, amino acid, and sugar metabolism of avocado tissues during PLS can encourage these by-products exploitation in multiple industries.

Differences in the Abundance of Auxin Homeostasis Proteins Suggest Their Central Roles for In Vitro Tissue Differentiation in Coffea arabica.

Coffea arabica is one of the most important crops worldwide. In vitro culture is an alternative for achieving Coffea regeneration, propagation, conservation, genetic improvement, and genome editing. The aim of this work was to identify proteins involved in auxin homeostasis by isobaric tandem mass tag (TMT) and the synchronous precursor selection (SPS)-based MS3 technology on the Orbitrap Fusion™ Tribrid mass spectrometer™ in three types of biological materials corresponding to C. arabica: plantlet leaves, calli, and suspension cultures. Proteins included in the ß-oxidation of indole butyric acid and in the signaling, transport, and conjugation of indole-3-acetic acid were identified, such as the indole butyric response (IBR), the auxin binding protein (ABP), the ATP-binding cassette transporters (ABC), the Gretchen-Hagen 3 proteins (GH3), and the indole-3-acetic-leucine-resistant proteins (ILR). A more significant accumulation of proteins involved in auxin homeostasis was found in the suspension cultures vs. the plantlet, followed by callus vs. plantlet and suspension culture vs. callus, suggesting important roles of these proteins in the cell differentiation process.