2.1. Sampling of Plant Materials

The leaf samples were harvested from matured 14-year-old T and NT sago palm (Fig. 1) in Pelita Mukah Sebakong Sago Plantation (GPS: 2.851847, 111.827095). The leaf samples were obtained from the third latest developed frond. The leaf samples were harvested in the morning and cut and kept immersed in RNA later (Ambion, USA) solution. Duplicate leaf samples from each plant were obtained for triplicates of T and NT sago palms. In the lab, the samples were removed from the solution and kept at −80°C until further processing.

Figure 1: (A) NT sago palm; bar equals 0.5 m. (B) T sago palm; bar equals 3 m. [Click here to view]

2.2. Isolation and Purification of Poly(A) mRNA From Various Plant Tissue Samples

Leaf tissues were ground into fine powder in the presence of liquid nitrogen. The RNA was extracted from the fine-powdered leaf tissues using extraction kits (RNA queous Total RNA extraction kit, Ambion, USA, and FavorPrep Plant Total RNA Mini extraction kit, Favorgen, Taiwan). Plant Isolation Aid (Ambion, USA) was added to the extraction buffer to counter problems caused by the presence of polysaccharides in tissue samples. The RNA purity was performed through A260/A280 ratio, with the ratio of 1.8 being considered good RNA quality to proceed to the next step.

2.3. First-Strand cDNA Synthesis and polymerase chain reaction (PCR)

The amount of poly(A) mRNA isolated was first adjusted to ensure that an equal amount of template from each sample is used in the differential display analysis. Reverse transcription of the poly(A) mRNA was performed using GeneFishing DEG Premix Kit (SeeGene, Korea) and MBiotech (Korea). A reverse transcription reaction includes 10 μM dT-ACP1 (5?-CTGTGAATGCTGCGACTACGATIIIII(T)18-3?, with I representing deoxyinosine) and poly(A) mRNA. The reaction was incubated at 80°C for 3 minutes and then chilled on ice for 2 minutes and spun briefly. Reagents (5 × RT buffer, 2 mM Deoxynucleoside triphosphate (dNTP), RNase inhibitor, and Moloney-Murine Leukemia Virus (M-MLV) reverse transcriptase) were added to the reverse transcription reaction and incubated at 42°C for 90 minutes. The reaction was then heated at 94°C for 2 minutes and chilled on ice for 2 minutes and spun briefly and DNase-free water was added to dilute the first-strand cDNA. The first-strand cDNA was stored at −20°C until use.

The first-strand cDNA was used as a template in the subsequent PCR reaction consisting of 10 μM dT-ACP2 (5?-CTGTGAATGCTGCGACTACGATIIIII(T)15-3?), 5 μM arbitrary ACP (5?-GTCTACCAGGCATTCGCTTCATIIIII-(10 random sequence)-3?) (Table 1), 2 × SeeAmp ACP master mix, and distilled water to a final volume of 20 μl volume. The PCR mix was placed in a 94°C preheated thermal cycler (Bio-Rad, USA), and the amplification followed the recommended PCR profile. Twenty different arbitrary ACPs provided in the GeneFishing DEG Premix Kit (SeeGene, Seoul, Korea) were used in this study.

2.4. Selection of Differentially Expressed Transcript (DET)

The PCR products were separated on 2% agarose gel and compared for the presence of bands and differences in the intensity between similar-sized bands. The selection of DETs for cloning was done by visual analysis of the transcript pattern on the gel image and the graph and referring to the relative value of band intensity. Images of the transcript were analysed using ImageJ version 1.44 [14], which translates the transcript patterns into graphs, identifying the presence of bands with peaks in the graph. The higher peaks indicated a higher intensity of the bands, while the width of peaks indicated the thickness of the bands. Band sizes among the transcripts were verified by the graph output, which converted the band intensity into a numerical value. The differentially expressed bands showing a similar pattern in the plant triplicates were chosen and recovered using the GF-1 Gel DNA recovery kit (Vivantis, Malaysia) following the manufacturer’s protocol.

Table 1: Arbitrary ACPs sequence used. Provided in the GeneFishing DEG Premix Kit (SeeGene, Seoul, Korea). [Click here to view]

2.5. Cloning of DETs

The DETs recovered were ligated into a pGEM-T easy cloning vector (Promega, USA). The ligation mix was added to 50 μl of competent cells (JM109 or XL-1Blue) and transformed using the heat-shock method. Preliminary screening of colonies for successful transformants was done by the blue/white screening method. Upon confirmation of successful clones, plasmid extraction using the alkaline lysis method was carried out on 1.5 ml of fresh overnight culture cell pellet. The resulting plasmid DNA pellet was dissolved in 50 μl of dH₂O and an aliquot of the clones was sequenced based on the dideoxy sequencing method.

2.6. Analysis and Characterization of DETs

The sequencing results of the DETs were checked for reliability by checking the chromatogram and performing alignment between the forward and reverse sequences using the ClustalW [15]. Before a BLAST search for sequence alignment, the sequences were screened for vector sequences using the online VecScreen program (http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html). Further removal of sequences from dt-ACP2 and the respective arbitrary primer was then performed manually. Further analysis included searching the screened sequences for open reading frames using the search tool Open Reading Frame (ORF) Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and the standalone software CLC Sequence Viewer version 6 (CLC Bio). The outcome of the search was translated into protein sequence and searched against the online database for similarity using the BLASTP search tool (http://blast.ncbi.nlm.nih.gov/blastp.cgi).

3.1. Differential Expression Analyses

The transcript patterns for the PCR products separated on a 2% agarose gel showed some differentially expressed bands with sizes ranging between 100 and 900 bp. Five DETs selected for their presence in either one sample had over 92% difference in the intensity values between NT and T. Out of these, two contained unannotated sequence. Seven DETs with a clear difference in band intensity had 55% to over 200% difference in intensity values between T and NT. The remaining 11 DETs had a range of 16%–127% difference in intensity between T and NT, and a further two DETs had 4% and 9% difference. The bands that appear more reproducible in the different batches of amplification were desired. Excision was difficult for closely situated bands and priority was placed on the larger sized DETs of over 300 bp. When the cloned sequences were cleaned up by the removal of vector sequence as well as the tagging sequence from the GeneFishing primers, the real sequences were about 100 bp less than the size indicated during gel electrophoresis. Examples of differently expressed bands and their relative intensity values are shown in Figures 2 and 3. Bands that were differentially expressed in T and NT are summarized in Table 2.

A low restriction search on the successfully cloned transcripts found multiple ORFs within a stretch of the DET sequence. Some other sequences did not contain an ORF. The results were double-checked using the standalone software, CLC Sequence Viewer. These ORFs amounted to 185 sequences, annotated and unannotated. The ORF sequences were aligned to the online database using BLASTP. The list of ORF sequences identified in the DETs and the resulting identity matches are presented in Table 2. The ORFs are listed with details such as the location of sequence and length in amino acids (aa), while the alignment details are accompanied by a max score and an expectation value (e-value) to indicate the significance of the match. A high max score and low e-value present higher reliability of the sequence match.

Figure 2: Transcript pattern for amplification with ACP1. (A) Gel photos. Lanes: M, 100 bp marker; 2, NT sample; 3 and 4, T sample; 5, rosette sample. (B) Graphic representation of ACP1 transcripts. Graphs represent transcripts in the respective lanes. The arrows indicate bands that show differences between T and NT; 1t1 and 1t2 DETs were selected and excised for identification. The unselected DET is labeled as ns. [Click here to view]

Table 2: Differences in DETs’ intensity value between T and NT sample (%). [Click here to view]

Figure 3: Transcript pattern for amplification with ACP11. (A) Gel photos. Lanes: M, 100 bp marker; 2, NT sample; 3, T sample. (B) Graphic representation of ACP11 transcripts. Graphs represent transcripts in the lanes. The arrows indicate bands that show differences between T and NT; 11nt1, 11t1, and 11t3 DETs were selected and excised for identification. [Click here to view]

The 43 annotated sequences made up a functionally diverse group of proteins and enzymes, with roles such as redox regulators and roles in biosynthesis and plant defense mechanism, among others. Table 2 shows a compilation of the identity matches for the ORF, which will be discussed about the calculated differential expression. ORF sequences that lack similarity to the sequences in the database, matching nonannotated proteins and with low-similarity matches, present the possibility of being novel genes.

3.2. Functional Analyses of DETs

DET profiles showed the gene expression of T and NT sago palm leaves. Comparison of different profiles would allow the determination of potential genes responsible for trunk formation in the sago palm. This provides baseline information about the molecular studies of the sago palm while imparting future researchers with a list of target sequences for further works of annotating possible novel genes and other proteomic studies. The DETs with identity matches were results of amplification using ACP 1, 2, 4, 5, 8, 9, 10, 11, 13, 14, 15, 17, and 19. Examples of the DET profiles are shown in Figure 4. The different ORF sequences in a single DET also presented identity matches with a variety of protein functions. These include functions in regulating metabolic pathways, in the intercellular and intracellular signaling and transport of molecules, and in catalyzing various biosynthetic reactions in studied organisms ranging from prokaryotes to eukaryotes. However, only identities with known presence in plants are included in the discussion.

3.3. DETs with Redox-Related Functions

Compounds such as reactive oxygen species (ROS) and free radicals are present in all living organisms. Along with other electron-related species, these compounds act as messengers via redox signaling. The high presence of ROS in some parts of the cell, such as in mitochondria, can be attributed to roles carried by the organelles involved, and these by-products of respiration reactions are harmful when accumulated. Linked to its regulation and other redox signaling roles are the following identified DETs.

The DETs encoding for short-chain dehydrogenase/reductase (SDR) and Crp/Fnr family transcriptional regulator were both higher in the T sample by 47% and 49%, respectively. Both proteins are functionally versatile due to their structures. The SDRsuperfamily (SDR) is a group of oxidoreductases that are Nicotinamide Adenine Dinucleotide (Phosphate) (Reduced) Nicotinamide Adenine Dinucleotide (Phosphate) (Reduced) (NAD(P)(H))-dependent. The characteristic Rossmann fold of the SDR protein structure enables it to act as scaffolds for various binding activities of NAD(P)(H) [16]. SDR is often expressed during development at certain stages in specific tissues, for example, tuber, glandular hair of leaves, roots, flowers, and fruits [16,17]. Owing to the conserved motifs in the enzyme, SDR proteins are involved in the redox sensor system, which is linked to regulating the metabolism of amino acid, carbohydrate, cofactor, hormone, lipid, and xenobiotic, as well as in the transcription and signaling. The protein Crp/Fnr transcriptional regulators belong to a large family characterized by an amino-terminal cyclic nucleotide-binding [c? nucleotide monophosphate] domain followed by a specific DNA-binding domain comprised of helix-to-helix motif [18]. The protein Crp/Fnr family of transcriptional regulator functions in the areas of sensory mechanism, DNA recognition, and RNA polymerase interaction [19] and response to various signals, intracellular and exogenous, to include cAMP, anoxia, redox state, oxidative stress, and nitrosative stress [18].

Figure 4: Transcript pattern for amplification with ACP2, ACP3, APC4, ACP5, ACP6, ACP8, ACP9, ACP10, and ACP17. Lanes: M, 100 bp marker; 2, NT sample; 3, T sample. [Click here to view]

Ascorbate peroxidase (APX) is a redox-regulating protein that uses the ascorbate group to reduce peroxide, a species of ROS, to water. The transcript for cytosolic APX was found with a higher abundance in T samples (81%). In other studies, a few types of APXs accumulate in stress-induced plants [20,21]. APX activity was also reported to upregulate in response to heavy metal, drought, water, and heat stress [22,23]. The cytosolic APX specified the expression in cytosols. Out of the few types identified, the cytosolic APX was recommended to be involved in combination stresses. While the APXs may seem readily present in nonstressed plants, this is probably due to the differences in roles played by the APX types.

Accompanying various redox reactions are the electron acceptors and oxidoreductases. The DET encoding for both NADH-dehydrogenase subunit 2 and thioredoxin m₃ [Thioredoxin (TRX)-m₃] was found to be more abundantly expressed in NT (116%). NADH-dehydrogenase subunit 2 is one of the many subunits making up the proton-pumping NADH-dehydrogenase, also called NADH-quinone reductase, NDH-1, or complex I. No specific role is attached to the subunit but as a whole, the protein is involved in aerobic respiration and photosynthesis via NADH-quinone reductase, capable of oxidizing NADH, reducing quinone, and pumping proton [24]. By binding quinone, a peroxide source, it affects the production of ROS. The defective chloroplast NDH in rice mutant shows that the plant growth and PsII yield were slightly impaired in low light and is essential in improving oxidative stress in variable light [25,26]. Other studies identified more NDH subunits in plants and have reported their functions in energy metabolism in the plastid [27]. The other redox-related protein, TRX-m₃, regulates redox homeostasis, and this role was also found to relate to the cellular transport regulating functions. TRX-m₃ reduces plastid glucose-6-phosphate dehydrogenase (G6PDH), one of the enzymes in the oxidative pentose phosphate pathway [28]. The reaction then allows for NADPH regeneration, which is essential in preventing oxidative stress.

Based on the roles of these redox-related proteins, a few interpretations can be made with regard to the activities in the sampled sago palm. The earlier discussion on APX identifies it as a peroxide-reducing protein, with peroxide as a type of ROS and an important messenger in the signaling mechanism. The high transcript levels of APX and SDR in the T sample show an active signaling mechanism occurring, possibly due to the stresses sensed by the palm. This active signaling possibly prevented further accumulation of ROS from affecting the sago palm growth and development. On the other hand, the APX may signify a higher respiration rate in the T palm, indicated by the possibly higher presence of peroxide. Likewise, the two proteins identified in the NT sample, TRX-m₃ and NADH-dehydrogenase subunit 2, were found to take part in the respiratory chain, but without the direct ROS-binding roles as seen with the APX. TRX-m₃ is involved in a reaction that indirectly links it to redox-regulating roles. A list of the proteins linked to redox signaling and regulation is shown in Table 3.

3.4. DETs with Roles in Ca²⁺ and N Signaling and Regulation

Calcium and nitrogen are elements that are also involved in a plant’s signaling system. ROS generated in the various metabolic and respiratory processes not only causes oxidative stress when accumulated but is also found to activate the Ca-permeable channel and K-permeable channel. In such conditions, a high concentration of free Ca²⁺ in the cytosols can be observed. The presence of Ca²⁺ in cytosols also influences the uptake of Na⁺ and K⁺. Sodium (Na) is an element that may be toxic with high levels in the cytosols. In the K, Na, and Ca network, it is found that a low K⁺ level or a high Na⁺ concentration can trigger a change in the Ca²⁺ level [29]. The Ca²⁺ sensors respond by regulating the uptake and distribution of K⁺ and Na⁺. This illustrates the interrelatedness and dependency of the various networks in the plant system.

Proteins involved in the regulative processes involving calcium as the secondary messenger were found with higher abundance in NT samples. DETs encoding calmodulin and Ca²⁺/Na⁺ antiporter sodium/calcium exchanger (NCX) were expressed with intensity differences as high as 123% and 47%, respectively. In Ca-signaling, a stimulus produces a pattern of spikes in Ca²⁺ concentration, with differing frequency and amplitude. This is interpreted by Ca²⁺ sensors such as calmodulin. The complex formed between Ca²⁺ and the Ca²⁺ sensor binds and changes the conformation and activity of the target protein and may also regulate gene expression, indirectly triggering cell response [30]. In plants, Magnesium Proton Exchangers (MHX), which is the homolog of NCX, functions in regulating the Zn²⁺ and Mg²⁺ level with H⁺ gradient as the driving force [31]. A study on sago palm in peat soil found these two micronutrients to be of importance to the palm’s productivity [40]. Mg²⁺ is required for starch synthesis processes. The deficiency of Mg²⁺ was found to inhibit carbohydrate transport, whereas, in excess, it inhibited photosynthesis. Zinc deficiency meanwhile will result in enhanced RNA degradation and reduced rate of protein synthesis, whereas, in toxic levels, photosynthesis and root growth are reduced.

Transcriptional regulator asparagine synthetase C(AsnC) and nonsymbiotic hemoglobin 1 (nsHb) are two proteins involved in regulating nitrogen-related reactions. The DET encoding for AsnC was found with higher abundance in the T sample (56%), whereas the DET encoding for nsHb was only expressed in the NT sample. AsnC is a member of the leucine-responsive regulatory protein family. This transcriptional regulator activates the protein asparagine synthetase A (AsnA), which catalyzes the formation of asparagine from ammonia and aspartic acid, in the presence of Adenosine Triphosphate (ATP) [35]. The involvement of AsnC in the formation of asparagine, indirectly optimizing the utilization of nitrogen in plants [36], can be seen as a mechanism being expressed during nitrosative stress. Asparagine Synthetase (ASN) genes are readily present in plants and are differentially expressed throughout plant development. The roles of the gene products are not limited to recycling nitrogen in the vegetative organs [36]. The other protein, nsHb, is one of the three types of Hb found in plants. These proteins that are expressed in osmotic stress, nutrient deprivation, nitric oxide exposure, and even rhizobial infection act together with APX [37]. The nsHb is classified into class 1 and class 2 based on the oxygen affinity, and of these two, nsHb-1 is more studied. Findings for nsHb-1 include its functions in nitrogen oxide (NO)-related metabolic processes and its expression during stress tolerance [38]. When under an oxygen-limited environment, the reaction between nsHb and NO helps regulate ATP level for mitochondrial electron transport processes [37]. By the presence of these proteins, it can be deduced that both T and NT sago palm were responding to nitrogen limitation.

Table 3: List of genes with redox sensor and regulator-related functions. [Click here to view]

Table 4: List of genes with Ca2+ and N sensor and regulatory-related functions. [Click here to view]

A comparison of the discussed proteins revealed the T sample as having efficient nitrogen utilization through asparagine formation by AsnC and the NT sample as having active nitrogen uptake through nsHb. The transcript in the NT sample also showed a sensor and antiporter functioning to ensure the proper level of Ca²⁺ and possibly Mg²⁺ and Zn²⁺ in the palm. Based on the above protein functions, NT palm can be portrayed as nutritionally challenged, which could be attributed to its growth condition in deep peat soil. A list of the proteins linked to Ca²⁺ and N signaling and regulation is compiled in the following Table 4.

3.5. Transport-Related Proteins

The functions of the transport-related proteins were mostly identified to transport various substrates through membranes. The transcript encoding for ER-Golgi Soluble N-ethylmale-imide-sensitive factor-attachment Protein Receptors (SNARE) complex subunit was found more abundant in the NT sample (127%) where the transcript for sorting nexin (SNX) is more in the T sample (65%). The endoplasmic reticulum-Golgi (ER-G) SNARE complex is involved in the transport between the ER and the G apparatus, functioning as coating proteins and facilitating the membrane fusion between the two organelles [23,41]. Meanwhile, the trafficking role of SNX is suggested to occur between endosomes and vacuole [42]. As a family of cellular trafficking proteins, SNX has general roles in protein–protein interaction via membrane association and possible roles in protein sorting [43,44]. It was initially presumed that since vesicular trafficking and intercellular transport mechanism are a natural process in all cells, their effects on this differential expression study in the NT and T samples would be negligible. Phan et al. [42], however, showed that these proteins are important in ensuring that the plant cellular trafficking system is regulated. In their study, the overexpression of the lipid-binding SNX in Arabidopsis was shown to result in an enlarged endosome formation, restricting the trafficking to vacuoles.

Another protein involved in regulating the plant trafficking system is the previously discussed TRX-m₃, also shown in the NT sample. In the extension of its role in redox homeostasis, TRX-m₃ was described to be responsible for regulating symplastic trafficking [45]. By linking ROS production with callose deposition, TRX-m₃ is suggested to regulate the plasmodesmata permeability and thus, responsible for regulating cellular signal and transport. The importance of this role is supported by the study of stunted growth and the effect of arrested symplastic trafficking [45]. The effect of redox homeostasis in carbon partitioning or callose production is also thought to affect carbon storage and hence the starch production [46].

Other DETs found involved in intracellular transport functions encode for peptide-2 ATP-binding cassette (ABC) transporter and glucosyltransferase 2. The transcript for peptide-2 ABC transporter was comparably abundant in the NT sample (45%), whereas it was glucosyltransferase 2 in the T sample (80%). ABC or ATP-binding cassette transporter is a protein that functions to transport various substrates across membranes, using the energy of adenosine triphosphate or ATP [47]. Based on the protein’s annotation, the function is described to involve peptide. Glucosyltransferase 2 is part of the glycosyltransferase (GT) family. While the GT family is identified with various functions to include signaling, storage, and biosynthesis [48], the role for the transcript isolated in this study was identified to involve the transfer of glucose.

Also, more abundant in the T sample were transcripts encoding for inorganic phosphate transporter 3 (PHT3) and cobalt transport protein (CbiO). Their band intensities in the T sample were higher by 49% and 35%, respectively. PHT3 is a protein that assists in the transport of inorganic phosphate. Under its family classification, its function is localized in mitochondria, mobilizing the mineral across the inner membrane of the organelle [49]. As for the cobalt transport protein, CbiO is one of the four components making up the protein, which include two transmembrane components, a membrane-bound component, and an ATP-binding protein [51]. It is identified as the ABC-type cobalt transport protein, which facilitates the cobalt ion uptake. As with the peptide-2 ABC transporter, the CbiO is the component that enables the trafficking function of the transporter protein. Phosphate and cobalt are both essential minerals required in plant metabolic pathways [51]. Inorganic phosphate, in particular, is one of the components involved in forming ATP. Therefore, the functionality of PHT3 is important for energy production and possibly for the functionality of the ABC-related proteins.

The above proteins were described to have transporting roles, with involvement in other aspects of the plant system. Other than the SNX, other proteins in the T sample are found to have their specific cargo such as peptides to glucose. The proteins PHT3 and CbiO were involved in roles that signify the elements phosphate and cobalt as being actively processed in the T sample. In the NT sample, the proteins were believed to be involved in regulating the trafficking system in the cell and tissue to ensure that protein interaction and the various signaling occur. The higher expression of ER-Golgi SNARE and TRX-m₃ in NT may be related to the other role of TRX-m₃, as discussed earlier. Table 5 shows the compilation of proteins discussed in this section.

3.6. Biosynthesis-Related and Other Proteins

The protein products described in this section were involved in the mechanisms such as plant defense, redox regulation, and cell regulation. In the plant defense mechanisms, the plant must first detect and recognize the threat before it can induce the appropriate responses. Cellular membrane and cell wall both act as the primary defense against invasion while sensing for threats and being involved in general signaling. The structures that signal invasion and trigger plant defenses include the lipopolysaccharide (LPS) and peptidoglycan, which are of bacterial origin. In this study, enzyme functioning in the biosynthetic pathway for LPS was detected, with the DET encoding it higher by 36% in intensity value in the T sample. The transcript identity, O-antigen ligase, functions by adding O-antigen to the glucose group [52]. In the peptidoglycan synthesis pathway, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-lysine ligase or MurE plays a role in the transfer of amino acids [53]. The transcript encoding for the enzyme was found to be more abundant in NT samples (116%). The previous study found the enzyme expressed in leaves and flowers, specifically in plastids, and suggested MurE being involved in the proper development of the chloroplast [54].

Other proteins involved in plant defense are beta-glucosidase and mitogen-activated protein kinase 4 (MAPK4). Detection of potential threat signals and induces the defenses in the form of chemicals and hormones. Portraying the characteristics of family 1 glycoside hydrolases, beta-glucosidase is found to influence the formation of intermediates in cell wall lignifications [56]. It is also stored in an inactive glycosylated form, released when the cell wall is ruptured, activating the chemicals defenses (phytoanticipins) and phytohormones of the plants [57]. The DET encoding for beta-glucosidase was found comparatively abundant in the T sample (16%). The DET encoding for the other protein, MAPK4, was more abundant in the NT sample (529%). The roles of MAPK4 include repressing systemic acquired resistance (SAR) and inducing jasmonic acid (JA)-responsive gene expression [58]. The transcript abundance presents speculation of the high occurrence of signaling and JA-responsive gene expression. Due to the opposing roles of MAPK4 towards SA- and JA-dependent defense pathway, it is indeterminate whether its presence in the NT sample is defense-related.

The following proteins, membrane-associated zinc metallopeptidase and riboflavin synthase, are proteins with roles in energy metabolism. The DETs encoding for these proteins are from the NT sample, with higher band intensity of 123% and 47%, respectively. The membrane-associated zinc metallopeptidase is suggested to have cleaving functions at the trans-membrane [59]. Several homologs of the metallopeptidase have been identified in Arabidopsis using the bioinformatics approach [60]. One of the many homologs is found to be involved in the site-2 protease (S2P) signaling cascade, with roles in regulating the chloroplast development [16]. Proper development of the thylakoid grana and lamellae and accumulation of chlorophyll is important for a functional chloroplast. As with an earlier mentioned protein, membrane-associated zinc metallopeptidase is expected to regulate against abnormal development of chloroplast. As for riboflavin synthase subunit alpha, its role in the formation of riboflavin (B₂) [61] showed that the transcript is expressed in many organisms. Riboflavin is important as flavocoenzymes in energy production. The enzyme is therefore located in the plastids, cytosols, and mitochondria [62].

Table 5: List of genes with transport-related functions. [Click here to view]

In other biosynthetic roles, the glycerol kinase and Phosphotransferase System (PTS) system transcriptional activator are some proteins involved in the synthesis of glycerol and sugar. Both DETs are more abundant in T samples (57% and 4%, respectively) and partly function to regulate the cellular concentration of related substrates. Glycerol kinase is an enzyme involved in the biosynthesis of glycerol, triglyceride, and glycerophospholipids [63]. The protein product, glycerol, is a compound involved in metabolism and cell signaling, particularly in osmotic stress signaling. Glycerol helps maintain the water balance in cells and enhances resistance to osmotic stress [64,65]. Its other products appear to be involved in reactions related to energy metabolism and structure formation. A triglyceride is a form of energy reserve in plants, whereas glycerophospholipids make for a key component in the cell structure. PTS or phosphotransferase system transcriptional activator is one of the many enzymes which function to regulate sugar biosynthesis as a sensor and phosphorylating agent [66].

The following transcript identities were proteins without any similarity in functions or roles with each other. Deoxyhypusine synthase (DHS) was expressed to be 80% higher in the T sample. Its role in adding hypusine to eukaryotic translational initiator factor 5A (eIF-5A) precursor protein is imperative for activating the eIF-5A complex [67]. The eIF-5A has been identified in plants with differing functions among its isoforms, but the activation of the protein relies on DHS. Among the many roles of eIF-5A in plant growth and development, of particular interest are the roles of the isoforms in cell division and senescence [69]. The expression of DHS and an eIF-5A isoform was found to increase in senescing parts of a plant. While identifying this process as a stress tolerance mechanism, a study found that suppression of these proteins prevented senescence and caused plant growth can be severely stunted [70]. The observation provided a possible scenario explaining the stunted disposition of NT sago palm. Finally, isolated in the NT sample with a 127% higher intensity value is GntR transcriptional regulator. The protein was identified as a transaminase that functions in processes such as amino acid metabolism. Studies on this protein identified many other roles which are specific to bacteria but identified as a member of the MocR subfamily [68,71], the protein may be involved in the synthesis and catabolism of various compounds such as rhizopine [72] and pyridoxal phosphate [73,74].

Table 6: List of genes with biosynthesis-related functions and other proteins. [Click here to view]

In a summary, the T sample has a higher expression of defense-related and regulative proteins. Based on the earlier described roles of glycerol kinase and its products, it is presumed that the T sample had the advantage of enhanced tolerance towards osmotic stress and better water stability. The enzyme expressing plant defense and structure-forming roles is beta-glucosidase. The activation of eIF-5A via DHS in the T sample ensures the proper growth via mRNA metabolism and trafficking. On the contrary, its suppression enhances stress tolerance. The NT sample has a higher expression of proteins involved in energy metabolism. The synthesis of riboflavin supports the production of flavocoenzymes needed in the cellular respiration reaction, whereas zinc metallopeptidase and MurE ensure proper synthesis and regulation of the chloroplast. The higher transcript expression of the chloroplast-regulating proteins in NT does not necessarily enhance the energy-generating potential of the organelle in the plant. Rather, their expression may be in response to other circumstances which involve the chloroplast. MAPK4 is the only transcript in the NT sample with regulative roles in the signaling of plant-pathogen defense. The protein is similarly induced in other stress signaling pathways. The transcript identities discussed in this section are presented in Table 6.