Exploring the edible gum (galactomannan) biosynthesis and its regulation during pod developmental stages in clusterbean using comparative transcriptomic approach

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Morpho-physiochemical analysis and galactomannan estimation in RGC-936 and M-83

Morphological observation revealed that RGC-936 has hairy rough leaf surface, purple flower color, 3–4 cluster plant−1, and 4–7 pods cluster−1, 100 seed weight (2.6 g), small and round seed with taller plant stature. On the contrary, M-83 variety has smooth leaf surface, white flower color, 2–3 cluster plant−1, 2–4 pods cluster−1, 100 seed weight (2.2 g), big and flat seeds, and shorter plant stature (Fig. 1A,B). Also, M-83 recorded higher chlorophyll content in comparison to RGC-936. Gum content at 39DAF was found to be 24–26% and 14–16% in RGC-936 and M-83 respectively.

Figure 1
figure1

(A) Pod development stages (25, 39 and 50 DAF). (B) Morphological difference between two genotypes RGC-936 and M-83 and (C) Transcriptome analysis pipeline for identification of DEGs genes related to galactomannan biosynthesis in clusterbean.

Summary of transcriptome sequencing analysis

For transcriptome analysis, RNA extracts from two genotypes (RGC-936 and M-83) during different pod development stages were subjected to Illumina Hiseq X Ten sequencing, which generated a total of 286.85 Mb raw sequencing reads from 12 RNA libraries. After filtering out low quality reads and adaptor removal, 285.71 Mb clean reads were obtained. A total of 86.05 GB data was obtained from the transcriptome sequencing (150 × 2) bp. Finally, through this high-quality data, 422,998 contigs were assembled with an average length of 201 bp and an N50 value of 1,069 bp (Table 1, Supplementary Table 1A). The sequence reads have been submitted to NCBI-SRA database (Submission ID: SUB5664803, BioProject: PRJNA545776). Differentially expressed genes (DEGs) of the fifteen possible combinations viz. R25-M25, R39-M39, R50-M50, R25-M39, R25-M50, R39-M50, M25-R39, M25-R50, M39-R50, R25-R39, R25-R50, R39-R50, M25-M39, M25-M50, M39-M50 were filtered at log2fold change and significantly DEGs (p ≤ 0.01) were found to be 1489, 4417, 1198, 3180, 1958, 5044, 2778, 1710, 3202, 3766, 1964, 4849, 1997, 2450 and 4192 unigenes, respectively. Transcriptome analysis pipeline for identification of DEGs related to galactomannan biosynthesis in clusterbean has been shown in Fig. 1C.

Table 1 Statistical analysis of non-redundant assembly and average mapping percentage (with two biological replicates) at three pod developmental stages (25DAF, 39DAF and 50DAF) in two clusterbean genotypes RGC-936 and M-83.

GO classification and KEGG enrichment analysis of DEGs

GO annotation of the DEGs from the 15 pairwise comparisons (mentioned above) were used to classify genes into three main GO categories (present in the gene ontology annotations) based on total gene count and their role in different functional processes: information storage and processing category, metabolic and cellular function. KEGG enrichment analysis allowed mapping of DEGs to top 20 pathways. At 25DAF (early development), highly expressed genes belonged to metabolic pathways, ribosome, oxidative phosphorylation, photosynthesis and secondary metabolite biosynthesis. At 39DAF (mid development), higher expression was observed for genes involved in metabolic pathways, glutathione biosynthesis, secondary metabolite biosynthesis and cysteine methionine metabolism; while late developmental stage i.e., 50DAF showed higher expression of genes involved in metabolic pathways, secondary metabolite biosynthesis, antibiotic biosynthesis, carbon metabolism, oxidative phosphorylation, amino acid biosynthesis, and carboxy acid metabolism. Thus, genes involved in metabolic pathways and secondary metabolite biosynthesis were common to all the three seed developmental stages, while oxidative phosphorylation genes were prominent at 25 and 50 DAF.

Identification and comparison of number of DEGs potentially involved in galactomannan biosynthesis at three pod developmental stages in clusterbean

Total 209,525, 375,595 and 255,401 unigenes were found at three-time points i.e., R25-M25, R39-M39 and R50-M50 with a higher number of genes at 39DAF followed by 50DAF. Differential gene expression analysis showed a total of 2384 DEGs (1131 up-regulated and 1253 down-regulated), 4780 DEGs (3934 up-regulated and 846 down-regulated) and 1792 DEGs (1238 up-regulated and 554 down-regulated) between R25-M25, R39-M39 and R50-M50 groups respectively. Results showed that highest number of DEGs were expressed during the middle stage of pod development i.e., 39DAF, followed by 25 DAF and 50 DAF (Supplementary Table 1B).

To elucidate the potential pathways involved in galactomannan biosynthesis, we used annotated galactomannan pathway genes in soybean (Glycine max) as a reference sequence. (Supplementary Table 2). Systematic illustration of galactomannan metabolism pathway in clusterbean has been shown in Fig. 2. These included genes encoding for invertase, sucrose synthase, cellulose synthase, GDP- mannose pyrophosphorylase, phoshphomannomutase, phosphomannoisomerase, UDP-galactose-4-epimerase, mannan synthase, glycosyltransferase, galactosyltransferase, endo-β-1,4-mannanase and β mannosidase. Most of the predicted gum related genes showed highest expression in 39DAF followed by 50DAF and least expression in 25DAF. A total of 53, 51, and 43 unigenes, related to sucrose synthase were identified at 25DAF, 39DAF, and 50DAF respectively in clusterbean.

Figure 2
figure2

Galactomannan biosynthesis pathway and galactomannan related gene expression and their correlation with transcription factor in clusterbean. Red arrow denotes relative expression in RGC-936 and black arrow denotes relative expression in M-83 genotype at 39DAF pod developmental stage (using Microsoft Powerpoint based on transcriptome data).

No ESTs related to Phosphomannoisomerase (EC 5.3.1.8) were detected in any of the developmental stages studied. However, nine unigenes which resembled phosphomannomutase (EC 5.4.2.8) (catalyses the reversible conversion of D-mannose 6-phosphate to α-D-mannose 1-phosphate) were identified at 25DAF, while eleven such unigenes were found at 39DAF and nine unigenes at 50DAF. GDP-D-mannose and UDP-D-galactose, the direct precursors for galactomannan biosynthesis are synthesized by enzyme GDP mannose phosphorylase (EC 2.7.7.22) and UDP-galactose 4-epimerase (EC 5.1.3.2) respectively. A total of 27, 33, and 26 unigenes of UDP-glucose-4-epimerase were detected at 25DAF, 39DAF, and 50DAF respectively, while 9 unigenes were observed for GDP-mannose transporter at 25DAF and 21 unigenes at both 39DAF and 50DAF stages.

For endo-β-mannanase, a total of 21, 24, and 23 unigenes, while for cellulose synthase like genes, 165, 161 and 156 unigenes were identified at 25DAF, 39DAF, and 50DAF respectively.

For Mannan synthase (ManS), a key enzyme of galactomannan metabolism, 6 unigenes were found at 25DAF while 2 unigenes were found at both 39DAF and 50DAF. Further, we identified a total of 0, 36 and 39 unigenes related to glycosyltransferase, 135, 136 and 127 unigenes related to α-glucosyltransferase and β-glucosyltransferase, 44, 54 and 53 unigenes related to α-galactosidase, and 137, 145 and 146 β-galactosidase unigenes at 25DAF, 39DAF and 50DAF respectively. Details have been provided in Supplementary Table 3. Out of these unigenes, 1367 (at 25DAF), 2715 (at 39DAF) and 1065 (at 50DAF) unigenes were found to be differentially expressed between two genotypes (Fig. 4B).

Co-expression of gum synthesis genes with identified TFs

Seed development is a highly coordinated process controlled by various TFs. A total of 15 combinations among three developmental stages (25, 39 and 50DAF) were analyzed for TFs/genes related to gum synthesis. Total numbers of annotated genes were found to be 50,275, 49,376, 49,656, 49,376, 49,686, 48,287, 50,095, 49,686, 48,075, 49,999, 49,475, 49,194, 49,622, 49,968, and 48,287 in 15 combinations viz. R25-M25, R39-M39, R50-M50, R25-M39, R25-M50, R39-M50, M25-R39, M25-R50, M39-R50, R25-R39, R25-R50, R39-R50, M25-M39, M25-M50, M39-M50 respectively. However, after filtering the fold change value (p ≤ 0.01 and padj ≤ 0.01), the highest number of genes were identified in M39-M50 (1566) followed by M39-R50 (1218). Stage-specific TFs were also identified such as BHLH family protein, MYB related family protein, LBD, BZIP, NAC, and C2H2. Members of the bZIP, NAC, WRKY and C2H2 families were specifically found to be highly expressed at 39DAF in RGC-936. A model for WRKY and other TFs regulating galactomannan biosynthesis in clusterbean is illustrated in Fig. 3.

Figure 3
figure3

Model for WRKY and other TF regulating galactomannan biosynthesis in clusterbean: WRKY targets 3 genes (Mannan synthase, galactosyl transferase and cellulose synthase) related to galactomannan metabolism. Bold arrow shows enzyme targeted by TF, while dotted blue arrow shows indirect involvement and red arrow shows synergistic role (using Microsoft Powerpoint based on transcriptome data).

Some of the in-silico TFs that were found to be co-expressed with the genes regulating galactomannan biosynthesis were validated using real time PCR. Cellulose synthase expression was found to be highly correlated with LBD, G2-Like and WRKY TFs. β-galactose was found to be co-expressed with NAC TF; epimerase with BHLH TF; mannan synthase with WRKY; galactosyltransferase with BHLH, WRKY TF; α galactosyltransferase with BHLH and WRKY TF; and sucrose synthase with bZIP TF (Fig. 2).

Expression profiling of genes involved in galactomannan biosynthesis using the heat map

MeV software was used for Hierarchical Clustering Analysis (HCA) of DEGs according to their transcription profile. The HCA was performed to see the distribution of DEGs present in the transcriptome data at three pod development stages. Interestingly, we found that the highly expressed genes (indicated by red color) showing annotation of the important genes involved in galactomannan biosynthesis are grouped in cluster II mainly at mid and late development stages in RGC-936 (Fig. 4C). No expression was observed at 25DAF in both the genotypes.

Figure 4
figure4

Differential expressed genes at three pod developmental stages (25DAF, 39DAF and 50DAF) between two contrasting genotypes RGC-936 and M-83 in clusterbean as shown by (A) Volcano plot (using R package), where red color dots represent expressed genes and black color dots represent non expressed genes, (B) Venn diagram (using Venny software) and (C) Heat map diagram (using R package).

Real time PCR validation and spatiotemporal expression profiles of genes potentially involved in galactomannan biosynthesis

A total of six RNA samples with two biological replicates each were isolated from pod tissues at different developmental stages (25DAF, 39DAF and 50DAF) from RGC-936 and M-83. The expression profile of the enzymes directly related to galactomannan biosynthesis was validated by RT-PCR at 39DAF in RGC-936 which correlated well with in-silico data, except for sucrose synthase which showed higher expression in M-83 at 39DAF. The relative expression of α-galactosidase, mannan synthase, glycosyltransferase, cellulose synthase, Mannan endo 1,4 β mannosidase, and phosphomannomutase were found to be significantly higher at 39DAF and 50DAF in RGC-936 as compared to M-83 (Fig. 5). For sucrose synthase, relative expression was found to be 5.4-fold at 39DAF and 0.9-fold at 50DAF in RGC-936, while it was 39.9-fold at 39DAF and 0.8-fold at 50DAF in M-83 (Fig. 5).

Figure 5
figure5

The expression levels and patterns of 15 selected unigenes associated with galactomannan biosynthesis in RGC-936 and M-83 at different pod developmental stages 25DAF, 39DAF and 50DAF in clusterbean were confirmed by qRT-PCR (Using Microsoft Excel).

Real time PCR validation and spatiotemporal expression profiles of TFs potentially involved in galactomannan biosynthesis

The relative expression level of TFs including WRKY, BHLH was found to be higher at 39DAF in RGC-936, while expression of NAC, G2-Like, bZIP was higher in M-83 at 39DAF. MYB relative expression was found to be 3.9-fold at 39DAF and 1.2-fold at 50DAF in RGC-936, while it was 1.5-fold at 39DAF and 621.7-fold at 50DAF in M-83. Expression of LBD, G2 Like, and BHLH was found to be higher at 39 DAF in both the genotypes. bZIP relative expression was found to be 1.1-fold at 39DAF and 1.9-fold at 50DAF in RGC-936, while 148.2-fold at 39DAF and 0.7-fold at 50DAF in M-83 (Fig. 5).

Electron microscopy analysis of seeds of RGC-936 and M-83 genotypes

Scanning electron microscopy (SEM) micrographs of the mature seeds showed larger cuticle structure with more space in RGC-936 as compared to smaller cuticles in M-83. Further, the mature seed coat of RGC-936 (Fig. 6) in surface view showed epidermal as well as hypodermal wall thickenings underneath, a well-defined sub-epidermal layer, and a larger endosperm than M-83. RGC-936 had thicker seed coat (163.8 µm), rich in protein bodies with compact and uniform distribution of aleuroplast cells, and contained a greater number of globoids as compared to M-83 which had moderately thick (158.8 µm) seed coat, scattered aleuroplast, large number of oil bodies and intracellular spaces due to the larger vacuole. The starch content was lower in M-83 seeds with a greater number of oil bodies as compared to RGC-936.

Figure 6
figure6

Comparative electron micrograph and light microscopic cross sectioned seed images of M-83 and RGC-936 genotypes. (AC) represent scanning electron microscope of M-83. (DF) represent cross sectioned image of RGC-936. G represent light microscopic images of M-83 stained with toluidine blue while (H,I) represent Transmission electron micrograph of M-83 seeds. (J) represent light microscopic images of RGC-936 stained with toluidine blue while (K,L) represent Transmission electron micrograph of RGC-936 seeds. Hg hourglass cells, pc palisade cells, sep sub epidermal pillar cells, en endosperm, ep epidermis, sc seed coat, g globoids, v vacuole, a alurone, ob oil bodies, is intracellular spaces, cw cell wall.

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