Increased Seed Size Mediated by Cyamopsis Tetragonoloba Big SEEDS LIKE Silencing Is Positively Co-related to Galactomannan Content, Phytochemical Biosynthesis and Anti-diabetic Potential of Guar


 In higher plants, seed size is an important agronomic trait that determines the crop yield and evolutionary fitness. Guar is an economically important legume crop because of the presence of galactomannan in its seed endosperm which is used as a stabilizer and thickener in pharmaceutical, paper, cosmetic and textile industries. The current study demonstrate the role of CtBSL, in regulation of seed size, weight and galactomannan content in two independent BSL12-25-15-31-3 and BSL70-21-25-4-16 T4 transgenic lines of guar, via. pCMKCtBSL mediated post transcriptional CtBSL silencing approach. The upregulation of CtCYCA2;4, CtCYCD3;2, CtCDKE-1, CtCYCT1;5, CtH2A, CtH2B, CtH3, CtH4, CtERF, CtWRKY43, CtGRF5 and CtGIF1 genes in transgenic lines of guar contributed to enhanced cell proliferation and final seed size which was further strongly and positively correlated to phytochemical biosynthesis and their anti-diabetic, anti-AGEs and lipase inhibitory potential against α-glucosidase, α-amylase, AGEs and lipase enzymes, respectively. Therefore, here we conclude the potential role of CtBSL silencing in improving the crop yield and therapeutic treatment of diabetes and obesity by positive regulation of seed size and phytochemical biosynthesis, respectively, in guar.


Introduction
Clusterbean (Cyamopsis tetragonoloba (L) Taub) commonly known as guar, is an economical legume crop because of the presence of galactomannan or guar gum in its seed endosperm (Thakur and Prasad, 2020). The galactomannan, composed of linear chain of β (1->4) mannose residues cross-linked to galactose residues by α (1->6) linkages, is used as a cost-effective natural thickener, stabilizer and emusil er in paper, pharmaceutical, textile, food, and cosmetic industries and additionally for the treatment of irritable bowel syndrome, high cholesterol and diarrhea (Thakur and Randhawa, 2018).
Therefore, due to global demand of galactomannan there is a necessity to produce the high yielding guar varieties which can tolerate adverse climatic conditions. Seed size is an important agronomic trait, selected during the process of domestication, that determines seed yield, evolutionary tness and stress responses in crop plants. Large sized seeds are preferred over smaller seed size due to higher availability of reserves and better seedling survivorship Gresta et al., 2013). Seed size is a complex agronomic trait that is controlled by quantitative trait loci (QTLs) and affected by several environmental factors. However, signalling pathways, transcription factors and to some extent accumulation of seed reserves like storage proteins, sugars and fatty acids also plays a major role in determination of nal seed size in angiosperms (Sreenivasulu and Wobus, 2013;Na and Yunhai, 2015).
Plants also produce secondary metabolities or phytochemicals in adverse environmental conditions, as a defense mechanism, for their normal growth and development. These phytochemicals, mainly phenolics, avanoids, carbohydrates, terpenoids and tannins, holds a great pharmacological value in modern medicine as immune-modulators, antibiotics and therapeutic drugs for treatment of various diseases (Thirumurugan et al., 2018). Diabetes mellitus and obesity are among the most prevalent health disorders worldwide that leads to several micro and macro-vascular complications in various individuals (Umar et al., 2018). In a study, about 366 million are expected to be diabetic by 2030, out of which 79.4 million are projected to be in India only (Firdous, 2014). Several compounds extracted from plant sources have been previously reported to show antidiabetic activity and are used for discovery and development of new type of antidiabetic molecule.
In current study we have demonstrated the role of Cyamopsis tetragonoloba BIG SEEDS LIKE (CtBSL) gene, in regulation of seed size, weight, sugars and other reserves, through RNAi mediated gene silencing approach in guar. Further the seed size is positively correlated to phytochemical biosynthesis which has anti-diabetic, anti-AGEs and anti-lipase bioactivity. Therefore, we expect this study to be useful for improving the seed yield, an important agronomic and economical trait, in guar and development of novel drug candidates for treatment of diabetes and obesity.

Transformation, screening and phenotypic characterization
Fifteen days old cotyledon explants of HG 2-20 variety of guar transformed by Agrobacterium-mediated transformation were grown in selective medium containing 50 mg/L kanamycin. After the screening of putative transgenics the in vitro grown plantlets were transferred after 30 days to agro-peat, vermiculite and sand mixture ( Fig S2). The acclimatized T 0 generation guar lines, BSL12-25-15-31-3-3 and BSL70-21-25-4-16-5, grown upto T 4 generation were phenotypically characterized for seed size and weight. The nontransformed in vitro grown acclimatized guar plantlets were used as control. All the images were analyzed using ImageJ (https://imagej.nih.gov/ij/) software.

SEM and FE-SEM
For SEM and FE-SEM analysis, thin seed sections of non transformed control (NTC),  generation guar lines were xed in formaldehyde (3.7 %) at 37 0 C for 14 h followed by OsO 4 (1 % ) at 4 0 C for 1 h. The thin seed sections dehydrated with series of ethanol washing up to critical dried point were imaged under SEM and FE-SEM QUANTA 200 FEG for measurement cell size and area (Huang et al., 2008). All the images were analyzed using NIH ImageJ software (https://imagej.nih.gov/).

Quantitative RT-PCR
For RT-qPCR analysis, 1 µg of total RNA isolated using Qiagen RNeasy Plant Mini Kit from seeds of NTC and BSL12-25-15-31-3-3 and BSL70-21-25-4-16-5 T 4 generation guar lines at different developmental stages was subsequently used for rst strand cDNA synthesis using Superscript II Invitrogen kit as per the manufacturer's instructions. The triplicate reaction was performed in Applied biosystems real-time PCR (Thermo Fisher) and the data was normalized using ΔΔ Ct method. GAPDH was used as internal control.
The RT-qPCR analysis was performed using Applied biosystems real-time PCR (Thermo Fisher) and the data was normalized using ΔΔ Ct method. GAPDH was used as an internal control. Followinf primer pairs were used for the reaction, CtBSL and GAPDH (FP-5′ATGTTCATCGAGAATTTTAAGGTT3′/ RP-5′ATCACTTGTACTCGAGAATCAT3′ and 5′AAGCCAGCATCCTATGACAGATTC3′/ RP-5′AAGCCAGCATCCTATGACAGATTG3′),performed in triplicates with the following cycling conditions: 94°C for 2 min followed by 35 cycles of 94°C for 30 sec, 58°C for 30 sec and 94°C for 30 sec.

Quanti cation of proteins, sugars and galactomannan
Bicinchoninic acid assay was used to determine the protein content in seeds of NTC, BSL12-25-15-31-3-3 and BSL70-21-25-4-16-5 T 4 generation guar lines. Brie y, 100 µg of powdered seeds mixed with sodium bicinchoninic and CuSO 4 .5H 2 O mixture (10:1) were incubated at 60 0 C for 35 mins. After cooling the solution's absorbance was measured at 562 nm. One mg/ml bovine serum albumin (BSA) was used as standard. For quanti cation of reducing and total soluble sugars, powdered seeds incubated in 80 % ethanol (70 0 C for 90 min) were centrifuged at 14000 rpm for 10 min followed by supernatant extraction in rotary evaporator. The residuals obtained were dissolved in 0.5 mg/ml de-ionized water and the absorbance was noted at 620 nm and 490 nm, respectively (Nelson 1944;Somogyi 1952;Focks and Benning, 1998). For determination of galactomannan content, the dried residuals of powdered seeds extracted in phosphorus pentoxide (P 2 O 5 ) and ethanol were hydrolyzed in 2 M tri uoroacetic acid (TFA) followed by membrane lteration and de-ionization in C 18 column. The hydrolyzed galactose and mannose residues were separated using reverse phase high performance liquid chromatography (RP-HPLC) in water (H 2 O) and acetonitrile (ACN) gradient elution mixture, where the ACN concentration increases from 5 % to 50 % (Tapie et al., 2008).

CtBSL sub-cellular localization
The CtBSL protein encoding gene was cloned in pENTRY-D (Invitrogen) vector containing green uorescent protein (GFP) and subsequently sub-cloned in a gateway destination vector pMDC83, using LR reaction. The GFP signals were imaged using the Leica SP2 laser confocal microscope in tobacco epidermal cells as described by (Tian et al., 2014).

Yeast two hybrid assay
The full length CtBSL (1006 bp), CtJAZ3 (1241 bp) and CtNINJA (1361 bp), CtMYC2 (1062 bp) gene sequences were cloned in pGBKT7 (bait) and pGADT7 (prey) plasmids, respectively, and subsequently transformed into yeast two hybrid GOLD strain (Clontech), using a Frozen-EZ Yeast Transformation II Kit (Zymo Research). The protein interactions were analyzed on SD medium containing X-α-Gal (40 ng/mL) and Aureobasidin A (125 ng/mL), using quadruple dropout method (Lai and Lau, 2017). The list of primers used in this study is given in Table S1.
Quercetin was used as standard and the TFC expressed as quercetin equivalent (QE/g). The total avonoid production (TFP) was calculated by the formula: TFP (mg/L) = dry weight (g/L) × TFC (mg/g).
2.9. Anti-diabetic, anti-advanced glycation end products and lipase inhibition assay The α-glucosidase and α-amylase enzymatic activity of methanol extract of dried seeds of NTC, BSL12-25-15-31-3-3 and BSL70-21-25-4-16-5 T 4 generation guar lines was measured at 405 nm using chromogenic method as described by Hano et al. (2013). The percentage α-glucosidase and α-amylase inhibition was calculated as: absorbance values in the presence of callus extracts -absorbance values in the absence of callus extracts. The inhibitory activity of anti-advanced glycation end products (AGEs) was determined as described by Kaewseejan and Siriamornpun (2015) at 330 nm and 410 nm excitation and emission wavelength, respectively, and the results were expressed as percent inhibition relative to equal volume of DMSO control. The lipase inhibitory activity of methanol extracts was determined as per Chen et al. (2018). The change in absorbance value (K) was determined at 400 nm and the percentage inhibition was calculated as: K (normal value) -K (experimental value) / K (normal value) × 100.

Statistical analysis
All the experiments were performed in triplicates. The means calculated from replicated data were analyzed by analysis of variance one-way analysis of variance (ANOVA) and compared by Student's t tests using IBM Statistical Package for the Social Sciences [SPSS] software, New York, USA at P ≤ 0.05. Statistically signi cant differences were determined at a 5% level of probability for all comparisons. The results were expressed as mean ± SD of total experiments.
The change in seed size could be due to cell proliferation or cell expansion or both. So to understand the underlying mechanism we observed the thin section of cotyledon and seed coats under scanning electron microscope (SEM) and found a signi cant increase in cell number rather than cell size, which was nearly indistinguishable, in seeds of BSL12-25-15-31-3-3 and BSL70-21-25-4-16-5 transgenic lines of guar as compared to NTC (Fig. 5, 6).

CtBSL forms a complex with repressor proteins
The yeast two-hybrid assay con rmed strong interaction of CtBSL protein with CtNINJA (repressor) protein, whereas, a very weak or no interaction was seen with another CtMYC2 (activator) protein. To con rm these results, we analyzed the interaction of CtJAZ3, a positive control, with both CtNINJA and CtMYC2 proteins. As shown in Fig. 8, CtJAZ3 showed a strong interaction with both activator and repressor proteins suggesting the formation of a repressor complex between CtBSL and other repressor proteins (Fig. 8).

Discussion
Guar or clusterbean is an economically important crop because of the presence of galactomannan in its seed endosperm which is used in paper, textile, pharmaceutical and cosmetic industries as a natural thickener and stabilizer. India is the largest producer of guar with 80 % of total world's production followed by the United States of America and Pakistan. However, recently a decline in its yield has been observed due to various biotic and abiotic stresses.
Seed size and weight are two major agronomic traits which are positively correlated to tness of crop and its yield. The seedlings obtained from large sized seeds have better seedling survivorship as compared to smaller sized seeds (Orozco-Arroyo et al., 2015;Punjabi et al., 2018).
For crop improvement, conventional breeding programes are challenging in guar due to cleistagamous nature of owers. Hence in this study we have used a reverse genetics approach to understand the role of CtBSL gene in guar plants. CtBSL is a member of BIG SEEDS gene family, having a conserved TIFY domain (Fig. S4). We observed that arti cial RNAi mediated CtBSL silencing had a profound impact on cotyledon size, seed size, seed weight and other reserves in transgenic guar plants, which is consistent with the notion that seed size in legumes is correlated with cotyledon size (Lemontey et al., 2000). Further, the increase in cotyledon size of guar was mainly due to cell proliferation which is again in accordance with the previous studies in other plants (White 2006;Gonzalez et al., 2010Gonzalez et al., , 2015Hepworth and Lenhard, 2014).
Through in silico and in vivo studies in yeast, we observed that CtBSL a member of TIFY protein family interacts with repressor and co-repressor proteins to form a repressor complex which negatively regulates the expression of growth factors and other cell cycle genes in guar seeds. This con rms that in transgenic guar plants due to CtBSL silencing there is no formation of repressor complex which lead to increased expression or up-regulation of CtCYCA2;4, CtCYCD3;2, CtCDKE-1, CtCYCT1;5 cell cycle genes, CtH2A, CtH2B, CtH3, CtH4 histone genes and CtERF, CtWRKY43, CtGRF5 and CtGIF1 transcription factors might have lead to increased primary cell proliferation and enhanced seed size in transgenic guar plants.
Further the sub-cellular localization of CtBSL-GFP fusion protein (Fig. S5) con rms the role of CtBSL as putative transcription regulator. Similar results were also reported in previous studies where certain transcription factors negatively regulate the organ growth in certain plants including model plant Arabidopsis (Horiguchi et al. 2005;Pauwels et al., 2010;Cuéllar Pérez et al., 2014;Ge et al., 2016).
Plant produces various phytochemicals (phenolics and avonoids), in response to external biotic and abiotic stress as a defense mechanism for their normal growth and development (Ali et al., 2006), which play therapeutic role in development of relevant medicines (Mohale et al., 2014;Tungmunnithum et al., 2018). Diabetes is one the major health concern and formation of AGEs is one of the major complications faced by diabetic patients in hyperglycemic conditions. Another big concern is obesity, as per world health organization (WHO) it leads to 3.4 million deaths every year, as per WHO (Ezzati et al., 2002;Chen et al., 2017). One way to control obesity is to inhibit pancreatic lipase activity which results in 50-70 % fat decomposition (Chen et al., 2017). In this study, we have reported the anti-diabetic, antiadvanced glycation end products (AGEs) and anti-lipase activity of phytochemicals (phenolics and avonoids) extracted from CtBSL silenced transgenic guar plants. We observed that phytochemicals showed higher α-amylase inhibitory activity, which contributed more to its anti-diabetic potential, as compared to α-glucosidase.
So collectively, on the basis of results obtained we can conclude that CtBSL gene expression is strongly correlated to seed size in guar, which further linked to enhanced phytochemical biosynthesis and its antidiabetic, anti-AGEs and anti-lipase potential.

Conclusion
The post-transcriptional silencing of CtBSL results in favourable agronomic traits like increased seed size and weight along with the enhanced accumulation of commercially important galactomannan in seeds of transgenic clusterbean plants. Further we observed a strong positive co-relation between seed size and phytochemical biosynthesis which is directly related to enhanced anti-diabetic, anti-AGEs and lipase inhibitory activities. Thus our nding paved a way of improving the yield of agronomically and economically important clusterbean and galactomannan, respectively. Simultaneously our results indicate the potential of the phytochemicals obtained from clusterbean for the treatment of two important health issues, diabetes and obesity, which would be useful in development of effective strategies to signi cantly use clusterbean for therapeutic purposes.        Anti-diabetic, anti-AGEs, lipase inhibitory potential of NTC, wild, BSL12-25-15-31-3 and BSL70-21-25-4-16 transgenic lines of clusterbean.

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