Characterization of rice straw lignin phenolics and evaluation of their role in pollen tube growth in Cucurbita pepo L.

Abstract Rice straw lignin was extracted via alkaline hydrolysis and structurally characterized using FT-IR and 1H NMR spectra. Ethyl acetate extract of acid solubilized lignin was found to contain p-coumaric acid, ferulic acid and caffeic acid as major phenolic acids which were isolated and characterized using spectral data. Amides of isolated phenolic acids were synthesized by their reaction with propyl and butyl amines using microwave irradiation and analysed using spectral studies. Phenolic acids and amides were evaluated for their effect on pollen germination and tube growth in pumpkin. Pollen tube length was significantly increased with N-butyl-3-(3, 4-dihydroxyphenyl) acrylamide and N-butyl-3-(4-hydroxyphenyl) acrylamide at 5 ppm concentration than the control. These results could be utilised in increasing pollen tube length of Cucurbita pepo while making interspecific cross between C. moschata and C. pepo in order to transfer hull-less character of C. pepo to virus resistant C. moschata genotypes. Graphical Abstract


Introduction
Rice straw is an abundant and bio-renewable biomass produced after the harvesting of rice crop.In India, about 23 million tons of rice residue, also called rice straw, is produced annually (Zaidi 2021).Due to its short turn-around time for decomposition, farmers prefer to burn it and this practice is dramatically increasing for over the last decade.Burning of rice straw causes air pollution by the release of harmful gases leading to global warming.Its inefficient burning also causes loss of plant nutrients, soil fertility and respiratory problems.Therefore, the utilization of rice straw is necessary to combat these serious environmental issues.
straw is made up of approximately 30-45% cellulose, 17.5-25% hemicellulose and 5-23% lignin (sharma et al. 2020).Cellulose and hemicelluloses have already been exploited for their potential for conversion into high-value chemicals and materials (Huang et al. 2021;Kaur and Kaur 2021).In recent years, researchers have started focusing on exploring lignin (Kaur and Goyal 2021), which entangles cellulose and hemicellulose fibres and is chemically different from them.lignin is a highly complex network composed of three cross-linked monolignol monomers i.e. coniferyl alcohol, p-coumaryl alcohol and sinapyl alcohol incorporated as guaiacyl, p-hydroxyphenyl and syringyl units, respectively (Zakzeski et al. 2010).Due to its unique structure and occurrence of diverse phenolic and aliphatic hydroxyl reactive groups, lignin renders a great opportunity for the manufacturing of various high-value products and renewable chemicals, particularly aromatic compounds.Phenolics comprising lignin are, in majority, hydroxycinnamic acids, which are produced as simple esters with glucose or hydroxy carboxylic acids.Hydroxycinnamic acids like p-coumaric acid, caffeic acid, ferulic acid and sinapic acids are found generally in lignocellulosic materials.These lignin phenolics are reported to possess good anti-inflammatory, choleretic (Bjorsvik and liguori 2002), antihypertensive, antiplasmodic, antimutagenic, antioxidant (Elzaawel et al. 2017) and antibacterial properties (Kaur et al. 2017).Different derivatives of these phenolic compounds have been synthesized including formation of aldehydes, Scheme 1. synthesis of phenolic amides from isolated hydroxycinnamic acids.
esters, metal complexes, schiff bases and amides (Kaur et al. 2018) etc., which have been found to show diverse pharmaceutical, health and biological applications (Garrido et al. 2012;Grzesik et al. 2018).
The presence of phenolamides has also been reported in plants (Roumani et al. 2021).Phenolics play a vital role in selective promotion and inhibition of pollen grain on the stigma.Pollen tubes of olive and date palm treated with phenolics were found to be longer than those in control (Ateyyeh 2016).Till date, quantitative phenolics have been estimated (Chauhan et al. 2004) in stigma of different plants at different stages, whereas few efforts are made for its application in increasing pollen tube length.The smaller pollen tube length of Cucurbita pepo is found to act as obstacle for interspecific hybridization between Cucurbita moschata and Cucurbita pepo as pollen tube length and style length are positively correlated (Kaur et al. 2021).C. pepo genotypes are known for its hull-less seeds which skip the decortication, make it available as snacks and also reduce the cost of oil processing.While, these genotypes are susceptible to viral diseases and due to high temperature sensitivity, grown in summer only in North India.Cucurbita moschata genotypes can be grown in high temperature and also have large seed cavity, vigorous growth, resistant to viral diseases but lack hull-less seed trait.The transfer of hull-less trait of C. pepo into C. moschata is restricted by short pollen tube length.Therefore, in this study, rice straw lignin phenolics and their derivatives were evaluated for their effect on in vitro germination and pollen tube growth in Cucurbita pepo which has very small pollen tube than the length of C. moschata style.

Results and discussion
Rice straw was found to constitute 41.00, 24.00, 9.00, 20.00 and 6.00% cellulose, hemicellulose, lignin, extractives and acid-insoluble ash (silica), respectively.Acid pretreatment hydrolyzes hemicellulose followed by delignification with alkaline solvents which disrupts lignocellulosic cell wall of plants.The lignin was extracted with a yield of 7.34 ± 1.2 mg per unit gram of rice straw.The major phenolic acids that were isolated from ethyl acetate extract of lignin using preparative thin layer chromatography were p-coumaric acid, ferulic acid and caffeic acid (Kaur et al. 2018).Preparative thin layer chromatography was chosen over column chromatography to extract and purify hydroxycinnamic acids because of minute difference between the polarities of these hydroxycinnamic acids.p-Coumaric acid (1), ferulic acid (2) and caffeic acid (3) were extracted in yields 9.79 ± 2.1, 6.55 ± 1.3 and 2.35 ± 1.7 mg per unit gram of rice straw, respectively.Amides were synthesized from isolated phenolic acids and reactions were monitored through thin layer chromatography (Figure s1).The physical parameters of isolated phenolic acids and amides are listed in Tables s1 and s2, respectively.The IR, 1 H NMR spectra of lignin, isolated phenolic acids (1, 2, 3) and synthesized amides (1a, 1b, 2a, 2b, 3a, 3b) are presented in Table s3 and Figures s2-s14.
Germination of pumpkin pollens varied when treated with different media with different concentrations of 5 and 10 ppm (Table 1).After 2 h of germination, it was observed that there was a 1.5 and 1.44 fold increase in pollen germination percentage in treatments T17 and T18 respectively as compared to the control.Whereas, treatments T1, T9, T13, and T15 showed 1.29, 1.31, 1.31 and 1.27 fold increase respectively in germination percentage as compared to control.However, treatments T2, T5, T7, T10, T11, T14 and T16 depicted almost similar germination percentage of pollens as in control.Also, there was a 1.03, 1.08, 1.01, 1, 1.22 fold decrease in pollen germination percentage of T3, T4, T12, T6 and T8 with respect to control (Table 1).overall, germination percentage was greater than the control for all acids and amides but the result was found to be non-significant statistically.
The preparation of media has an important role to play in in vitro germination of pollens (Mondal and Ghanta 2012).has been reported that pollen germination and tube growth were enhanced at lower concentrations of growth medium (Fragallah et al. 2019).At 5 ppm concentration, germination percentage was higher than 10 ppm concentration in case of all phenolic acids as well as their amides.The results were supported by Chauhan et al.(2004) who reported that germination was enhanced at lower concentrations (5 and 10 mg/l) of caffeic acid in the growth medium whereas higher concentrations of the same reduced germination.It was also reported that at very high concentration (50 mg/l) of caffeic acid in the growth medium completely inhibited pollen germination.Caffeic acid, p-coumaric acid and their amides showed higher germination percentage than ferulic acid and its amides.All phenolic acids at 5 ppm concentration showed higher germination percentage than control.Ateyyeh (2016) reported the role of phenolics in pollen germination and stated that germination percentage varied under different concentrations.significant improvement in vitro pollen germination of olive pollens was reported using caffeic acid and ferulic acid at low concentrations.
After two hours of germination, maximum pollen tube length was observed in T17 (1127.25 µm) and minimum pollen tube length was observed in T8 (105.12 µm).It was observed that treatments T17, T5, T13 and T1 showed much greater pollen tube lengths than the control by 11.96, 11.84, 8.80 and 7.95 fold.Also, treatments T18, T15, T16, T11, T3, T9 and T6 showed 6. 038, 5.34, 3.884, 3.21, 3.03, 2.53 and 2.26 fold longer tube lengths than control respectively (Figure s15).Treatments T2, T4, T7, T12 and T14 depicted almost similar pollen tube lengths as in control.However, treatments T8 and T10 showed 1.73 and 1.3 fold shorter tube lengths than the control (Table 1).Among the different treatments, T5 and T17 showed a significant increase in tube length of Cucurbita moschata.At 5 ppm concentration, pollen tube length was greater than 10 ppm concentration in case of all synthesized amides, indicating pronounced tube growth at lower concentrations (Chauhan et al. 2004) Phenolics provided nutrition for the growth of pollen tube and were larger than the control.In vitro pollen germination studies indicated the inhibition of pollen germination and pollen tube growth at higher concentrations of caffeic acid, ferulic acid and other phenolics in the basal medium.Also, caffeic acid amides and p-coumaric acid amides showed longer tube length than ferulic acid amides.This may be due to the presence of methoxy group in ferulic acid which is known to have minimal effect on pollen growth (Forbes et al. 2014).All amides having butyl chain showed longer tube lengths at 5 ppm concentration than propyl amides as well as control.This was supported by the studies which stated that longer alkyl chains help in hydration and pollen adhesion which helps to overcome male sterility (Yu et al. 2019;Ni et al.2018).

Conclusions
The present investigation involved the isolation of phenolics from rice straw lignin followed by synthesis of their derivatives and evaluation of their effect on in vitro pollen germination and tube growth in Cucurbita pepo.Pollen germination was increased with all the phenolics and amides than control but increase was non-significant.Pollen tube length was significantly increased with N-butyl-3-(3,4-dihydroxyphenyl) acrylamide and N-butyl-3-(4-hydroxyphenyl) acrylamide at 5 ppm concentration.The use of these compounds could be utilized in increasing pollen tube length of C. pepo which acts as a barrier for the transfer of hull-less seed trait of C. pepo into virus tolerant C. moschata genotypes.