Isolation, cloning and characterization of cinnamate 4- hydroxylase (c4h) gene from leucaena leucocephala and its expression studies

Abstract

Leucaena leucocephala is one of the most versatile, fast growing commercially important trees for paper and pulp industry in India, contributing for about 1/4th of the total raw material supplied to the industry. L. leucocephala is traditionally high in lignin content which must be removed from pulp involving various chemical and energy intensive processes, which in turn release various toxic pollutants and damage the polysaccharide components of wood. There is currently intense interest to reduce, modify or alter lignin content of L. leucocephala to be used as model plant in paper industry. Phenylpropanoid pathway is operative in plants and responsible for the production of a variety of compounds including lignin. Cinnamate 4-Hydroxylase (C4H) catalyzes the second step of phenylpropanoid pathway and hydroxylates trans-cinnamic acid at the para position. C4H plays central role in lignification as it catalyzes slow reaction early in the pathway, which branches after the action of C4H. To have a better understanding of the role of C4H in L. leucocephala, three C4H isoforms (LlC4H) were isolated from L. leucocephala and characterized. The isoforms share more than 98% sequence identity at amino acid level to each other. These isoforms contained all the conserved sequences found in P450 superfamily and C4H from other plants. Phylogenetic analysis grouped LlC4H with class I C4Hs, some members of which are functionally well characterized. Southern hybridization study suggested that there may be 4-6 C4H isoforms in Leucaena genome with some isoforms may be tandemely arranged. Three isoforms of C4H were expressed in Escherichia coli, strain Rosetta (DE3) and one isoform was purified from inclusion body. LlC4H proteins are highly hydrophobic and hence they profusely formed inclusion body in bacteria. C4H bound very weakly to the Ni-NTA matrix and was difficult to obtain in pure form from inclusion body. Under the optimized conditions, yield of purified C4H from inclusion body varied between 50-100 μg/mL. Secondary and vi tertiary structure comparison between the LlC4H isoforms suggested that all the isoforms are identical in their secondary and tertiary structure. Tissue and age specific Quantitative Real Time (Q-RT) PCR study suggested that C4H transcripts were highly abundant in root tissues followed by stem and leaves. Maximum transcript level at any time in any tissue was observed in 30 day old root tissue. Among the tissues investigated, demonstrable C4H activity was found maximum in 1 year old stem tissues. C4H was found to be stress responsive and its transcript level increased in response to external abiotic stimuli (salt stress, methyl jasmonate stress and UV-C stress). Tissue-wise quantitative comparison of lignin from developing seedling stage to one year old tree stage indicated that while acid insoluble lignin increased with age, acid soluble lignin first decreased and then slightly increased. Transgenic L. leucocephala and tobacco plants with reduced C4H expression were raised using modified pCAMBIA1301 vector harbouring partial L. leucocephala C4H in antisense orientation. Careful study of transcript level of different phenylpropanoid pathway genes in transgenics, down-regulated for C4H activity showed coordinated down-regulation and gave indication that C4H might be catalyzing rate limiting step early in the pathway. Transgenic plants had reduced lignin content consistent with its role in lignification.