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Fulltext Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway

Ong SS, Wickneswari R

BMC Genomics, 2011 Nov 30;12 Suppl 3(Suppl 3):S13.
PMID: 22369296 DOI: 10.1186/1471-2164-12-S3-S13

BACKGROUND: Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem.
RESULTS: In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in epigenetic silencing. Differential expression of the small RNAs between secondary xylem tissues with contrasting lignin content suggests that a cascade of miRNAs play an interconnected role in regulating the lignin biosynthetic pathway in Acacia species.
CONCLUSIONS: Our study critically demonstrated the roles of small RNAs during secondary wall formation. Comparison of the expression pattern of small RNAs between secondary xylem tissues with contrasting lignin content strongly indicated that small RNAs play a key regulatory role during lignin biosynthesis. Our analyses suggest an evolutionary mechanism for miRNA targets on the basis of the length of their 5' and 3' UTRs and their cellular roles. The results obtained can be used to better understand the roles of small RNAs during lignin biosynthesis and for the development of gene constructs for silencing of specific genes involved in monolignol biosynthesis with minimal effect on plant fitness and viability. For the first time, small RNAs were proven to play an important regulatory role during lignin biosynthesis in A. mangium.

Matched MeSH terms: Acacia/metabolism
Fulltext Characterization of microRNAs expressed during secondary wall biosynthesis in Acacia mangium

Ong SS, Wickneswari R

PLoS One, 2012;7(11):e49662.
PMID: 23251324 DOI: 10.1371/journal.pone.0049662

MicroRNAs (miRNAs) play critical regulatory roles by acting as sequence specific guide during secondary wall formation in woody and non-woody species. Although thousands of plant miRNAs have been sequenced, there is no comprehensive view of miRNA mediated gene regulatory network to provide profound biological insights into the regulation of xylem development. Herein, we report the involvement of six highly conserved amg-miRNA families (amg-miR166, amg-miR172, amg-miR168, amg-miR159, amg-miR394, and amg-miR156) as the potential regulatory sequences of secondary cell wall biosynthesis. Within this highly conserved amg-miRNA family, only amg-miR166 exhibited strong differences in expression between phloem and xylem tissue. The functional characterization of amg-miR166 targets in various tissues revealed three groups of HD-ZIP III: ATHB8, ATHB15, and REVOLUTA which play pivotal roles in xylem development. Although these three groups vary in their functions, -psRNA target analysis indicated that miRNA target sequences of the nine different members of HD-ZIP III are always conserved. We found that precursor structures of amg-miR166 undergo exhaustive sequence variation even within members of the same family. Gene expression analysis showed three key lignin pathway genes: C4H, CAD, and CCoAOMT were upregulated in compression wood where a cascade of miRNAs was downregulated. This study offers a comprehensive analysis on the involvement of highly conserved miRNAs implicated in the secondary wall formation of woody plants.

Matched MeSH terms: Acacia/metabolism
Fulltext Isolation and characterization of CCoAOMT in interspecific hybrid of Acacia auriculiformis x Acacia mangium--a key gene in lignin biosynthesis

Pang SL, Ong SS, Lee HH, Zamri Z, Kandasamy KI, Choong CY, et al.

Genet. Mol. Res., 2014;13(3):7217-38.
PMID: 25222227 DOI: 10.4238/2014.September.5.7

This study was directed at the understanding of the function of CCoAOMT isolated from Acacia auriculiformis x Acacia mangium. Full length cDNA of the Acacia hybrid CCoAOMT (AhCCoAOMT) was 1024-bp long, containing 750-bp coding regions, with one major open reading frame of 249 amino acids. On the other hand, full length genomic sequence of the CCoAOMT (AhgflCCoAOMT) was 2548 bp long, containing three introns and four exons with a 5' untranslated region (5'UTR) of 391 bp in length. The 5'UTR of the characterized CCoAOMT gene contains various regulatory elements. Southern analysis revealed that the Acacia hybrid has more than three copies of the CCoAOMT gene. Real-time PCR showed that this gene was expressed in root, inner bark, leaf, flower and seed pod of the Acacia hybrid. Downregulation of the homologous CCoAOMT gene in tobacco by antisense (AS) and intron-containing hairpin (IHP) constructs containing partial AhCCoAOMT led to reduction in lignin content. Expression of the CCoAOMT in AS line (pART-HAS78-03) and IHP line (pART-HIHP78-06) was reduced respectively by 37 and 75% compared to the control, resulting in a decrease in the estimated lignin content by 24 and 56%, respectively. AhCCoAOMT was found to have altered not only S and G units but also total lignin content, which is of economic value to the pulp industry. Subsequent polymorphism analysis of this gene across eight different genetic backgrounds each of A. mangium and A. auriculiformis revealed 47 single nucleotide polymorphisms (SNPs) in A. auriculiformis CCoAOMT and 30 SNPs in A. mangium CCoAOMT.

Matched MeSH terms: Acacia/metabolism*
Fulltext Identification of lignin genes and regulatory sequences involved in secondary cell wall formation in Acacia auriculiformis and Acacia mangium via de novo transcriptome sequencing

Wong MM, Cannon CH, Wickneswari R

BMC Genomics, 2011;12:342.
PMID: 21729267 DOI: 10.1186/1471-2164-12-342

Acacia auriculiformis × Acacia mangium hybrids are commercially important trees for the timber and pulp industry in Southeast Asia. Increasing pulp yield while reducing pulping costs are major objectives of tree breeding programs. The general monolignol biosynthesis and secondary cell wall formation pathways are well-characterized but genes in these pathways are poorly characterized in Acacia hybrids. RNA-seq on short-read platforms is a rapid approach for obtaining comprehensive transcriptomic data and to discover informative sequence variants.

Matched MeSH terms: Acacia/metabolism*
Fulltext The effects of acacia honey on in vitro corneal abrasion wound healing model

Ker-Woon C, Abd Ghafar N, Hui CK, Mohd Yusof YA, Wan Ngah WZ

BMC Cell Biol., 2015;16:2.
PMID: 25887200 DOI: 10.1186/s12860-015-0053-9

Acacia honey (AH) has been proven to improve skin wound healing, but its therapeutic effects on corneal epithelium has not been elucidated to date. This study aimed to investigate the effects of AH on cultured corneal epithelial cells (CEC) on in vitro corneal abrasion wound healing model. Six New Zealand white rabbits' CEC were isolated and cultured until passage 1. Circular wound area was created onto a confluent monolayer CEC using a corneal trephine which mimicked corneal abrasion and treated with 0.025% AH supplemented in basal medium (BM) and complete cornea medium (CCM). Wound healing was measured as the percentage of wound closure by the migration of CEC on day 0, day 3 and day 6, post wound creation. The morphological changes of CEC were assessed via phase contrast microscopy. Gene and protein expressions of cytokeratin (CK3), fibronectin and cluster of differentiation 44 (CD44) in AH treated groups and control groups were determined by real-time PCR and immunocytochemistry, respectively.

Matched MeSH terms: Acacia/metabolism