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Title: Modeling of cellulose, hemicellulose and lignin-carbohydrate complex formation and regulation to understand plant cell wall structure

Abstract

The goal of this project is to increase the fundamental understanding of the plant secondary cell wall in wood formation, using Populus trichocarpa as the model species. The project is built on a systematic transgenic strategy to perturb the expression of targeted cell-wall component genes and transcription factors (TFs). We used a systems biology approach that integrates this genome-wide transgenesis with full genome information to advance our understanding of wood formation and how to engineer woody plants to create robust feedstocks that maximize beneficial traits for biofuel and material production. This work has resulted in 14 peer reviewed publications in journals such as Plant Cell, Proceedings of the National Academy of Sciences, Nature Protocols, Journal of Proteome Research, Plant Biotechnology Journal, etc. and in 13 conference presentations. The project’s full genome RNA-seq data sets are available through Gene Expression Omnibus (GSE49911 and GSE81077). 7 PhD and 2 MS graduate students received degrees under full or partial support of this grant. We also supported 3 undergraduates, 5 post-docs and 3 visiting scientists. Total 30 RNAi and artificial microRNA (amiRNA) transgene constructs were prepared for knocking down the genes involved in cellulose and hemicellulose biosynthesis and constructs for overexpressing TFs that controlmore » wood formation. The project generated over 1,000 transgenic P. trichocarpa and made many novel contributions to the research field, as outline below. We established a simple ihigh-throughput xylem protoplast system for studying wood formation. The system can be used as a cellular model to study gene transactivation and nucleocytoplasmic protein trafficking, and is particularly useful for studies where stable transgenics and mutants are unavailable. Our system is markedly faster and provides better yields than previous protocols. We further developed the first chromatin immunoprecipitation (ChIP) procedures for P. trichocarpa and many other woody species. We used this protocol to identify genome-wide specific TF-DNA interactions and histone modifications associated with wood formation. Our protocol is suitable for many tissue types and is so far the only working ChIP system for wood-forming tissue. We discovered a stem-differentiating xylem (SDX)-specific alternative SND1 transcription factor (TF) splice variant, PtrSND1-A2IR, that acts as a dominant negative of SND1 transcriptional network genes in Populus trichocarpa. PtrSND1-A2IR is exclusively in cytoplasmic foci but translocated into the nucleus exclusively as a heterodimeric partner with full-size PtrSND1s. The translocated PtrSND1-A2IR can disrupt the function of full-size PtrSND1s, making them nonproductive through heterodimerization, and thereby modulating the SND1 transcriptional network. We further discovered another splice variant, PtrVND6-C1IR, derived from PtrVND6-C1. Both PtrVND6-C1IR and PtrSND1-A2IR cannot suppress their cognate transcription factors but can suppress all members of the other family. However, these splice variants from the PtrVND6 and PtrSND1 families may exert reciprocal cross-regulation for complete transcriptional regulation of these two families in wood formation. This reciprocal cross-regulation between families suggests a general mechanism among NAC domain proteins and likely other transcription factors, where intron-retained splice variants provide an additional level of regulation. We next developed a mass spectrometry (MS) based absolute quantification of SDX proteins including TFs, which are normal present in very low concentration. With our digestion optimization, subcellular fractionation of SDX nuclei, and improved instrumentation in the quadrupole orbitrap MS, we were able to identify over 6,000 unique proteins, including cellulose and hemicellulose biosynthesis proteins as well as many SND and VND TF members. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa and used these transgenics built a hierarchical genetic regulatory network (GRN), which demonstrated that ptr-miR397a is a negative regulator of laccases for lignin biosynthesis. The GRN further identified previously undisclosed TFs and their targets including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. We also analyzed the transcriptomes of 5 tissues (xylem, phloem, shoot, leaf, and root) and 2 wood forming cell-types (fiber and vessel) of P. trichocarpa to assemble gene co-expression subnetworks. We identified 165 TFs that showed xylem-, fiber-, and vessel-specific expression. Of the 165 TFs, 101 co-expressed with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Our co-expression network suggests a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.« less

Authors:
ORCiD logo
Publication Date:
Research Org.:
North Carolina State Univ., Raleigh, NC (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1420055
Report Number(s):
DOE-NCSU-0006691-final product
DOE Contract Number:  
SC0006691
Resource Type:
Technical Report
Resource Relation:
Related Information: RNA-seq data sets: Gene Expression Omnibus GSE49911 and GSE81077Publications of the DOE DE-SC-0006691 projectShi R, Wang JP, Lin YC, Li Q, Sun YH, Chen H, Sederoff RR, Chiang VL (2017). Planta 245: 927-938.Wang JP, Tunlaya-Anukit S, Shi R, Yeh TF, Chuang L, Isik F, Yang C, Liu J, Li Q, Loziuk PL, Naik PP, Muddiman DC, Ducoste JJ, Williams CM, Sederoff RR, Chiang VL (2017). In: Quideau S & Yoshida K (eds) Recent Advances in Polyphenol Research (Volume 5).Loziuk PL, Hecht ES, Muddiman DC (2017). Anal Bioanal Chem. 409(2):487-497.Philip L. Loziuk, Jennifer Parker, Wei Li, Chien-Yuan Lin, Jack P. Wang, Quanzi Li, Ronald R. Sederoff, Vincent L. Chiang, and David C. Muddiman (2015). J Proteome Res. 14: 4158-4168.Li Q, Song J, Peng S, Wang JP, Qu G-Z, Sederoff RR,. Chinag VL (2014). Plant Biotechnol. J. 12: 1174-1192.Loziuk PL, Sederoff RR, Chiang VL, Muddiman DC (2014). Analyst. 139(21): 5439-5450.Lin Y-C, Li W, Chen H, Li Q, Sun Y-H, Shi R, Lin C-Y, Wang JP, Chen H-C, Chuang L, Qu G-Z, Sederoff RR, Chiang VL (2014). Nature Protocols 9: 2194-2205.Li W, Lin Y-C, Li Q, Shi R, Lin C-Y, Chen H, Chuang L, Qu G-Z, Sederoff RR, Chiang VL (2014). Nature Protocols 9: 2180-2193.Lin Y-C, Li W, Sun Y-H, Kumari S, Wei H, Li Q, Tunlaya-Anukit S, Sederoff RR and Chiang VL (2013). Plant Cell 25: 4324-4341.Loziuk PL, Wang J, Li Q, Sederoff RR, Chiang VL, Muddiman DC (2013). J Proteome Res. 12(12): 5820-58299.Lu, S., Li, Q., Wei, H., Chang, M., Tunlaya-Anukit, S., Kim, H., Liu, J., Song, J., Sun, Y. H., Yuan, L., Yeh, T. F., Peszlen, I., Ralph, J., Sederoff, R. R., & Chiang, V. L. (2013). Proceedings of the National Academy of Sciences, USA, 110, 10848-10853.Quanzi Li, Ying-Chung Lin, Ying-Hsuan Sun, Jian Song, Hao Chen, Xing-Hai Zhang, Ronald R. Sederoff, and Vincent L. Chiang. (2012). Proceedings of the National Academy of Sciences, USA. 109: 14699-14704.Min D, Li Q, Jameel H, Chiang V, Chang HM. (2012). Appl Biochem Biotechnol. 168:947-55.Li Q, Min D, Wang JP, Peszlen I, Horvath L, Horvath B, Nishimura Y, Jameel H, Chang HM, Chiang VL (2011). Tree Physiol. 31(2): 226-236.
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 09 BIOMASS FUELS; Populus trichocarpa; wood formation; cell-wall component genes; transcription factors; artificial microRNA; mass spectrometry; lignin, cellulose and hemicellulose biosynthesis; RNA-seq; ChIP-seq; xylem protoplasts; splice variant; transcriptional regulatory network; hierarchical genetic regulatory network

Citation Formats

Chiang, Vincent L. Modeling of cellulose, hemicellulose and lignin-carbohydrate complex formation and regulation to understand plant cell wall structure. United States: N. p., 2018. Web. doi:10.2172/1420055.
Chiang, Vincent L. Modeling of cellulose, hemicellulose and lignin-carbohydrate complex formation and regulation to understand plant cell wall structure. United States. doi:10.2172/1420055.
Chiang, Vincent L. Sun . "Modeling of cellulose, hemicellulose and lignin-carbohydrate complex formation and regulation to understand plant cell wall structure". United States. doi:10.2172/1420055. https://www.osti.gov/servlets/purl/1420055.
@article{osti_1420055,
title = {Modeling of cellulose, hemicellulose and lignin-carbohydrate complex formation and regulation to understand plant cell wall structure},
author = {Chiang, Vincent L},
abstractNote = {The goal of this project is to increase the fundamental understanding of the plant secondary cell wall in wood formation, using Populus trichocarpa as the model species. The project is built on a systematic transgenic strategy to perturb the expression of targeted cell-wall component genes and transcription factors (TFs). We used a systems biology approach that integrates this genome-wide transgenesis with full genome information to advance our understanding of wood formation and how to engineer woody plants to create robust feedstocks that maximize beneficial traits for biofuel and material production. This work has resulted in 14 peer reviewed publications in journals such as Plant Cell, Proceedings of the National Academy of Sciences, Nature Protocols, Journal of Proteome Research, Plant Biotechnology Journal, etc. and in 13 conference presentations. The project’s full genome RNA-seq data sets are available through Gene Expression Omnibus (GSE49911 and GSE81077). 7 PhD and 2 MS graduate students received degrees under full or partial support of this grant. We also supported 3 undergraduates, 5 post-docs and 3 visiting scientists. Total 30 RNAi and artificial microRNA (amiRNA) transgene constructs were prepared for knocking down the genes involved in cellulose and hemicellulose biosynthesis and constructs for overexpressing TFs that control wood formation. The project generated over 1,000 transgenic P. trichocarpa and made many novel contributions to the research field, as outline below. We established a simple ihigh-throughput xylem protoplast system for studying wood formation. The system can be used as a cellular model to study gene transactivation and nucleocytoplasmic protein trafficking, and is particularly useful for studies where stable transgenics and mutants are unavailable. Our system is markedly faster and provides better yields than previous protocols. We further developed the first chromatin immunoprecipitation (ChIP) procedures for P. trichocarpa and many other woody species. We used this protocol to identify genome-wide specific TF-DNA interactions and histone modifications associated with wood formation. Our protocol is suitable for many tissue types and is so far the only working ChIP system for wood-forming tissue. We discovered a stem-differentiating xylem (SDX)-specific alternative SND1 transcription factor (TF) splice variant, PtrSND1-A2IR, that acts as a dominant negative of SND1 transcriptional network genes in Populus trichocarpa. PtrSND1-A2IR is exclusively in cytoplasmic foci but translocated into the nucleus exclusively as a heterodimeric partner with full-size PtrSND1s. The translocated PtrSND1-A2IR can disrupt the function of full-size PtrSND1s, making them nonproductive through heterodimerization, and thereby modulating the SND1 transcriptional network. We further discovered another splice variant, PtrVND6-C1IR, derived from PtrVND6-C1. Both PtrVND6-C1IR and PtrSND1-A2IR cannot suppress their cognate transcription factors but can suppress all members of the other family. However, these splice variants from the PtrVND6 and PtrSND1 families may exert reciprocal cross-regulation for complete transcriptional regulation of these two families in wood formation. This reciprocal cross-regulation between families suggests a general mechanism among NAC domain proteins and likely other transcription factors, where intron-retained splice variants provide an additional level of regulation. We next developed a mass spectrometry (MS) based absolute quantification of SDX proteins including TFs, which are normal present in very low concentration. With our digestion optimization, subcellular fractionation of SDX nuclei, and improved instrumentation in the quadrupole orbitrap MS, we were able to identify over 6,000 unique proteins, including cellulose and hemicellulose biosynthesis proteins as well as many SND and VND TF members. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa and used these transgenics built a hierarchical genetic regulatory network (GRN), which demonstrated that ptr-miR397a is a negative regulator of laccases for lignin biosynthesis. The GRN further identified previously undisclosed TFs and their targets including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. We also analyzed the transcriptomes of 5 tissues (xylem, phloem, shoot, leaf, and root) and 2 wood forming cell-types (fiber and vessel) of P. trichocarpa to assemble gene co-expression subnetworks. We identified 165 TFs that showed xylem-, fiber-, and vessel-specific expression. Of the 165 TFs, 101 co-expressed with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Our co-expression network suggests a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.},
doi = {10.2172/1420055},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2018},
month = {2}
}