Functional Characterisation of the Poplar Atypical Aspartic Protease Gene PtAP66 in Wood Secondary Cell Wall Deposition

Secondary cell wall (SCW) deposition is an important process during wood formation. Although aspartic proteases (APs) have been reported to have regulatory roles in herbaceous plants, the involvement of atypical APs in SCW deposition in trees has not been reported. In this study, we characterised the Populus trichocarpa atypical AP gene PtAP66, which is involved in wood SCW deposition. Transcriptome data from the AspWood resource showed that in the secondary xylem of P. trichocarpa, PtAP66 transcripts increased from the vascular cambium to the xylem cell expansion region and maintained high levels in the SCW formation region. Fluorescent signals from transgenic Arabidopsis plant roots and transiently transformed P. trichocarpa leaf protoplasts strongly suggested that the PtAP66-fused fluorescent protein (PtAP66-GFP or PtAP66-YFP) localised in the plasma membrane. Compared with the wild-type plants, the Cas9/gRNA-induced PtAP66 mutants exhibited reduced SCW thickness of secondary xylem fibres, as suggested by the scanning electron microscopy (SEM) data. In addition, wood composition assays revealed that the cellulose content in the mutants decreased by 4.90–5.57%. Transcription analysis further showed that a loss of PtAP66 downregulated the expression of several SCW synthesis-related genes, including cellulose and hemicellulose synthesis enzyme-encoding genes. Altogether, these findings indicate that atypical PtAP66 plays an important role in SCW deposition during wood formation.

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The R2R3 MYB transcription factor MYB189 negatively regulates secondary cell wall biosynthesis in Populus

Tree Physiology ◽

10.1093/treephys/tpz040 ◽

2019 ◽

Vol 39 (7) ◽

pp. 1187-1200 ◽

Cited By ~ 1

Author(s):

Bo Jiao ◽

Xin Zhao ◽

Wanxiang Lu ◽

Li Guo ◽

Keming Luo

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Secondary Wall ◽

Secondary Cell Wall ◽

Populus Trichocarpa ◽

Wood Formation ◽

Myb Transcription Factor ◽

Polymerase Chain ◽

Xylem Fibers ◽

R2r3 Myb

Abstract Secondary cell wall (SCW) biosynthesis during wood formation in trees is controlled by a multilevel regulatory network that coordinates the expression of substantial genes. However, few transcription factors involved in the negative regulation of secondary wall biosynthesis have been characterized in tree species. In this study, we isolated an R2R3 MYB transcription factor MYB189 from Populus trichocarpa, which is expressed predominantly in secondary vascular tissues, especially in the xylem. A novel repression motif was identified in the C-terminal region of MYB189, which indicates this factor was a transcriptional repressor. Overexpression (OE) of MYB189 in Arabidopsis and poplar resulted in a significant reduction in the contents of lignin, cellulose and hemicelluloses. Vascular development in stems of MYB189 OE lines was markedly inhibited, leading to a dramatic decrease in SCW thickness of xylem fibers. Gene expression analyses showed that most of the structural genes involved in the biosynthesis of lignin, cellulose and xylans were significantly downregulated in MYB189-overexpressing poplars compared with the wild-type control. Chromatin immunoprecipitation-quantitative real-time polymerase chain reaction and transient expression assays revealed that MYB189 could directly bind to the promoters of secondary wall biosynthetic genes to repress their expression. Together, these data suggest that MYB189 acts as a repressor to regulate SCW biosynthesis in poplar.

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Functional understanding of secondary cell wall cellulose synthases in Populus trichocarpa via the Cas9/gRNA‐induced gene knockouts

New Phytologist ◽

10.1111/nph.17338 ◽

2021 ◽

Author(s):

Wenjing Xu ◽

Hao Cheng ◽

Siran Zhu ◽

Jiyao Cheng ◽

Huanhuan Ji ◽

...

Keyword(s):

Cell Wall ◽

Secondary Cell Wall ◽

Populus Trichocarpa ◽

Functional Understanding ◽

Gene Knockouts ◽

Cellulose Synthases

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Involvement of CesA4, CesA7-A/B and CesA8-A/B in secondary wall formation in Populus trichocarpa wood

Tree Physiology ◽

10.1093/treephys/tpz020 ◽

2019 ◽

Vol 40 (1) ◽

pp. 73-89 ◽

Cited By ~ 1

Author(s):

Manzar Abbas ◽

Ilona Peszlen ◽

Rui Shi ◽

Hoon Kim ◽

Rui Katahira ◽

...

Keyword(s):

Solid State ◽

Mechanical Strength ◽

Cell Walls ◽

Secondary Wall ◽

Populus Trichocarpa ◽

Wood Formation ◽

Fiber Cell ◽

Modulus Of Rupture ◽

Cellulose Content ◽

Wall Formation

Abstract Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4, PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesAs did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation.

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Lignification of spruce tracheid secondary cell walls related to longitudinal hardness and modulus of elasticity using nano-indentation

Canadian Journal of Botany ◽

10.1139/b02-091 ◽

2002 ◽

Vol 80 (10) ◽

pp. 1029-1033 ◽

Cited By ~ 41

Author(s):

W Gindl ◽

H S Gupta ◽

C Grünwald

Keyword(s):

Cell Wall ◽

Modulus Of Elasticity ◽

Cell Walls ◽

Secondary Cell Wall ◽

Lignin Content ◽

Wood Formation ◽

Nano Indentation ◽

Secondary Cell Walls ◽

Cell Wall Development ◽

The One

The lignin content and the mechanical properties of lignifying and fully lignified spruce tracheid secondary cell walls were determined using UV microscopy and nano-indentation, respectively. The average lignin content of developing tracheids was 0.10 g·g1, as compared with 0.21 g·g1 in mature tracheids. The modulus of elasticity of developing cells was on average 22% lower than the one measured in mature, fully lignified cells. For the longitudinal hardness, a larger difference of 26% was observed. As lignifying cells in the cambial zone are undergoing cell wall development, spaces in the cellulosehemicellulose structure are filled with lignin and the density of the cell wall is believed to increase. It is therefore suggested that the observed difference in modulus of elasticity between developing and fully lignified cell walls is due to the filling of spaces with lignin and an increase of the packing density of the cell wall during lignification. Although remarkably less stiff than the composite polysaccharide structure in the secondary cell wall, lignin may be considered equally hard. Therefore, the observed increase in lignin content may contribute directly to the measured increase of hardness.Key words: secondary cell wall, hardness, lignin, modulus of elasticity, wood formation.

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Analysis of xylem formation in pine by cDNA sequencing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.16.9693 ◽

1998 ◽

Vol 95 (16) ◽

pp. 9693-9698 ◽

Cited By ~ 209

Author(s):

Isabel Allona ◽

Michelle Quinn ◽

Elizabeth Shoop ◽

Kristi Swope ◽

Sheila St. Cyr ◽

...

Keyword(s):

Cell Wall ◽

Loblolly Pine ◽

Secondary Xylem ◽

Expression Patterns ◽

Wood Formation ◽

Xylem Differentiation ◽

Cell Wall Proteins ◽

Powerful Means ◽

Xylem Formation ◽

Enzymes Of Carbohydrate Metabolism

Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (Pinus taeda L.). A total of 1,097 single-pass sequences were obtained from 5′ ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.

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Clone and Analysis of Secondary Xylem Cellulose Synthase Gene Fragment from Populus ussuriensis

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.113-116.1908 ◽

2010 ◽

Vol 113-116 ◽

pp. 1908-1913

Author(s):

Jiang Tao Shi ◽

Jian Li ◽

Yi Xing Liu ◽

Lei Xu

Keyword(s):

Populus Tremuloides ◽

Gene Sequence ◽

Secondary Xylem ◽

Sequence Similarity ◽

Populus Trichocarpa ◽

Wood Formation ◽

Cellulose Synthase ◽

Cellulose Synthase Gene ◽

Populus Ussuriensis ◽

Close Genetic Relationship

wood is the most abundant renewable resource and environmentally friendly energy source on the earth, it not only provides industrial raw materials for economic and social sustainable development, meanwhile, the biological process of wood formation which is mainly to sink the excessive carbon dioxide in the atmosphere can also play an active role in reducing “greenhouse effect”, so it is the contributor of green environment and human heath. Therefore, it is of great importance to explore the biosynthesis process and the wood formation mechanism of woods cellulose. This study adopted RT-PCR to clone gene fragments from the total RNA of populus ussuriensis secondary xylem, through sequence analysis, we found that its size was 487bp, which was named as PusC1，by means of blast comparative analysis, we found that the gene sequence similarity of this fragment and Populus trichocarpa cellulose synthase (XM 002305024.1) reached 94%, and its gene sequence similarity with Populus tremuloides PtrCesA1 and Populus tremula × Populus tremuloides xylem specificity cellulose synthase genes (AY573574.1) sequence could also reach 92%. Therefore, it is inferred to be populus ussuriensis xylem specificity cellulose synthase gene fragment. Through sequence similarity, we can also infer that populus ussuriensis has a close genetic relationship with Populus trichocarpa and belongs to cathay poplar species; while Populus tremuloides belongs to white poplar species and has a close genetic relationship with populus ussuriensis.

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Golgi-localized GALT7 and GALT8 participate in cellulose biosynthesis

10.1101/2021.04.21.440809 ◽

2021 ◽

Author(s):

Pieter Nibbering ◽

Romain Castilleux ◽

Gunnar Wingsle ◽

Totte Niittylä

Keyword(s):

Cell Wall ◽

Golgi Apparatus ◽

Secondary Cell Wall ◽

Plant Cell Wall ◽

Arabinogalactan Protein ◽

Primary Cell Wall ◽

Cellulose Content ◽

Cellulose Biosynthesis ◽

Cell Wall Assembly ◽

Protein Levels

AbstractArabinogalactan protein (AGP) glycan biosynthesis in the Golgi apparatus contributes to plant cell wall assembly, but the mechanisms underlying this process are largely unknown. Here, we show that two putative galactosyltransferases -named GALT7 and GALT8 -from the glycosyltransferase family 31 (GT31) of Arabidopsis thaliana participate in cellulose biosynthesis. galt7galt8 mutants show primary cell wall defects manifesting as impaired growth and cell expansion in seedlings and etiolated hypocotyls, along with secondary cell wall defects, apparent as collapsed xylem vessels and reduced xylem wall thickness in the inflorescence stem. These phenotypes were associated with a ∼30% reduction in cellulose content, a ∼50% reduction in secondary cell wall CELLULOSE SYNTHASE (CESA) protein levels and reduced cellulose biosynthesis rate. CESA transcript levels were not significantly altered in galt7galt8 mutants, suggesting that the reduction in CESA levels was caused by a post-transcriptional mechanism. We provide evidence that both GALT7 and GALT8 localise to the Golgi apparatus, while quantitative proteomics experiments revealed reduced levels of the entire FLA subgroup B in the galt7galt8 mutants. This leads us to hypothesize that a defect in FLA subgroup B glycan biosynthesis reduces cellulose biosynthesis rate in galt7galt8 mutants.

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PmMYB4, a Transcriptional Activator from Pinus massoniana, Regulates Secondary Cell Wall Formation and Lignin Biosynthesis

Forests ◽

10.3390/f12121618 ◽

2021 ◽

Vol 12 (12) ◽

pp. 1618

Author(s):

Sheng Yao ◽

Peizhen Chen ◽

Ye Yu ◽

Mengyang Zhang ◽

Dengbao Wang ◽

...

Keyword(s):

Cell Wall ◽

Secondary Cell Wall ◽

Paper Industry ◽

Lignin Biosynthesis ◽

Pinus Massoniana ◽

Wood Formation ◽

Genetically Engineered ◽

Cellulose Synthesis ◽

Masson Pine ◽

Reading Frame

Wood formation originates in the biosynthesis of lignin and further leads to secondary cell wall (SCW) biosynthesis in woody plants. Masson pine (Pinus massoniana Lamb) is an economically important industrial timber tree, and its wood yield affects the stable development of the paper industry. However, the regulatory mechanisms of SCW formation in Masson pine are still unclear. In this study, we characterized PmMYB4, which is a Pinus massoniana MYB gene involved in SCW biosynthesis. The open reading frame (ORF) of PmMYB4 was 1473 bp, which encoded a 490 aa protein and contained two distinctive R2 and R3 MYB domains. It was shown to be a transcription factor, with the highest expression in semi-lignified stems. We overexpressed PmMYB4 in tobacco. The results indicated that PmMYB4 overexpression increased lignin deposition, SCW thickness, and the expression of genes involved in SCW formation. Further analysis indicated that PmMYB4 bound to AC-box motifs and might directly activate the promoters of genes (PmPAL and PmCCoAOMT) involved in SCW biosynthesis. In addition, PmMYB4-OE(over expression) transgenic lines had higher lignin and cellulose contents and gene expression than control plants, indicating that PmMYB4 regulates SCW mainly by targeting lignin biosynthetic genes. In summary, this study illustrated the MYB-induced SCW mechanism in Masson pine and will facilitate enhanced lignin and cellulose synthesis in genetically engineered trees.

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