Dongliang Song

Characterization of cellulose synthase complexes in Populus xylem differentiation

*It is generally hypothesized that the synthesis of cellulose in higher plants is mediated by cellulose synthase complexes (CSCs) localized on the plasma membrane. However, CSCs have not been investigated thoroughly through their isolation. The availability of ample Populus tissue allowed Populus CSCs to be isolated and characterized in association with xylem differentiation. *The methods used here included co-immunoprecipitation, proteomic analysis, laser microdissection, immunolocalization and others. *Western blot analysis of the immunoprecipitated CSCs led to the identification of at least two types of CSC in the membrane protein of Populus xylem tissue. Proteomic analysis further revealed that the two types of CSC were assembled from different cellulose synthase proteins. Immunolocalization confirmed that both types of CSC were involved in secondary cell wall formation. In addition, a number of noncellulose synthase proteins were also identified in association with CSC precipitation. *The results indicate that two types of CSC participate in secondary wall formation in Populus, suggesting a new mechanism of cellulose formation involved in the thickening of wood cell walls. This study also suggests that the CSC machinery may be aided by other proteins in addition to cellulose synthase proteins.

Populus endo-beta-mannanase PtrMAN6 plays a role in coordinating cell wall remodeling with suppression of secondary wall thickening through generation of oligosaccharide signals

Endo-1,4-β-mannanase is known to able to hydrolyze mannan-type polysaccharides in cell wall remodeling, but its function in regulating wall thickening has been little studied. Here we show that a Populus endo-1,4-β-mannanase gene, named PtrMAN6, suppresses cell wall thickening during xylem differentiation. PtrMAN6 is expressed specifically in xylem tissue and its encoded protein localizes to developing vessel cells. Overexpression of PtrMAN6 enhanced wall loosening as well as suppressed secondary wall thickening, whilst knockdown of its expression promoted secondary wall thickening. Transcriptional analysis revealed that PtrMAN6 overexpression downregulated the transcriptional program of secondary cell wall thickening, whilst PtrMAN6 knockdown upregulated transcriptional activities toward secondary wall formation. Activity of PtrMAN6 hydrolysis resulted in the generation of oligosaccharide compounds from cell wall polysaccharides. Application of the oligosaccharides resulted in cellular and transcriptional changes that were similar to those found in PtrMAN6 overexpressed transgenic plants. Overall, our results demonstrated that PtrMAN6 plays a role in hydrolysis of mannan-type wall polysaccharides to produce oligosaccharides that may serve as signaling molecules to suppress cell wall thickening during wood xylem cell differentiation.

PtrHB7, a class III HD-Zip Gene, Plays a Critical Role in Regulation of Vascular Cambium Differentiation in Populus

A key question in the secondary growth of trees is how differentiation of the vascular cambium cells is directed to concurrently form two different tissues: xylem or phloem. class III homeodomain-leucine zipper (HD-Zip III) genes are known to play critical roles in the initiation, patterning, and differentiation of the vascular system in the process of primary and secondary growth. However, the mechanism of how these genes control secondary vascular differentiation is unknown. Here, we show that a Populus class III HD-Zip gene, PtrHB7, was preferentially expressed in cambial zone. PtrHB7-suppressed plants displayed significant changes in vascular tissues with a reduction in xylem but increase in phloem. Transcriptional analysis revealed that genes regulating xylem differentiation were down-regulated, whereas genes regulating phloem differentiation were up-regulated. Correspondingly, PtrHB7 overexpression enhanced differentiation of cambial cells toward xylem cells but inhibited phloem differentiation. PtrHB7 regulation on cambial cell differentiation was associated with its transcript abundance. Together, the results demonstrated that PtrHB7 plays a critical role in controlling a balanced differentiation between secondary xylem and phloem tissues in the process of Populus secondary growth in a dosage-dependent manner.

Characterization of the plasma membrane proteins and receptor-like kinases associated with secondary vascular differentiation in poplar

The constituents of plasma membrane proteins, particularly the integral membrane proteins, are closely associated with the differentiation of plant cells. Secondary vascular differentiation, which gives rise to the increase in plant stem diameter, is the key process by which the volume of the plant body grows. However, little is known about the plasma membrane proteins that specifically function in the vascular differentiation process. Proteomic analysis of the membrane proteins in poplar differentiating secondary vascular tissues led to the identification 226 integral proteins in differentiating xylem and phloem tissues. A majority of the integral proteins identified were receptors (55 proteins), transporters (34 proteins), cell wall formation related (27 proteins) or intracellular trafficking (17 proteins) proteins. Gene expression analysis in developing vascular cells further demonstrated that cambium differentiation involves the expression of a group of receptor kinases which mediate an array of signaling pathways during secondary vascular differentiation. This paper provides an outline of the protein composition of the plasma membrane in differentiating secondary vascular tissues and sheds light on the role of receptor kinases during secondary vascular development.

Conservation and functional influence of alternative splicing in wood formation of Populus and Eucalyptus

BackgroundWood formation in tree species is regulated by multiple factors at various layers. Alternative splicing (AS) occurs within a large number of genes in wood formation. However, the functional implications and conservation of the AS occurrence are not well understood.ResultsIn this study, we profiled AS events in wood-forming tissues of Populus and Eucalyptus, and analyzed their functional implications as well as inter-species conservation. 28.3% and 20.7% of highly expressed transcripts in the developing xylem of Populus and Eucalyptus respectively were affected by AS events. Around 42% of the AS events resulted in changes to the original reading frame. 25.0% (in Populus) and 26.8% (in Eucalyptus) of the AS events may cause protein domain modification. In the process of wood formation, about 28% of AS-occurring genes were putative orthologs and 71 conserved AS events were identified in the two species.ConclusionThrough analysis of AS events in developing xylem of two tree species, this study reveals an array of new information regarding AS occurrence and function in tree development.

Diverse roles of PtrDUF579 proteins in Populus and PtrDUF579‑1 function in vascular cambium proliferation during secondary growth

DUF579 (domain of unknown function 579) family proteins contain a DUF579 domain structure but vary greatly in their overall sequence similarity. Several DUF579 proteins have been found to play a role in cell wall biosynthesis in Arabidopsis, while DUF579 family genes have not yet been systematically investigated in Populus. In this study, the Populus DUF579 family proteins were found to be localized in different cell types and subcellular locations. The diverse expression patterns of the proteins indicate that they may perform different functions in Populus. Among the DUF579 family members, PtrDUF579-1 is found to be specifically expressed in vascular cambium zone cells where it is localized in the Golgi apparatus. Suppression of PtrDUF579-1 expression reduced plant height and stem diameter size. Cambium cell division and xylem tissue growth was inhibited while secondary cell wall formation was unchanged in PtrDUF579-1 suppressed plants. Cell walls analysis showed that the composition of the pectin fraction of the cambium cell wall was altered while other polysaccharides were not affected in PtrDUF579-1 suppressed plants. This observation suggest cambium expressed PtrDUF579-1 may affect cell wall biosynthesis and be involved in cambium cell proliferation in Populus. Overall, DUF579 family proteins play a diverse set of roles in Populus.

Suppression of PtrDUF579-3 Expression Causes Structural Changes of the Glucuronoxylan in Populus.

DUF579 (domain unknown function 579) genes have been reported to play diverse roles in cell wall biosynthesis, such as in glucuronoxylan (GX) synthesis. As GX is a major type of hemicelluloses in hard wood species, how DUF579 genes function in wood formation remains to be demonstrated in planta. This study reports a Populus DUF579 gene, PtrDUF579-3, which is characterized for its function in wood cell wall formation. PtrDUF579-3 is localized in Golgi apparatus and catalyzes methylation of the glucuronic acid (GlcA) in GX biosynthesis. Suppression of PtrDUF579-3 expression in Populus caused a reduction in both the GlcA side chain number and GlcA side chain methylation on the GX backbone. The modified GX polymer through PtrDUF579-3 suppression was more susceptible to acid treatment and the PtrDUF579-3 suppressed plants displayed enhanced cellulose digestibility. These results suggest that PtrDUF579-3 is involved in GX biosynthesis and GX structure can be modified through PtrDUF579-3 suppression in Populus.

Formation of wood secondary cell wall may involve two type cellulose synthase complexes in Populus.

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.

A HD-ZIP III gene, PtrHB4, is required for interfascicular cambium development in Populus

Summary Wood production is dependent on the activity of the vascular cambium, which develops from the fascicular and interfascicular cambia. However, little is known about the mechanisms controlling how the vascular cambium is developed in woody species. Here, we show that PtrHB4, belonging to the Populus HD‐ZIP III family, plays a critical role in the process of vascular cambium development. PtrHB4 was specifically expressed in shoot tip and stem vascular tissue at an early developmental stage. Repression of PtrHB4 caused defects in the development of the secondary vascular system due to failures in interfascicular cambium formation. By contrast, overexpression of PtrHB4 induced cambium activity and xylem differentiation during secondary vascular development. Transcriptional analysis of PtrHB4 repressed plants indicated that auxin response and cell proliferation were affected in the formation of the interfascicular cambium. Taken together, these results suggest that PtrHB4 is required for interfascicular cambium formation to develop the vascular cambium in woody species.