Laigeng Li

Functional Characterization of Evolutionarily Divergent 4-Coumarate:Coenzyme A Ligases in Rice

4-Coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) is a key enzyme in the phenylpropanoid metabolic pathways for monolignol and flavonoid biosynthesis. 4CL has been much studied in dicotyledons, but its function is not completely understood in monocotyledons, which display a different monolignol composition and phenylpropanoid profile. In this study, five members of the 4CL gene family in the rice (Oryza sativa) genome were cloned and analyzed. Biochemical characterization of the 4CL recombinant proteins revealed that the rice 4CL isoforms displayed different substrate specificities and catalytic turnover rates. Among them, Os4CL3 exhibited the highest turnover rate. No apparent tissue-specific expression of the five 4CLs was observed, but significant differences in their expression levels were detected. The rank in order of transcript abundance was Os4CL3 > Os4CL5 > Os4CL1 > Os4CL4 > Os4CL2. Suppression of Os4CL3 expression resulted in significant lignin reduction, shorter plant growth, and other morphological changes. The 4CL-suppressed transgenics also displayed decreased panicle fertility, which may be attributed to abnormal anther development as a result of disrupted lignin synthesis. This study demonstrates that the rice 4CLs exhibit different in vitro catalytic properties from those in dicots and that 4CL-mediated metabolism in vivo may play important roles in regulating a broad range of biological events over the course of rice growth and development.

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.

Rapid characterization of woody biomass digestibility and chemical composition using near-infrared spectroscopy.

Rapid determination of the properties of lignocellulosic material is highly desirable for biomass production and utilization. In the present study, measurements of woody biomass digestibility and chemical composition using near-infrared reflectance (NIR) spectroscopy were calibrated. Poplar and eucalyptus materials were recorded in NIR spectrum as well as determined for their chemical compositions of Klason lignin, α-cellulose, holocellulose, lignin syringyl/guaiacyl (S/G) ratio and enzymatic digestibility. Fitting of the NIR information with chemical properties and digestibility by partial least-squares (PLS) regression generated a group of trained NIR models that were able to be used for rapid biomass measurement. Applying the models for woody biomass measurements led to a reliable evaluation of the chemical composition and digestibility, suggesting the feasibility of using NIR spectroscopy in the rapid characterization of biomass properties.

Microwave Pretreatment of Switchgrass to Enhance Enzymatic Hydrolysis

Switchgrass is a promising lignocellulosic biomass for fuel-ethanol production. However, pretreatment of lignocellulosic materials is necessary to improve its susceptibility to enzymatic hydrolysis. The objectives of this study were to examine the feasibility of microwave pretreatment to enhance enzymatic hydrolysis of switchgrass and to determine the optimal pretreatment conditions. Switchgrass samples immersed in water, dilute sulfuric acid and dilute sodium hydroxide solutions were exposed to microwave radiation at varying levels of radiation power and residence time. Pretreated solids were enzymatically hydrolyzed and reducing sugars in the hydrolysate were analyzed. Microwave radiation of switchgrass at lower power levels resulted in more efficient enzymatic hydrolysis. The application of microwave radiation for 10 minutes at 250 watts to switchgrass immersed in 3% sodium hydroxide solution (w/v) produced the highest yields of reducing sugar. Results were comparable to conventional 60 minute sodium hydroxide pretreatment of switchgrass. The findings suggest that combined microwave-alkali is a promising pretreatment method to enhance enzymatic hydrolysis of switchgrass.

Genome-wide analysis revealed the complex regulatory network of brassinosteroid effects in photomorphogenesis.

Light and brassinosteroids (BRs) have been proved to be crucial in regulating plant growth and development; however, the mechanism of how they synergistically function is still largely unknown. To explore the underlying mechanisms in photomorphogenesis, genome-wide analyses were carried out through examining the gene expressions of the dark-grown WT or BR biosynthesis-defective mutant det2 seedlings in the presence of light stimuli or exogenous Brassinolide (BL). Results showed that BR deficiency stimulates, while BL treatment suppresses, the expressions of light-responsive genes and photomorphogenesis, confirming the negative effects of BR in photomorphogenesis. This is consistent with the specific effects of BR on the expression of genes involved in cell wall modification, cellular metabolism and energy utilization during dark-light transition. Further analysis revealed that hormone biosynthesis and signaling-related genes, especially those of auxin, were altered under BL treatment or light stimuli, indicating that BR may modulate photomorphogenesis through synergetic regulation with other hormones. Additionally, suppressed ubiquitin-cycle pathway during light-dark transition hinted the presence of a complicated network among light, hormone, and protein degradation. The study provides the direct evidence of BR effects in photomorphogenesis and identified the genes involved in BR and light signaling pathway, which will help to elucidate the molecular mechanism of plant photomorphogenesis.

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.

Intron-Mediated Alternative Splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B Regulates Cell Wall Thickening during Fiber Development in Populus Species

Alternative splicing of a transcription factor generates two isoforms which play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in planta. Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation.

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.

Grain setting defect1, encoding a remorin protein, affects the grain setting in rice through regulating plasmodesmatal conductance.

A remorin protein plays a role in regulating photoassimilate translocation by modulating plasmodesmata permeability in rice. Effective grain filling is one of the key determinants of grain setting in rice (Oryza sativa). Grain setting defect1 (GSD1), which encodes a putative remorin protein, was found to affect grain setting in rice. Investigation of the phenotype of a transfer DNA insertion mutant (gsd1-Dominant) with enhanced GSD1 expression revealed abnormalities including a reduced grain setting rate, accumulation of carbohydrates in leaves, and lower soluble sugar content in the phloem exudates. GSD1 was found to be specifically expressed in the plasma membrane and plasmodesmata (PD) of phloem companion cells. Experimental evidence suggests that the phenotype of the gsd1-Dominant mutant is caused by defects in the grain-filling process as a result of the impaired transport of carbohydrates from the photosynthetic site to the phloem. GSD1 functioned in affecting PD conductance by interacting with rice ACTIN1 in association with the PD callose binding protein1. Together, our results suggest that GSD1 may play a role in regulating photoassimilate translocation through the symplastic pathway to impact grain setting in rice.

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.