- Prof. Dr. Muhammad Iqbal
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The hydrolyzate was removed and filtered through dry, pre-weighed, medium coarseness sintered-glass crucible to estimate the content of acid-insoluble lignin. Acid-soluble lignin was determined by absorbance at nm on the Bio UV-visible spectrophotometer using quartz cuvettes Varian, Inc. Holocellulose and alpha-cellulose analysis 2. Extract-free wood samples mg were used for holocellulose quantification. The reaction was quenched by placing it into an ice bath to cool.
The weight of the crucible was determined after drying, to quantify holocellulose content Yokoyama et al. Approximately mg of holocellulose was used for alpha-cellulose isolation. Samples were reacted for 30 minutes after adding 8 mL of After 29 minutes, the retentate was filtered through a pre-weighed coarse sintered-glass crucible by washing with distilled water. The crucibles were soaked in 1.
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The dry weight of crucibles was used to quantify the alpha-cellulose contents. Wood quality 2. Measurements were taken from three separate samples. Bark to bark samples were first precision cut to 1. Microfibril angle was determined by the average T-value of the two diffraction arc peaks according to Megraw et al. Statistical analysis 2. Fibers confer the supportive strength for xylem development, while vessels facilitate water conduction by forming long hollow conduits that are composed of dead lignified cells.
Vessel characteristics affect the movement of water, with larger diameter providing higher hydraulic conductance. However, large vessels must balance conductivity with resistance to cavitation which can result in a discontinuous water column Sperry, Thinner vessel cell walls are vulnerable to collapse, as witnessed in irregular xylem irx mutants of Arabidopsis thaliana with defects in secondary cell wall deposition Turner and Somerville, ; Jones et al.
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Vessels arise from the xylem mother cells in the vascular cambium, and are fully differentiated after cell expansion, cell wall deposition, lignification, and programmed cell death. During vessel cell expansion, the rapid cell enlargement requires vessel cells to generate turgor pressure, and adjust cell wall plasticity Ray et al. Vessel cells build the hydraulic system by attracting osmotically active ions such as potassium ions to power cell expansion Fromm, After cell expansion, the vessel secondary cell wall is deposited, which is ultimately followed by programmed cell death.
Vessels have a shorter life span than fibers or tracheids, which can live for up to one month Courtois-Moreau et al. As a consequence of the numerous cell types in the cambial zone, their distinct cellular activities and short lifespan, the mechanism s of vessel differentiation have been difficult to study. In addition, genes encoding aquaporins, expansin, pectin methylesterase, and xyloglucan endotransglycosylase can be detected during cambial cell division and expansion Guglielmino et al. Although some genes responsible for cell wall modification are easily reconciled, there are several arabinogalactan proteins AGPs detected during xylem development whose functional roles are yet to be assigned.
These transcriptome data are consistent with the previous results which showed that AGP9 was expressed during cell expansion in developing poplar xylem by microarray analysis Hertzberg et al.
PtAGP9 was shown to be highly expressed in the developing xylem, especially in the middle stem Figure 3. Figure 3. Identical conserved amino acids are in black shading, non-identical amino acids with similar properties are in grey shading. Transcript abundance in each tissue is based on the comparative threshold cycle value relative to the housekeeping gene actin. Roles of AGP9 in xylem development in poplar 3. Since the introduction of an extra copy of an endogenous gene commonly leads to post-transcriptional gene silencing in plant Vaucheret et al.
However, when the morphology of developing xylem at the base of stem was examined, the RNAi-PtAGP9 poplar lines had smaller, more abundant vessels, and the vessels appeared to group together relative to the vessels in wild-type trees P39 Figure 3. In Figure 3. Given the Relative expression level of PtAGP9 73 visual observations, the number and size of vessels were quantified in all lines, with measurements restricted to the first 30 cell files initiating from the vascular cambium. By quantifying the characteristics of the vessels at base of the stem in cross section , statistically significant differences were observed in the average area occupied by the vessels in three of five transgenic lines overexpressing the Arabidopsis ortholog Figure 3.
In those same lines, the number of vessels per area was reduced from a mean value of 52 to a mean value of 42 Figure 3. These findings suggest that AGP9 has an impact on the vessel development in poplar. Given the vessel phenotype, we also investigated the impact of AGP9 on fiber development in these same transgenic poplar trees.
The bases of poplar stems were sectioned, stained with toluidine blue O and observed via a light microscope. Panels A-C. Wild type from three biological replicates. Vessels groups are depicted by red circle in D and E.
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Early initiation of vessel differentiation in cambial zone F is indicated by blue arrow compared with wild type in C. The cambium is indicated by yellow arrows. Wild-type vessel is indicated by black arrow. CA: cambium; V: vessel. The stem base of fresh trees was sectioned and stained with toluidine blue O. For each line, stem cross section images from three biological replicates were captured by microscopy from several aspects of one section omitting tension wood sections. The size of the vessels was quantified in the first 30 layers of fiber cells from the vascular cambium.
Approximately vessels were measured for each line.
The asterisks indicate a significant difference relative to wild type wt. For each line, stem cross-section images from three biological replicates were captured by microscope from several aspects of one section omitting tension wood sections. The vessel numbers per field of view were quantified in the first 30 layers of cells from the vascular cambium in stem cross sections. Around 20 stem cross sections were used for each line.
The Project Gutenberg eBook of Encyclopædia Britannica, Volume XII Slice VII - Gyantse to Hallel.
The asterisks indicate a significant difference relative to wild type. The middle stem and stem base of trees were sectioned and stained with toluidine blue O and observed via a light microscope. Middle stem cross section of wild type. Larger, fewer and irregular vessels shown in middle stem cross sections of transgenic poplar line 3 B and line 2 C compared with wild type A.
Base of stem cross section of wild type. Larger and fewer vessels shown in base of stem cross section of transgenic poplar line 3 E and line 2 F relative to the wild type D. Irregular vessel phenotype is indicted by blue arrow in B and C. Cambial zone is indicated by a yellow arrow. V: vessel.
The stem base of trees was sectioned and stained with toluidine blue O. Transgenic line 2, 3, 6 and wild type wt-1 were grown in September Transgenic line 7, 8 and wild type wt-2 were grown in February For each line, stem cross section images from three biological replicates were captured by microscopy from several aspects of one section excluding tension wood.
The size of the vessels was quantified in the first 30 layers of fiber cells from the vascular cambium in stem cross sections. The asterisks indicate a statistically significant difference relative to wild type. For each line, stem cross section images from three biological replicates were captured by microscope from several aspects of one section except tension wood.
The vessel number per field of view was quantified in the first 30 layers of fiber cells from the vascular cambium in stem cross sections. Approximately 20 stem cross sections were used for each line. For each line, three biological replicates and 10, fibers for each replicate were measured per tree by fiber quality analyzer. A B 81 AGP9 in cell wall traits in poplar 3. These findings imply that the defects in vessel development were not correlated with any obvious changes in cell wall chemistry. Moreover, wood density was shown not to be significantly changed in the transgenic lines compared to the wild-type trees Figure 3.
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Data represent the average of three biological replicates. Measurements were based on the dry weight of cell wall material. The asterisk indicates a statistically significant difference relative to wild type. The roles of AGP9 in cell expansion 3. Dark-grown hypocotyls are a model system for cell expansion because their elongation has been shown to be strictly due to expansion and not cell division Gendreau et al. To test if dark-grown hypocotyls were a suitable system to study the role of AGP9 in cell expansion, it was necessary to first demonstrate AGP9 expression in Arabidopsis hypocotyls.
As such, the AtAGP9 promoter was fused to the GUS reporter gene, and shown to be present in elongating hypocotyls after three and seven day dark cultivation Figure 3.