Characterising the roles of the members of the nuclear-encoded Rubisco small subunit family in Arabidopsis thaliana

Rubisco is the enzyme responsible for the net photosynthetic carbon fixation in plants. The enzyme consists of chloroplast-encoded large subunits and a family of nuclear-encoded small subunits (SSUs). SSUs are essential for the assembly of the Rubisco enzyme in higher plants and have been shown to influence catalytic activities of Rubisco (Atkinson et al., 2017; Laterre et al., 2017). This study aims to characterise the roles of SSUs in the model plant Arabidopsis thaliana (Arabidopsis). Arabidopsis contains four SSU (RbcS) genes, RbcS1A (1A hereafter) on chromosome 1, and RbcS1B, RbcS2B and RbcS3B (1B, 2B, 3B, respectively hereafter) located in tandem on chromosome 5. To characterise the roles of SSU, I have i) generated SSU knock-out mutants using CRISPR/Cas9 editing and analyse the impact of single and multiple SSU knock-outs on plant growth and fitness; ii) measured RbcS expression under different light qualities, quantities and temperatures; iii) performed growth analyses under different environmental conditions using CRISPR/Cas9 rbcs mutants; and iv) complemented the triple mutant 1a2b3b (BigBoi) with SSU from Chlamydomonas reinhardtii (Chlamydomonas) and performed growth analyses on the complemented lines. To generate SSU knock-out mutants via CRISPR/Cas9, two pairs of gRNAs were designed to target each RbcS gene. The efficiency of the gRNAs pairs was evaluated in mesophyll protoplasts and it was found that at least one pair for each gene was able to induce large deletions of 96-180 bp in RbcS. Constructs containing Cas9 nuclease and gRNAs were stably transformed in Arabidopsis by floral dipping. The T1 progeny containing the transgene were screened for large deletions by PCR and small insertions/deletions (indels) by Sanger sequencing. PCR screening showed that large deletions were induced in planta, but they were chimeric deletions and not transmittable to the T2 progeny. On the other hand, indels of 1-7 bp occurred at a higher rate (12% and 7%, respectively) and were heritable. Analyses of the T2 progeny revealed that heterozygous mutation was the most common type of mutation in 1B and 2B but chimeric mutation was the most common in 1A and 3B. The heritability rates for 1a, 1b, 2b and 3b were 4%, 20%, 8% and 6%, respectively. To measure the RbcS expression under different environmental conditions, the arrhythmic clock mutant prr5/7/9 mutant was grown under constant light for 14 d. The mutant was kept in darkness for 24 h before exposing to white light for 12 h. Transcript analysis was performed and the result showed that i) all RbcS genes were induced by light and the total transcript abundance increased when light was turned on and decreased after light was turned off; and ii) each RbcS had different induction and degradation rates. 1A was induced the most quickly and degraded most rapidly while 1B was induced the most slowly and 2B was the most stable transcript after light was turned off. The experiment was repeated but with different light qualities (blue, red, and far-red), light quantities (high (1000 μmol photon m-2 s-1), medium (200 μmol photon m-2 s-1) and low (50 μmol m-2 s- 1) light), and temperatures (high (30oC) and low (10oC), white light at 200 μmol m-2 s-1). Transcript analyses showed that blue light induced the highest level of increase among light qualities followed by red light and far-red light. High light induced the highest level of transcript abundance followed by medium light and low light. Under high temperature, the expression of 2B and 3B increased significantly and 3B was the major isoform. On the other hand, the expression of 2B and 3B were suppressed under low temperature and 1A was the major isoform. This suggested that 2B and 3B were the most sensitive temperature mediators of the RbcS gene family. Based on the transcript analyses, the high light, high temperature and low temperature conditions were chosen for growth experiments. WT, T-DNA and CRISPR/Cas9 mutants were used to test the following hypotheses: i) under the light saturating condition (high light), Rubisco becomes limiting and plants with reduced Rubisco content (1a, 3b, 2b3b and 1a2b) would grow more slowly than WT; ii) under high temperature where 3B is the major isoform, plants lacking 3B (3b, 2b3b) would suffer a reduced growth rate relative to WT; iii) under low temperature where 1A is the major isoform, plants lacking 1A (1a, 1a2b) would suffer a reduced growth rate relative to WT. Growth under high light was able to differentiate the areas of 1a, 2b3b and 1a2b mutant in comparison to WT, but not 3b mutant. However, the weight of 3b mutant was significantly lower than that of WT, suggesting the leaves of 3b were thinner. Under high temperature, 3b and 2b3b mutants were not significantly different from WT. This was due to 3B accounting for ca 50% of the transcript abundance under high temperature and growth was found to be unaffected at this level of RbcS decrease. Under low temperature, the areas of 1a and 1a2b were not significantly different from that of WT, but weights were significantly lower. This was similar to 3b under high light and suggested that leaves of plants with significant Rubisco reduction become thinner first and further decrease in Rubisco resulted in the loss of leaf area. Altogether, this study showed that RbcS genes collectively contribute to the overall transcript abundance and 2B and 3B genes are most susceptible to the changes in temperature. The triple mutant 1a2b3b generated in this study was used as a model to study the effects of heterologous SSUs to growth. After complementation with Chlamydomonas SSU, seven independent complemented lines were identified and the slow-growing phenotype was rescued in all lines. The area of complemented plants ranged from 8-34% of WT compared to 1% of the triple mutant on day 28. This study showed that the triple mutant could be used as an Arabidopsis platform to study the effects of heterologous SSUs to Rubisco catalytic activities

Anti-acne activity of Garcinia mangostana L.: A review

Garcinia mangostana L. or mangosteen of the Clusiaceae has traditionally been employed as medicinal drugs for decades. A plethora of compounds are responsible for a wide range of medicinal properties and biological activities. The ethanol extract of the mangosteen rind has been found to be anti-inflammatory, antioxidant and anti-acne-causing bacteria. Many research studies have confirmed its potency, with the ethanol extract of the rind being able to inhibit pro-inflammatory cytokines (TNF-?) at relatively low concentration. DPPH assay also revealed its potent radical scavenging activity. The compound responsible for the antibacterial activity, a-mangostin, was especially potent and one of the compounds responsible for the anti-bacterial activity

Physical and Biophysical Pretreatment of Water Hyacinth Biomass for Cellulase Enzyme Production

One practical way to control water hyacinth overgrowth due to eutrophication is by utilizing it as a substrate to produce cellulase. Water hyacinth was subjected to pretreatment to degrade lignin and improve microbes’ accessibility to cellulose. Physical and biophysical pretreatment methods were investigated. Biomass size reduction was performed in the physical pretreatment whereas white rot fungus (Ganoderma boninense) was used in the biophysical pretreatment. Cellulase-producing fungi, Aspergillus niger and Trichoderma reesei, were exploited in this study. Although lignin content was reduced by two-fold after the biophysical pretreatment, the maximum production of cellulase occurred when only the physical pretreatment was employed on the substrate. It may be because the higher apparent crystallinity of cellulose in physical pretreatment triggers more cellulase production compared to that in biophysical pretreatment. The maximum cellulase activity was found to be 1.035 IU mL–1 when water hyacinth was only physically pretreated

Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis

The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded for by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (ca. 3% relative to WT) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests this is a viable approach for exploring the functional roles of SSU isoforms in plants

Apolipoprotein E LDL receptor-binding domain-containing high-density lipoprotein: A nanovehicle to transport curcumin, an antioxidant and anti-amyloid bioflavonoid

AbstractCurcumin is an antioxidant and anti-inflammatory bioflavonoid that has been recently identified as an anti-amyloid agent as well. To make it more available in its potent form as a potential amyloid disaggregation agent, we employed high-density lipoproteins (HDL), which are lipid–protein complexes that transport plasma cholesterol, to transport curcumin. The objective of this study was to employ reconstituted HDL containing human apoE3 N-terminal (NT) domain, as a vehicle to transport curcumin. The NT domain serves as a ligand to mediate binding and uptake of lipoprotein complexes via the low-density lipoprotein receptor (LDLr) family of proteins located at the cell surface. Reconstituted HDL was prepared with phospholipids and recombinant apoE3-NT domain in the absence or presence of curcumin. Non-denaturing polyacrylamide gel electrophoresis indicated that the molecular mass and Stokes' diameter of HDL bearing curcumin were ~670kDa and ~17nm, respectively, while electron microscopy revealed the presence of discoidal particles. Fluorescence emission spectra of HDL bearing (the intrinsically fluorescent) curcumin indicated that the wavelength of maximal fluorescence emission (λmax) of curcumin was ~495nm, which is highly blue-shifted compared to λmax of curcumin in solvents of varying polarity (λmax ranging from 515–575nm) or in aqueous buffers. In addition, an enormous enhancement in fluorescence emission intensity was noted in curcumin-containing HDL compared to curcumin in aqueous buffers. Curcumin fluorescence emission was quenched to a significant extent by lipid-based quenchers but not by aqueous quenchers. These observations indicate that curcumin has partitioned efficiently into the hydrophobic milieu of the phospholipid bilayer of HDL. Functional assays indicated that the LDLr-binding ability of curcumin-containing HDL with apoE3-NT is similar to that of HDL without curcumin. Taken together, we report that apoE-containing HDL has a tremendous potential as a ‘nanovehicle’ with a homing device to transport curcumin to target sites

Optimal Production of <i>Ganoderma formosanum</i> Mycelium with Anti-Melanogenic Activity

Ganoderma formosanum is a medicinal mushroom endemic to Taiwan. The extraction of G. formosanum mycelium using ethyl acetate showed a significant reduction of melanin activity due to the inhibition of tyrosinase. To optimize the production of G. formosanum mycelium with anti-melanogenic activity, different variables (carbon sources and concentrations, initial pH value, and temperature) were investigated. According to the results, the optimal conditions included the utilization of 50 g/L lactose as the carbon source at pH 7 and 25 °C for 9 days. Afterward, the G. formosanum ethanolic extracts-optimized (GFE-O) was used to study the anti-melanogenic activity in vitro and in vivo. In B16-F10 melanoma cells treated with GFE-O (0.1 mg/mL), the intracellular melanin content was reduced to 76% compared to the control group. By applying GFE-O (0.05 mg/mL) in vivo on zebrafish embryo, hypopigmentation was observed, and the melanin content was reduced to 62% compared to the control with no toxicological effects. The results showed that the optimal inoculation conditions can provide the basis for future large-scale production of G. formosanum mycelium to maximize the extraction of anti-melanogenic ingredients