Acceptability of malunggay (Moringa oleifera) and squash (Cucurbita moschata) cookies

The dried leaf powder of malunggay (Moringa oleifera) contains an excellent nutritional value, and this tropical plant is widely available in the Philippines. On the other hand, squash (Cucurbita moschata) is an incredibly beneficial food with high amounts of vitamins. The nutrients from Malunggay and squash can help prevent deficiencies in children. The main focus of the research was to determine the acceptability of the different formulations of M. oleifera and C. moschata cookies. The study determined the most preferred cookie formulation based on sensory evaluation using descriptive testing of the product's appearance, aroma, taste, sweetness, texture, and acceptability rating of the formulations. The respondents were 20 food experts and 40 non-experts. The Analysis of Variance was used to examine the statistical significance of mean differences among the distinct groups. Subsequently, Tukey's Honest Significant Difference (HSD) test was employed to ascertain the significance of differences between pairs of group means, providing a more nuanced understanding of the variations observed within these groups. Results showed that among the three cookie formulations, F3 got the highest mean score of the different attributes, namely appearance (x̄= 4.45; x̄= 4.6) aroma (x̄ = 4.9; x̄= 4.7), taste (x̄= 4.95; x̄= 4.93), sweetness (x̄= 4.9; x̄=4.75), and texture (x̄= 4.8; x̄= 4.68), as perceived by the expert and non-expert, respectively. This implies that respondents prefer cookie formulation F3 over the two other formulations.

Wound-induced PAL activity is suppressed by heat-shock treatments that induce the synthesis of heat-shock proteins

Wounding lettuce leaves induces the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the accumulation of phenolic compounds, and subsequent tissue browning. A brief heat-shock at 45degreesC reduces the rise in wound-induced PAL, the accumulation of phenolic compounds, and tissue browning. The activity of PAL measured 24 h after wounding and the content of phenolic compounds (absorbance of methanol extract at 320 nm) measured 48 h after wounding was highly correlated (R-2 > 0.90) in tissue developing the normal wound response and in tissue subjected to 0-180 s of heat-shock after wounding. The synthesis of a unique set of proteins called heat-shock proteins (hsps) is induced by these heat-shock treatments. Western-blot analyses of proteins isolated from wounded and heat-shocked Iceberg and Romaine lettuce mid-rib leaf tissue was done using antibodies against hsp 23. Only those heat-shock treatments that were effective at inducing the synthesis of hsp 23 were effective in reducing the activity of PAL induced by wounding and the subsequent accumulation of phenolic compounds. Hsps induced in non-wounded, whole leaves by exposure to 45degreesC for 150 s did not significantly interact with PAL previously synthesized in non-heat-shocked wounded leaves to limit its activity. The preferential synthesis of hsps over that of wound-induced PAL, rather than the presence of hsps, may be responsible for the ability of a heat-shock treatment to reduce the wound-induced increase in PAL activity. Our results support this novel concept, and the possibility that heat-shock treatments can have significant physiological effects on the response of the tissue to other stresses, not because of the specific genes they induce or repress, or the products they cause to be synthesized, but by their secondary action of influencing the synthesis of other proteins (e.g. PAL) by the suppression of non-hsps protein synthesis

A Chaperonin Subunit with Unique Structures Is Essential for Folding of a Specific Substrate

Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60β4 forms a heterooligomeric Cpn60 complex with Cpn60α1 and Cpn60β1–β3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60β subunits cannot complement the function of Cpn60β4. Furthermore, the unique C-terminus of Cpn60β4 is required for the full activity of the unique Cpn60 complex containing Cpn60β4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates

Manipulating the Biosynthesis of Bioactive Compound Alkaloids for Next-Generation Metabolic Engineering in Opium Poppy Using CRISPR-Cas 9 Genome Editing Technology

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) endonuclease system is a powerful RNA-guided genome editing tool. CRISPR/Cas9 has been well studied in model plant species for targeted genome editing. However, few studies have been reported on plant species without whole genome sequence information. Currently, no study has been performed to manipulate metabolic pathways using CRISPR/Cas9. In this study, the type II CRISPR/SpCas9 system was used to knock out, via nonhomologous end-joining genome repair, the 4′OMT2 in opium poppy (Papaver somniferum L.), a gene which regulates the biosythesis of benzylisoquinoline alkaloids (BIAs). For sgRNA transcription, viral-based TRV and synthetic binary plasmids were designed and delivered into plant cells with a Cas9 encoding-synthetic vector by Agrobacterium-mediated transformation. InDels formed by CRISPR/Cas9 were detected by sequence analysis. Our results showed that the biosynthesis of BIAs (e.g. morphine, thebaine) was significantly reduced in the transgenic plants suggesting that 4′OMT2 was efficiently knocked-out by our CRISPR-Cas9 genome editing approach. In addition, a novel uncharacterized alkaloid was observed only in CRISPR/Cas9 edited plants. Thus, the applicabilitiy of the CRISPR/Cas9 system was demonstrated for the first time for medicinal aromatic plants by sgRNAs transcribed from both synthetic and viral vectors to regulate BIA metabolism and biosynthesis

Vesicle Transport with Emphasis on Chloroplasts

The plants on which we depend for food and oxygen need photosynthesis to prepare their own food. Photosynthesis takes place in the chloroplast. Inside chloroplasts is a specialized membrane called the thylakoids in which the photosynthesis activity takes place. The thylakoid membrane does not produce its own lipids, so instead they are transported from the envelope membrane to the thylakoid. Similarly, most of the proteins needed for maintenance of the photosynthetic apparatus and thylakoids are imported into the chloroplast across the envelope membrane and transported to the thylakoid. The lipids produced in the envelope membrane were suggested to be transported in three ways; through diffusion, through contact between thylakoids and the envelope or with the help of vesicles. The thylakoids and envelope membrane are well separated from each other by an aqueous solution, the stroma, which makes it hard for the lipids to move between the two compartments. Biochemical and ultrastructure data show vesicle transport inside chloroplasts. One of the vesicle functions in chloroplasts is to transport lipids from the envelope to the thylakoid to maintain its membrane structure. Proteins transported from the envelope to the thylakoids take four routes (Sec, Tat, SRP and Spontaneous pathways). Only a few proteins have been shown or hypothesized to follow these pathways. For many proteins it is unclear how they are transported to the thylakoids. It has been shown that vesicle transport in the chloroplast is similar to the cytosolic secretory system, which transports both lipids and proteins between different compartments in the cytosol. This hypothesis became more likely when putative protein components of the COPII transport pathway i.e. Sec23/Sec24, Sec13/Sec31 and Sar1 (which operate between the endoplasmic reticulum and the Golgi apparatus) were suggested to exist in chloroplasts. This thesis reports that indeed vesicle transport inside the chloroplast is similar to that of the cytosolic secretory system. The Sar1 homologue CPSAR1 (CP = chloroplast localized) has been characterized and shown to be important for embryo development and thylakoid biogenesis. Other studies have already shown that proteins such as VIPP1, THF1, ADL and FZL in the chloroplast do have an impact on vesicle transport and are also involved in thylakoid maintenance and biogenesis. This gives an indication that CPSAR1 could be involved in vesicular transport as well as collaborating with these proteins. Indeed it has been shown that CPSAR1 is found in low temperature induced vesicles and there are indications that CPSAR1 and THF1 may interact. CPSAR1 could be involved in several functions. Previous data shows its involvement in ribosome biogenesis, which is also indicated by genes co-expressed with CPSAR1 (on the publically available ATTED-II database) that have roles in protein synthesis. If there is a functional vesicle transport system in chloroplasts we expect there to be more components that are similar to vesicle transport in the cytosol. A bioinformatics approach predicted components like tethering factors, SNAREs, Rab GTPase, etc., to be present in chloroplasts. It was also proposed that the transport of cargo proteins in vesicles from the envelope to thylakoids would occur in a similar way to the secretory system in cytosol. One of the Rab GTPases, CPRabA5e has been found in the chloroplast and is localized in the stroma and thylakoids. It has been suggested that it binds to the thylakoid in its active form and has a role in vesicle tethering and fusion similarly to its homologue in yeast. Ultrastructure analysis of CPRabA5e mutant chloroplasts shows accumulation of vesicles at low temperature compared to wild type indicating a role in vesicle transport. Furthermore, CPRabA5e has been shown to have a role in seed germination, oxidative stress and maintaining the size of plastoglobuli. There has been clear evidence of vesicle transport inside chloroplasts and this transport is related to the secretory system in the cytosol. Two proteins in the chloroplast similar to proteins found in the secretory system are CPSAR1 and CPRabA5e, whose roles have been further characterized in chloroplast vesicle transport. At the same time other predicted components need confirmation of their localization. Finally, the cargo protein transport using vesicles need experimental verification to fill the model of vesicle transport inside chloroplasts