Photosynthesis sustains almost all of the living world directly or indirectly. It is the process of converting light energy to chemical energy of sugars and other organic compounds. The process depends on the interaction between two sets of reactions, namely the light-dependent and light-independent reactions. The light-dependent reactions convert low energy electrons from splitting of water to a high energy state in the form of NADPH. This process is also coupled with ATP synthesis. The photosynthetic electron transport occurs in thylakoid membranes inside chloroplasts.
It was first demonstrated by Robert Hill that photosynthesis could be “uncoupled” if the thylakoid membranes and the stroma components of chloroplast are separated via cell fractionation. The Hill reaction is also referred to as photolysis of water with the subsequent evolution of oxygen. Isolated thylakoids do not contain much of their complement of electron acceptors hence a lot of artificial electron acceptors are used to study the Hill reaction in vitro (the final electron acceptor in vivo is NADP). Many of these artificial electron acceptors accept electrons at different sites along the transport pathway.
This study used DPIP dye as an artificial electron acceptor in determining the effects of two herbicides on photosynthetic electron transport by isolated thylakoids of Zea mays leaves. DPIP in analysed by color changes it exhibits – it is blue in its oxidized form and turns colorless when reduced. The shikimate pathway is a biosynthetic sequence in plants to produce the aromatic amino acids phenylalanine, tyrosine, and tryptophan. One of the enzymes of the pathway, EPSP (5-enolpyruvylshikimate-3-phosphate) synthase, is often targeted by herbicides inhibiting its function, consequently shutting down the entire pathway.
With this, plants can no longer produce amino acids important for their diet. As a result, plants die after some time. Since only plants undergo shikimate pathway, animals are not affected by the action of the active ingredient of the herbicide. Zea mays, or commonly called mais in the Philippines, is an erect, fast-growing, short-lived annual plant. It has a single main culm and has one or occasionally two branches in the leaf axils in the upper part. The leaves are alternate with broad, sword-shaped leaf blades, parallel veins with a prominent mid-rib, and small ligules. Z. ays is extensively cultivated in the Philippines, and is a staple food is some parts of the country. Its uses include edibility of pollen, seed, and stem; and are a good source of carbohydrates, vitamins A, B, C, potassium, and zinc. The embryo is rich in oil and used widely for cooking, and manufacturing of soaps. Other products made from corn are starch, glue, alcohol, and silk. ClearOut® 41 Plus is a water soluble herbicide solution used for non-selective weed control in cropland systems and non-cropland areas. Its active ingredient is glyphosate as isopropylamine salt.
Glyophosate verges on a perfect herbicide more than any other herbicide currently on the market, and was named as the best herbicide in the Philippines due to its affordable price. The herbicidal activity of glyophosate focuses on the inactivation EPSP (5-enolpyruvylshikimate-3-phosphate) synthase enzyme of the shikimate pathway. This pathway does not occur in animals, hence it is harmless and safe to nontarget organisms. Symptoms observed after using ClearOut® 41 Plus include gradual wilting and yellowing (chlorosis) of the plant, at most 4 days after application to annual weeds and at most 7 days on perennial weeds.
DCMU or Diuron 50 WP (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is an herbicide used to block photosynthetic electron transport of plants by inhibiting Photosystem II, to plastoquinone. This block halts electron transport chain in photosynthesis hence it reduces the ability of plant to obtain ATP and sugars. The general objective of this study is to establish the effects of two herbicides (ClearOut 41® Plus and Diuron 50 WP) on Hill reaction in corn leaves. Specifically, this study aims to: 1. isolate membranes from corn leaves that consist mainly of thylakoid membrane vesicles by differential centrifugation; 2. rack and locate electron flow from Photosystem II; and 3. construct a graph of the Hill reaction activity. MATERIALS AND METHODS Three leaves of corn were randomly obtained from fully grown corn plants at the Barangay Organic Garden of Deca Homes, Tacunan, Davao City. These were brought to the laboratory and were deveined and washed using distilled water. Ten grams of deveine[pd leaves were placed in an ice-cold blender added with 50 mL cold 0. 35 M NaCl solution. Then, it was blended at high speed for 30 seconds and cooled in an ice bukcet for 30 seconds, both were done three times.
The extract was strained using a double-layered cheesecloth into an ice-cold beaker. The suspension was transferred into four cold 15-mL centrifue tubes having equal weights and were centrifuged at 200 x g for 5 minutes. Then, the supernatants were poured into four clean 15-mL centrifuge tubes while the pellets were discarded. The obtained supernatant was centrifuged again, however, at 1400 x g for 15 minutes. The supernatant was discarded and the pellets were resuspended using 5 mL 0. 35 M NaCl to each tube. Afterwards, all the suspensions were collected in a small beaker immersed in an ice bath.
The determination of chlorophyll concentration was done by first diluting 0. 1 mL of the prepared chloroplast suspension to 20 mL (1:200 dilution) of 80% acetone to extract the chlorophyll. Then, the suspension was subjected to spectrophotometer at 652 nm (wavelength). The spectrophotometer was zeroed with a blank containing 80% acetone. The absorbance value of the chlorophyll solution was recorded as shown in Appendix Table 1. The amount of total chlorophyll per mL of the chloroplast suspension was computed using the equation adapted from Reamillo et al. (2009): (ab) At 652 nm, the molar extinction coefficients of chl a and b are both equal to 34. 5. The original suspension was diluted with —— cold 0. 35 M NaCl solution to obtain a concentration of 0. 05 mg chlorophyll/mL. Fifteen milliliters of 0. 05 mg chlorophyll/mL suspension was then placed in a test tube wrapped with aluminum foil and chilled in an ice bucket. Listed in Table 1 are tubes containing different contents to be used for spectrophotometric determination of Hill reaction. The spectrophotometer was zeroed using the blank and was set at 605 nm wavelength. Initial readings were taken.
The absorbance values of the two experimental setups were read every 5 minutes for 30 minutes. The data obtained is plotted in Figure 1. Table 1. Test tube contents for the spectrophotometric determination of the Hill reaction. Contents| Volume Added (mL)| | Blank| Experimental Setups| | | Control| Herbicide 1(Clear out)| Herbecide 2(Diuron)| 0. 2 M phosphate buffer| 1. 5| 1. 5| 1. 5| 1. 5| 0. 35 M NaCl| 3. 0| 2. 0| 1. 0| 1. 0| Chloroplast suspension| 1. 0| 1. 0| 1. 0| 1. 0| Herbicide| -| -| 1. 0| 1. 0| 0. 01% DPIP (added last prior to 1st reading)| -| 1. 0| 1. 0| 1. 0|