In class, we learned that a gene is "a discrete unit of hereditary information consisting of a specific nucleotide sequence in DNA (or RNA, in some virus)" (Glossary, Campbell Biology, 12th edition). They physically reside in chromosomes and are segments of nucleotides that contain either regulatory instructions or building instructions for one or more molecules that help the body to work. Imagine genes as tiny apartments in a huge skyscraper called the chromosome, which is composed of chains of apartments. Each apartment houses a unique tenant, a sequence of nucleotides. They're not just any tenants though. They're special because they carry the instruction manual for how to run the complex machine that is our body. Here's where things get really interesting. When we zoom in on these nucleotides, we see a funky pattern. Instead of seeing just a random repetition of the four nucleotide bases - adenine (A), cytosine (C), guanine (G), and thymine (T) - we find specific regions with unique patterns. It's like finding hidden messages or secret codes. 

While some regions may seem repetitive (they used to be called "junk DNA"); however there are regions, the "nucleotide blocks", demonstrating clear patterns that could convey particular functions, such as recruiting specific nuclease or DNA-binding proteins. Therefore, people grouped these sequences based on their determined functions and for fun names like "promoter", "5' untranslated region", "exon", "intron", "3' untranslated region", and "terminator", etc. Sometimes, you can tell their functions simply from these names, like the ones that I have listed above. There are times it is not easy, like "TATAAT Box". So there you have it - genes are more than just random strings of letters. They're an incredible mix of patterns, codes, and functions that keep us running smoothly. Check this out if you are interested.

These "nucleotide blocks" are like the ultimate bio-Legos. When they're arranged in a specific order, AND flipped in a certain direction, AND placed at just the right distance from each other, it's like building a Lego masterpiece! The final structure isn't just for show - it performs a specific job in our bodies, much like how different parts of a Lego NASA Apollo Saturn V rocket each have their own function. I built one in 3 hours! The joy of building with Legos is a bit like the thrill of understanding our genes - with every piece clicked into place, we get closer to understanding the big picture of how life works. When placed in a particular order AND orientation AND distance, the assembled pieces could function together as a whole to convey certain biological functions. 

Just like what is shown in the top sub-figure of Fig. 1, the 35S promoter of cauliflower mosaic virus, the 5'UTR from cowpea mosaic virus RNA-2, target gene coding sequence (converted to DNA), 3'-UTR from vowpea mosaic virus RNA-2 and Nopaline synthase terminator are placed in a linear order in the plasmid. This topology/design of the plasmid used to over-express "Target gene" in plants driven by the 35S Promoter. 

This means the authors have generated three different plasmids with the pEAQ-HT backbone (*Academic rule: write gene names and plasmid names in Italic, not protein names): 

• pEAQ-HT-E

• pEAT-HT-M 

• pEAT-HT-S

Therefore, the coding sequences of E, or M, or S protein could be fused downstream of the 35S promoter and upstream of the NOS Terminator to construct the final binary plasmid. (Note: these sequences are DNA; while, in coronavirus, they are RNA). Following the process of agro-infiltration, wherein plants such as tobacco are genetically modified, the coding sequence contained within the plasmid is transcribed into matching mRNA molecules. Subsequently, these mRNA molecules serve as templates for translation, enabling the synthesis of corresponding proteins.

ATTENTION: Whenever we are looking at research data, I request you to the followings in order:

(1) First, ask yourselves one question: "What is the hypothesis being tested here?" ;

(2) Then, think of the second question next: "What is the alternative hypothesis?";

(3) Next follow the Material and Methods for this figure, get all the simple, stand-alone data points that you can get from justing interpreting the figure, forget about the "hypothesis" that you concluded at the first step;, 

(4) Draw whatever complete conclusions  

First, in the experiments of Fig. 2, the authors tested whether the in vitro introduced proteins were actually expressed or not. They examined total protein extracts from infiltrated leaves expressing either S, or EMS. They examined it through SDS-PAGE, Commassie Blue Staining

Q: When conducting the SDS-PAGE analysis, why is it necessary to boil the protein samples?

A: The reason scientists usually don't run SDS-PAGE without boiling the samples first is because proteins aren't just lines, they're more like crumpled balls of paper or a tangled ball of yarn in their natural state. Proteins in their natural or "native" state have a complex three-dimensional structure (remember the secondary and tertiary structure of proteins?). The way a protein folds up into this structure can vary a lot between different proteins, and it depends on factors like the sequence of amino acids (the building blocks of proteins, primary sequence) and the environment the protein is in. The issue is that these folded structures can cause proteins to move differently through the SDS-PAGE gel. Two proteins of the same size might move at different speeds because one is folded up tightly and the other is more loose. This makes it really hard to compare different proteins, because the results aren't just about size anymore, they're also about shape. By boiling the samples, we unfold these proteins into a straight line, so they all have a similar shape. This makes it a fair race where the fastest protein is simply the smallest one, not the one with the most aerodynamic shape. So boiling isn't about making the proteins hot, it's about giving them a shape that makes the results of SDS-PAGE easier to interpret and compare. Like straightening out all the runners before a race, so we can see who's actually the fastest!

Q: Why is it necessary to boil protein samples WITH beta-mercaptoethanol before conducting the SDS-PAGE analysis?

A: Sure! As we mentioned earlier, when scientists want to study proteins, one of the tools they use is called SDS-PAGE. This is like a super small obstacle race for proteins that helps scientists figure out how big or small the proteins are. But for the race to be fair and just measure size, we need all the proteins to be in the same shape - like straight lines - so they don't get tangled up or stuck. That's where boiling comes in.  Think about it like a really messy ball of yarn. If you just try to pull on it, it'll get stuck and tangled, but if you can straighten it out, you can easily see how long the piece of yarn is. Boiling is like that - it "unwinds" the proteins and straightens them out. Now, about beta-mercaptoethanol (let's call it ß-ME for short), it's like a tiny pair of scissors. Some proteins are like several pieces of yarn tied together. If we boil them without ß-ME, they'll still be stuck together, and we won't get an accurate size. ß-ME "cuts" these ties (which are actually called disulfide bonds), so each protein is a single, straight line, which can then run the race! So, to sum it up, we boil protein samples with ß-ME to untangle them and get them ready for the SDS-PAGE race. Without this step, we wouldn't be able to figure out the size of the proteins correctly!

Q: Why is it necessary to boil protein samples WITHOUT beta-mercaptoethanol before conducing the SDS-PAGE analysis?

A: While, this is indeed a great question! Now, imagine you're back in the obstacle race, but this time, you want to know who's running the race as a solo player and who's part of a relay team. For this, you need to keep the teams or "linked proteins" together. Remember those ties or bonds that ß-ME (beta-mercaptoethanol) cuts? Those are like the batons in a relay race that link team members together. If we use ß-ME, it's like removing the batons, and we won't be able to tell who was running together. So in this case, we boil the samples but do NOT add ß-ME. When we boil proteins without ß-ME, the heat straightens out the proteins like before, but it leaves the "batons" or disulfide bonds intact. This allows us to see proteins that are connected or function together, which can be super important for understanding how they work inside our bodies. So, sometimes scientists boil samples without ß-ME to keep these protein "teams" together. This way, they can study how proteins interact and cooperate, just like a relay race team in our race!

Q: In Figure 2a, we notice several distinct bands of varying intensities under both reducing and non-reducing conditions in the EV, S and EMS lanes. What do these bands represent? Do each of these clear bands indicate a unique protein? There are notably three major intense bands approximately at 97 kDa, 51 kDa, and 14 kDa. What proteins could they possibly correspond to?

A: Fig.2a showed an SDS-PAGE analysis for the total proteins extracted from tobacco leaf tissues. In this figure, since all the samples have been boiled for 5 minutes before loading on the gel; therefore, each distinct band on the gel signifies a unique protein or a group of proteins with similar molecular weights. The intensity (darkness) of the band provides an estimate of the amount of that protein, with darker bands indicating higher amounts. When you see several bands, this is showing you the range of different proteins that are present in the sample. Also, it is possible that the observed bands are degraded proteins. Again, each band could represent one or multiple proteins that have a similar size, and the intensity of the band corresponds to the abundance of that protein or proteins in the sample. The Fig. 2a examined leaf tissues, which are typically enriched with a few types of proteins: RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase), Photosystem proteins, storage proteins, chlorophyll-binding proteins, heat shock proteins, cytoskleton proteins and cell wall associated proteins.

Regarding the three intense bands that you're observing at around 97 kDa, 51 kDa, and 14 kDa in the total proteins extracted from tobacco leaves, the one around 51 kDa is very likely correspond to the large subunit of RuBisCO, a key enzyme involved in photosynthesis. RuBisCO is often one of the most abundant proteins in leaf tissue, and the large subunit has a size of approximately 55 kDa. The difference could be due to slight variations in the measurement, or to post-translational modifications of the protein. While, for the band around 14 kDa is likely due to the small subunit of RuBisCO. The complete RuBisCO enzyme is a hexadecamer, consisting of 8 large subunits and 8 small subunits. The large subunit has a molecular weight of about 55 kDa and the small subunit around 15 kDa. The whole active complex weighs approximately 560 kDa. The RuBisCO is estimated the MOST ABUNDANT protein on our planet Earth! While, the intensive band around 97 kDa may represent several proteins with similar molecular weight.  Some possibilities might include larger enzymes involved in primary metabolic processes or multi-subunit protein complexes, such as subunits of Photosystem I and II, Glycosylphosphatidylinositol (GPI)-anchored proteins, Extensins, pectin methylesterase, certain cellulose synthase proteins, etc.

It's important to note, however, that while SDS-PAGE can give you valuable information about the size and abundance of different proteins in a sample, further analysis is usually required to definitively identify these proteins. Techniques that could be used for this purpose include mass spectrometry or immunoblotting with antibodies specific for the proteins of interest (just like in Fig. 2b). Additionally, factors such as protein modifications and the formation of protein complexes can influence the apparent molecular weight of proteins on an SDS-PAGE gel.

Plant necrosis refers to the death of plant tissue, usually observed as areas of browning, blackening, wilting, or withering. It's often a visible symptom of disease or stress, and can be caused by a variety of factors. In simpler words, it is like the plant version of a bruiser or a bad sunburn. It's when parts of the plant turn brown or black, wilt. or shrivel up because the cells in that area have died. It's the plants' way of shouting, "Come on, something's not right here!" Please bear in mind, this is not an accident. but an active response by the plant to prevent further damage and maintain overall plant health.

In the above Fig. 1S (Note: This is the abbreviated way to say Supplemental Figure 1), comparing to the negative control EV, various degrees of necrosis was observed in leaves over-expressing different foreign proteins driven by the 35S promoter. In the case of necrosis due to over-expression of a foreign protein (like in a genetically modified plant), the necrosis is essentially a sign that the plant cells are under stress and dying because they can't properly manage the overproduction of the foreign protein. Over-expressing foreign proteins in plants is a bit like hosting a huge, non-stop party in your house. While a party can be fun, a non-stop one can cause a lot of problems. 

Similarly, when a plant cell has to make lots of a foreign protein, it can run into some serious trouble:

1. Protein Traffic Jam: Imagine your house getting so crowded that people start to trip over each other, creating chaos. Something similar happens when too much of a foreign protein gets made in a plant cell. Proteins might not fold up right and can stick together, causing a sort of traffic jam inside the cell. This can mess with the cell's normal routine and even cause the cell to die off, leading to what we call 'necrosis'.

2. Energy Drain: Hosting a big party needs a lot of energy and resources. Similarly, producing lots of foreign protein uses up the plant cell's resources and energy. The cell could get so overwhelmed keeping up with the protein production that it can't do its normal activities properly. This could stress out the cell and, again, lead to necrosis.

3. Identity Crisis: Sometimes, the foreign protein might be seen as an invader or a threat by the plant's defense system. The defense system might then kick in and start damaging its own cells, causing necrosis.

Q: Why does transformed tobacco show necrosis actively or passively?

Plant cells respond to extreme stress, whether it's from overproduction of foreign proteins or environmental factors, by undergoing a process called programmed cell death (PCD), which results in necrosis. This isn't an accident, but an active response by the plant to prevent further damage and maintain overall plant health.

Imagine you're the captain of a big ship and suddenly, you notice water leaking into one compartment. If the leak is too large to repair, you might decide to seal off that compartment, even though it means the items inside will be lost. That's a tough decision, but it could prevent the whole ship from sinking and save the rest of the crew and cargo.

Now, let's translate this to our plant scenario:

1. Active Response (Captain's Order): When a plant cell is overstressed from overproduction of foreign proteins (the leak), it might decide to "seal off" or actively destroy itself in a process called programmed cell death. This helps prevent the problem from spreading to other cells (sinking the ship).

2. Passive Result (The Flooded Compartment): The result of this decision is necrosis, the visible sign of cell death in the plant tissue (the sealed, flooded compartment). 

It might seem harsh, but this active response of triggering cell death is often a plant's best strategy to protect the whole organism from further damage. But remember, in a well-managed experiment, scientists would aim to avoid such stress and prevent this from happening.