Molecular Genetics for plants

What is stress in plant biology?

Any change in environmental conditions that might reduce or adversely change a plant's growth or development.

What is abiotic stress?

The negative impact of non-living factors on living organisms in a specific environment, such as salinity, drought, and heat.

List some effects of abiotic stress on plants.

Reduction of photosynthesis, oxidative stress, reduction of growth, alteration of phenology, improper development, yield reduction, and inhibition of seed germination.

What is biotic stress?

Stress experienced by an organism due to damage caused by other living organisms, including bacteria, fungi, viruses, parasites, insects, and plants.

What is the point of no return in drought stress?

A critical threshold beyond which recovery from stress or damage is no longer possible.

What are the two main strategies plants use to adapt to drought?

Drought escape and drought avoidance.

What is drought escape?

Plants avoid prolonged periods of water scarcity by changing the timing of their growth and reproduction, such as early flowering.

What is drought avoidance?

Physiological and structural adaptations that minimize water loss and maximize water uptake to sustain hydration under drought conditions.

Describe the taproot system.

A root system characterized by the presence of a primary or dominant root, typically one deep main root.

Describe the fibrous root system.

A root system that develops from the stem in the form of thin and moderately branching roots without any primary root.

What are root hairs?

Cylindrical extensions of root epidermal cells that absorb water and nutrients.

How do plants control water loss during drought?

By closing their stomata to reduce water loss through transpiration.

What is the cost of reduced stomatal density in plants?

It can lead to reduced CO2 uptake, photosynthesis, and growth.

What are key biochemical mechanisms for drought tolerance?

Antioxidant defense, osmotic adjustment, and protective protein synthesis.

What are the key molecular processes involved in drought response?

Signal transduction pathways (ABA-dependent and independent) and gene regulation by transcription factors such as DREBs, ERFs, and NACs.

What are Reactive Oxygen Species (ROS)?

Highly reactive molecules produced during oxygen metabolism that can damage macromolecules.

What is hydrogen peroxide in relation to ROS?

A type of ROS with a longer half-life, produced in all cellular compartments, and has signaling functions.

How do ROS damage DNA?

By causing oxidative DNA lesions, such as transversion mutations where guanine pairs incorrectly with adenine instead of cytosine.

How do ROS damage proteins?

By oxidizing cysteine thiols, leading to the formation of disulfide linkages that alter protein structure and function.

What role do transcription factors play in plant stress responses?

They regulate gene expression involved in stress responses, including drought tolerance mechanisms.

Where are reactive oxygen species (ROS) produced in plant cells?

ROS are produced in chloroplasts, peroxisomes, and apoplasts.

What role do antioxidants and anti-oxidative enzymes play in plants?

They detoxify ROS and help reduce or neutralize their harmful effects.

What causes oxidative stress in plants?

The imbalance between ROS production and their detoxification by enzymatic and non-enzymatic reactions.

What is glutathione (GSH) and its function in plants?

GSH is a non-enzymatic antioxidant that oxidizes itself to reduce other molecules and serves as a measure for oxidative stress.

What is proline and its significance in plant stress responses?

Proline is an amino acid that scavenges ROS, is important for drought stress, and helps balance osmotic pressure.

What is the role of abscisic acid (ABA) in plants?

ABA regulates plant growth and stress responses, including the closure of stomata and activation of drought-responsive transcription factors.

Where is abscisic acid (ABA) synthesized and transported in plants?

ABA is synthesized in roots in response to stress and transported to leaves, where it can also be produced.

What is JUB1 and its role in plant stress responses?

JUB1 is a NAC transcription factor that regulates gene expression, suppresses growth hormone biosynthesis, and activates ROS scavenging enzymes.

How do microbes enhance plant drought resilience?

Microbes increase stress tolerance by forming biofilms, facilitating water access, and secreting chemicals that mimic plant hormones.

What is a transcription factor (TF)?

A protein involved in the transcription of DNA into RNA, possessing a DNA-binding domain to bind to cis-regulatory elements.

What method can be used to determine promoter activity?

Promoter-reporter gene fusion, which involves cloning a promoter into a plasmid with a reporter gene and measuring reporter activity.

What characteristics should a reporter gene have for promoter studies?

It should not be naturally expressed in the host, be easily detectable, non-toxic, and sensitive to promoter activity.

What is the role of β-Glucuronidase (GUS) in plant research?

GUS is a hydrolytic enzyme used to visualize gene expression in plants, derived from Escherichia coli.

How is GUS activity detected?

GUS activity can be visualized through histochemical staining with a substrate that produces a colored product upon enzyme cleavage.

What is luciferase and its application in research?

Luciferase is a reporter gene used in both plant and animal research to measure gene expression.

What are the components of proline's role in osmotic balance?

Proline accumulates in the vacuole to reduce osmotic potential, helping cells retain water during dehydration or high salinity.

What is the significance of DREB2A in plant stress responses?

DREB2A is a transcription factor that stimulates the expression of ROS scavenging enzymes, enhancing stress tolerance.

How does JUB1 affect cell growth and stress tolerance?

JUB1 suppresses growth hormone biosynthesis and activates ROS scavenging, promoting stress tolerance.

What is the role of biofilms formed by microbes in plant stress resilience?

Biofilms enhance water retention and facilitate access to limited water resources.

What is 5-bromo-4-chloro-3-indolyl glucuronid (X-Gluc) used for?

It is a colorless substrate that reacts to produce a blue dye, indicating active GUS enzyme and promoter activity.

What is the significance of the blue color produced in the X-Gluc reaction?

The blue color indicates the presence of an active GUS enzyme.

What is the MUG assay and its primary substrate?

The MUG assay is a fluorometric method for quantitative analysis of GUS activity, using 4-Methylumbelliferyl-beta-D-glucuronid as the substrate.

What happens when MUG is cleaved?

It yields a fluorescent product that emits blue fluorescence under UV light.

List some advantages of using GUS as a reporter gene.

Easy detection (colorimetric or fluorometric), sensitive and stable, allows quantitative analysis, provides tissue resolution, and is cost-effective.

What are some disadvantages of using GUS as a reporter gene?

It is destructive and lacks single-cell resolution, as the stain may diffuse into neighboring cells.

What is b-Galactosidase (lacZ) and its origin?

b-Galactosidase is an enzyme commonly used as a reporter gene, derived from the bacteria Escherichia coli.

How is b-Galactosidase (lacZ) detected?

Detection is achieved through the formation of a blue precipitate when it hydrolyzes X-Gal.

What are the advantages of using b-Galactosidase as a reporter gene?

Easy detection, sensitive, stable, allows for quantitative analysis, and is cost-effective.

What are the limitations of using b-Galactosidase as a reporter gene?

It is destructive and has a limited dynamic range, potentially saturating at high levels of gene expression.

What is Green Fluorescent Protein (GFP) and its source?

GFP is a protein naturally produced by jellyfish that emits green light when excited by blue or UV light.

What are some applications of GFP?

GFP is used to measure promoter activity and detect subcellular localization of proteins. Mainly in plant and animal research

What are the advantages of using GFP as a reporter gene?

It is noninvasive, does not require substrates or co-factors, can be detected in single cells, and allows for easy detection.

What are the disadvantages of GFP?

GFP detection is equipment dependent, can suffer from background autofluorescence, and has limitations in dynamic range.

What is luciferase and its primary function?

Luciferase is a light-producing enzyme that generates bioluminescence, primarily found in insects.

How is luciferase activity detected?

Luciferase activity is measured using a luminometer, which detects photon emission.

What are the advantages of using luciferase as a reporter gene?

It allows for quantitative expression detection, has high sensitivity, reflects current promoter activity closely, and is suitable for dynamic processes.

What are the disadvantages of using luciferase?

It requires substrates and co-factors, and cannot be detected in a single cell due to sensitivity limitations.

What is Reverse Transcription PCR (RT-PCR)?

RT-PCR is a semi-quantitative technique that involves reverse transcribing RNA into complementary DNA (cDNA) and then using cDNA for PCR.

What are the main steps involved in RT-PCR?

Isolation of mRNA, hybridization with oligo-dT primer, conversion of mRNA into cDNA, degradation of the RNA strand, and PCR amplification.

What is the purpose of detecting and quantifying PCR amplicons in RT-PCR?

To monitor the presence and quantity of specific DNA sequences throughout the PCR reaction.

What is the purpose of using a fluorometer in detection chemistry?

To measure the amount of fluorescence in every cycle.

What is SYBR Green?

A fluorescent dye that binds to/intercalates into double-stranded DNA.

What light does SYBR Green absorb and emit?

Absorbs blue light (λmax = 498 nm) and emits green light (Λmax = 522 nm).

How does SYBR Green fluorescence change during PCR amplification?

As DNA is amplified, SYBR Green fluorescence increases proportionally.

What is the threshold line in qRT-PCR?

The point at which a reaction reaches a fluorescent intensity above background levels.

What does the CT value represent in qRT-PCR?

The number of cycles where the PCR kinetic curve reaches a threshold amount of fluorescence.

How is the CT value related to the initial amount of cDNA?

The higher the amount of cDNA at the start, the lower the CT value (inverse correlation).

What are the steps to compare the expression of DREB2B between two Arabidopsis samples?

1. Harvest leaves, 2. Isolate RNA, 3. Make cDNA, 4. Perform qRT-PCR.

Why is it important to include reference genes in qRT-PCR?

To control for variability not related to the actual gene expression of interest.

What is the formula to calculate fold change in gene expression?

Fold change = 2^(-ΔΔCT).

What does Tm stand for in the context of melting curve qRT-PCR?

Temperature of Melting, defined as the temperature at which 50% of double-stranded DNA is changed to single-stranded DNA.

What factors influence the melting temperature (Tm) of DNA?

The greater the guanine-cytosine content, the higher the melting temperature.

What are the ideal parameters for primer design in qPCR?

60-150 bp amplicon length, 40-60% GC content, and 60-64 °C melting temperature.

What is a microarray?

A tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.

How are different tissues labeled in microarray experiments?

With different colored dyes, typically Cy3 (green) and Cy5 (red).

What technology is used in Affymetrix microarrays?

Direct synthesis of oligonucleotides (probes) on the glass surface using photolithographic technology.

What is RNA-seq?

An unbiased approach to measure the abundance of transcripts, including those not previously known.

What are the typical steps of RNA-seq?

1. Design experiment, 2. RNA preparation, 3. Prepare libraries, 4. Sequence, 5. Analyze.

What is the role of the DNA-binding domain (DBD) in transcription factors (TFs)?

It binds the TF to the cis-regulatory elements in promoters.

What is the function of the Nuclear Localization Signal (NLS) in transcription factors?

It serves as a localization signal for the transport of the TF into the cell nucleus.

What is the activation domain (AD) in transcription factors?

Required for interaction with proteins of the basal transcription machinery and other regulators for transcription activation.

What is the significance of the dimerization domain in transcription factors?

TFs often interact with other TFs to form homo- or heterodimers.

What is the role of repression domains (RD) in transcription factors?

Repression domains enable transcription factors to interact with other proteins to suppress the initiation or progression of gene transcription.

How can a transcription factor (TF) without a nuclear localization signal (NLS) be transported to the nucleus?

It may be transported by dimerization with another TF that has an NLS.

What distinguishes a transcription factor (TF) from a transcription regulator (TR)?

A TF is a DNA-binding protein that regulates gene expression, while a TR contributes to gene regulation without directly binding to DNA.

What are bZIP transcription factors and where are they found?

bZIP transcription factors are a large family found in all eukaryotes, relevant in development and stress responses.

What is the structural motif of bHLH transcription factors?

bHLH transcription factors have a helix-loop-helix structure, consisting of two amphipathic alpha-helices separated by a linker.

What is the significance of the helix-turn-helix (HTH) structure in homeobox proteins?

The third alpha-helix in the HTH structure is responsible for contact with the major groove of DNA.

What are NAC transcription factors and their role in plants?

NAC transcription factors are one of the largest TF families in plants, regulating aging/senescence and responses to environmental stress.

How many members typically comprise the NAC transcription factor family in each plant species?

Typically 100-150 members.

What methodologies can be used to identify target genes of transcription factors?

Methods include IOE-RNA seq, DAP-seq, ChIP-seq, and EMSA.

What does IOE-RNA seq stand for and what does it identify?

Inducible Over Expression + RNA-sequencing; it identifies early responsive genes that are rapidly regulated by a TF.

What is DAP-seq used for?

DNA affinity Purification sequencing is used to identify TF binding sites.

What is the purpose of ChIP-seq?

Chromatin ImmunoPrecipitation-DNA sequencing is used to confirm the binding of TFs to their target gene promoters in vivo.

What does EMSA stand for and what is its purpose?

Electrophoretic Mobility Shift Assay; it is used to study TF binding in vitro.

What are Co-IP and BiFC used for in the context of transcription factors?

Co-Immunoprecipitation (Co-IP) is used to identify interacting partners of a TF, while Bimolecular Fluorescence Complementation (BiFC) also identifies interactions.

What is the difference between immunoprecipitation (IP) and pulldown?

IP is used when a tag is a protein or peptide, while pulldown is used when a fusion tag is not a protein.

What is the difference between in vitro and in vivo experiments?

In vitro experiments are conducted outside living organisms, while in vivo experiments take place within living organisms.

What is inducible overexpression (IOE)?

A method to control the expression of a gene in a regulated manner, allowing increased expression at specific times or conditions.

What are some examples of inducible systems used in plants?

Dexamethasone (DEX) Inducible System, Heat Shock Inducible System, Alcohol Inducible System, Estradiol Inducible System.