(oct 20) Plant RNA Silencing, Epigenetics, and Transformation: Mechanisms & Case Studies

What is the primary mechanism by which small RNAs regulate gene expression in RNA interference?

They guide silencing protein machinery to complementary target RNA sequences.

From what precursor structure is a plant microRNA (miRNA) processed?

A hairpin RNA transcribed from a MIR gene.

What is the primary role of short interfering RNAs (siRNAs) generated from introduced viral double-stranded RNA?

They guide silencing protein machinery to destroy matching viral RNA transcripts.

Which Dicer-like enzyme in Arabidopsis is specifically responsible for processing microRNA hairpin precursors?

DCL1

In Arabidopsis, what distinct role is performed by the DCL3 enzyme?

It cuts double-stranded RNA to produce 24-nucleotide siRNAs for the DNA methylation pathway.

What is the enzymatic function of RDR2 in plant RNA-directed DNA methylation?

It reads single-stranded RNA as a template to synthesize complementary RNA, creating double-stranded RNA.

Why are 24-nucleotide short interfering RNAs heavily concentrated in pericentromeric regions of the Arabidopsis genome?

Pericentromeric regions are rich in transposons and repetitive sequences, which serve as the templates for these siRNAs.

What fundamental concept about RNA was established by the discovery of regulatory small RNAs?

RNA can directly regulate gene expression without being translated into protein.

Which class of small regulatory RNAs is unique to animals and acts primarily in the germline to silence transposons?

PIWI-interacting RNA (piRNA)

What direct consequence occurs in the plant methylation pathway if Polymerase V cannot produce scaffold transcripts?

The loaded AGO4-small RNA complex cannot anchor to the target genomic locus.

How does the silencing of transposable elements promote overall genome stability?

It limits transposon mobility that could otherwise mutate active genes or alter genomic structure.

What typically happens to the duplex strands generated during microRNA biogenesis?

The functional strand is loaded into Argonaute, while the partner strand is removed and degraded.

How does inverted duplication lead to the birth of a new plant microRNA gene?

A gene segment duplicates in reverse orientation, producing a self-complementary transcript that folds into a hairpin structure.

Why do sessile organisms like plants rely so heavily on microRNA-mediated gene regulation?

It enables rapid, reversible physiological and developmental adjustments to environmental stress without relying on motility.

How do conserved microRNA families differ from species-specific microRNA families in evolutionary age and scope?

Conserved microRNAs are ancient and regulate fundamental developmental processes, while species-specific microRNAs are young, lower-expressed, and drive regulatory innovation.

What precursor RNA shape distinguishes short interfering RNAs (siRNAs) from microRNAs (miRNAs)?

siRNAs derive from long double-stranded RNA, whereas miRNAs derive from single-stranded hairpin RNA loops.

How does target matching generally differ between plant microRNAs and animal microRNAs?

Plant microRNAs match their target transcripts nearly perfectly (causing cleavage), whereas animal microRNAs match imperfectly (repressing translation).

What is the T-DNA component of the Agrobacterium tumefaciens Ti plasmid?

A defined DNA segment cut from the plasmid that is transferred into plant cells and integrated into host chromosomes.

How was the Ti plasmid modified for use in plant biotechnology?

Tumour-inducing and opine-producing genes were removed from T-DNA and replaced with the gene of interest.

What is the delivery mechanism of the biolistic (gene gun) transformation method?

Heavy metal microparticles (gold or tungsten) coated with DNA are fired at plant tissue at high velocity.

In plant tissue culture, what is a callus?

An undifferentiated mass of dividing plant cells grown on a nutrient medium.

What is totipotency in plant biology?

The capacity of a differentiated plant cell to return to an undifferentiated state and regenerate a complete organism.

Why is a selectable marker gene linked to a gene of interest during plant transformation?

It allows survival of only the rare transformed cells when a selective chemical agent (such as an antibiotic) is applied.

What enzyme was targeted in the petunia flower color experiment that led to the discovery of co-suppression?

Chalcone synthase (CHS)

What unexpected flower phenotype resulted from introducing extra sense copies of the chalcone synthase gene into petunias?

White and variegated flowers due to the simultaneous silencing of both introduced and endogenous CHS genes.

What observation ruled out permanent DNA mutation or deletion as the cause of co-suppression in petunias?

Reversion, where pigment synthesis returned in specific sectors of the plant despite the foreign transgene remaining present.

What key finding from the antisense chalcone synthase experiment proved that protein overproduction was not the trigger for silencing?

Antisense constructs produced no protein yet still triggered identical silencing, showing that matching RNA sequence was the trigger.

What molecular trigger for gene silencing was identified by Andrew Fire and Craig Mello in C. elegans?

Double-stranded RNA (dsRNA)

What structural arrangement at the soybean I locus causes natural chalcone synthase silencing?

An inverted repeat cluster of chalcone synthase genes that forms hairpins and generates short interfering RNAs.

What evidence demonstrates that soybean I locus silencing is tissue-specific?

Small RNAs targeting chalcone synthase are abundant in the seed coat but scarce in other tissues of the plant.

What genome rearrangement processes typically combine to create functional inverted repeat loci?

Unequal crossing over (duplication) followed by a chromosomal inversion.

What is epigenetics?

The study of heritable changes in gene expression that occur without any change in the DNA sequence.

How does DNA cytosine methylation lead to transcriptional silencing?

Methylated cytosines recruit binding proteins that compact chromatin, rendering DNA inaccessible to transcription machinery.

How does condensed chromatin affect genetic recombination frequency?

It suppresses recombination because the recombination protein machinery cannot physically access the tightly packed DNA.

How does epigenetic inheritance violate a fundamental assumption of Mendelian genetics?

Alleles can emerge from a heterozygote altered in expression state (e.g., methylated) despite no change in DNA sequence.

What is the main operational difference between mitotic and meiotic epigenetic inheritance?

Mitotic inheritance spreads state changes across somatic cell divisions within an individual, while meiotic inheritance passes state changes through gametes to offspring.

Why is meiotic epigenetic inheritance more common in plants than in mammals?

Plants do not set aside a germline early in development and instead produce gametes from somatic cells that underwent many divisions.

What mechanism causes the patchy coat pattern in female calico cats?

Random mitotic X-chromosome inactivation occurring early in embryonic development.

Why are calico and tortoiseshell cats almost exclusively female?

The coat pigment gene is X-linked, requiring two X chromosomes to simultaneously carry and express both orange and black variants in different patches.

What is paramutation?

An epigenetic interaction where a silent allele heritably converts an active partner allele into a silent state without changing its DNA sequence.

How do the B I and B' epialleles at the maize b1 locus compare in DNA sequence?

They are identical in DNA sequence across approximately 150,000 base pairs, differing only in methylation and chromatin state.

What upstream non-coding element is required for paramutation at the maize b1 locus?

Seven tandem copies of an 853-base-pair sequence located ~100,000 base pairs upstream of the gene.

What is the role of the mop1 gene in maize paramutation?

It encodes an RNA-dependent RNA polymerase required to produce small RNAs from upstream tandem repeats to direct cross-allele methylation.

What mobile signal transfers the silent epigenetic state from one allele to another during paramutation?

Diffusible small RNAs generated from transcribed repeat sequences.

Why does the paramutation pathway require multiple biochemical steps and a high repeat copy threshold?

To safeguard against accidental, irreversible locus silencing triggered by stray single-stranded transcripts or minor duplications.

In Agouti mice, how does the methylation state of a nearby transposable element alter phenotype?

Unmethylated element causes continuous expression (yellow fur, obesity, diabetes risk); methylated element silences it (brown, healthy fur).

What effect did supplementing pregnant Agouti mice with methyl-donor nutrients (like folic acid and B12) have on offspring?

It increased transposable element methylation, shifting offspring phenotypes from yellow to brown.

What key correlation was found in children evaluated 13 years after Project Ice Storm (1998)?

Maternal hardship during pregnancy correlated with long-lasting DNA methylation differences in children's immune and metabolic genes.

How is promoter DNA methylation relevant to cancer therapy?

Tumour suppressor genes are often silenced by promoter methylation rather than mutated, allowing potential reactivation using methylation-inhibiting drugs.

How do microRNA (miRNA) and messenger RNA (mRNA) differ in function?

miRNA is a non-coding regulatory RNA (~21 nt), whereas mRNA is a protein-coding transcript translated by ribosomes.

What is the distinction between Arabidopsis DCL1 and DCL4 enzymes?

DCL1 processes microRNA precursors, whereas DCL4 produces small interfering RNAs involved in transcript silencing.

In plant RNA-directed DNA methylation, what distinct role is played by Polymerase IV versus Polymerase V?

Polymerase IV generates single-stranded precursors for small RNAs, whereas Polymerase V transcribes scaffold transcripts at target loci.

Which Argonaute protein specifically functions in the plant RNA-directed DNA methylation pathway?

AGO4

Why is gold preferred over tungsten as a microparticle substrate in biolistic gene delivery?

Gold is significantly less toxic to plant cells than tungsten.

What antibiotic is commonly used as a selectable marker in plant genetic transformation experiments?

Kanamycin

How does RNA interference therapy utilize small RNAs in modern clinical medicine?

Synthetic small RNAs are designed to complement specific disease-causing mRNA transcripts and target them for degradation or silencing.

What is the definition of totipotency?

The capacity of a differentiated cell to return to an undifferentiated state and regenerate an entire functional organism.