pmb 160 midterm 3, pre-assignment

stomatal closure, inhibition of photosynthesis, reduced cell division

increased ABA synthesis and transport

PYR/PYL/RCAR genes, PP2C

natural variation, transposon mutagenesis, T-DNA mutagenesis, chemical mutagenesis, gene editing (CRISPR)

variation is already created, can do GWAS, for some species published datasets already available

many genetic differences requires mapping, mutations that cause a loss in fitness likely don't exist, requires germplasm collection

induces random single nucleotide mutations in the genome, usually G to A or T to C

random positions; silent

soak WT seeds in EMS, plant seeds to get F1 (heterozygous), screen for dominant mutations and allow those to self-pollinate; screen for recessive mutation in F2

no genome bias, can generate mutations in many plants at once

finding mutations difficult, may be multiple mutations in a singular plant, requires multiple rounds of back-crossing to eliminate non-causal mutation

copy-and-paste mechanism using RNA

cut-and-paste mechanism, no RNA intermediate

transposase

Ac; transposase

inverted repeats at the end

reverse transcriptase; integrase

1. insertion into gene may abolish its action. 2. insertion near a gene may disrupt epigenetic regulation. 3. can indirectly lead to gene duplication

first transposon misalligned to second transposon during meiosis, leading to unequal crossing over, and 1 chromosome is extra short and the other has both copies of the gene.

introduce lines with a Ds in a different location for each line, then cross with Ac elements to activate Ds. Use PCR to screen for mutations

start with pools of gDNA from many individual lines, and select only positive pools to move forward with

little genome bias, generate many mutations in many plants, more likely to cause LOF mutations than EMS

not precise; Ds could theoretically become active again

are artificial restricion enzymes generated by fusing zinc finger DNA-binding domains to a DNA cleaveage domain

REs that can be engineered to cut specific DNA sequences by fusing a Tal Effector DNA-binding domain to a DNA cleavage domain.

bacterial immune system used to cleave foreign DNA using CRISPR sequences to guide the Cas9 recognition and cleavage protein

guide RNA leads Cas9 to target, where it cleaves the sequence, where DNA machinery will "fix" the DNA with insertion of a new sequence

catalytically dead dCas9 proteins have been fused to a cytidine or adenine deaminase base editors, so no DSB

targeted/precise; can create variety of mutation types; ideal when target sequence known

requires plant transformation, which doesn't work in some plant systems; not helpful if you don't know what sequence you want to target

auxin, cytokinin, octopine, nopaline

virulant has t-DNA, avirulant does not

plasmid DNA has been transferred to the plant

start with denatured ti plasmid as template, PCR with primers matching ti DNA plus radioactively labeled dATP nucleotide --> results in double stranded radiolabeled probe

isolate DNA from tissues, run on gel, transfer to nitrocellulose filter paper, probes hybridize to DNA on filter paper, wash unhybridized probe and expose to X-ray film

transposon mutagenesis

perceive plant and begin VIR transcription

excises single-stranded T-DNA and become "leader" protein

Forms the bacterial export channel used by the T-DNA to exit the Agro cell

coat the single-stranded T-DNA and protect it from DNA nucleases and/or help with nuclear import

VirA senses plant, phophorylates VirG, VirG activates VirD and VirB, VirD excises and leads T-DNA out, VirB forms the channel to get out, and VirE protects the T-DNA+VirD

shooty

rooty

too big, don't need nopaline synthesis or tumor, hard to track

remove hormone and nopaline synthesis genes, split the plasmid into a T-DNA shuttle vector and helper plasmid with Vir genes, add antibiotic resistance genes to track (one for bacteria one for plants), add unique restriction site for adding gene of interest

dip arabidopsis flowers, self-pollinate and grow F1 seeds

When a mutated plant contains a T-DNA inserted into a gene, the gene is said to be “tagged”

EMS screen

recessive loss of function; has long roots when WT does not

E1 ubiquitin activating enzyme

T-DNA, same as axr

F-box protein, the part of the E3 ubiquitin proteinligase that directly touches the tagged protein

protein that produces bioluminescence, used for tagging

2

DNA footprinting; the site where a protein binds to DNA can be identified because that region is protected from DNase endonuclease digestion. Running the DNA on a gel will reveal regions that were protected by protein as a lack of bands in that area

ARF1 binds the ARE sequence

SNRK2; drought response genes

gravitropism; sources of water

hydropatterning

movement away from high salinity environments

amyloplasts in the root tip reposition to the new "bottom" based on gravity, directing PIN proteins to transport auxin to grow down

gravitropism; transmembrane domain and a nuclear localization signal

BRXL4; plasma membrane; 5

amyloplasts are degraded through autophagy

increase in auxin transport and concentration on the upper face of the root surface, causing bending

no

OSCA1; EMS

protein that in the presence of Ca+ leads to fluoresence

lower levels of aequorin and hence Ca+, under high osmotic stress; severe root and leaf phenotype under osmotic stress

membrane bound ion channel

physical barrier between adjacent endodermal cells

a coating that occurs across the entire endodermal cell layer

younger root tissue; elongation zone

older root tissue; the maturation zone

the transcellular pathway; it surrounds the entire endodermal cell wall

apoplastic transport

not within cells

all within cells

in and out of cells

stain that when applied outside a root will pass through the root tissue into the vasculature through the apoplastic transport pathway

casparian strip

endodermis; casparian strip formation

cortex side of endodermal cells; SGN3

small signalling peptides; xylem

if casparian strip leaks; causes additional lignin and suberin to be deposited creating a new addition to the casparian strip

casparian strip

over lignification and suberization

Suberization of the root endodermis

Nitrogen, Phosphorous, Iron, Calcium

Soil erosion and overtillage; Nutrient leaching and excess irrigation; Biological cycling and gaseous emission

pH, water content, microbial activity, nutrient form (ex: nitrogen unfixed)

increased initiation of root hair formation, and root hair elongation

1) a hair cell always contacts two interior cortical cells; 2) a non-hair cell contacts only one interior cortical cell

makes too many root hairs; GL2 represses root hair formation

root hari fate; GL2

root hair cell fate; non-hair cells

non-hair; hair cells; GL2 promoter

CPC

receptor kinase protein; Transduces a signal from cortex cells to epidermal cells

low

high

regulating root hair development under phosphate stress

more root hair cells than usual

GL2

specifically induced by phosphate stress

auxin and ethylene

BR and ABA

improve nitrogen-fixing bacteria; make crops better hosts for nitrogen-fixing bacteria; allow crops to fix nitrogen without microbes