Plant biology

Hormone 1

Auxin and Gibberellin

Hormone 2

Salicylic Acid

Hormone 3

Jasmonate (JA)

Hormone 4

Brassinosteroids and peptide hormones

Hormone 5

ABA and ethylene

Hormone 6

Cytokinin

Forward genetics

To discover the genes responsible for a particular phenotype

Reverse genetics

To understand the function of a specific gene

Solar tracking

Leave bend toward the sun through the day

Photoblasty

Light requirement for seed to germinate

Photomorphogenesis

Light regulates phenotypic changes

Phototropism

Growth toward the light

Nyctinasty/photonasty

Folding of leave at night/opening at dawn

Photoperiodism

Regulation of flowering based on length of day

Action spectra

Graph that displays wavelength and response

Photoreceptor

These molecules absorb light and initiate a photoresponse

ex:

Phytochromes

Cryptochromes

Photochromes

UVR8

Absorption spectra

show which wavelengths a molecule absorbs

Phytochromes (PHY)

Mediate responses to red/far-red light, characterized by their photoreversibility between a red light-absorbing form (Pr) and a far-red light-absorbing form (Pfr), with Pfr being the physiologically active form

Cryptochromes (CRY)

Blue-light photoreceptors binding FAD and MTHF as chromophores, with FAD being the primary regulator. Arabidopsis has three cryptochromes (CRY1, CRY2, CRY3).

Phototropins

Blue-light photoreceptors (phot1 and phot2 in angiosperms) associated with the plasma membrane, functioning as light-activated serine/threonine kinases. They contain LOV domains binding FMN. Phototropins regulate chloroplast movement and stomatal opening.

UVR8

The UV-B photoreceptor, unique in lacking a chromophore. It functions as a dimer, with Trp residues serving as UV sensors whose structural changes upon UV illumination break salt bridges within the dimer.

Shade Avoidance

Enhanced stem elongation in response to shading by other plants, perceived as a low red:far-red (R:FR) ratio. Phytochromes (mainly phyB), gibberellins, and brassinosteroids are central to this response

Gravity Sensing

Plants sense gravity through statoliths, dense starch-containing amyloplasts located in specialized gravity-sensing cells called statocytes

Plant Hormones (Phytohormones)

Organic substances that regulate plant growth, development, stress tolerance, defense, etc

Auxin examples

Indole-3-acetic acid (IAA) is the primary plant auxin

2,4-D are used as growth regulators and herbicides

Auxin signaling - Ubiquitination

Ubiquitination is the covalent attachment of a small peptide (ubiquitin) to a target protein, which frequently leads to its degradation by the 26S proteasome. Ubiquitination occurs through an enzymatic cascade comprising three enzymes: an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase. 3

Auxin signalling - AUX/IAA

transcriptional repressors (of auxin-responsive genes) - they keep the auxin response off in the absence of the hormone

In the absence of AUX/IAA, auxin responsive genes can be expressed.

Auxin-regulated genes

mediated by ARF transcriptional factors

ARFs can heterodimerize with AUX/IAA repressors, which repress the transcriptional activation

Auxin transport

only plant hormone polarly transported from cell to cell in an energy-dependent manner

Auxin transport step 1

IAA enters the cell either passivley in the undissociated form or by the secondary active cotransport

Auxin transport step 2

The cell wall is maintained at an acidic pH by the activity of the plasma membrane H-ATPase

Auxin transport step 3

In the cytosol which as a neutral pH, the anionic form (IAA) predominates

Auxin transport step 4

The anions exit the cell via auxin anion efflux carriers that are concentrated at the basal ends of each cell in the longitudinal pathway

Auxin reporters

DR5 (a synthetic auxin-responsive promoter) and DII-Venus (an AUX/IAA-based sensor) are used to visualize auxin responses and levels

Auxin function

including apical dominance, stem elongation (via the "acid growth hypothesis" involving expansins), phototropism (requiring auxin redistribution), gravitropism, and tissue-specific responses mediated by ARF transcription factors

Gibberellins Biosynthesis

Synthesized in developing/germinating seeds, leaves, and elongating internodes, involving plastids, ER, and cytosol. GGPP is the linear precursor.

Gibberellin biosynthetic pathways

Plastids, the ER, and the cytosol

GA response

Is regulated by a series of feedback mechanisms involving components of GA signal transduction and biosynthesis

Gibberellins signaling

Involves DELLA proteins, which are transcriptional repressors of the GA response

Gibberellins Function

Regulate stem elongation, promote seed germination, induce flowering, repress senescence, promote shade avoidance, and regulate flower/fruit/seed development

Gibberellins biotechnology

GA3/GA1 is sprayed on grapes for larger, seedless fruits. GA3 promotes elongation in dwarf species and induces flowering. GA biosynthesis inhibitors are used to decrease stalk growth in cereals (part of the "green revolution" through dwarf wheat lines with mutated GA signaling genes) and keep indoor plants small

Jasmonates (JA) Summary

A family of oxylipins derived from oxygenated fatty acids, including jasmonic acid (JA), methyl jasmonate (MeJA), and jasmonoyl-isoleucine (JA-Ile). Methyl jasmonate is present in jasmine oil.

Jasmonates Biosynthesis

Membrane-derived linolenic acid is converted to jasmonic acid. Initial steps occur in chloroplasts, producing OPDA, which is transported to peroxisomes for further conversion by β-oxidation

Jasmonates signaling

COI1 is the jasmonate receptor. COI1 is an F-box protein that is part of the SCF(COI1) E3 ubiquitin ligase complex. JA-Ile binds to COI1 and acts as a "molecular glue," promoting interaction between COI1 and JAZ proteins. JAZ proteins are negative regulators of jasmonate-responsive gene transcription. The SCF(COI1) complex ubiquitinates JAZ proteins, leading to their degradation by the proteasome, thereby activating JA responses mediated by transcription factors like MYC2.

Jasmonate Functions

role in plant defense against insect herbivores and confer resistance against necrotrophic pathogens. They are also essential for stamen development and regulate petiole and seed size

What do jasmonates do in plant defence responses

Jasmonates play an important role in plant defence against herbivores and necrotrophic pathogens

What do jasmonates do in development?

Jasmonates are essential for stamen development. (They also regulate petiole size and seed size.)

What is the jasmonate receptor?

The jasmonate receptor is the COI1 protein, which is an F-box protein that acts as adaptor for the E3 ubiquitin ligase SCFCOI1. JA-Ile can be bound by COI1 and act as molecular glue to promote the interaction between COI1 and JAZ proteins, which are negative regulators of the transcription of jasmonate-responsive genes. As a result, JAZ proteins get ubiquitinated and degraded by the 26S proteasome.

Salicylic acid

Has been used to treat pains and fevers for a long time. Used today in asprin. Its levels rise in plants after a pathogen infection.

Salicylic acid biosynthesis

Made by 2 alternative pathways

1. Isochorismate synthase (ICS)

2. Phenylalanine ammonia lyase (PAL) pathway

ICS pathway

Predominant during pathogen attack.

SA accumulates in the cytosol after biosynthesis in the plastid.

In Arabidopsis, over 90% of SA is derived from the ICS pathway during immune responses.

SID2=ICS1

PAL pathway

Part of the phenylpropanoid pathway, which also leads to lignin and flavonoid production.

May play a bigger role in basal SA levels or in species with a less active ICS pathway.

More common in rice

SA homeostasis

Most plants maintain relatively low SA levels during normal growth and development.

Pathogen infections usually lead to a rapid increase in SA levels.

The dynamic changes in SA levels are a result of tight regulation of SA production and metabolism

SA reacts to a pathogen

In Arabidopsis there is a strong expression of: ICS1, EDS5, and PBS3

Their transcription depends on the action of SARD1 and CBP60g

SA modification

SA can undergo multiple chemical modifications, including hydroxylation, glycosylation, methylation, and amino acid conjugation, which contribute to the dynamics of SA levels in plants and play important roles in SA homeostasis control.

To promote their virulence, pathogens can also modify SA. Some bacterial and fungal pathogens possess SA hydroxylases (e.g. NahG) for inactivating SA

NahG from Pseudomonas putida

SA degradation: The enzyme hydroxylates and decarboxylates SA to form catechol, thus reducing SA levels.

SA perception: NPR1

Forward genetic screens were carried out to search for SA-insensitive mutations

NPR1 was isolated, suggesting it plays a role in connecting SA and downstream receptors

NPR1 exists as an oligomer (inactive form) in the cytoplasm.

Oligomerization is maintained through disulfide bonds (oxidizing conditions).

Transcription of SA-responsive genes is repressed

Upon Pathogen Infection → SA Accumulation

SA levels rise in the plant.

This leads to a reduction in the cellular redox potential.

Disulfide bonds in NPR1 are broken → monomeric NPR1 forms.

Monomeric NPR1 translocates to the nucleus.

3. In the Nucleus

NPR1 interacts with TGA transcription factors (e.g., TGA2, TGA5, TGA6).

This complex activates transcription of defense genes, such as PR1, PR2, and PR5.

Result: Systemic Acquired Resistance (SAR) is established.

SA perception: NPR3 and NPR4

same as NPR1

SA does not affect the interactions between NPR3/NPR4 and TGAs. THe binding of them to promoters of their target genes is not affected by SA treatment.

How SA inhibits the transcriptional repression activity of NPR3 and NPR4 is still unknown

SA signalling in plant immunity

SA confers resistance against biotrophic pathogens.

SA induces closure of stomata and plasmodesmata

SA signalling: Systemic resistance

Two types of systemic resistance (SAR/ISR) a long lasting memory effect for previous resistance responses

SAR

triggered by necrotising pathogens, wounding and feeding insects. More efficient against biotrophic pathogens

ISR

triggered upon colonisation of roots by non-pathogenic rhizobacteria. More efficient against necrotrophic pathogens

Other roles of SA

regulates senescence

SA and JA crosstalk

SA and JA often antagonize each other—when one pathway is strongly activated, the other is often suppressed.

Gibberellin

Regulate stem elongation.

promote seed germination.

induce flowering.

repress senescence.

promote shade avoidance.

regulate the development of flowers, fruits, and seeds

Gibberellin Signalling

The GA response is regulated by a series of feedback mechanisms involving components of GA signal transduction and biosynthesis

Biotechnological applications of Gibberellins

GA is sprayed onto grape vines to produce large, seedless grapes. It causes elongation of the cells and parthenocarpy. Used to promote elongation in dwarf species or to induce flowering. Substances that inhibit the synthesis are sprayed to keep certain plants small.

Sunlight uses

Energy source for photosynthesis

External cue that regulates development

A plant grown in light

Expanded cotyledons

Green cotyledons

short hypocotyl

Photomorphogenesis

A plant grown in dark

Cotyledons in the apical hook

non-photosynthetic cotyledons

elongated hypocotyl

Skotomorphogenesis

Co-suppression

Altered pigmentation only occurred when the transgene was present and active

PTGS as an anti-viral mechanism

Plants expressing a viral transgene recovered from the viral infection. ▪ This resistance was virus-specific. ▪ The resistance phenotype was functional at the single-cell level.

▪ Steady-state levels of transgene mRNA were 12- to 22-fold less in recovered tissue. ▪ Transgene transcription rates were not changed in recovered tissue.

Southern blot

DNA

Northern blot

RNA

Western blot

proteins (incubated with an antibody instead of a nucleic acid probe)

RNA silencing

refers to a number of related mechanisms in which Argonaute (AGO) family proteins are effectors that are guided by small RNA molecules to their nucleic acid (RNA or DNA) targets

Outcome of RNAi/RNA silencing

Gene silencing

through epigenetic mechanisms if the target is DNA

through various post-translational mechanisms for DNA

RNA silencing mechanism

involve the cleavage of double-stranded RNA (dsRNA) by an RNase III-like protein, known as Dicer, into 21-28 nucleotide (nt) short RNAs (sRNAs). ▪ The two strands of these sRNAs are then separated, and one of the two strands is recruited as the guide RNA of a silencing effector complex containing an Argonaute RNA-binding protein (AGO).

Variations in the RNA silencing mechanism

There are variations on this basic silencing pathway: the dsRNA input, for example, can be derived from inverted-repeat transcripts from complementary RNAs that anneal by base pairing or, in worms, fungi, and plants from single stranded RNA that is copied into a duplex by an RNA-dependent RNA polymerase (RDR). ▪ Silencing pathways can also vary because there are different types of effector complex that recruit the sRNAs. In the best characterized of these effector complexes, the AGO protein is a ribonuclease known as Slicer that degrades the target RNA. Other sRNA-containing effector complexes silence at the translational level or at the transcriptional level by targeting DNA

RNA interference (RNAi) / RNA silencing

The RNAi pathway is a set of cellular reactions to the presence of double-stranded (ds)RNA molecules, which involves small RNA molecules that do not code for proteins (noncoding RNAs - ncRNAs)

dsRNA

1. microRNAs genetically encoded

2. siRNAs

3. Synthesis by RNA-dependent RNA polymerases(RdRP)

PTGS

Post-transcriptional gene silencing mechanism.

Transcript degradation

Translational inhibition

TGS

transcriptional gene silencing

DNA methylation

Histone modification

Synthesis and action of miRNAs

Plants contain hundreds of genes encoding miRNAs, which lead to degradation or inhibition of translation of their target mRNAs

RISC

RNA induced silencing complexes

Synthesis and action of trans-acting siRNA (tasiRNAs)

tasiRNAs are plant-specific. ▪ tasiRNAs are at the convergence between the miRNA and the siRNA pathways. ▪ Pre-tasiRNA transcripts are produced from endogenous TAS loci (TAS1-4 in Arabidopsis). ▪ tasiRNA biogenesis requires microRNA (miRNA)-mediated cleavage of transcripts from the TAS loci (pre-tasiRNA), which triggers the production of dsRNA by RDR6. The dsRNA is diced into 21-nucleotide tasiRNAs by DCL4 and acts through either AGO1 or AGO7. ▪ TAS3-derived tasiRNAs degrade ARF transcripts (encoding auxin responsive factors)

DICER proteins

Rnas III-like protein

it cleaves dsRNA into 21-28 nt sRNAs

The RNA-dependent DNA methylation (RdDM) pathway

RdDM is a plant-specific pathway that leads to DNA methylation (repressive mark; cytosine residues in CG, CHG, and CHH contexts) and defends against invading nucleic acids. RdDM mediates transcriptional gene silencing (TGS)

RNAi as a defence against invading nucleic acids

A role of silencing is to protect cells against invading nucleic acids (viruses or mobile genetic elements). ▪ dsRNA corresponding to the invader is either directly available or produced by an RDR, and it is then cleaved into sRNAs that target DNA or RNA of the invader. ▪ RNA silencing is an optimal defence mechanism. The possibility of non-specific side effects is minimized because it is sequence-specific. In addition, the mechanism has an inherent amplification property and, consequently, is effective against invaders that replicate and spread rapidly

Viral counterdefence: viral suppressors of RNA silencing (VSRs

Viruses encode viral suppressors of RNA silencing (VSR), which are viral proteins capable of suppressing RNA silencing. ▪ All plant viruses known to date encode at least one VSR. ▪ VSRs are independently evolved and interfere with different steps of the RNA silencing pathway

Brassinosteroids (BRs) are involved in

Growth •Cell division, elongation, and differentiation •Stress tolerance •Reproductive development •many more.

Genes that showed BRs are essential

det2 and cpd

BR-synthesis enzymes

cytrochrome P450s (CYPs)

What encodes CYP C22-oxidase

DWF4, Loss-of-function mutants are dwarfed

BR deactivation

glucosylation and other reactions

When overexpressed, the UDP-glycosyltransferase UGT73C5 deactivates brassinosteroids and limits plant growth

reversible conjugation and irreversible modifications

BR synthesized

Made at their site of action, of evidence for long dsitance transport of endogenous BRs

BR synthesis regulation

regulated by negative feedback, light and circadian cycles, and interactions with other hormones

BRI1 BRASSINOSTEROID INSENSITIVE

encodes the main BR receptor

BRI1 type of receptor

plasma-membrane localized receptor kinase

Leucine-rich repeat - receptor kinase (LRR-RK) (also known as a LRR Receptor-Like Kinase (LRR-RLK

LRR-RLKs need to know

CLV1; FLS2; EFR; BRI1; BAK1; ERECTA. are abundant in plants and bind a wide variety of signaling molecules