1. Plant hormone signaling I 1.What is a plant hormone? How to determine if a molecule functions as a hormone or not? Several criteria: 1) produced by plant and has profound effect on growth and development 2) transportable (from one site to another inside the plant) 3) functions at a low level (often micromolar or less) 4) level regulated tightly by biosynthesis and degradation. Currently accepted families: auxin, GA, cytokinin, ethylene, abscisic acid (ABA), steroids, jasmonic acid, peptides 2. Auxin 1)Overview: IAA is the major endogenous form of auxin, produced mainly at rapidly growing tissues like meristems, young leaves, transported polarly from top to bottom of the plants.

2. 2) Biological functions: enhance cell elongation, apical dominance, Promote lateral root formation, vascular tissue differentiation… 3) Signaling: how does auxin work? A)Two responses have been used as models to study auxin function: cell elongation and gene expression B)Auxin receptor? One candidate is ABP (auxin-binding protein): identified by photoaffinity labeling technique (auxin-photoactive group that will be attached to an amino acid group in a protein when this protein gets very close to the auxin molecule). ABP is a small protein about 200 AA long and has a KDEL sequence at the C-terminus. Considering its location in the cell, how could this protein be auxin receptor? What is the evidence for ABP function? Essential gene for embryo development (knockout embryo cannot develop to mature stage).

3. C) How does ABP regulate auxin-induced cell elongation? Auxin has been shown to enhance cell wall acidification by promoting H- ATPase activity. Some studies showed That ABP regulates H-pump activity. BUT it is not clear if ABP is a true receptor for auxin. Certainly, ABP is not the ONLY Receptor.

4. D) From auxin to gene expression a)Auxin-induced genes and promoter analysis Promoter transcribed region mRNA Gene/DNA Detect gene expression at mRNA level: northern blot An typical pattern of auxin-induced gene expression 0 1h 2h 3h 4h IAA - + - + - + - + - +

5. PromoterGUS/reporter IAA ++-++- TGTCTC --AuxRE Isolation of proteins bound to this sequence Resulting in discovery of ARF—auxin response factor, a transcription factor controling auxin-induced gene expression

6. b) Auxin-induced genes: early genes encoding inhibitors of ARF. These proteins are called IAA/Aux. They were first discovered in Theologis lab as they worked on “rapid” auxin-induced genes. The IAA proteins form dimers with other IAA proteins and with ARF. When they bind ARF, ARF activity is suppressed so that gene expression is shut down. Promoter transcribed region mRNA Gene/DNA ARF ON OFF Promoter transcribed region Gene/DNA ARF IAA/Aux

7. c) The genetics analysis further revealed how auxin, the hormone, may regulate the gene expression process Several auxin-insensitive mutants were identified and they all represent genes encoding IAA/Aux or components in the ubiquitin-dependent protein degradation pathway. For example, axr2 and axr3 (auxin-resistant mutants) have mutation in the IAA/Aux genes. Tir1and ask1 are mutants that have mutation in genes for the ubiquitin pathway (the E3 enzyme complex).

8. The summary: how does auxin regulate gene expression?

9. 3. Gibberellins 1)overview: gibberillic acid discovered as a fungal compound that enhances rice growth but reduce seed production—”folish seedling disease. Later on similar compounds were identified in plants and defined as a growth hormone with many functions such as promoting stem growth, seed germination, flowering etc.

10. 2) Signal transduction for GA responses A)In the arabidopsis seedlings: mutant selection for plants that are more or less sensitive to GA. Several categories have been isolated: --GA-insensitive dwarf mutants, --suppressors of GA-deficient dwarf, and constitutive GA- response mutants (slender mutants). What are the genes ? GAI—GA Insensitive: a transcription factor that repress GA-induced gene expression RGA—Repressor of ga mutants (ga standing for GA-deficient). Similar to GAI. SPY (spindly)—slender plant mutant Encodes glycosylation enzyme

11. B) How does GA work to regulate a gene expression? Again protein degradation pathway is the key control point! It turns out that GA works to degrade GAI/RGA repressors. Evidence for the involvement of protein degradation: the gai and rga mutants all inhibited the GA-induced degradation of RGA/GAI proteins. Recent studies identified a ubiquitin-ligase for GAI/RGA proteins. It is likely that GA signaling may follow a similar paradigm as IAA signaling

12. C) GA signaling in germination of monocot plants --a story from studies of aleurone cells embryo endosperm aleurone Germination: GA is produced in the embryo, released to the endosperm, and diffused to the Aleurone cells where GA induces gene expression for alpha-amylase synthesis. The enzyme is then secreted into endosperm to digest the starch to produce sugar for the growth of embryo into a seedling. Q: How does GA induce gene expression in the aleurone cells? a)Where is the receptor for GA? Some evidence indicates that it is located at the cell surface (plasma membrane) but not identified at molecular level. b) A number of components may serve as intermediates that pass the GA signal to the nucleus. These include G-proteins, calcium, protein kinases/phosphatases,

13. c) Common signaling components found between germination and stem growth For example, the RGA/GAI proteins also prove to be repressors for alpha-amylase expression in aleurone cells; the GA-Myb type of transcription factors required for aleurone gene expression is also positive regulator of stem growth. d) Transcription factor genes like GA-Myb are activated before the alpha-amylase gene to establish a gene expression cascade: e) calcium-dependent pathway is required for alpha-amylase secretion through vesicular transport and Ca- independent pathway (involves cGMP) is essential for GA-induced gene expression cascade. f) Genetic and biochemical evidence for involvement of G protein: rice dwarf1 mutant has mutation in the gene encoding the G alpha subunit; G protein inhibitors block while activators enhance aleurone gene expression

14. 1.GA binding to receptor 2.GA receptor may interact with G protein that leads to two branches of signaling, one is calcium-dependent and other is Ca-independent 3-6 A second messenger (possibly cGMP) may work in the calcium independent pathway to lead to degradation of repressor proteins (RGA/GAI) and turns on GA-Myb expression. 7-8 GA-myb activate alpha- amylase gene expression 9-10 alpha-amylase is synthesized at ER and secreted through the vesicular trafficking 11 A Ca-dependent pathway regulates the secretion.