Stress responses/stress avoidance Plan C We will pick a problem in plant biology and see where it takes us. Plant products Climate/CO2 change Stress responses/stress avoidance

Plants & products chosen Capsaicin Capsicum sp. Glucosinolates Radishes (Raphanus sativus) Mustard (Brassica juncea) Alliin -> Allicin Garlic (Allium sativum)

Other candidates Propanethial-S-oxide Onions (Allium cepa) Portulacanones Purslane (Portulaca oleracea) Allyl isothiocyanate Horseradish (Armoracia rusticana) Wasabi (Eutrema japonicum) eugenol (and others) Basil (Ocimum basilicum)

Stresses Chosen Climate change Temperature Increased pCO2 Drought pH Nutrient deprivation Metal toxicity (a result of low pH) Predation Shaking

Next Friday Present a plant stressor, what is known about it, and why it might affect plant 2˚ compounds in an ~ 10 minute presentation. Alternative: present another good plant/stressor response to study and why we should choose it over the ones already chosen.

Nutrient transport in roots Move from soil to endodermis in apoplast Move from endodermis to xylem in symplast

Nutrient uptake Taken into endodermis by H+ symporters, channels, pumps

Nutrient transport in roots Move from endodermis to xylem in symplast Transported into xylem by H+ antiporters, channels, pumps

Transport to shoot Nutrients move up plant in xylem sap

Nutrient transport in leaves Xylem sap moves through apoplast Leaf cells take up what they want

Nutrient assimilation Assimilating N and S is very expensive! Reducing NO3- to NH4+ costs 8 e- (1 NADPH + 6 Fd) Assimilating NH4+ into amino acids also costs ATP + e- Nitrogen fixation costs 16 ATP + 8 e- SO42- reduction to S2- costs 8 e- + 2ATP S2- assimilation into Cysteine costs 2 more e- Most explosives are based on N or S!

Nutrient assimilation Most explosives are based on N or S! Most nutrient assimilation occurs in source leaves!

N cycle Must convert N2 to a form that can be assimilated N2 -> NO3- occurs in atmosphere: lightning (8%) & Photochemistry (2%) of annual total fixed Remaining 90% comes from biological fixation to NH4+

N cycle Soil bacteria denitrify NO3- & NH4+ back to N2 Plants must act fast! Take up NO3- & NH4+ but generally prefer NO3- Main form available due to bacteria

N assimilation by non-N fixers Nitrate reductase in cytoplasm reduces NO3- to NO2- NO3- + NADPH = NO2- + NADP+ large enzyme with FAD & Mo cofactors

N assimilation by non-N fixers Nitrate reductase in cytoplasm reduces NO3- to NO2- NO3- + NADPH = NO2- + NADP+ large enzyme with FAD & Mo cofactors NO2- is imported to plastids & reduced to NH4+ by nitrite reductase

N assimilation by non-N fixers NO2- is imported to plastids & reduced to NH4+ by nitrite reductase NO2- + 6 Fdred + 8 H+ = NH4+ + 6 Fdox + 2 H2O 0.2% of NO2- is released as N2O : another reason rape & corn biofuels increase global warming

N assimilation by non-N fixers NO2- is imported to plastids & reduced to NH4+ by nitrite reductase NO2- + 6 Fdred + 8 H+ = NH4+ + 6 Fdox + 2 H2O 0.2% of NO2- is released as N2O : another reason rape & corn biofuels increase global warming Regulated at NO3- reductase; always << NO2- reductase NO2- is toxic!

N assimilation by non-N fixers Regulated at NO3- reductase; always << NO2- reductase NO2- is toxic! NR induced by light & nitrate

N assimilation by non-N fixers Regulated at NO3- reductase; always << NO2- reductase NO2- is toxic! NR induced by light & nitrate Regulated by kinase in dark, dephosphorylation in day

NH4 assimilation GS -> GOGAT Glutamate + NH4+ + ATP <=> Glutamine + ADP +Pi

GS -> GOGAT Glutamate + NH4+ + ATP <=> Glutamine + ADP +Pi Glutamine + a-ketoglutarate + NADH/2 Fdred <=> 2 Glutamate + NAD+/ 2 Fdox

GS -> GOGAT Glutamate + NH4+ + ATP <=> Glutamine + ADP +Pi Glutamine + a-ketoglutarate + NADH/2 Fdred <=> 2 Glutamate + NAD+/ 2 Fdox 3. Fd GOGAT lives in source cp

GS -> GOGAT Fd GOGAT lives in source cp NADH GOGAT lives in sinks

GS -> GOGAT Glutamate + NH4+ + ATP <=> Glutamine + ADP +Pi Glutamine + a-ketoglutarate + NADH/2 Fdred <=> 2 Glutamate + NAD+/ 2 Fdox 3. Use glutamate to make other a.a. by transamination

GS -> GOGAT 3. Use glutamate to make other a.a. by transamination Glutamate, aspartate & alanine can be converted to the other a.a.

N assimilation by N fixers Exclusively performed by prokaryotes Dramatically improve the growth of many plants

N assimilation by N fixers Exclusively done by prokaryotes Most are free-living in soil or water

N assimilation by N fixers Exclusively done by prokaryotes Most are free-living in soil or water Some form symbioses with plants

N assimilation by N fixers Exclusively done by prokaryotes Most are free-living in soil or water Some form symbioses with plants Legumes are best-known, but many others including mosses, ferns, lichens

N assimilation by N fixers Exclusively done by prokaryotes Most are free-living in soil or water Some form symbioses with plants Legumes are best-known, but many others including mosses, ferns, lichens Also have associations where N-fixers form films on leaves or roots and are fed by plant

N assimilation by N fixers Exclusively done by prokaryotes Also have associations where N-fixers form films on leaves or roots and are fed by plant All must form O2-free environment for nitrogenase

N assimilation by N fixers All must form O2-free environment for nitrogenase O2 binds & inactivates e-transfer sites

N assimilation by N fixers O2 binds & inactivates e-transfer sites Heterocysts lack PSII, have other mechs to lower pO2

N assimilation by N fixers Heterocysts lack PSII, have other mechs to lower O2 Nodules have special structure + leghemoglobin to protect from O2

Nodule formation Nodules have special structure + leghemoglobin to protect from O2 Bacteria induce the plant to form nodules

Nodule formation Bacteria induce the plant to form nodules Root hairs secrete chemicals that attract N-fixers

Nodule formation Bacteria induce the plant to form nodules Root hairs secrete chemicals that attract N-fixers Bacteria secrete Nod factors that induce root hair coiling Nod factors determine species-specificity

Nodule formation Root hairs secrete chemicals that attract N-fixers Bacteria secrete Nod factors that induce root hair coiling Nod factors determine species-specificity Nod factors induce degradation of root cell wall

Nodule formation Nod factors induce degradation of root cell wall Plant forms "infection thread"= internal protusion of plasma membrane that grows into cell When reaches end of cell bacteria are released into apoplast and repeat the process on inner cells

Nodule formation When reaches end of cell bacteria are released into apoplast and repeat the process on inner cells Cortical cells near xylem form a nodule primordium

Nodule formation When reaches end of cell bacteria are released into apoplast and repeat the process on inner cells Cortical cells near xylem form a nodule primordium When bacteria reach these cells the infection thread breaks off, forming vesicles with bacteria inside

Nodule formation When bacteria reach these cells the infection thread breaks off, forming vesicles with bacteria inside Vesicles fuse, form the peribacteroid membrane and bacteria differentiate into bacteroids.

Nodule formation Vesicles fuse, form the peribacteroid membrane and bacteria differentiate into bacteroids. Plant cells differentiate into nodules

Nodule formation Vesicles fuse, form the peribacteroid membrane and bacteria differentiate into bacteroids. Plant cells differentiate into nodules: have layer of cells to exclude O2 & vasculature to exchange nutrients

Nodule formation Plant cells differentiate into nodules: have layer of cells to exclude O2 & vasculature to exchange nutrients Complex process that is difficult to engineer: 21 non-legume plant genera have N-fixers

N2 + 8H+ + 8e− + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi Nitrogen fixation N2 + 8H+ + 8e− + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi Catalysed by nitrogenase, a very complex enzyme!

N2 + 8H+ + 8e− + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi Nitrogen fixation N2 + 8H+ + 8e− + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi Catalysed by nitrogenase, a very complex enzyme! Also catalyzes many other reactions Usually assayed by acetylene reduction

N2 + 8H+ + 8e− + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi Nitrogen fixation N2 + 8H+ + 8e− + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi Usually assayed by acetylene reduction Sequentially adds 2 H per cycle until reach NH3

Nitrogen fixation Sequentially adds 2 H per cycle until reach NH3 May then be exported to cytosol & assimilated by GS/GOGAT or assimilated inside bacteroid

Nitrogen fixation Sequentially adds 2 H per cycle until reach NH3 May then be exported to cytosol & assimilated by GS/GOGAT or assimilated inside bacteroid Are then converted to amides or ureides & exported to rest of plant in the xylem!

S assimilation SO42- comes from weathering or from rain: now an important source! Main cause of rain acid!

S assimilation S is used in cysteine & methionine

S assimilation S is used in cysteine & methionine Also used in CoA, S-adenosylmethionine

S assimilation S is used in cysteine & methionine Also used in CoA, S-adenosylmethionine Also used in sulphoquinovosyl-diacylglycerol

S assimilation S is used in cysteine & methionine Also used in CoA, S-adenosylmethionine Also used in sulphoquinovosyl-diacylglycerol And in many storage compounds: eg allicin (garlic)

S assimilation SO42- comes from weathering or from rain: now an important source! Main thing that makes rain acid! Some bacteria use SO42- as e- acceptor -> H2S

S assimilation SO42- comes from weathering or from rain: now an important source! Main thing that makes rain acid! Some bacteria use SO42- as e- acceptor -> H2S Some photosynthetic bacteria use reduced S as e- donor!

S assimilation SO42- comes from weathering or from rain: now an important source! Main thing that makes rain acid! Some bacteria use SO42- as e- acceptor -> H2S Some photosynthetic bacteria use reduced S as e- donor! Now that acid rain has declined in N. Europe Brassica & wheat need S in many places

S assimilation SO4 2- is taken up by roots & transported to leaves in xylem Most is reduced in cp

S assimilation SO4 2- is taken up by roots & transported to leaves in xylem Most is reduced in cp 1. add SO4 2- to ATP -> APS

S assimilation add SO4 2- to ATP -> APS Transfer S to Glutathione -> S-sulfoglutathione

S assimilation add SO4 2- to ATP -> APS Transfer S to Glutathione -> S-sulfoglutathione S-sulfoglutathione + GSH -> SO32- + GSSG

S assimilation add SO4 2- to ATP -> APS Transfer S to Glutathione -> S-sulfoglutathione S-sulfoglutathione + GSH -> SO32- + GSSG Sulfite + 6 Fd -> Sulfide

S assimilation add SO4 2- to ATP -> APS Transfer S to Glutathione -> S-sulfoglutathione S-sulfoglutathione + GSH -> SO32- + GSSG Sulfite + 6 Fd -> Sulfide Sulfide + O-acetylserine -> cysteine + acetate O-acetylserine was made from serine + acetyl-CoA

S assimilation Most cysteine is converted to glutathione or methionine

S assimilation Most cysteine is converted to glutathione or methionine Glutathione is main form exported

S assimilation Most cysteine is converted to glutathione or methionine Glutathione is main form exported Also used to make many other S-compounds

S assimilation Most cysteine is converted to glutathione or methionine Glutathione is main form exported Also used to make many other S-compounds Methionine also has many uses besides protein synthesis

S assimilation Most cysteine is converted to glutathione or methionine Cys + homoserine -> cystathione

S assimilation Most cysteine is converted to glutathione or methionine Cys + homoserine -> cystathione Cystathione -> homocysteine + Pyruvate + NH4+

S assimilation Most cysteine is converted to glutathione or methionine Cys + homoserine -> cystathione Cystathione -> homocysteine + Pyruvate + NH4+ Homocysteine + CH2=THF -> Met + THF 80% of met is converted to S-adenosylmethionine & used for biosyntheses

S assimilation Most cysteine is converted to glutathione or methionine Glutathione is made enzymatically! Glutamate + Cysteine -> g-glutamyl cysteine

S assimilation Glutathione (GluCysGly) is made enzymatically! Glutamate + Cysteine -> g-glutamyl cysteine g-glutamyl cysteine + glycine -> glutathionine

S assimilation Glutathione (GluCysGly) is made enzymatically! Glutamate + Cysteine -> g-glutamyl cysteine g-glutamyl cysteine + glycine -> glutathionine Glutathione is precursor for many chems, eg phytochelatins

S assimilation Glutathione (GluCysGly) is made enzymatically! Glutamate + Cysteine -> g-glutamyl cysteine g-glutamyl cysteine + glycine -> glutathionine Glutathione is precursor for many chemicals, eg phytochelatins SAM & glutathione are also precursors for many cell wall components

Secondary Metabolites 1˚ metabolites are essential for growth & development therefore present in all plants 2˚ metabolites “not” required for growth & development therefore not present in all plants

1˚ metabolites are essential for growth & development Natural Products 1˚ metabolites are essential for growth & development therefore present in all plants 2˚ metabolites “not” required for growth & development therefore not present in all plants Often only found in one or a few spp. capsaicin Nepetalactone, menthol Nepetalactone Menthol

Natural Products 1˚ metabolites are essential for growth & development therefore present in all plants 2˚ metabolites “not” required for growth & development therefore not present in all plants Often only found in one or a few spp. Derived from 1˚ metabs

Natural Products 2˚ metabolites “not” required for growth & development therefore not present in all plants Often only found in one or a few spp. Derived from 1˚ metabs, can be hard to draw line

Natural Products 2˚ metabolites “not” required for growth & development therefore not present in all plants Often only found in one or a few spp. Derived from 1˚ metabs, can be hard to draw line

Natural Products 2˚ metabolites not present in all plants Often only found in one or a few spp. Derived from 1˚ metabs, can be hard to draw line Can be up to 3% of dry weight: very expensive to the the plant!

>100,000 types; tremendous variety of functions Defense Herbivores Natural Products >100,000 types; tremendous variety of functions Defense Herbivores nicotine

>100,000 types; tremendous variety of functions Defense Herbivores Natural Products >100,000 types; tremendous variety of functions Defense Herbivores Pathogens Resveratrol Brassilexin = antifungal Berberine = antibacterial

>100,000 types; tremendous variety of functions Defense Herbivores Natural Products >100,000 types; tremendous variety of functions Defense Herbivores Pathogens Other plants leptospermone leptospermone

>100,000 types; tremendous variety of functions Defense Attractant Natural Products >100,000 types; tremendous variety of functions Defense Attractant Pollinators Odors Colors Limalool smells nice Cyanidin colors blueberries, grapes, etc

Natural Products >100,000 types; tremendous variety of functions Defense Attractant Pollinators Symbionts

Natural Products >100,000 types; tremendous variety of functions Defense Attractant Pollinators Symbionts Seed dispersers Methyl anthranilate(strawberries) Pentyl butyrate (pears, apricots)

Natural Products >100,000 types; tremendous variety of functions Defense Attractant Pollinators Symbionts Seed dispersers “protectors” (tritrophic interactions)

Natural Products >100,000 types; tremendous variety of functions Defense Attractant Protecting from UV

Natural Products >100,000 types; tremendous variety of functions Defense Attractant Protecting from UV metal uptake and transport

Natural Products >100,000 types; tremendous variety of functions Defense Attractant Protecting from UV metal uptake and transport Hardening cell walls

Natural Products >100,000 types; tremendous variety of functions Some are made constitutively

Natural Products >100,000 types; tremendous variety of functions Some are made constitutively Others are induced, especially defense compounds

Natural Products >100,000 types; tremendous variety of functions Some are made constitutively, others are induced Made in many cellular locations Cytoplasm: most hydrophilics

Natural Products >100,000 types; tremendous variety of functions Some are made constitutively, others are induced Made in many cellular locations Cytoplasm: most hydrophilics Chloroplasts: terpenoids and some alkaloids

Natural Products >100,000 types; tremendous variety of functions Some are made constitutively, others are induced Made in many cellular locations Cytoplasm: most hydrophilics Chloroplasts: terpenoids and some alkaloids Mitochondria: some alkaloids and some amines

Natural Products >100,000 types; tremendous variety of functions Some are made constitutively, others are induced Made in many cellular locations Cytoplasm: most hydrophilics Chloroplasts: terpenoids and some alkaloids Mitochondria: some alkaloids and some amines Peroxisomes: some terpenoids, convert glucosinolates to active form upon pathogen attack

Natural Products >100,000 types; tremendous variety of functions Some are made constitutively, others are induced Made in many cellular locations Cytoplasm: most hydrophilics Chloroplasts: terpenoids and some alkaloids Mitochondria: some alkaloids and some amines Peroxisomes: some terpenoids, convert glucosinolates to active form upon pathogen attack Endoplasmic reticulum: fatty acid derivatives, other nonpolars

Natural Products >100,000 types; tremendous variety of functions Made in many cellular locations Made in many cell types Some in all

Natural Products >100,000 types; tremendous variety of functions Made in many cellular locations Made in many cell types Some in all Others in specialized cells or tissues

Natural Products >100,000 types; tremendous variety of functions Made in many cellular locations Made in many cell types Some in all Others in specialized cells or tissues Some are transported in xylem or phloem from site of synthesis to where they are stored

Natural Products >100,000 types; tremendous variety of functions Made in many cellular locations Made in many cell types Many are stored in vacuole

Natural Products >100,000 types; tremendous variety of functions Made in many cellular locations Made in many cell types Many are stored in vacuole Others in plastids or ER

Natural Products >100,000 types; tremendous variety of functions Made in many cellular locations Made in many cell types Some are stored in special cell types, eg trichomes

>100,000 types; tremendous variety of functions Natural Products >100,000 types; tremendous variety of functions Made in many cellular locations Made in many cell types Some are stored in special cell types Others in special cavities or ducts Left = lemon secretory cavity Right = pine resin duct Secretory cavity in lemon Pine resin duct

>100,000 types; tremendous variety of functions Natural Products >100,000 types; tremendous variety of functions Made in many cellular locations Made in many cell types Some are stored in special cell types Others in special cavities or ducts Others in flowers, leaves or fruits Left = lemon secretory cavity Right = pine resin duct

Natural Products >100,000 types; 3 main groups Terpenoids Alkaloids Phenolics