Advance Advance BiochemistryBiochemistry

IntroductionIntroduction GoalsGoals

• To cover aspects of biochemistry unique and important To cover aspects of biochemistry unique and important to plantsto plants

Sometimes will involve bacterial biochemistrySometimes will involve bacterial biochemistry

• See some of the many biochemical pathways critical to See some of the many biochemical pathways critical to plants (Structures will be shown!)plants (Structures will be shown!)

• Hear about techniques important in plant biochemistryHear about techniques important in plant biochemistry Molecular biology, mass spectrometry etc.Molecular biology, mass spectrometry etc.

• Major emphasis on regulationMajor emphasis on regulation GradingGrading

• Three one hour exams (in class, Tuesdays) (90 points Three one hour exams (in class, Tuesdays) (90 points each)each)

• 30 points for homework assignments30 points for homework assignments• Total points at the end used to determine grade Total points at the end used to determine grade

Textbook and readingTextbook and reading

• Biochemistry &Molecular Biochemistry &Molecular Biology of Plants, ASPBBiology of Plants, ASPB

• Plant Biochemistry and Plant Biochemistry and Molecular BiologyMolecular Biology by Hans-Walter by Hans-Walter HeldtHeldt

• Readings from original literature Readings from original literature (PDFs supplied for UW-Madison (PDFs supplied for UW-Madison licensed materials)licensed materials)

Overall planOverall plan Cell and Cellular ConstituentsCell and Cellular Constituents

Cell structure and functionsCell structure and functions

Water and solutionsWater and solutions

CarbohydratesCarbohydrates

Fatty acids and lipidsFatty acids and lipids

Amino acids and proteinAmino acids and protein

EnzymesEnzymes

Vitamins and mineralsVitamins and minerals MetabolismMetabolism Strategy for Processing of Nutrients in PlantsStrategy for Processing of Nutrients in Plants Applied BiochemistryApplied Biochemistry

Overall planOverall plan

PhotosynthesisPhotosynthesis• Carbon metabolism, Electron transportCarbon metabolism, Electron transport• Nitrogen, reduction and metabolismNitrogen, reduction and metabolism• Carbon end productsCarbon end products

Cell constituentsCell constituents• Membranes, Cell wallsMembranes, Cell walls

Cellular metabolismCellular metabolism• Ion pumpsIon pumps• Protein turnoverProtein turnover

Symbiotic nitrogen fixationSymbiotic nitrogen fixation

Regulation of MetabolismRegulation of Metabolism

Plant cells do a wide range of Plant cells do a wide range of biochemistrybiochemistry

Regulation of metabolismRegulation of metabolism• Stoichiometric requirements (e.g. amino Stoichiometric requirements (e.g. amino

acids)acids)• Avoid waste (energy that is needed when it is Avoid waste (energy that is needed when it is

needed)needed)• Directionality of metabolismDirectionality of metabolism

Most reactions are reversibleMost reactions are reversible The cytoplasm as a soup, how does anything get The cytoplasm as a soup, how does anything get

done?done?

http://www.sigmaaldrich.com/img/assets/4202/sigma_metabolic_path-new.pdf

Plant and animal biochemistry Plant and animal biochemistry sometimes differsometimes differ

Methods of regulationMethods of regulation

1.1. Properties of enzymesProperties of enzymes

2.2. CompartmentationCompartmentation

3.3. Gene expressionGene expression

Methods of regulationMethods of regulation

Properties of enzymesProperties of enzymes• Affinity for substrate, inherent catalytic Affinity for substrate, inherent catalytic

capacitycapacity• Feedback regulation/feedforward/loopgainFeedback regulation/feedforward/loopgain• Allosteric effects, competitive versus non-Allosteric effects, competitive versus non-

competitive inhibitioncompetitive inhibition Fructose 2,6-bisphosphate as an exampleFructose 2,6-bisphosphate as an example

• Redox control of enzymes (vicinal cysteines Redox control of enzymes (vicinal cysteines can become cystine)can become cystine)

• pH and Mg regulationpH and Mg regulation Especially chloroplast enzymesEspecially chloroplast enzymes

Methods of regulationMethods of regulation

Properties of enzymes (Post-translational Properties of enzymes (Post-translational regulation)regulation)• Phosphorylation Phosphorylation

Protein kinases and phosphatasesProtein kinases and phosphatases Turns enzymes on or off, can affect sensitivity to effectors (SPS)Turns enzymes on or off, can affect sensitivity to effectors (SPS)

• Fatty acidsFatty acids Palmitic acid in a regulatory way, myristic acid is non-Palmitic acid in a regulatory way, myristic acid is non-

regulatoryregulatory

• PrenylationPrenylation Fanesylation (3 isoprenoids, 15 C) CaaX C-terminusFanesylation (3 isoprenoids, 15 C) CaaX C-terminus Geranylgeranylation (20 carbons) CaaL C-terminusGeranylgeranylation (20 carbons) CaaL C-terminus

• Fatty acids and prenylation anchors proteins to Fatty acids and prenylation anchors proteins to membranes or to other proteinsmembranes or to other proteins

Anchoring proteins to Anchoring proteins to membranesmembranes

Buchannan et al. (ASPB book) Fig. 1.10 page 9

Methods of regulationMethods of regulation

Cellular compartmentationCellular compartmentation• Hallmark of eukaryotic cellsHallmark of eukaryotic cells• Oxygen reactions mostly in mitochondria and Oxygen reactions mostly in mitochondria and

chloroplastschloroplasts• Chloroplasts – more generally plastids – are Chloroplasts – more generally plastids – are

what make plants uniquewhat make plants unique Cell walls, vacuoles also distinctive but not uniqueCell walls, vacuoles also distinctive but not unique Plastids are biochemical powerhousesPlastids are biochemical powerhouses

I hope this course will leave you with an I hope this course will leave you with an appreciation for the unique biochemistry appreciation for the unique biochemistry of plants, and where in the cell it happensof plants, and where in the cell it happens

The family of plastidsThe family of plastids

Buchannan et al. Fig. 1.44

EndosymbiosisEndosymbiosis

Well accepted that chloroplasts and Well accepted that chloroplasts and mitochondria were once free living bacteriamitochondria were once free living bacteria

Their metabolism is bacterial (e.g. Their metabolism is bacterial (e.g. photosynthesis)photosynthesis)

Retain some DNA (circular chromosome)Retain some DNA (circular chromosome)• Protein synthesis sensitive to chloramphenicolProtein synthesis sensitive to chloramphenicol• Cytosolic P synthesis sensitive to cycloheximideCytosolic P synthesis sensitive to cycloheximide

Most genes transferred from symbiont to Most genes transferred from symbiont to nucleusnucleus• Requires protein tagetingRequires protein tageting

PhylogenetiPhylogenetic location of c location of chloroplasts chloroplasts

and and mitochondrimitochondri

aa

DNA for chloroplast proteins DNA for chloroplast proteins can be in the nucleus or can be in the nucleus or

chloroplast genomechloroplast genome

Buchannan et al. Fig. 4.4

Import of Import of proteins proteins

into into chloroplaschloroplas

tsts

Buchannan et al. Fig. 4.6

Biochemistry inside plastidsBiochemistry inside plastids

Photosynthesis – reduction of C, N, and SPhotosynthesis – reduction of C, N, and S Amino acids, essential amino acid synthesis Amino acids, essential amino acid synthesis

restricted to plastidsrestricted to plastids• Phenylpropanoid amino acids and secondary Phenylpropanoid amino acids and secondary

compounds start in the plastids (shikimic acid compounds start in the plastids (shikimic acid pathway)pathway)

• Site of action of several herbicides, including Site of action of several herbicides, including glyphosateglyphosate

• Branched-chain amino acidsBranched-chain amino acids• Sulfur amino acidsSulfur amino acids

Fatty acids – all fatty acids in plants made in Fatty acids – all fatty acids in plants made in plastidsplastids

Biochemistry inside plastidsBiochemistry inside plastids

Carotenoids – source of vitamin ACarotenoids – source of vitamin A Thiamin and pyridoxal, B vitamins Thiamin and pyridoxal, B vitamins Ascorbic acid – vitamin CAscorbic acid – vitamin C Tocopherol – vitamin E Tocopherol – vitamin E Phylloquinone (an electron accepttor Phylloquinone (an electron accepttor

in PS I – vitamin K)in PS I – vitamin K)

Photorespiration is highly Photorespiration is highly compartmentalizedcompartmentalized

Buchannan et al. Fig. 1.40

Methods of regulationMethods of regulation

Gene expressionGene expression• Normally slow relative to metabolic control that Normally slow relative to metabolic control that

will be discussed most of the time in this coursewill be discussed most of the time in this course• Allows metabolism to be changed in response Allows metabolism to be changed in response

to environmental factorsto environmental factors• Transcriptional control most commonTranscriptional control most common

Sometimes variation in transcription rate not Sometimes variation in transcription rate not reflected in enzyme amountreflected in enzyme amount

• Translational control also foundTranslational control also found No change in mRNA levels but changes in protein No change in mRNA levels but changes in protein

amountsamounts

Gene structure relevant to metabolic Gene structure relevant to metabolic regulationregulation

PromotersPromoters

Exploring metabolism by Exploring metabolism by genetic methodsgenetic methods

Antisense – what happens when the amount of Antisense – what happens when the amount of an enzyme is reducedan enzyme is reduced• not clear how antisense worksnot clear how antisense works

KnockoutsKnockouts• Often more clear-cut since all of the enzyme is goneOften more clear-cut since all of the enzyme is gone• Use of t-DNA, Salk linesUse of t-DNA, Salk lines

OverexpressionOverexpression• Use an unregulated version of the protein or Use an unregulated version of the protein or

express on a strong promoterexpress on a strong promoter• Sometimes leads to cosuppressionSometimes leads to cosuppression

RNA interferenceRNA interference• 21 to 26 mers seem very effective in regulating 21 to 26 mers seem very effective in regulating

translationtranslation

What do we expect for the What do we expect for the reaction of metabolism to reaction of metabolism to changes in amount of an changes in amount of an

enzyme?enzyme? Is subtracting 50% likely to give exactly the Is subtracting 50% likely to give exactly the

opposite result as adding 50%?opposite result as adding 50%? Are there threshholds?Are there threshholds? Are there optimal amounts?Are there optimal amounts? Are there compensatory pathways?Are there compensatory pathways? Are there compensatory regulatory mechanisms?Are there compensatory regulatory mechanisms? Kacser H, Porteous JW. Control of metabolism: Kacser H, Porteous JW. Control of metabolism:

what do we have to measure. Trends Biochem.Sci. what do we have to measure. Trends Biochem.Sci. 1987;12:5-14.1987;12:5-14.

Koshland DE. Switches, thresholds and Koshland DE. Switches, thresholds and ultrasensitivity. Trends Biochem.Sci. 1987;12:225-ultrasensitivity. Trends Biochem.Sci. 1987;12:225-9.9.