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Advance Biochemistry. Introduction. Goals To cover aspects of biochemistry unique and important to plants Sometimes will involve bacterial biochemistry See some of the many biochemical pathways critical to plants (Structures will be shown!)

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Advance Biochemistry

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Advance Biochemistry


Introduction

  • Goals

    • To cover aspects of biochemistry unique and important to plants

      • Sometimes will involve bacterial biochemistry

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

    • Hear about techniques important in plant biochemistry

      • Molecular biology, mass spectrometry etc.

    • Major emphasis on regulation

  • Grading

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

    • 30 points for homework assignments

    • Total points at the end used to determine grade


Textbook and reading

  • Biochemistry &Molecular Biology of Plants, ASPB

  • Plant Biochemistry and Molecular Biology by Hans-Walter Heldt

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


Overall plan

  • Cell and Cellular Constituents

    Cell structure and functions

    Water and solutions

    Carbohydrates

    Fatty acids and lipids

    Amino acids and protein

    Enzymes

    Vitamins and minerals

  • Metabolism

  • Strategy for Processing of Nutrients in Plants

  • Applied Biochemistry


Overall plan

  • Photosynthesis

    • Carbon metabolism, Electron transport

    • Nitrogen, reduction and metabolism

    • Carbon end products

  • Cell constituents

    • Membranes, Cell walls

  • Cellular metabolism

    • Ion pumps

    • Protein turnover

  • Symbiotic nitrogen fixation


Regulation of Metabolism

  • Plant cells do a wide range of biochemistry

  • Regulation of metabolism

    • Stoichiometric requirements (e.g. amino acids)

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

    • Directionality of metabolism

      • Most reactions are reversible

      • The cytoplasm as a soup, how does anything get done?


http://www.sigmaaldrich.com/img/assets/

4202/sigma_metabolic_path-new.pdf


Plant and animal biochemistry sometimes differ


Methods of regulation

  • Properties of enzymes

  • Compartmentation

  • Gene expression


Methods of regulation

  • Properties of enzymes

    • Affinity for substrate, inherent catalytic capacity

    • Feedback regulation/feedforward/loopgain

    • Allosteric effects, competitive versus non-competitive inhibition

      • Fructose 2,6-bisphosphate as an example

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

    • pH and Mg regulation

      • Especially chloroplast enzymes


Methods of regulation

  • Properties of enzymes (Post-translational regulation)

    • Phosphorylation

      • Protein kinases and phosphatases

      • Turns enzymes on or off, can affect sensitivity to effectors (SPS)

    • Fatty acids

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

    • Prenylation

      • Fanesylation (3 isoprenoids, 15 C) CaaX C-terminus

      • Geranylgeranylation (20 carbons) CaaL C-terminus

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


Anchoring proteins to membranes

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


Methods of regulation

  • Cellular compartmentation

    • Hallmark of eukaryotic cells

    • Oxygen reactions mostly in mitochondria and chloroplasts

    • Chloroplasts – more generally plastids – are what make plants unique

      • Cell walls, vacuoles also distinctive but not unique

      • Plastids are biochemical powerhouses

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


The family of plastids

Buchannan et al. Fig. 1.44


Endosymbiosis

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

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

  • Retain some DNA (circular chromosome)

    • Protein synthesis sensitive to chloramphenicol

    • Cytosolic P synthesis sensitive to cycloheximide

  • Most genes transferred from symbiont to nucleus

    • Requires protein tageting


Phylogenetic location of chloroplasts and mitochondria


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

Buchannan et al. Fig. 4.4


Import of proteins into chloroplasts

Buchannan et al. Fig. 4.6


Biochemistry inside plastids

  • Photosynthesis – reduction of C, N, and S

  • Amino acids, essential amino acid synthesis restricted to plastids

    • Phenylpropanoid amino acids and secondary compounds start in the plastids (shikimic acid pathway)

    • Site of action of several herbicides, including glyphosate

    • Branched-chain amino acids

    • Sulfur amino acids

  • Fatty acids – all fatty acids in plants made in plastids


Biochemistry inside plastids

  • Carotenoids – source of vitamin A

  • Thiamin and pyridoxal, B vitamins

  • Ascorbic acid – vitamin C

  • Tocopherol – vitamin E

  • Phylloquinone (an electron accepttor in PS I – vitamin K)


Photorespiration is highly compartmentalized

Buchannan et al. Fig. 1.40


Methods of regulation

  • Gene expression

    • Normally slow relative to metabolic control that will be discussed most of the time in this course

    • Allows metabolism to be changed in response to environmental factors

    • Transcriptional control most common

      • Sometimes variation in transcription rate not reflected in enzyme amount

    • Translational control also found

      • No change in mRNA levels but changes in protein amounts


Gene structure relevant to metabolic regulation


Promoters


Exploring metabolism by genetic methods

  • Antisense – what happens when the amount of an enzyme is reduced

    • not clear how antisense works

  • Knockouts

    • Often more clear-cut since all of the enzyme is gone

    • Use of t-DNA, Salk lines

  • Overexpression

    • Use an unregulated version of the protein or express on a strong promoter

    • Sometimes leads to cosuppression

  • RNA interference

    • 21 to 26 mers seem very effective in regulating translation


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

  • Is subtracting 50% likely to give exactly the opposite result as adding 50%?

  • Are there threshholds?

  • Are there optimal amounts?

  • Are there compensatory pathways?

  • Are there compensatory regulatory mechanisms?

  • Kacser H, Porteous JW. Control of metabolism: what do we have to measure. Trends Biochem.Sci. 1987;12:5-14.

  • Koshland DE. Switches, thresholds and ultrasensitivity. Trends Biochem.Sci. 1987;12:225-9.


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