EFB530 Plant Physiology

Plant cells-structure and organelles

Cell wall

• Cellulose
• Hemicellulose-xyloglucans
• Pectins-polygalacturonic acid
• Proteins-extensin

Plasma membrane

• Bilipid layer w/protein

Vacuole

• Membrane=tonoplast

Peroxisome

• Involved in photorespiration and oxidative reactions

Endoplasmic reticulum/Golgi body

• Protein trafficking
• Site of membrane synthesis
• Vesicles carry non-cellulosic cell wall precursors

Mitochondria

• Carry DNA-encodes some of the mito proteins
• Main site of energy conversion=glucose to ATP

Plastids

• Proplastids->etioplasts, chloroplasts, leucoplasts, amyloplasts, chromoplasts
• Carry DNA-encodes some of the plastid proteins-LSU
• Evolved from cyanobacterial precursor/endosymbiont
• Site of photosynthesis, also amino acid synthesis

Nucleus

• Surrounded by double membrane envelope w/pores
• Chromatin=DNA + protein

Cytoskeleton

• Proteinaceous-microtubules (tubulin); microfilaments (actin)
• Microtubules involved in mitosis & cytokinesis
• Microfilaments=streaming, tip growth

Plasmodesmata

• Desmotubule is extension from ER
• PM and cytoplasm contiguous

Physiology and levels of regulation

Physiology=the organic processes and phenomena of an organism or of any of its parts or of a particular bodily process

Biochemical components and organization of metabolic processes, also the regulation and integration of those processes

Levels of regulation:

Transcription

• Promoter- RNA polymerase II
• Transcription start site
• NO operons, each gene regulated independently
• Regulatory elements-cis & trans
• Constitutive vs. regulated expression
• Regulation-up or down
• through development-organ or cell localization
• by stimuli

Post-transcription

• Exons/introns
• RNA processing-m⁷G cap; splicing; poly(A) tail
• RNA stability
• Differential splicing

Translation

• Ribosomes
• Translation start/stop
• Codon usage/bias
• Translation efficiency

Post-translational

• Trafficking / compartmentalization / cleavage
• Protein modification-glycosylation
• Protein turnover-ubiquitin / proteases

Enzymes & enzyme activity:

Characteristics of enzymes

• Catalysts-participate in rxn, but are not changed
• Lower the free energy of activation
• Highly specific
• Highly efficient
• Saturable
• Sensitive to pH and temp

Protein structure

• 1=sequence of amino acids
• 2- alpha-helix, beta-sheet (local, predictable structures)
• 3- protein folding of entire polypeptide
• 4- subunit assembly=folding of multiple polypeptides

Active site

• Binding site, catalytic site

Kinetics

• Michaelis-Menten
• V_max, K_m

Regulation of enzyme activity

• Amount of enzyme
• Compartmentalization
• Covalent modification
• phosphorylation
• regulatory domain
• Feedback inhibition
• Regulatory subunit
• Cofactor binding
• pH, temperature

How do we study proteins and measure activity?

Extractions from plants

• takes some effort to break open cell walls - grinding, mortar/pestle, high pressure

Separations = Organelle fractionation

• separate by centrifugation techniques - based on density
• particular organelles will form a pellet at a particular centrifugal force (speed in RPM) - others stay in supernatant
• can also use a dense solution (like concentrated sucrose) - organelles separate by the way they "float" in the sucrose

Protein purification - need to obtain a single, pure enzyme to study it best

• can use different types of matrix, which will bind to particular proteins (others stay in solution) - wash away non-binding proteins, then elute the particular proteins that were bound to the matrix
• matrix can select for proteins according to: size, hydrophobicity, positive charge, negative charge, specific binding sites

Enzyme assays

• mix an amount of enzyme with increasing amounts of substrate
• measure the amount of product generated over time = activity (reaction velocity, V)
• V increases linearly at low substrate conc., then levels out as enzyme binding sites are saturated

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