Plant Biology Fall 2006

1. BISC 367 - Plant Physiology Lab Spring 2009 Plant Biology Fall 2006 • Notices: • Photosynthesis lab report due Feb. 09 • Lecture test Feb 10 • Please email water relations data to Doug Wilson & myself • Reading material (Taiz & Zeiger): • Chapter 12 assimilation of mineral nutrients

2. Water relations data

3. Measuring Yw Relative water content • Assesses the water content of plant tissues as a fraction of the fully turgid water content • relevant when considering metabolic / physiological aspects of water deficit stress • Considered to be a better indicator of water status and physiological activity • Captures effects of osmotic adjustment • Osmotic adjustment lowers the Yw at which a given RWC is reached • Simple technique: • Leaf disks are excised, weighed (W) then allowed to reach full turgidity and re-weighed (TW). Leaf disks are dried to obtain their dry weight (DW). • RWC (%) = [(W – DW) / (TW – DW)] X 100

4. Water uptake by roots • Water crosses the roots using 3 possible pathways • Apoplastic pathway • Water moves via cell walls • Symplastic pathway • Water moves through the cells passing through the plasmodesmata • Transmembrane pathway • Water moves through cells but independently enters and exits each cell

5. Water uptake by roots • Casparian strip forces water to enter endodermal cells • must cross plasma membrane • Allows plant to select what can pass on to the xylem • Important for discrimination against toxic ions etc. • Usually consider a single hydraulic conductance for entire root

6. Water movement - an overview

7. Inorganic ions in the soil • Soil particles carry a negative charge • Bind cations • Anions are not readily bound • NO3- is soluble • PO42- binds to Al3+ or Fe3+ and can be unavailable • SO42- reacts with Ca2+ to form gypsum (CaSO4)

8. Ion transport across the root • Ions can cross the root in the apoplast or symplast • All ions enter the symplast at the endodermis before entering the stele (vascular tissue) • To enter the cells of the xylem ions must move back to the apoplast Note: the casparian strip: • prevents outward movement of ions • Can allow a higher level of ions to build in the xylem relative to the soil

9. Ion uptake into a cell • Driving force for ion uptake is the electrochemical gradient • Conc. gradient across membrane • Electrical gradient across membrane • At eqm the conc. difference across the membrane is balanced by the electrical difference • Calculate electric potential for given ion using Nernst equation • All living cells have an electrical difference across the membrane - membrane potential

10. Ion uptake into a cell • Membrane potential is established by several ions coming to “eqm” • Ability to come to eqm (or steady state) is influenced by membrane transport processes • Only K+ is close to eqm. • Anions have a higher than predicted conc • Cations have a lower than predicted conc

11. Ion uptake into a cell • Membrane potential is set by: • Passive diffusion • Electrogenic pumping (primarily H+) • H+-ATPases • Located on PM (plasma membrane) - P-ATPases • Pump H+ into cell wall • and tonoplast (membrane surrounding vacuole) - V-ATPases • Pump H+ into vacuole

12. Ion uptake into a cell • H+ gradients drive 2o transport across PM and tonoplast • In vacuole [H+] is high: • Anions move in to balance charge • Ys falls • Water moves in - turgor increases • H+-pyrophosphatase also moves H+ into vacuole • Utilize energy of PPi hydrolysis

13. Ion Composition • K+ acquired passively • Na+ actively pumped out to apoplast and vacuole • H+ actively pumped out to apoplast and vacuole • Acidic apoplast and vacuole, neutral cytoplasm (regulates cell pH) • Anions are actively acquired • Ca2+ is actively pumped out Passive transport Active transport

14. Nitrogen assimilation • Only C, H, and O are more abundant in plants than N • N is abundant in the atmosphere as N2 • not readily available • Triple N-N bond needs lots of NRG to break

15. Nutrient assimilation • Energetically costly! • NO3- reduction to NH4+ utilizes 25% of a plants NRG requirements • Requires large amounts of reductant • Most occurs in stroma of chloroplast (cp) • Dependent on photosynthetic e- transport • photoassimilation

16. Nitrogen assimilation • Nitrate uptake is inducible: • Low and high affinity carriers exist • Carriers are synthesized in response to external NO3 and is influenced by: • plant N status • form of N available in the soil • Sustained protein synthesis is necessary • NO3 that enters root cells has 3 fates • Storage in the vacuole • Assimilation in root cells • Translocation in the xylem for assim. in leaf cells

17. Nitrogen assimilation • Assimilation of N via reduction of NO3 • NO3 NO2 NH4+ NH2 group of amino acidNRG cost = 12 ATP

18. Nitrate assimilation • Nitrate absorbed by the soil is reduced in the cytosol by nitrate reductase (NR) • NO3- + NAD(P)H + 2H+ NO2- + NAD(P)+ + H2O • NR is the major Molybdenum containing enzyme in plants

19. Nitrate assimilation • NR is tightly regulated: • Gene transcription and enzyme activation are stimulated by: • NO3 • Light • enhances activation by NO3 • links NO3 assimilation with NRG • CHO • Inactivation of NR is stimulated by: • Dark • Mg2+ • NR is regulated by a NR kinase • phosphorylated and non-phosphorylated states are active • if the phosphorylated form is transferred to darkness an inhibitor switches NR off • activity is restored in the light by: • inhibitor release • phosphatase

20. Model for the post-translational modulation of NR Kaiser, W. M. et al. J. Exp. Bot. 2001 52:1981-1989; doi:10.1093/jexbot/52.363.1981

21. Nitrate assimilation • NO2- is toxic and must be utilized immediately • Transported to cp (leaf) or plastid (root) • Reduced by nitrite reductase (NiR) • NO2- + 6 Ferredoxinred + 8 H+ NH4+ + 6 Fdox + 2 H2O • NiR is regulated by light/NO3 (inducers), and by amino acids (repressors) • NiR levels are higher than NR Reduction of NO2- Relies on e- produced by photosynthesis

22. Ammonium assimilation • NH4+ is toxic and must be utilized rapidly • Dissipates pH gradients

23. Ammonium assimilation • Glutamine synthetase (GS) combines NH4+ and glutamate Glu + NH4+ + ATP Glutamine + ADP + Pi • Glutamate synthase (GOGAT) transfers the amide of glutamine to 2-oxoglutarate Glutamine + 2-oxoglutarate + Fdred/NADH 2 glutamate + Fdox • Transamination rxns transfer amide N to other amino acids Glu + oxaloacetate aspartate and 2-oxoglutarate