1. BISC 367 Plant Biology Fall 2006 BISC 367 - Plant Physiology Lab Spring 2009 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 PlantTreatmentPressure Bomb (Individual readings) Pressure Bomb (MPa) (average) Leaf Press (psi) Individual readings Leaf Press (MPa) Average Osmometer Reading (mmol/Kg) Osomometer Reading (mmol/Kg) Average Poplardry1.80314 and 3.039, 2.335,39,550.2965804, 1007905.5 wet1.26625/1.16495/0.9117, 1.251.114360,85,52, 29, 300.4528632, 589, 588603 Geraniumdry0.75975/0.82053/0.729360.7698873,85, 600.5447294, 293, 274, 298289.75 wet.38494/.51663/.48624.0.46260333390,58,62, 450.4826280, 263, 299, 272278.5 Beandry0.86105 420.2896405 wet0.35455 370.2551353 Corndry ---- wet ---- 1psi=0.00689476MPa 350.2413 Convert to Ys using van’t Hoffs eqtn 390.2689 550.3792 600.4137 Poplar, bean Ys is lower for dry but not for gernanium – why? The units are MPa850.5861 The sign is NEGATIVE520.3585 730.5033 850.5861 These readings look good900.6205 Combine with data from other group580.3999 620.4275 420.2896 370.2551 ???This data doesn't jive with the pressure bomb I agree! BISC 367

3. Measuring  w 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  w 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 Relative water content

4. BISC 367 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 Water uptake by roots

5. BISC 367 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. BISC 367 Water movement - an overview

7. Inorganic ions in the soil Soil particles carry a negative charge –Bind cations Anions are not readily bound NO 3 - is soluble PO 4 2- binds to Al 3+ or Fe 3+ and can be unavailable SO 4 2- reacts with Ca 2+ to form gypsum (CaSO 4 )

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

11. 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 Ion uptake into a cell

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

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 Ca 2+ 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 N 2 not readily available Triple N-N bond needs lots of NRG to break

15. Nutrient assimilation Energetically costly! –NO 3 - reduction to NH 4 + 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 NO 3 and is influenced by: plant N status form of N available in the soil Sustained protein synthesis is necessary NO 3 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 NO 3 NO 3 NO 2 NH 4 + NH 2 group of amino acid NRG cost = 12 ATP

18. Nitrate assimilation Nitrate absorbed by the soil is reduced in the cytosol by nitrate reductase (NR) NO 3 - + NAD(P)H + 2H + NO 2 - + NAD(P) + + H 2 O NR is the major Molybdenum containing enzyme in plants

19. Nitrate assimilation NR is tightly regulated: Gene transcription and enzyme activation are stimulated by: NO 3 Light enhances activation by NO 3 links NO 3 assimilation with NRG CHO Inactivation of NR is stimulated by: Dark Mg 2+ 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. BISC 367 Kaiser, W. M. et al. J. Exp. Bot. 2001 52:1981-1989; doi:10.1093/jexbot/52.363.1981 Model for the post-translational modulation of NR

21. Nitrate assimilation NO 2 - is toxic and must be utilized immediately Transported to cp (leaf) or plastid (root) Reduced by nitrite reductase (NiR) NO 2 - + 6 Ferredoxin red + 8 H + NH 4 + + 6 Fd ox + 2 H 2 O NiR is regulated by light/NO 3 (inducers), and by amino acids (repressors) NiR levels are higher than NR Reduction of NO 2 - Relies on e - produced by photosynthesis

22. NH 4 + is toxic and must be utilized rapidly –Dissipates pH gradients Ammonium assimilation

23. Glutamine synthetase (GS) combines NH 4 + and glutamate Glu + NH 4 + + ATP Glutamine + ADP + Pi Glutamate synthase (GOGAT) transfers the amide of glutamine to 2- oxoglutarate Glutamine + 2-oxoglutarate + Fd red /NADH 2 glutamate + Fd ox Transamination rxns transfer amide N to other amino acids Glu + oxaloacetate aspartate and 2-oxoglutarate Ammonium assimilation