Lecture 12 - Getting and Using Nitrogen as a Building Block Last week- getting carbon and energy

1. Lecture 12 - Getting and Using Nitrogen as a Building Block Last week- getting carbon and energy This week’s reading: “Life together in the underground” From Plants, Genes and Agriculture (p.219-229) on Reserve 1. What atomic features make Nitrogen such an important atom build complex molecules? 2. Nitrogen is found in most complex molecules of life: Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.788 ASPP, Rockville MD, 2000 In plants, a major demand for Nitrogen is Rubisco (50-60% of leaf protein). Slide 12.1

2. 3. N2 gas makes up 80% of the Earth's atmosphere, and yet Nitrogen is the most limiting nutrient in temperate agricultural systems!! Why is Nitrogen so limiting for agricultural plants?? a) Atomic structure: b) Soil composition: c) Usable forms present a limitation: Forms of Nitrogen: •N2 - inorganic nitrogen -- a gas in the atmosphere - NOT usable by non-bacteria •However, the N-H or N-0 bonds require much less energy to break to allow Nitrogen to be used by a wide variety of enzymes to incorporate N into complex molecules: •NH3, NH4+ - ammonia, ammonium -- usable form that can be taken up by plants •NO2-, NO3- - nitrite, nitrate -- usable form that can be taken up by plants •Therefore, the triple N bond must be broken and the molecule reduced using electrons to form N-H or N-O bonds. d) Nitrite and Nitrate are both negatively charged. Many soil particles are also slightly negatively charged. This means that these forms of organic nitrogen are not as well retained by soil for use by roots and are leached, especially after rainfall. Leaching is therefore another problem for getting adequate amounts of usable Nitrogen for agriculture. Slide 12.2

3. e) How does life create usable forms of Nitrogen from N2 gas??? Evolution created an enzyme, Nitrogenase, that can convert inorganic N2 gas into organic nitrogen, ammonia (NH3), It can break the triple bond in N2 gas. Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.792 ASPP, Rockville MD, 2000 No plant genome encodes the nitrogenase enzyme. Why not?? 4. However, some bacteria ("Nitrogen-fixing bacteria") did evolve this enzyme and the ability to split N2. This is likely because they could survive anaerobic respiration conditions (especially if they evolved in the early atmosphere). By the time multicellular plants evolved, atmospheric oxygen concentrations had already increased. Plants are large, and they need large amounts of energy from mitochondrial respiration, which requires O2. Slide 12.3

4. 5. "Breaking the triple N2 bond". Multicellular plants could not have evolved if they didn't find a way to get nitrogen. What strategies were adapted? a) Attracting free-living microbes to build a "Rhizosphere" around plant roots. -carbohydrates from sloughed-off root tip cells, slime (mucilage) -15% of all photosynthate is deposited in the soil Plants, Genes and Agriculture,pp.220 M. Chrispeels and D.Sadava Jones and Bartlett Publishers, Boston, 1994 Decomposers of organic matter -- fungi and bacteria Direct methods? Indirect methods? Slide 12.4

5. To facilitate contact between the roots and these organic stores of Nitrogen, plant roots increase their surface area: i) Roots grow continuously (5-6 days spent). This uses 30% of the photosynthate. Why are aerated soils so important? Soybean Root system Plants, Genes and Agriculture,p.204-205 M. Chrispeels and D.Sadava Jones and Bartlett Publishers, Boston, 1994 ii) Root hairs iii) Roots are associated with fungal hyphae (mycorrhizae) Slide 12.5

6. b) A more evolved (and rare) strategy, symbiotic relationships between N-fixing bacteria (such as Rhizobium) and "legume" plants. In soybeans and peanuts, up to 1/3 of the nitrogen comes from this process. How and why does this work?? Nodule - a plant Organ From Biology of Plants p. 603 P.Raven, R. Evert, and S. Eichhorn Worth Publishers, New York, 1992 Nitrogen-fixing Rhizobium bacteroids inside plant root nodule Plants, Genes and Agriculture,pp.227 M. Chrispeels and D.Sadava Jones and Bartlett Publishers, Boston, 1994 i) Creating a near-anaerobic environment. How?? -bacteroids - -leghaemoglobin - How do the bacteria produce energy (respiration) in such a low oxygen environment?? Slide 12.6

7. ii) The nodule - a plant organ that is only formed when Rhizobium signal plant genes to turn on in a coordinated manner. Images from: Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.801, 805 ASPP, Rockville MD, 2000 iii) Signalling iv) A symbiotic relationship What are the costs and benefits of these adaptive strategies to each organism?? ***It is energetically expensive for Nitrogenase to break N2 into NH3 (it takes 16 ATP molecules for 1 molecule of N2 to be broken).**** < 20% of the photosynthate produced by legumes are used for N fixation Slide 12.7

8. 5c) A synthetic way to break the N2 triple bond is the Haber-Bosch process, perhaps the most important single advance in chemistry in the 20th century. This is the basis of fertilizer. What are the advantages and disadvantages of Nitrogen fertilizer? i) Impact on agriculture Plants, Genes and Agriculture,p.49 M. Chrispeels and D.Sadava Jones and Bartlett Publishers, Boston, 1994 ii) Costs - requires high temperature (400-650C) and high pressure (consumes much fossil fuel) iii) The Environment (two views on this) iv) There are environmentally-friendly alternatives to Nitrogen- fertilizer. What are these? -crop rotation, intercropping, green manure eg. Azolla-Anabaena for rice Plants, Genes and Agriculture,p.212 M. Chrispeels and D.Sadava Jones and Bartlett Publishers, Boston, 1994 Slide 12.8

9. 6. The uptake and assimilation of Nitrogen by plants. a) Uptake from the soil. •Transpiration, but the ammonia/nitrate molecules form hydrogen bonds to H20 = hydrophilic (charged) so cannot pass through the hydrophobic lipid membranes of root cells. •Therefore, specific channels are used, called nitrogen transporters. •This is an active process, uses energy (ATP) to allow these transporters to open, close and change conformation. •Therefore, organic nitrogen uptake by plants expends energy. . Nitrogen uptake Nitrogen Assimilation Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.816 and p. 366 ASPP, Rockville MD, 2000 b) How is Nitrogen assimilated? Into what molecule? Slide 12.9

10. c) In carbon fixation, sucrose (12C) is the transport version which delivers Carbon and C-C bond energy to sinks such as seeds and roots. Starch (6C+6C+6C...) is the storage form for carbon. For Nitrogen, what are the transport and storage molecules? Why? i) Transport? ii) Storage? - Prolamins, Globulins storage proteins inside organelles (= "protein storage bodies"), •Dicots (eg. soybeans, peas) = cotyledons •monocots (cereals, corn, wheat, rice) = Endosperm of the seed . Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.18 ASPP, Rockville MD, 2000 •on average legume seeds store 2X more protein than cereal grains •the storage proteins in the seeds are named after the species: Zea mays (corn) = zein Phaseolus (bean) = phaseolin Hordeum (barley) = hordein, etc. During seed germination, these storage reserves are used for rapid growth and development. In agriculture, why might rapid early growth be so important?? Slide 12.10

11. 7. Protein and human and animal nutrition •8 amino acids cannot be synthesized by humans •cereals are deficient in 2-3 amino acids (corn in Lysine + Tryptophan) amino acids cannot be stored, so ancient societies complemented their foods (a cereal with a legume): Mexico and South America -- beans and tortillas Middle East - chickpeas (hummus, falafel) - Pita breads South Asia - rice and lentils East Asia - soybean (tofu) and noodles/rice Plants, Genes and Agriculture,p.94-95 M. Chrispeels and D.Sadava Jones and Bartlett Publishers, Boston, 1994 Slide 12.11

12. The Protein Content of Foods Plants, Genes and Agriculture,p.113 M. Chrispeels and D.Sadava Jones and Bartlett Publishers, Boston, 1994 However, CIMMYT scientists have released high protein quality maize (in 1998) = seed has the same protein quality as cow's milk = mutation in a transcription factor for amino acid synthesis the photosynthate. Slide 12.12

13. 8. The control and regulation of plant Nitrogen Recycling and remobilizing Nitrogen: -the best crops are those that can recycle the proteins from aging leaves and stalks and transport the amino acids to the developing seeds (sinks) •Nitrogen is an energetically expensive molecule to fix by legumes •12 to 17g of carbohydrates are consume for each 1g of Nitrogen that is fixed by Rhizobium •NH4/NO3 is an energetically expensive molecule to uptake from the soil and to assimilate into amino acids. •In addition, to assimilate nitrogen into an amino acid, there must be enough carbon skeletons available. •50-60% of leaf nitrogen is consumed just be Rubisco and other photosynthetic enzymes consume much organic nitrogen. •For these reasons, evolution has evolved very tight regulation between Nitrogen fixation and assimilation vs Photosynthesis and carbon-fixation. i) Examples of this regulation? -light and sucrose upregulate N assimilation -darkness + sucrose repress N assimilation genes -repression of N assimilation when organic N is high relative to carbon ii) How is the ratio of C to N adjusted?. - when organic N is high relative to Carbon, then the N is shunted and stored as the amino Acid Asparagine which has a higher N:C ration than Glutamine, and thus can store organic N more efficiently when carbon skeletons arer limited Slide 12.13