READING ASSIGNMENTS:
pages 323-330 (Taiz and Zeiger)
Kaiser,WM and Huber,SC. (1994). Posttranslational regulation of nitrate reductase in higher plants. Plant Physiol. 106:817-821
INTRODUCTION
Elemental analysis reveals that 2 to 6 % of plant dry matter is composed of N in the form of amino acids, proteins and nucleic acids. In these compounds nitrogen exists in its most reduced state (-3). Yet the principal form in which N is absorbed by agricultural plants is in the form of nitrate (NO3-), in which N is in its most oxidised form (+5). This is because agricultural soils are usually well aerated and the process of mineralization and nitrification result in the conversion of amino compounds to NH4+ and ultimately NO3-, respectively. Thus, nitrate that is absorbed by plant roots must be reduced from the +5 to the -3 state, by enzyme-mediated redox reactions. Actually, while most textbooks state that nitrate is the principal form of N available to plants they are perhaps guilty of nitrate chauvinism, probably because of the bias towards agriculture. In many forest soils, cold or waterlogged soils, nitrate is not the principal form of N available, rather, NH4+ and amino acids are. There are well-characterized transport systems for amino acids in plant roots but to date there have been virtually no careful ecological studies of the importance of amino acids as sources of N for plants. Since writing this last year there has been increasing interest in amino acid absorption by plant roots and one study from Sweden demonstrated that amino acids can be absorbed intact in natural soils. I emphasize intact because until recently it was not known whether the amino acids were first broken down in the soil to yield NH4+ or NO3- and then absorbed. By using amino acids labelled in the N atoms with 15N and in the carbon atoms with 13C, it was shown that the amino acids could be taken up intact. This does not preclude the simultaneous breakdown of amino acids by microorganisms. Furthermore, one very important agricultural crop, namely rice, obtains most of its N in the form of NH4+ and also possibly in the form of amino acids.
Mineralization (ammonification) and Nitrification:
The amino compounds in soil organic nitrogen derivatives (proteins, amino acids and amino sugars), as well as fertilizer N (in the form of urea) are released as NH4+ through the activities of soil microorganisms. Proteins are hydrolyzed to amino acids and then the amino acids are oxidatively-deaminated to produce keto acids and NH3. Unless the soil pH is very high (the pKa of NH3/NH4+ conjugate pair is 9.25) the NH3 is protonated to generate NH4+.
The NH4+ generated can bind to soil colloids, enter into soil solution or be absorbed by plant roots. It can also be converted to NO2- and finally NO3-, by soil microorganisms, a process called nitrification.
NH4+---------> NH2OH------->[HNO]----------->NO2------------> NO3-
The NO3-, thus generated through mineralization and nitrification, can be absorbed by plant roots. From a study of 36 agricultural soils from America, Australia and New Zealand (Wolt, 1994), I calculated that the average soil solution [NO3-] was 6 mM, while the average [NH4+] was around 0.7 mM. No data were given for amino acid concentrations but they are ususally quite low (~20 mM). Having absorbed the available NO3-, the plant has to reverse the oxidation steps that created the NO3- in order to build up amino acids again. This is done, step by step, by means of enzyme reactions; NO3- is first converted to NO2- and subsequently to NH4+, before incorporation into amino acids. In cold soils, waterlogged soils or strongly acidic soils, NO3- is usually present in very low amounts. For a long time it was claimed that these harsh conditions inhibited the activities of nitrifying organisms and hence no nitrification took place. However, a recent study of about 11 forests in the USA (Stark and Hart, 1997) claims that there is intense nitrification in mature forests but that the NO3-is immediately absorbed by the microorganisms. Therefore, the end result is the same, i.e. virtually no NO3- accumulates in soil solution, and plants in these environments must absorb NH4+, or amino acids. It is important to remember that in these habitats mycorrhizae are probably largely responsible for accumulating nutrients. In turn the mycorrhizae pass the nutrients on to the plant via the fungal/plant connections.
1. THE REDUCTION STEP FROM NO3- TO NO2-: Nitrate reductase
Through the activity of the nitrate transporters, [NO3-] in the cytosol can reach values of between 1 to 30 mM. Several fates may await the absorbed NO3-:
a. transport across the tonoplast and storage in the vacuole.
b. transport across the root and delivery to the xylem to be translocated to the shoot.
c. biochemical reduction to NO2- in the root or the shoot through the activity of the enzyme nitrate reductase (N.R.).
From Crawford & Glass, 1998
Reductant
The reducing power for this reduction comes from reduced forms of the coenzymes NADH or NADPH . In barley and maize an NADH-specific enzyme is found in roots and shoots, whereas a second N.R., an NAD(P)H-bispecific enzyme, is found in roots, but is (usually) absent from green tissues. The latter enzyme can use either NADH or NADPHas the source of reducing power. The NADH form of N.R. predominates. The redox state of N in NO3- is +5, and in order to convert NO3- to NO2- (redox state +3), a 2 electron transfer is required:
NO3- + 2H+ + 2e- -----------------------> NO2- +H2O
The nitrate reductase (NR) enzymes are particularly important because this reaction is the first step in NO3- assimilation, and, therefore, a likely location for regulation. Secondly it is energy-consuming; Remember that 1 mole of NADH can be oxidized inside mitochondria to generate 2.5 to 3 moles of ATP. The complete reduction of N from +5 (NO3-)to -3 (NH4+) requires 8 electrons, and it is estimated that 20% of the electrons produced through photosynthesis are consumed for nitrate reduction. Thirdly, and, perhaps most importantly of all, the reduction of NO3- generates a cytotoxic, potentially mutagenic product, namely, NO2-. Hence, there is another important reason to regulate this step via controls exerted on NR. The control of this enzyme is so efficient that typically it is impossible to detect NO2- in plant tissues, because the production of NO2- and its subsequent conversion to NH4+, are so well co-ordinated with the plantís capacity to assimilate NO3-. Since carbon skeletons are needed to accept the ammonium resulting from nitrite reduction for the formation of amino acids, carbon and nitrogen metabolism need to be well integrated. Since both carbon reduction and nitrate reduction require reducing power, these processes could be viewed as in competition. However, we know that carbon reduction takes the main share of the reducing power generated in photosynthesis and so it is unlikely that these two processes compete. Can you think of conditions where they might compete for reducing power ?
Location of the enzyme
There is general agreement that the NADH and NAD(P)H enzymes are localized in the cytoplasm. As stated above, many plants reduce nitrate both in the root and in the shoot, with the bulk of the reduction occurring in the shoot. Other species limit their reduction to the shoot, while in other species most of the reduction occurs within the root. Interestingly, in barley when the NADH requiring N.R. is mutated the NAD(P)H requiring enzyme is expressed in shoots as well as in roots. Thus, it serves like a back-up enzyme when the main enzyme is not functional in the shoot.
Prosthetic Groups.
All known N.R. enzymes have three prosthetic groups:-
1. FAD (flavin adenine dinucleotide)
2. heme
3. molybdenum complex
The overall reaction for reduction of NO3- to NO2- is as follows :-
Molybdenum is therefore an essential element for this and other Mo-requiring reactions in which it serves as a redox centre. Nitrate reduction is, therefore, severely reduced in Mo-deficient plants or in plants pretreated with tungstate (WO3-) an analogue of molybdate, which displaces Mo from the reaction centre. For this reason, tungstate is often used as an inhibitor of the enzyme NR.
Molecular genetics.
Nigel Crawford (at San Diego) has cloned and sequenced the gene coding for NADH-requiring N.R. from Arabidopsis. Knowing that the enzyme has FAD, heme and Mo as prosthetic groups, he compared the known sequence of his N.R. enzyme with sequences of enzymes known to bind FAD, heme and Mo. He defined regions of the NR which corresponded to an FAD domain (at the C-terminus), a heme domain (mid-region) and a Mo domain (at the N-terminus) of the protein. The functional enzyme is believed to operate as a homodimer (ie. two identical subunits) with total M.W. = 200,000-270,000 with a Km around 0.75 mM. A model of the enzyme dimer is shown on the hand-out and in Fig 12.3 of Taiz and Zeiger.
Regulation
TRANSCRIPTIONAL CONTROL
1.The NR enzyme is inducible by NO3- (Tang and Wu :1957). It is also inducible by NO2- in barley (Aslam et al., 1987).
2. Induction occurs at the level of transcription ; within 40 min of exposure to NO3- increased levels of mRNA for N.R. could be detected in barley roots (Kleinhofs et al., 1989: see pp327 Fig 12.4 of Taiz and Zeiger).By 2 h mRNA levels for the enzyme were at a maximum
3. Enzyme levels depend on the balance between synthesis and degradation. According to Ann Oaks at Guelph, the half-life of the enzyme is but a few hours.
4. For maximum induction both light and nitrate are required. If we
remember that carbon skeletons are needed for assimilating the NH4+
resulting from nitrate reduction and that 20% of the electrons resulting
from the light reaction are consumed in reducing NO3-
to NH4+, it is not surprising that induction of the
enzyme should be responsive to light.
POSTTRANSLATIONAL CONTROL
1. In illuminated spinach, extractable NR activity was 20 m mol NO2- g-1h-1 (about 10% of C fixation). If the leaves were darkened first, the N.R., activity dropped by 50 to 85% with a half time of 2 to 15 min.
2. Rapid wilting of leaves also caused reduced N.R. activity.
3. Turgid leaves exposed to CO2-free air likewise showed decreased N.R. activity.
THE INESCAPABLE CONCLUSION DRAWN FROM THESE RESULTS IS THAT N.R. IS MODULATED IN PARALLEL WITH PHOTOSYNTHESIS.
Mechanism of control: The current hypothesis is that phosphorylation/dephosphorylation of the enzyme nitrate reductase is responsible for posttranslational control. The paper by Kaiser et al., (see recommended reading ) describers the experimental work.
2. FROM NO2- TO NH4+: Nitrite reductase
a. Location of the enzyme
This reaction is thought to occur within plastids, i.e. within the chloroplasts of photosynthetic tissue and within proplastids in roots. Nitrous acid HNO2 has a pKa of 3.3,
hence it has been suggested that this uncharged chemical species might travers the membrane of chloroplasts by diffusion. Pamela Brunswick ( from South Africa) established that uptake of NO2- by chloroplasts was saturable and induced by growth on NO3-. When chloroplasts were isolated from plants grown on urea, the chloroplasts had no capacity to absorb NO2-. Can you offer an explanation for this observation ?
b. The redox change
The redox state of N in NO2- (+3) requires a 6 electron transfer to reach the -3 state of N in NH4+. The reducing power is provided by reduced ferredoxin, a product of the light reactions of photosynthesis. Can you now offer a suggestion why (NOT HOW) the enzyme NR is switched off rapidly after leaves are darkened ? You should also be able to say how it happens.
The reduction of to NO2- to NH4+ reaction is as follows:-
NO2- + 6Fdred + 8H+------------------------------------->NH4+ + 6Fdox +2H2O
In non-green tissues NADPH is the source of reducing power, although there is a ferredoxin-like protein which shares antigenic properties in common with the leaf ferredoxin that is thought to be the immediate donor of electrons to NO2-. So it seems that a sequence from NADPH to the Fd-like protein to NO2- serves to transfer electrons to NO2-.
NO2- + 3NADPH + 5H+------------------------------------->NH4+ + 3NADP+ +2H2O
Spinach NiR is probably the best studied. It consists of a single polypeptide of M.W. 60,000, with two prosthetic groups :-
a. an Fe4S4 cluster
b. a specialised heme molecule.
Probable reaction in chloroplasts is given in Fig 12.5 p 327 of Taiz and Zeiger:
Although the enzyme functions inside the chloroplast, it is nuclear encoded. Hence, a transit peptide is involved in traversing the chloroplast double membrane. The enzyme from wheat has a precursor form with M.W. = 64,000. After transit through the membrane this is reduced to the active enzyme with M.W. 60,500.