Progress 10/01/99 to 09/30/04
Outputs
Basically, the energy for both nitrogen (N) and carbon (C) assimilation is provided, directly or indirectly, by light. Some believe that the role of light in N assimilation is via supply of carbohydrates which are used as a source of energy. Others believe that light is obligatory for nitrate assimilation. This project was the first to recognize that perhaps both observations were correct depending on the plant species (C3 or C4) used for experimentation. Earlier this project reported the novel finding that carbohydrate-depleted corn leaves, but not barley, were able to reduce nitrate under photosynthesis-inhibited (DCMU treatment) conditions in the presence of light. This has raised important questions about the possibilities of a new mechanism by which corn leaves may be able to utilize light energy directly for supporting N assimilation without having to go through photosynthetic CO2 fixation. The results also indicate that enzyme capacity was not limiting nitrate
reduction. These observations suggest that in corn both C and N assimilation processes may be driven directly by light whereas in barley light seems to be utilized only for C assimilation and the assimilated carbohydrates in turn drive N assimilation via glycolytic reactions. Clearly, the C4 system would be more efficient as it would harvest a greater amount of light energy falling on the leaf by using two separate and dedicated electron flow routes. Lately, we have been probing the possible mechanism(s) by which corn leaves may be using light energy directly for N assimilation. It has been puzzling to note that although corn leaves assimilated nitrate under photosynthesis-inhibited conditions in the presence of light, the presence of CO2 was still required. During the past year, we learned that it is possible for plants to possess alternate routes of photosynthetic electron transport, which may bye-pass the classical Z scheme of electron flow. Moreover, recent reports show that HCO3
ions (CO2 requirement) are an essential intermediate in the photosynthetic electron flow from hydrolysis. These reports explain the observations discovered in this project. Considering that the basic role of light in photosynthesis is splitting the water into oxygen and protons, our approach now has been to pursue photosynthetic reactions by monitoring oxygen evolution. We are working with the hypothesis that if corn leaves are harvesting light in the presence of DCMU, oxygen must be evolved. This would show that an alternate path of electron flow for N assimilation exists. However, measuring photosynthetic oxygen evolution is not easy and requires special equipment. It basically translates into the ability of measuring a few ppm oxygen differential in a background of 209,000 ppm. Preliminary data generated by using borrowed equipment are very promising; an aggressive effort to purchase this equipment is currently underway. A parallel approach currently being tested is to use the
inhibitors of the enzyme carbonic anhydrase in an attempt to inhibit photosynthetic oxygen evolution in the presence of DCMU.
Impacts
We now feel confident that corn leaves possess a separate, parallel, and perhaps dedicated route of photosynthetic electron flow for N assimilation. This mechanism ultimately results in harvesting of larger quantities of light energy by corn leaves relative to those of C3 species. These findings will explain the basis of response of C4 crops, such as corn, to significantly greater applications of nitrogen, and hence the greater productivity potential of C4 crops relative to those of C3 crops, such as wheat and barley. Once the entire pathway for the N assimilation electron flow in corn is understood, we should be able to clone the gene(s) and incorporate them into C3 plants, which would greatly enhance their productivity potential.
Publications
- Sehtiya, H.L. and S. S. Goyal (2000) Comparative uptake of nitrate by intact seedlings of C3 (barley) and C4 (corn) plants: Effect of light and exogenously supplied sucrose. Plant and Soil, 227:185 190.
- Goyal, S. S. and H. L. Sehtiya (2002). Evidence for Differential Photo-regulation of in vivo Nitrate Assimilation in Barley (C3) and Corn (C4) Seedlings.Ind. J. Pl. Physiol. 7:1-8.
- Basra, A. S., A. K. Dhawan, and S. S. Goyal (2002). DCMU inhibits in vivo nitrate reduction in illuminated barley (C3) leaves but not in maize (C4): A new mechanism for the role of light? Planta. 215:855-861.
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Progress 01/01/03 to 12/31/03
Outputs
Nitrogen (N) deficiency is perhaps the biggest factor limiting agricultural productivity worldwide. The superior productivity potential of C4 plants relative to C3 has been substantiated and is fact that the leaves of C4 plants have superior metabolic efficiency for photosynthetic carbon (C) and for N assimilation, compared to C3 plants. Role of light in C assimilation is understood, its role in N assimilation is controversial. Some believe that the role of light in N assimilation is via supply of carbohydrates which can then be used as a source of energy and carbon skeletons. Others believe light is obligatory for nitrate assimilation with a direct role in its assimilation. This project was the first to recognize that perhaps both observations were correct depending on the plant species (C3 or C4) used for experimentation. Our focus has been on the comparative regulation in C4 (corn) vs C3 (barley) plants. Corn (C4), relative to barley (C3), consistently assimilated
a significantly greater percentage of nitrate taken up. This project has reported the novel finding that carbohydrate-depleted corn leaves were able to reduce nitrate under photosynthesis-inhibited (DCMU treatment) conditions in the presence of light. This finding has raised some very important questions about the possibilities of a new mechanism by which corn leaves may be able to utilize light energy directly for supporting nitrate reduction without first having to go through photosynthetic CO2 fixation. On the other hand, barley leaves were unable to carry out any in vivo nitrate assimilation, whatsoever, under these conditions. The results also indicate that enzyme capacity was not limiting for nitrate reduction in leaves, as the NR activity was higher in barley than in corn. These observations suggest that in corn (C4) both C and N assimilation (at least partly) processes can be driven directly by light energy whereas in barley (C3) light energy seem to be utilized only for C
assimilation and the assimilated carbohydrates in turn drive N assimilation via glycolytic reactions. The C4 system would be more efficient because it would be able to harvest a greater amount of light energy falling on the leaf by using two separate and dedicated electron flow routes. Professor Surinder Sawhney, a pioneer and internationally known scientist in nitrate assimilation joined this project. We have learned that it is possible for plants to possess alternate and parallel routes of photosynthetic electron transport, which may bye-pass the classical Z scheme of electron flow. Recent reports have shown that bicarbonate ions (CO2 would be required for this) are an essential intermediate in the photosynthetic electron flow from hydrolysis of water. These explanations suggest interesting possibilities that explain the observations discovered in this project.
Impacts
We now feel confident that corn leaves possess a separate, parallel, and perhaps dedicated route of photosynthetic electron flow for N assimilation. This ultimately results in harvesting of larger quantities of light energy. Once the entire pathway for the N assimilation electron flow in corn is understood, we should be able to clone the gene(s) and incorporate them into C3 plants, which would greatly enhance their productivity potential.
Publications
- Sehtiya, H.L. and Goyal, S. S. (2003) Comparative uptake and assimilation of nitrate by excised roots and leaves from seedlings of C3 (barley) and C4 (corn) plants: effect of light, ambient CO2 and exogenously supplied sucrose. Ind. J. Pl. Physiol. 7: 203-210.
- Dhawan, A. K. and Goyal, S. S. (2003) Nitrate Assimilation Efficiency in Excised Leaves of C3 and C4 Species: Role of Photorespiration. Physiology and Biochemistry of Plants. 10:1-6.
- Goyal, S. S. (2003). Is nitrogen assimilation mechanism in C4 plants different than in C3 plants? In: R. P. Singh, N. Shanker, and P. K. Jaiwal (Eds), Nitrogen Nutrition and Plant productivity. Srivastava Society for Science and Society. In Press.
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Progress 01/01/02 to 12/31/02
Outputs
Carbon (C) and nitrogen (N) assimilation are the two most fundamental processes for plant growth and development. The energy for both is provided, directly or indirectly, by light. While the role of light in C assimilation is well understood, a controversy about its role in N (nitrate) assimilation still exists. Some believe that the role of light in N assimilation is via supply of carbohydrates which can be used as a source of energy. Others believe that light is obligatory for nitrate assimilation. This project was the first to recognize that perhaps both observations were correct depending on the plant species (C3 or C4) used. This project has now provided the first evidence for differential photo-regulation of nitrate assimilation in barley vs. corn plants. The plants with C4 metabolism (e.g., corn) are superior producers relative to the ones with C3 pathway. A grain yield of 7-9 tons per acre for corn may be normal whereas most C3 plants cannot produce even close
to that. It is well known that the leaves of C4 plants possess a superior photosynthetic C assimilation as compared to the C3. However, a superior C assimilation alone cannot account for the superior plant productivity, which must be accompanied by superior N assimilation. Hence, the C4 plants must also have a superior N assimilation, the physiology of which unknown. That is the subject and aim of this project. Numerous experiments, targeted on enhancing our understanding of the regulation of nitrate uptake and assimilation, have been conducted. Our focus has been on the comparative regulation in C4 (corn) vs C3 (barley) plants. Corn (C4), relative to barley (C3), consistently assimilated a significantly greater percentage of nitrate taken up. The observation was true even when the nitrate uptake was equated in both species by adjusting the substrate concentration. Therefore, the nitrate use efficiency was always higher in corn relative to that in barley. This project has provided the
first evidence for differential photo-regulation of nitrate reduction in barley vs. corn plants, which may help explain the superior N use efficiency, and hence superior productivity, of corn plants. The novel finding that carbohydrate-depleted corn leaves were able to reduce nitrate under photosynthesis-inhibited (DCMU treatment) conditions in the presence of light, suggests exciting possibilities of a new mechanism by which corn leaves may be able to utilize light energy directly for nitrate reduction. But barley leaves were unable to carry out any in vivo nitrate assimilation under these conditions. Moreover, in barley leaves, nitrate reductase (NR) activity and activation state remained unaffected due to DCMU but both were up-regulated in corn. These observations suggest that in corn (C4) both C and N assimilation (at least partly) processes can be driven directly by light energy whereas in barley (C3) light energy seem to be utilized only for C assimilation and the assimilated
carbohydrates in turn drive N assimilation via glycolytic reactions. Clearly, the C4 system would be more efficient probably because it would be able to harvest a greater amount of light energy falling on the leaf.
Impacts
If and when the entire pathways and contrasts between C4 and C3 are fully understood, we may be able to clone the critical genes and incorporate them into C3 plants. The knowledge resulting from this project is expected to have an economic as well as environmental impact. We expect it to reduce the amount of N-fertilizer currently used in managed cropping systems which in turn would lead to reduced fertilizer costs for farmers while reducing the amount of nitrate leaching into the ground water.
Publications
- S. S. Goyal and Sehtiya, H. L. (2002). Evidence for Differential Photo-regulation of in vivo Nitrate Assimilation in Barley (C3) and Corn (C4) Seedlings. Indian J. Plant Physiology. 7: 1-8.
- Basra, A. S., Dhawan, A. K. and Goyal, S. S. (2002). DCMU inhibits in vivo nitrate reduction in illuminated barley (C3) leaves but not in maize (C4): A new mechanism for the role of light? Planta. 215: 855-861.
- Dhawan, A. K. and Goyal, S. S. (2002) Nitrate Assimilation Efficiency in Excised Leaves of C3 and C4 Species: Role of Photorespiration. Submitted.
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Progress 01/01/01 to 12/31/01
Outputs
The leaves of C4 plants possess a superior metabolic efficiency not only in terms of photosynthetic carbon assimilation, but also in terms of inorganic nitrogen assimilation, when compared to C3 plants. However, the underlying physiological and biochemical mechanisms are not understood and that is the subject and aim of this project. Hundreds of experiments, targeted on enhancing our understanding of the regulation of nitrate uptake and assimilation, have been conducted. Our focus has been on the comparative regulation in C4 (corn) vs C3 (barley) plants, utilizing intact seedlings as well as excised leaves of these two species. Sharp contrasts in the nitrate uptake and assimilation regulatory mechanisms of the two species have been observed. Nitrate uptake was significantly higher in barley as compared to corn but its assimilation was much greater in corn. Consequently, corn, relative to barley, consistently assimilated a significantly greater percentage of nitrate
taken up. The observation was true even when the nitrate uptake was equated in both species by adjusting the substrate concentration. Therefore, the nitrate use efficiency (% assimilated of taken up) was always higher in corn relative to that in barley. In vivo nitrate assimilation efficiency of leaves is dependent on light, but the obligatory presence of light has been debated and its role remains confounded. This problem has not been addressed from the standpoint of C3 vs. C4 nature of the species investigated, which may actually hold the key to resolve the controversy. This project has provided first evidence for differential photo-regulation of leaf nitrate reduction in barley vs. corn plants, which may help explain the superior nitrogen use efficiency (and hence superior productivity) of corn plants. The novel finding that carbohydrate-depleted corn leaves were able to reduce nitrate under photosynthesis-inhibited (DCMU treatment) conditions in the presence of light, raises a
very important question about the possibilities of a new mechanism by which corn leaves may be able to utilize light energy directly for supporting nitrate reduction without first having to go through photosynthetic carbon dioxide fixation. On the other hand, barley leaves were unable to carry out any in vivo nitrate assimilation, whatsoever, under these conditions. We find another fundamental difference between the two species in terms of differential regulation of nitrate reductase (NR; EC 1.6.6.1). In barley leaves, NR activity and activation state remained unaffected due to DCMU, but in sharp contrast, both were appreciably upregulated in corn. Collectively, the results indicate that enzyme capacity is not limiting for nitrate reduction in leaves, as the NR activity was higher in barley than in corn. The corn leaves may have had a selective advantage due to C4 morphology/metabolism in terms of maintaining a better reductant/carbon skeleton supply for nitrate reduction.
Impacts
The knowledge resulting from this project is expected to have an economic as well as environmental impact. We expect it to reduce the amount of N-fertilizer currently used in managed cropping systems which would in turn lead to reduced fertilizer costs for farmers while reducing the amount of nitrate leaching into the ground water.
Publications
- Dhawan, A. K. And S. S. Goyal. 2002. Nitrate Assimilation Efficiency in Excised Leaves of C3 and C4 Species: Role of Photorespiration. Submitted.
- Sehtiya, H. L. and S. S. Goyal. 2002. Evidence for Differential Photo-regulation of in vivo Nitrate Assimilation in Barley (C3) and Corn (C4) Seedlings. Submitted.
- Basra, A. S., A. K. Dhawan, and S. S. Goyal. 2002. DCMU inhibits in vivo nitrate reduction in illuminated barley (C3) leaves but not in maize (C4): A new mechanism for the role of light. Submitted.
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Progress 01/01/00 to 12/31/00
Outputs
Literature shows that the C4 species, e.g., corn, are superior in terms of N utilization in addition to being superior in carbon utilization. However, the physiological and biochemical mechanisms for the superior N utilization in C4 plants is not understood. That is subject and objective of this project. Hundreds of experiments, targeted on enhancing our understanding of the regulation of nitrate uptake and assimilation, have been conducted. Our focus has been on the comparative regulation in C4 corn vs C3 barley plants. We have utilized intact seedlings as well as excised leaves of these two species. Sharp contrasts in the nitrate uptake and assimilation regulatory mechanisms of the two species have been observed. Nitrate uptake was significantly higher in barley as compared to corn but its assimilation was much greater in corn. Consequently, corn, relative to barley, consistently assimilated a significantly greater percentage of nitrate taken up. Our results
showthat etiolated seedlings of both barley and corn were able to uptake and assimilate nitrate in darkness. Hence, light, per se, was not obligatory for either nitrate uptake or assimilation and the carbohydrates supply may meet the requirements of energy supply needed for the uptake in both species. However, photo-regulation of NO3 - uptake and assimilation was different in corn than barley. Corn seedlings when shifted from darkness to light for uptake and in vivo NO3 - assimilation assay, took up and assimilated similar amounts of NO3 - as those held continuously in light and vice-versa. On the other hand, in barley such shifting resulted in NO3 - uptake and assimilation that was somewhat in the middle average of those held continuously in light or darkness. Clearly, NO3 - uptake and assimilation in corn depended on the light conditions only during the assay period, regardless of light conditions during the period preceding the assay whereas, in barley light conditions during both
periods influenced NO3 - uptake and assimilation. This is a significant observation which suggests two distinct possible photo-regulatory mechanisms for the two species: 1, since light was no more important during the assay than during the pretreatment, NO3 - uptake and assimilation in barley was perhaps driven by the overall carbohydrate pool of the seedlings and the role of light was simply to add carbohydrates in that pool via photosynthetic CO2 fixation. 2,whereas in corn, NR was activated by light probably via dephosphorylation process and/or the products of current CO2 fixation played a pivotal role in driving NO3-uptake and assimilation. The current photosynthates in corn could be important because NO3-assimilation occurs only in mesophyll cells which are not capable of carbon fixation and are dependent on bundle sheath cells for the needed carbon skeleton. Products of current CO2 fixation may also stimulate the post-translational activation of NR in corn. These observations
show that corn has a much more efficient regulatory mechanism of nitrate uptake and assimilation than barley which appears to be linked to light.
Impacts
The knowledge resulting from this project is expected to have an economic as well as environmental impact. We expect it to reduce the amount of N-fertilizer currently used in managed cropping systems which would in turn lead to reduced fertilizer costs for farmers while reducing the amount of nitrate leaching into the ground water.
Publications
- Goyal, S. S. and Dhawan, A. K. 1999. Effect of Factors Affecting Photoresporation on Comparative Nitrate Assimilation in Excised Leaves of Barley and Corn. Annual Meeting Abstracts. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. Madison, WI., pp. 87.
- Shetiya, H. L. and Goyal, S. S. 2000. Comparative uptake of nitrate by intact seedlings of barley and corn plants, effect of light and exogenously supplied sucrose. Plant and Soil, 227:185-190.
- Goyal, S. S. and Dhawan, A. K. 2000. Comparative in vivo nitrate assimilation efficiency of excised leaves of barley and corn when challenged with high substrate concentrations. Abstract Supplement. American Society of Plant Physiologists, Rockville, MD. pp. 123.
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