Acknowledgements

We thank CNPq/MCT/PRONEX for financial support.

MOLECULAR EVOLUTION OF URICASE II GENES AND CHARACTERIZATION OF TRANSGENIC LOTUS JAPONICUS CONTAINING ANTI-SENSE URICASE GENES

S. Tajima1, K. Shimomura1, M. Nomura1, K. Takane2, H. Kouchi2

'Dept Life Sci., Fac. Agri, Kagawa University, Miki-cho, Ikenobe, Kagawa, Japan 2Natl Inst. Agrobiol. Res., Kannondai, Tsukuba 305-8602, Japan

1. Introduction

Uricase (Nodulin 35) gene expression in uninfected cells of root nodules of tropical legumes like Glycine max, Phaseolus and Vigna has been studied as a typical case of symbiotically enhanced gene expression contributing to assimilation of fixed nitrogen. Uricase expression itself can be observed in various legume tissues, because the reaction introduces irreversible degradation of purine bases and the activity of very low intensity was detected in all senescent or dividing plant cells for recruiting nucleic acid-N. However, the molecular mechanism and physiological role of uricase over-expression that may be specific in tropical legumes is still obscure. In this report, we tried to identify the profile of the significance of uricase gene activation in legumes.

2. Materials and Methods

To analyze the mechanism, we compared the uricase gene structure and the expression profiles (in situ hybridization) after cDNA and genomic cloning of uricase genes from Glycine max (Takane et al. 1997), Medicago sativa (Cheng et at. 2000) and L. japonicus (Takane et al. MPMI2000).

In order to analyze the metabolic role of uricase gene in Lotus japonicus GIFU, we cloned the uricase gene from a cDNA library of Lotus japonicus nodules and introduced an anti-sense uricase gene for decreasing uricase content in uninfected cell of the nodules. Using an Agrobacterium tumefaciens-mediated gene transfer system, we have obtained over 20 independent transgenic plants. Southern blot analysis showed that these plants had introduced anti-sense uricase construct.

3. Results and Discussion

The result shows that legume uricases are divided into two major groups. Group-I is comprised of soybean, Vigna, and Phaseolus, which are well demonstrated to bear ureide-transporting determinate legumes (Streeter 1991). Canavalia bearing determinate nodules, which is also described as an ureide transporter (Kim and An 1993), is close to but distinct from group-I. Group-H is comprised of alfalfa, pea, Astragalus, and Lotus japonicus. Alfalfa and pea are well known as amide-transport legumes (Streeter 1991).

When we surveyed the gene expression by in situ hybridization technique, the data revealed that alfalfa uricase genes were expressed in uninfected cells of nodule tissue in small extent, and in Lotus japonicus nodules the magnitude was higher. The data suggested that cell type specific expression was observed with identical manners to that of Nod-35 in soybean nodules in various legumes, although the magnitude of the expression was much higher in Nod-35 in soybean nodules. The data also showed that enhancement of uricase gene expression was observed not only in tropical legumes but also in various non-ureide determinate type nodules.

An anti-sense RNA experiment using transgenic Lotus japonicus plants suggested uricase gene expression would influence to carbon metabolism of the plants because the transgenic plants were dwarfed under low light and grew normally under natural light. We also found that all of these transgenic plants grow more slowly than non-transgenic plants. After infection with M. loti, all of these transgenic plants were nodulated well.

A MODEL OF ADENYLATE GRADIENTS IN THE INFECTED CELLS OF LEGUME NODULES

H. Wei1, P.P. Thumfort2, C.A. Atkins2, D.B. Layzell1

'Biology, Queen's U, Kingston, ON, Canada K7L 3N6 2Botany, U Western Australia, Nedlands, WA 6009, Australia

1. Introduction

In metabolically active legume nodules, the adenylate energy charge (AEC = [ATP + 0.5ADP] / [ATP + ADP + AMP]) is low (0.70) compared with other aerobic tissues (typically > 0.80). Nonaqueous fractionation of soybean nodules showed that the low AEC was due to the plant fraction (0.66), not the bacteroids (0.76) (Kuzma et al. 1999). However, an O2 limitation of nodule metabolism reduced bacteroid AEC to 0.56 but had no effect on that of the plant fraction. Therefore, the bacteroids were O2 limited while the plant fraction was not. To explain why the plant fraction AEC was so low (0.66) when the oxygen supply to it was sufficient, we hypothesized that there were steep ATP gradients from the mitochondrial region near the gas space (i.e. the site of ATP synthesis) to the cytosol surrounding the symbiosomes. A mathematical model was developed to test this hypothesis.

2. Methods

The model assumed that the infected cells were tightly packed rhombic dodecahedra with intercellular gas spaces along the edges, from which O2 diffused to the center of the cell. The Thumfort et al. (2000) model was modified to predict the diffusion and gradients of O2 and ATP across the cell, assuming cytosolic ATP demand for NH<t+ assimilation, malate transport and plant growth + maintenance. The model was also used to simulate the effects of an Ar:02 atmosphere (5-7 min exposure), where the demand for NH/ assimilation was assumed to be zero.

3. Results and Discussion

The model supported the hypotheses, predicting that Fjgurel. Predicted adenylate gradients glutamine synthetase activity in the cytosol was largely m the bacteria infected cells, responsible for generating a gradient in plant AEC of 0.84

near the space to 0.55 near the center of the cell to achieve an average AEC of 0.66 (Figure 1). Under Ar:02, the adenylate gradients were predicted to be significantly reduced, resulting in an average plant fraction AEC of 0.71-0.75 (Figure 1). This prediction was consistent with previous studies of adenylates in whole nodules (deLima et al. 1994), and will be tested using non-aqueous fractionation of control and Ar:02 treated nodules.

4. References de Lima et al. (1994) Physiol. Plant. 91, 687-695 Kuzma et al. (1999) Plant Physiol. 119, 399-407 Thumfort et al. (2000) J. Theor. Biol. 204, 47-65

Predicted adenylate gradients across the infected cell mM r

in N2:02 in Ar02

400 200 0

Layer number

0 " "TO ~20 30 Distance from interface (um)

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