Materials and Methods

Alfalfa plants were grown in the glasshouse as previously described by Vance et al. (1979).

Procedures described by Gregerson et al. (1994) were used to isolate the alfalfa ne- and cMDH

genes. Comparable Medicago truncatula homologs were isolated by screening a M. truncatula BAC library (courtesy of Nevin Young) with radiolabeled M. sativa ne- and cMDH cDNAs (Miller et al. 1998). In situ hybridization and immunolocalizations were performed as described (Trepp et al. 1999a, 1999b). Chimeric c- and neMDH promoter::GUS reporter constructs were prepared and transformed into alfalfa according to Yoshioka et al. (1999). Plants overexpressing neMDH were developed essentially as described by Schulze et al. (1998) and Schoenbeck et al. (2000), except constructs were in the sense orientation.

3. Results

Protein immunoblots and RNA blots were used to evaluate expression of c- and neMDH. Similar to our previous report (Miller et al. 1998), cMDH protein and mRNA were expressed rather uniformly in all tissues and were unrelated to root nodule formation. By contrast, neMDH protein and mRNA were most highly expressed in effective root nodules, with maximum expression associated with effective nodule development.

An alfalfa genomic library was screened with P-labeled c- and neMDH cDNAs and two full-length genomic clones were isolated which encoded c- and neMDH, respectively. The alfalfa cMDH and neMDH were sequenced and 2 kb of the 5' upstream putative promoter regions defined for each. Cytosolic MDH was comprised of seven exons interrupted by six introns. This gene structure was conserved in the Arabidopsis genome, however, introns in Arabidopsis were much smaller than those in alfalfa. Preliminary sequencing of a Medicago truncatula cMDH showed that intron-exon structure and sequence similarity was very conserved between alfalfa and M. truncatula. By comparison, alfalfa neMDH contained only one intron and it was located in the 5'-untranslated region of the gene. Again intron structure was conserved between alfalfa, M. truncatula, and Arabidopsis.

The promoter region of cMDH and neMDH were translationally fused to (3-glucuronidase (cMDH::GUS; neMDH::GUS) to form a reporter construct and transformed into alfalfa. Both reporter genes directed GUS activity to root nodule but the pattern of staining varied. While neMDH::GUS plants showed staining throughout the nodule, cMDH::GUS nodules stained primarily in the nodule meristem and invasion zone. Interestingly, leaf stomata of cMDH::GUS plants showed intense staining while leaf stomata of neMDH:: GUS plants did not stain.

Protein immunolocalization and RNA in situ hybridization were used to evaluate protein and transcript location at the cellular level. Protein immunolocalization suggested that neMDH was localized in both infected and uninfected cells of root nodules. The most intense staining was at the periphery of infected cells and in amyloplasts of uninfected cells. Whereas, the most intense staining for cMDH appeared to be in uninfected cells of the nodule interior and inner cortex. Similar to protein localization, transcripts for neMDH were found primarily in the infected cells of the N2-fixing zone in nodules while those for cMDH were most apparent in the nodule meristem and inner cortex.

Transgenic alfalfa plants were produced that overexpress neMDH by fusing the CaMV35S promoter to the neMDH cDNA and transforming plants with Agrobacterium tumefaciens. Several independent transformants were evaluated for overexpression of neMDH through activity analysis and protein immunoblots. Two plants with greatest neMDH expression in roots were evaluated for N2 fixation using 15N2. Both plants (neMDH 10-20 and neMDH 16-27) had increased nodule efficiency as measured by 15N incorporation mg nodule"1 h"1. Nodule efficiency was increased more than 10%. However, little effect was seen on plant dry matter accumulation.

4. Discussion

We have extended the understanding of malate in root nodule symbiosis by showing c- and neMDH are encoded by separate distinct genes, and the promoters for these genes target expression to different cell types. The localization or targeting to different cell types was confirmed and strengthened by immunolocalization and in situ hybridization. The fact that neMDH appears to be localized to the symbiotic zone, particularly infected cells, suggests that this isoform is involved with providing malate to bacteroids and malate for amino acid synthesis. Initial subcellular localization of neMDH to plastids in the infected cell zone, along with the localization of aspartate aminotransferase and NADH-glutamate synthase to plastids (Robinson et al. 1996; Trepp et al. 1999a) provides good evidence for neMDH's involvement in nodule amino acid synthesis. This would be consistent with earlier radiolabeling studies showing incorporation of nodule fixed 14CC>2

and 14C-glutamate into aspartate (Rosendahl et al 1990). Likewise, rapid labeling of bacteroids by nodule fixed 14CC>2 and 14C-malate support a role for ne-MDH in providing bacteroids with energy.

The localization of cMDH protein to uninfected cells of the nodule inner cortex and N2-fixing zone along with the similar distribution pattern seen for nodule forms of carbonic anhydrase (CA) (Galvez et al. 2000) and phosphoenolpyruvate carboxylase (PEPC) (Pathirana et al. 1997) place the requisite carbon metabolism enzyme involved in osmoelectrical contraction of cells in the variable oxygen diffusion barrier. Moreover, the role of MDH, PEPC and CA in stomatal aperture and pulvinal function is well documented (Assman 1999). Consistent with an osmoelectrical function, the promoter of cMDH targets reporter gene expression to both leaf and stem stomata as well as the nodule meristem and inner cortex. Taken inclusively, our data demonstrate exquisite, independent cellular functions for neMDH and cMDH.

The unusual kinetics and high turnover rate for neMDH (Miller et al. 1998) made overexpression of this gene a potential target for improving N2 fixation. With plants overexpressing neMDH we did find an increase in nodule N2 fixation efficiency based upon 15N incorporation. However, there appeared to be little effect on total plant growth and N accumulation because transgenic plants produced fewer nodules. Similar types of molecular compensation have been noted when nodule aspartate aminotransferase was altered in alfalfa (Farnham et al. 1992).

Accompanying enhanced nodule efficiency in transgenic MDH plants was improved resistance to aluminum (Al) toxicity (Tesfaye et al. 2001). This increased Al resistance was attributed to enhanced synthesis of malate. This finding is consistent with enhanced resistance to Al in wheat and tobacco being related to higher amounts of malate and citrate, respectively, being released from roots.

From the studies reported here it is apparent that malate plays a pivotal role in legume N2 fixation and growth.

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