Expression levels of sucrose synthase and sugar transporters in roots and

VS

Figure 3.15. In situ staining of glucose and acid invertase activity in Medicago nodules show background in infected cells. Staining of root nodules for glucose (A) and invertase activity (C) in complete reaction mixtures led to the accumulation of brown precipitate in the infected cells in (C). Control staining without glucose oxidase for glucose (B) and invertase activity (D) led to similar amounts of brown precipitate in (D), but not in (B). For nodule structure see Figure 1.2. M – meristem; N – nitrogen-fixation zone; VS – vascular system.

3.3. Expression levels of sucrose synthase and sugar transporters in roots and nodules

Sucrose cleaving enzymes are regulated on the transcriptional and post-translational level, while for sugar transporters, so far only transcriptional regulation has been shown (Winter and Huber, 2000; Krausgrill et al., 1998; Ehneß and Roitsch, 1997; Truernit et al., 1996; Büttner et al., 2000). To understand which control mechanisms are employed to regulate sugar partitioning in nodules and roots, expression levels of SuSy and sugar transporters were examined by RNA gel blot hybridization analysis. Invertase expression could not be analysed this way, since invertases are encoded by large gene families with rather divergent sequences (see e.g. Godt and Roitsch, 1997; Tymowska-Lalanne and

Kreis, 1998), the characterization of which was not possible within the scope of this project.

3.3.1. Expression levels of sucrose synthase (RNA gel blot hybridization analysis)

Expression levels of sucrose synthase were compared in roots, nodules and leaves of the three model plants by RNA gel blot hybridization. The results are shown in Figure 3.16. In order to maximize the chance to detect all members of the sucrose synthase gene family, Medicago RNA was hybridized with a heterologous probe, namely a SuSy cDNA fragment from another legume, Vicia faba (Küster et al., 1993). Datisca and Casuarina RNA gel blots were hybridized with homologous cDNA probes (Wabnitz, 1998), since no cDNAs from close relatives were available.

In Medicago, SuSy expression was strongly increased in nodules compared to roots, while in Datisca, the expression of SuSy was only slightly induced in nodules compared to roots. The RNA gel blot hybridization results of Casuarina showed the opposite results: less SuSy transcripts were detected in nodules than in roots (Figure 3.16).

D a t i s c a glomerata

SuSy 28S

Figure 3.16. SuSy gene expression levels in Medicago, Datisca and Casuarina roots (R), nodules (N) and leaves (L). 28S rRNA served as a control for the amount of RNA per slot.

These experiments were performed at least three times. One representative result is shown.

No expression of SuSy was detected in Medicago leaves, and very low amounts of SuSy transcripts were detected in Datisca leaves. The lack of detectable SuSy transcripts in leaves is normal for source organs, where sucrose is synthesized, and not degraded (Sturm and Tang, 1999). However, the enzyme has also been localized in the sieve tube-companion cell complex of source leaves (Nolte and Koch, 1993), where it appears to be involved in the hydrolysis of a small proportion of the incoming sucrose to maintain a

proton gradient across the plasma membrane of companion cells for phloem loading (Lerchl et al., 1995). The relatively high expression levels of SuSy in Casuarina leaves are due to the fact that morphologically, these "leaves" represent branchlets with the true leaves reduced to tiny scales, i.e. mixed leaf and stem material. The role of SuSy in stems is not completely clear, because only hypocotyls of radish, sunflower and cotton were analysed with respect to SuSy (Rouhier and Usuda, 2001; Kutschera and Heiderich, 2002; Ruan et al., 1997). In sunflower hypocotyls the SuSy activity was implicated in cellulose synthesis (Kutschera and Heiderich, 2002) as it had already been shown for developing cotton seeds (Ruan et al., 1997). In hypocotyls of radish, high levels of SuSy were found in companion cells of the phloem (Rouhier and Usuda, 2001), where besides its potential function in the production of precursors for respiration, SuSy could also play a role in callose accumulation, and therefore in the regulation of both cell-to-cell and long-distance transport in plants. However SuSy protein was also found in the xylem parenchyma and some cortical cells, where its role remains unclear (Rouhier and Usuda, 2001).

3.3.2. Levels of sucrose synthase protein (Protein blot analysis)

Enzyme activity can be regulated both on the transcription level and posttranscriptionally.

SuSy is extensively regulated on all levels, including reversible protein phosphorylation and interaction with the actin cytoskeleton (Winter and Huber, 2000). The finding that similar levels of SuSy enzyme activity were found in roots and nodules of Medicago and Datisca, while the levels of SuSy mRNA were different in roots and nodules of all three model plants, raised the question of how SuSy activity is regulated at the posttranscriptional level.

glauca Figure 3.17. Immunodetection of sucrose synthase in total protein extracts from roots (R) and nodules (N) of Medicago, Datisca and Casuarina, separated by PAGE (2.24-2.25). 15 µg of total protein were applied per slot.

This experiment was performed three times. One representative result is shown.

The amounts of SuSy were analysed by the protein blot analysis of total protein extracts from roots and nodules (2.25) from the three plant systems with an antibody raised against SuSy from cotyledons of Vicia faba (Ross and Davies, 1992; kindly provided by N. Hohnjec, University of Bielefeld). The results are shown in Figure 3.17.

Increase of SuSy protein levels in nodules compared to roots could be found only in nodules of Datisca, which contain a single immunoreactive protein of approximately 92-kDa. For Casuarina glauca, also a single immunoreactive protein of the same mass was detected, but the amounts of SuSy were reduced in nodules compared to roots. So, for Datisca and Casuarina the amounts of protein are in accordance with mRNA expression levels. However, in Medicago nodules, only a slight increase in the amount of the immunoreactive 92 kDa protein was detected compared to roots. This does not reflect the mRNA levels, but is in accordance with SuSy enzyme activities in both organs (Figure 3.12A). A second immunoreactive protein of slightly higher molecular weight was detected in extracts of uninfected roots of Medicago, which is consistent with the results obtained by Hohnjec et al. (1999).

3.3.3. Expression levels of sugar transporters in roots, nodules and leaves

The organ-specific expression of sucrose and hexose transporters was analysed by RNA gel blot hybridisation in roots, nodules and leaves of the three model plants. For the sucrose transporter of Medicago, a heterologous probe from Vicia faba was used (Weber et al., 1997), while a homologous probe was used for the hexose transporter of Medicago (Wabnitz, 1998). The expression levels of the sucrose transporter (MtST) were strongly reduced in nodules compared to roots, and the expression levels of the hexose transporter (MtHT) in nodules were below the detection limit (Figure 3.18). For Datisca only homologous probes were used (Wabnitz, 1998). Here, the expression of the hexose transporter was strongly induced in nodules compared to roots, while sucrose transporter expression was below the detection limit in both nodules and roots as well as in leaves (Figure 3.18; data not shown for the sucrose transporter). For Casuarina, only hexose transporter expression was examined since no sucrose transporter cDNA fragment could be amplified by PCR with the degenerate primers that had been used successfully for Datisca (Wabnitz, 1998). Casuarina hexose transporter expression was strongly induced in nodules compared to roots, as in Datisca (Figure 3.18). Interestingly, in both Casuarina and Datisca, hexose transporter expression levels were very low in roots, while in Medicago roots high expression levels of both hexose and sucrose transporters were found, indicating that root sugar partitioning mechanisms differ significantly between the legume Medicago and the two actinorhizal plants.

D a t i s c a glomerata

28S ST HT

Figure 3.18. Sucrose transporter (ST) and hexose transporter (HT) gene expression in Medicago, Datisca and Casuarina roots (R), nodules (N) and leaves (L). 28S rRNA serves as a control for the amounts of RNA per slot. These experiments were performed at least three times. One representative result is shown.