**5.1 Characterization of chilling-induced tolerance traits in soybean**

We compared nodule structure, carbon/nitrogen interactions and respiration in PAN, which is chilling-sensitive and in HT, which is more chilling-resistant (van Heerden et al., 2008). Under optimal growth conditions, SNF began to decline after 9 weeks in PAN nodules and after 11 weeks in HT. A transcriptome analysis was performed on PAN and HT nodules harvested from plants at 3 until 15 weeks after germination. Of the genes that showed the same developmental pattern in both varieties transcripts encoding a cysteine proteinase gene (Gma.8481.1.S1\_at) that belongs to a subgroup a vacuolar processing enzymes (legumains) was up-regulated in the senescent nodules. Nodule numbers were unaffected by dark chilling in both genotypes. The abundance of the nitrogenase and leghemoglobin proteins was not changed as a result of dark chilling but nodule respiration rates, nitrogenase activity and NifH and NifK mRNAs were decreased while nodule starch, sucrose and glucose were increased. Chilling-induced decreases in nodule respiration continued in PAN nodules after return to optimal temperatures but respiration recovered in HT by the end of the chilling period. This recovery was associated with a large decrease in the area of the intercellular spaces in the nodule cortex and infected zone in HT. This acclimatory response was not seen in PAN nodules. We conclude that the ability to regulate the oxygen diffusion barrier is an important component of ability of nodules to tolerate stress (van Heerden et al., 2008). The HT nodules were able to regulate both respiration and the area of the intercellular spaces during chilling and so control the oxygen diffusion barrier. We conclude that chilling-induced inhibition of SNF in PAN nodules was caused by the inhibition of respiration coupled to the failure to regulate the oxygen diffusion barrier effectively (van Heerden et al., 2008). Furthermore, the stress-induced limitations in SNF make an important contribution the greater chilling-induced inhibition of photosynthesis in PAN than HT.
