3.3 CRIR1 plays a positive role in plant response to cold stress
To investigate the physiological function of CRIR1 in planta, we conducted genetic transformation experiments forgenerating transgenic cassava lines with knock-down or overexpression ofCRIR1 . Full-length sequences of antisense or sense CRIR1were transformed into cassava friable embryogenic calli under the control of the 35S Cauliflower Mosaic Virus (CaMV) promoter (35S ::anti-CRIR1 and 35S ::CRIR1 ). However, the calli carrying the 35S ::anti-CRIR1construct failed to regenerate into complete plantlets, so the basis for this phenomenon still needs to be investigated. Luckily, eight independent transgenic cassava lines carrying the35S ::CRIR1 construct were obtained and numbered from #1 to #8. The CRIR1 mRNA abundance in these transgenic lines was determined by qRT-PCR analysis. Six independent lines that showed higher expression levels of CRIR1 were obtained: #1-3, #5, #7, and #8 (Figure 3a). Then, the Southern blotting technique was applied to verify the integration events of 35S ::CRIR1 T-DNA in these lines. Two lines, #1 and #5, were detected as single-copy insertion into the genome and therefore selected for further investigation (Figure 3b).
In order to investigate whether overexpression of CRIR1 confers cold stress tolerance, five-week-old wild-type (WT) and CRIR1overexpression (OE) seedlings were incubated in growth chambers at 4°C for 2 days and then transferred to 26°C for 7 days. After the treatment, the WT plants showed withered, dry leaves and could not be recovered till death, whereas the OE seedlings were only slightly affected, with fewer leaves wilting and showed a relatively green and healthy appearance (Figure 3c). Proline, acting as a physico-chemical indicator during cold exposure, has been demonstrated to play an important role in protecting cellular enzymes from denaturation (Li, Yang, Iqbal, Qadri, Shi, Wang, Wu, Fan & Wu, 2019). Cold stress enhanced the accumulation of proline in the WT and OE lines, but the contents of proline in the transgenic lines were significantly higher than that in the wild type (Figure 3d). Malondialdehyde (MDA), which was considered to be an important indicator of cell membrane system injuries and cellular metabolism deterioration, has also been measured as well. Compared with WT, the MDA contents were consistently lower in OE lines (Figure 3e). In addition to morphological and physiological changes, a clear impact on the ultrastructure of chloroplasts membranes was also observed in leaves of cold-treated cassava plants to further assess the degree of cold stress-induced membrane injury (Figure 3f). Before cold treatment, the chloroplasts of leaf mesophyll cells had normal shapes with the typical arrangement of grana and stroma thylakoids and showed no significant differences between the WT and OE line. After cold treatment, the membrane system of chloroplasts in the WT plants showed disintegration with less organized thylakoids, indicating that chloroplasts were destroyed. In contrast, there were no significant alterations in the OE line, except for the appearance of the starch granules in chloroplasts (Figure 3f). These results indicate that CRIR1 plays a positive role in the response of cassava to cold stress through altering the accumulation of protectant metabolites and preventing the membrane system from cell damage under cold stress.