The ATPase function of GET3B is responsible for the growth phenotype
In order to investigate the restorative effects of GET3B expression in the alb4 srp54 get3b triple mutant, we complemented the triple mutant by transfecting the plants with a construct previously used (Anderson et al., 2021). In the T2 generation, 12 independent lines were identified carrying the ProUB10:GET3B-STREP transgene. Data are shown for 3 randomly chosen lines (Figure 5B-C, G). The rosette diameters of the three lines were significantly smaller (lines 2,3) as well as larger (line 1) than the alb4 srp54 double mutant (Figure 5A-C, G). However, in all of the lines, rosette diameters were more than three times larger than the alb4 srp54 get3b triple mutant, signifying that the transgene was able to rescue the triple mutant phenotype (Figure 5G, H).
To determine if the molecular complementation was due to the targeting or holdase function of GET3B, we introduced GET3B transgenes with D124N mutations into the alb4 srp54 get3b background (Figure 5D-F). These have also been previously used (Anderson et al., 2021) to eliminate the ATPase activity of GET3B (Mateja et al., 2009b). In the T2 generation 7 independent lines were identified that contained theProUB10:GET3B (D124N)-STREP transgene. Data are shown for 4 randomly chosen lines. One of the lines (line2) displayed a significantly larger rosette diameter than the triple mutant but all of the lines were approximately three times smaller than the alb4 srp54 double mutant (Figure 5D-H). Overall, the transgene with a D124N mutation was unable to rescue the alb4 srp54 get3b phenotype, illustrating that the ATP hydrolysis activity is crucial for complementation (Figure 5H).
To further investigate the functional consequences of the WT GET3B transgene and the ATP hydrolysis variant, we transfected these constructs into alb4 or stic2 backgrounds. In the T2 generation, we observed distinct phenotypic differences between the untransfected and transgenic lines (Figure 6). Transfection of the WT GET3B transgene into stic2 backgrounds did not result in significant phenotypic differences compared to stic2 plants (Figure 6C, D, G). In contrast, transfection of the ATP hydrolysis-deficient GET3B variant into the alb4 background led to a significantly smaller plant size in one of two randomly chosen lines (Figure 6E, H). This effect was more pronounced when the transgene was transfected into a stic2 background. Both of the two randomly chosen lines showed highly significant reductions in rosette diameter when compared to stic2 plants (Figure 6F, H).
These findings show that the loss of ATP hydrolysis of GET3B in conjunction with impairments of the STIC pathway result in stunted growth. This clearly illustrates the importance of both functioning pathways for normal plant growth and chloroplast function.