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.