The above-mentioned complementation lines were tested sequentially in three independent hyponastic growth experiments (Figure R25 and SR9, part I to III). While wildtype plants initiated hyponastic growth under low light conditions, and lowered their leaves when shifted back to control light conditions, tga1 tga4 mutants were compromised in this response. Similar effects were observed for the control complementation lines tga1 tga4 +
167 Figure R25. Analysis of hyponastic growth of the TGA1 complementation lines. Selected complementation lines of the tga1 tga4 mutant were grown together with the tga1 tga4 mutant and Col-0 wildtype plants for 4 weeks in 12 h day and then treated with CL, LL or first with LL followed by CL, as described in Methods (page 59). Pictures of the plants were taken with a Canon PowerShot G11 camera. Angles of leaf 7 or 8 were measured using ImageJ. Bars are mean ± SEM from at least 6 replicates. (A), (B), and (C) represent three independet experiments. CL – control light, LL – low light, LL to CL – shift from low light to control light. tga1 tga4 corresponds to tga1 tga4 + pB1xHA-TGA1g #22-7, which lost its transgene during segregation as a heterozygous plant.
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pB1xHA #10-3, #11-2, and #21-9. In contrast, tga1 tga4 mutants complemented with 1xHA-TGA1g or 1xHA-TGA1g C172N C260N C266S C287S showed a hyponastic response similar to the wildtype.
Complementation seemed sometimes partial (Figure R25B and C), which could be attributed to a reduced functionality of the resulting proteins or to differences in expression. In summary, it seemed that both, 1xHA-TGA1g and 1xHA-TGA1g C172N C260N C266S C287S-complemented the hyponastic growth deficit of the tga1 tga4 mutant, suggesting that the cysteine residues in TGA1 do not play a role during regulation of hyponasty.
To further substantiate these findings, the petiole length relative to the entire leaf length was determined. As shown in Figure R26 and SR10, Col-0 plants had petioles almost half as long as the entire leaf (ca. 49 %). The petioles of tga1 tga4 mutants and the internal control plant (tga1 tga4 + pB1xHA-TGA1g #22-7) were only 40 % as long as the whole leaf. This was also observed for the control complementation lines expressing only the 1xHA tag. When 1xHA-TGA1g or
Figure R26. Measurement of the relative petiole length of the TGA1 complementation lines. Col-0, tga1 tga4, a complementation line (TGA1g) from the Department of Botany (Prof.
Dr. Yuelin Zhang, UBC Vancouver [222,223,262]) and selected complementation lines constructed during this work were grown for 4 weeks in 12 h day. Either leaf 7 or 8 from at least 6 different plants was detached and photographed from the top with a Canon PowerShot G11 camera. The petiole length and the blade length were measured using Image J and the petiole length relative to the entire leaf length computed. Bars represent mean ± SEM.
Statistical analysis (One-way-ANOVA and Tukey test) was performed using GraphPad Prism 8.0.1 assuming normally distributed data. Adjusted p-values in comparison to Col-0 are indicated above the bars: ns – non-significant, p>0.05, * - p ≤ 0.05, ** - p-value ≤ 0.01, *** - p-value ≤ 0.001, **** - p-value ≤ 0.0001.
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1xHA-TGA1g C172N C260N C266S C287S were expressed in tga1 tga4, the resulting relative petiole length was in between that of the tga1 tga4 mutant and that of Col-0. However, this appeared to be only significant for two out of three 1xHA-TGA1g C172N C260N C266S C287S complementation lines. Including additional plants into the analysis revealed that both, 1xHA-TGA1g and TGA1 cysteine mutant transformants, varied in their capacitiy to complement the reduced petiole length of the tga1 tga4 mutant (Figure SR10). However, for the cysteine mutant complementation construct, more plants with a significantly reduced petiole length were observed than for the 1xHA-TGA1g complementation construct. Still, the differences in the petiole length between both types of complementation lines were not strong. Altogether, this might suggest only a partial complementation by both constructs. Thus, the complementation constructs might either not result in fully functional proteins or TGA1 might be redundant with TGA4 in controlling the petiole length. All in all, TGA1g seems to complement the phenotype independent of its cysteine residues at least partially.
In addition to controlling hyponasty and petiole length, the tga1 tga4 mutant was described to flower late [223]. Since this phenotype resembles the delay in flowering observed for ROXY9 overexpressing plants, it seems possible that ROXY9 represses TGA1 also during the induction of flowering [157]. Thus, the number of days, wildtype plants, tga1 tga4 mutants, and the internal control line TGA1g needed to start flowering was quantified along with those of the complementation lines generated for this study. Additionally, the number of leaves on the day of flowering was counted. The data is depicted in figures R27 and SR11, part I and II.
Col-0 flowered after ca. 35 days in this experiment and had ca. 19 leaves on the day of flowering. The tga1 tga4 mutant flowered three days later and had more leaves than the wildtype. As for the ROXY9 overexpressing plants, the formation of numerous smaller leaves among the counted leaves were observed. This indicates, that the late flowering phenotype of the tga1 tga4 mutant is not a result of a generally delayed development. However, the internal control line (#22-7) neither flowered significantly later than wildtype plants nor showed an increased leaf number. Similarly, only two out of three plants expressing the 1xHA tag showed a delay in flowering, and only one of them formed significantly more leaves. Consequently, flowering might be influenced by additional effects resulting e.g. from the construction process of these transgenic lines. Alternatively, the sample size used in this experiment might not have been high enough to clearly distinguish the small differences in the
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time point of flowering and leaf number between wildtype-like plants and tga1 tga4 mutant-like plants. This is supported by the observation of tendencies even in case of statistically insignificant differences, and the formation of additional, smaller leaves by tga1 tga4 + pB1xHA-TGA1g #22-7, and those plants expressing 1xHA. Despite these discrepancies for some control plants, the TGA1g complementation construct seemed to successfully restore the initiation of flowering after ca. 35 days. Interestingly, this was only observed for two of the three cysteine mutant complementation lines tested. The other transformant flowered significantly earlier than the wildtype. However, the leaf number did not differ from wildtype leaves for TGA1g- and TGA1g C172N C260N C266S C287S-complementation lines. Analysis of additional plant lines confirmed these results (Figure SR11, part I and II).
Considering the influence of additional factors and the small sample size, it seems that the tga1 tga4 mutant flowers later than Col-0 but is not generally delayed in development. Moreover, the 1xHA-TGA1g seems to complement the phenotype of the tga1 tga4 mutant. Similarly, the TGA1g complementation construct carrying the cysteine mutations complements the phenotype, and sometimes even causes an earlier flowering. However, because neither the TGA1 protein levels nor the influence of the tag and the effect of the cysteine mutations on the stability of TGA1 were analyzed in planta, the cause of this observation cannot be evaluated.
All in all, the results from analyzing hyponastic growth contrast with a redox regulation of TGA1 regarding hyponasty [155] whereas for the flowering timepoint, a minor influence of the cysteines cannot be ruled out.
171 Figure R27. Flowering time point and leave number at flowering of TGA1g complementation lines. Col-0, tga1 tga4, TGA1g [222,223,262] and tga1 tga4 mutants complemented with the constructs shown in Figure 24A were grown in a long day climate chamber. The number of days was determined until a floral stem of 1 cm lenght was visible (A). At this time point, the number of leaves was counted, as well (B). Bars are mean ± SEM from at least 5 replicates. Statistical analysis (One-way-ANOVA and Tukey test) was performed using GraphPad Prism 8.0.1 assuming normally distributed data. Adjusted p-values in comparison to Col-0 are indicated above the bars: ns – non-significant, p>0.05, * - p ≤ 0.05, **
- p-value ≤ 0.01, *** - p-value ≤ 0.001, **** - p-value ≤ 0.0001. a - smaller leaves in between counted leaves for all replicates.
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Discussion
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