DELIBERACIÓN CONSTITUCIONAL

5.2.9.1 Characterisation of dormancy phenotypes of linc1-2 seeds

A recent study has revealed a potential role for HOS1 as a component of the NPC (Tamura et al., 2010). LITTLE NUCLEI (LINC) are involved in regulating nuclear size and morphology and linc1 and linc2 nuclei are smaller than wild-type (Dittmer et al., 2007). Expression of LINC1, LINC2 and LINC3 is greater in dry seeds matured at 20°C

Cold Stratification Length (days)

0 1 2 3

GerminationFrequency(%)

0 20 40 60 80 100

Col 20°C arp6-1 20°C Col 15°C arp6-1 15°C

Chapter 5: The role of HOS1 in temperature regulation of seed dormancy

in comparison to those matured at 10°C (Kendall et al., 2011). A recent study has shown that dormancy levels are not correlated with nuclei size (van Zanten et al., 2011b). However, the effect of maturation temperature was not analysed in this study and so it is possible that temperature may have an effect on nuclear morphology.

Therefore, the ability to induce dormancy in linc1-2 loss-of-function seeds was tested to determine whether dormancy induction in response to low temperature was

compromised in linc1-2 mutant seeds. Germination of linc1-2 seeds was found to be high in response to maturation at 20°C, and this was higher than wild-type (Figure 5.14A). In this particular assay, germination of wild-type increased to approximately 50% when seeds were matured at 15°C and cold stratified for three days, whereas the germination of linc1-2 seeds remained significantly lower (Figure 5.14B). The

germination of wild-type seeds reached approximately 90% following seven days of cold stratification and secondary dormancy was induced in response to fourteen days of cold stratification or more (Figure 5.14B). In contrast, the germination of linc1-2 seeds was promoted to a maximum of 20% in response to fourteen days of cold stratification and the germination decreased in response to further lengths of cold stratification (Figure 5.14B). Together these results suggest that LINC1 may act as a negative regulator of dormancy in response to low temperature.

Chapter 5: The role of HOS1 in temperature regulation of seed dormancy

Figure 5.14: Germination of linc1-2 seeds.

Freshly harvested seeds matured at 20°C (A) or 15°C (B) were cold stratified for up to three days (A) or twenty eight days (B). Germination was scored as radical protrusion following seven days at 22°C in 12 hour white light/dark cycles. Data points are the average of five individual seed batches and error bars represent standard error.

5.2.9.2 Characterisation of nuclear volume in embryo and endosperm cells 5.2.9.2.1 Measurements in wild-type seeds

Following on from the observation that linc1-2 mutant seeds show increased dormancy in comparison to wild-type when matured at low temperature (Figure 5.14B), the prediction that alterations to nuclear size could be part of the mechanism by which temperature regulates dormancy was investigated. Very little is known about how nuclear size and shape is regulated. Studies on fission yeast reveal that nuclear size is independent of DNA content but is highly proportional to cell size (Neumann and Nurse, 2007). DAPI was used to stain nuclei in wild-type embryo and endosperm cells from seeds matured at 20°C and 15°C and hos1-3, hos1-4 and hos1-5 embryo and

endosperm cells from seeds matured at 15°C. Nuclear volume was calculated using 1D images and it was assumed that nuclei were spherical and so, the calculations are estimates.

Chapter 5: The role of HOS1 in temperature regulation of seed dormancy

The results showed that nuclei volume was unchanged by maturation temperature in embryo cells (Figure 5.15A, 5.16). In endosperm cells, nuclei from seeds matured at low temperature were significantly larger than nuclei from seeds matured at warm temperature (Figure 5.15B). Additionally, a comparison was made between nuclei circumference and cell circumference to determine if a positive correlation is present, as suggested by Neumann and Nurse (2007). The results show that there is no positive correlation between embryo nuclei circumference and embryo cell circumference in this experiment (R²=0.160) (Figure 5.17). Therefore, the results suggest that nuclei volume is not regulated by maturation temperature in embryo cells. However, in endosperm cells temperature did have an effect on nuclei size.

Figure 5.15: Measurements of nuclear volume.

Nuclear volume was calculated in DAPI stained wild-type and hos1 mutant embryo root tip cells (A) and endosperm cells (B). Data points are the average of approximately two hundred nuclei from five biological replicates and error bars represent standard error. * Indicates significant difference to wild-type when P≤0.05 by students t-test.

Col20

Chapter 5: The role of HOS1 in temperature regulation of seed dormancy

5.2.9.2.2 Measurements in hos1 seeds

Since a potential role for HOS1 as a component of the NPC has been proposed (Tamura et al., 2010), it was possible that the hos1 mutation was compromising the NPC and so it was hypothesised that nuclear volume could be altered in hos1 seeds.

Although the results showed no changes to nuclei volume by temperature in wild-type embryo cells, there were alterations in endosperm cells (Figure 5.15A, B). The data presented in Figure 5.5 show that the low dormancy phenotype of hos1 seeds is temperature independent and, therefore, it may not be surprising that the mechanism by which HOS1 acts through doesn’t show changes in response to temperature. When nuclei volume was measured in hos1 seeds that were matured at 15°C, the findings showed hos1-3 and hos1-4 embryo cells contained larger nuclei, but the difference was only significant for hos1-3 (Figure 5.15A, 5.16). The nuclei of hos1-5 cells were not different to wild-type. The situation with endosperm cells was different to embryo cells, with hos1-4 nuclei being larger than wild-type and hos1-5 nuclei being smaller, but these differences were not significant (Figure 5.15B). There was no difference in size of hos1-3 nuclei in comparison to wild-type. Together, these results show that there are alterations in nuclear volume in the hos1 mutants, but these aren’t consistent for all three alleles. For this reason, the mechanism through which HOS1 affects seed dormancy is unlikely to involve the regulation of nuclei volume.

Chapter 5: The role of HOS1 in temperature regulation of seed dormancy

Figure 5.16: DAPI staining of embryo nuclei in root top cells.

Embryo root tip cells from Col matured at 20°C (A), Col matured at 15°C (B), hos1-3 (C), hos1-4 (D) and hos1-5 (E) matured at 15°C. Scale bar represents 5µm.

Figure 5.17: Comparison of nuclei size and cell size.

Nuclei and cell circumferences measured in DAPI stained Col embryo root tips.

5.2.10 Characterisation of dormancy phenotypes of