Differential gene expression in germinating and thermoinhibited achenes of Tagetes minuta L.
When imbibed at their optimum germination temperature of 25°C, achenes of Tagetes minuta L. germinate over a period of approximately 48 h. At temperatures of between 35°C and 39°C, the achenes do not germinate but enter into a state of thermoinhibition. These supra-optimal conditions do not harm the achenes, however, and when the temperature is reduced below 35°C radicle emergence may be observed within 4 h. Achenes which have been thermoinhibited for periods of 24 h or more show "accelerated germination" which takes only 24 h, although the actual germination curve is identical to that of normally germinated achenes. This suggests that the achenes are metabolically active at thermoinhibitory temperatures and undergo most of the processes of normal germination, but that at some point any further development is halted, preventing radicle emergence. When the temperature is reduced, this block on germination is removed and since the achenes are already primed for germination, this occurs within a short time. An analysis of the proteins produced by germinating and thermoinhibited achenes was conducted using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This procedure was able to resolve approximately 40 different protein bands, but no differences were observed between thermoinhibited and germinating achenes. Two dimensional polyacrylamide gel electrophoresis (20-PAGE) was able to resolve approximately 200 individual polypeptides. The vast majority of polypeptides in T. minuta achenes are acidic, although the number of neutral to basic polypeptides increases as germination progresses. Ten polypeptides were identified which were specific to thermoinhibited achenes. These formed two distinct groups on the twodimensional gels. The larger group contained seven proteins, ranging in size from 22 kDa to 26.7 kDa and with isoelectric points of between 3.0 and 4.0. The smaller group contained three polypeptides with molecular weights of about 14 kDA and a pi of approximately 3.0. These polypeptides were all extremely specific to thermoinhibited achenes and declined rapidly when the incubation temperature was reduced, in a manner which correlated with an increase in the germinability of the achenes. Several characteristics of the expression of these polypeptides were similar to characteristics of embryo-dormancy in seeds where dormancy is thought to be actively imposed by the expression of specific dormancy-associated genes. This, along with the very tightly-regulated nature of these 10 polypeptides, suggests that thermoinhibition in T. minuta may be regulated through gene expression and that these ten polypeptides may represent the products of genes responsible for preventing radicle emergence at unfavourable temperatures. Since these polypeptides were only resolved using silver-staining and could not therefore be used for amino acid sequence analysis, this hypothesis was further investigated using differential display of mRNA to isolate genes which are expressed specifically in thermoinhibited achenes. A large number of cDNA fragments which were specific to either germinating or thermoinhibited achenes were identified and extracted from the differential display gels. Those cDNAs specific to the thermoinhibited achenes were taken for further analysis. Of the 62 fragments excised from the gels, 25 could be reamplified to generate single bands of the correct size on agarose gels. A further 22 cDNAs produced multiple bands, where one band was much brighter than the others and correlated with the size of the original fragment. Thirteen of the cDNAs which' generated single bands were cloned into the plasmid vector pGEM®-T Easy and transformed into either Escherichia coli JM109 or E. coli XL1-Blue. Recombinant colonies were identified using blue-white colour selection and the presence of the insert confirmed by colony blotting and restriction analysis. Three clones were chosen for each of the cDNAs. Reverse northern analysis confirmed that all 39 clones were specific to the mRNA pool of thermoinhibited achenes. High quality sequence data were obtained for 27 of the cDNA samples, the remainder appeared to have been degraded in transit. Alignment of the various sequences revealed that a total of 14 different sequences had been cloned, indicating that several of the bands isolated from the differential display gels contained multiple sequences. Electronic homology searches tentatively identified three of the sequences, whilst the remainder did not show significant homology to any known sequences. Of the cDNAs identified in this way, one may encode a plant transcription factor-like or nuclear RNA-binding protein whilst the other two may encode an RNase-L Inhibitor-like protein and a miraculin homologue. The potential roles of such genes in the imposition or maintenance of the thermoinhibited state are discussed. Although further research needs to be conducted to isolate full length cDNA sequences and to determine their exact expression patterns in germinating and thermoinhibited achenes, these results are consistent with the hypothesis that thermoinhibition in T. minuta achenes is under positive genetic control in a manner analogous to embryo dormancy. This thesis represents the first molecular study of thermoinhibition as well as the first report of active control over this phenomenon in any species. Since thermoinhibition, unlike dormancy, can be rapidly imposed and released under strictly controlled conditions without the need for any dormancy breaking treatment, T. minuta achenes represent an excellent model system for studies on the molecular control of seed germination.