Desiccation tolerance and sensitivity in Medicago truncatula and Inga vera seeds

Orthodox seeds acquire desiccation tolerance (DT) during their development which enables them to pass through the phase of maturation drying by the end of their development and enter a state of quiescence. After harvesting, these seeds can be dried further and stored for the long-term without significant loss of viability. On the other hand, there are many species that produce recalcitrant seeds, which have a developmental history and post-harvest behaviour rather opposite from the orthodox types. These are shed with high moisture contents (MCs), are metabolically active and can not be dried to MCs low enough to allow safe storage. Recalcitrant seeds thus represent a big challenge to those who need to store them in seed banks for germplasm conservation purposes. In order to gain insight into the recalcitrance phenomenon, this thesis addresses questions concerning the physiological, cytological and molecular aspects of desiccation sensitivity in developing and mature recalcitrant seeds of the tree species Inga vera Willd. subsp. affinis and in mature and germinating orthodox seeds of the model species Medicago truncatula Gaertn. cv. Jemalong A17. The desiccation sensitivity of I.vera seeds was analyzed in terms of water relations, DNA content and the microtubular cytoskeleton. Developing (6, 9 and 10 weeks after flowering - WAF) and mature seeds (14 WAF) were collected and processed, ending up with the naked embryos. Slight desiccation of immature embryos increased germination, but further drying resulted in a quick decline of germinability. Total loss of viability occurred while the embryos still showed high MCs (around 0.4 g H/g dry matter) and high water activity (a w ), around 0.88, irrespective of the developmental stage. During development the desiccation sensitivity of the embryos decreased slightly, while the percentage of cells with 4C DNA remained constant, around 15% in the root and 10% in the shoot, suggesting no relation between DT and DNA content. Immuno-histochemical detection of microtubules (MTs) in embryonic axes cells of mature embryos showed abundant cortical microtubule arrays, which were not affected by mild desiccation, but were totally dismantled by further drying. Upon rehydration, damaged cells were not able to reconstitute the microtubular cytoskeleton and this might be related to the viability loss during dehydration of the embryo. The desiccation sensitivity was also studied in seeds of M. truncatula during and after germination. When seedlings with a radicle length as short as 1 mm were dried back to the original MC found in dry seeds and rehydrated, only 15% survived. Seedlings with a radicle length equal or longer than 2 mm did not survive dehydration at all. By subjecting seedlings to an osmotic treatment with polyethylene glycol - PEG (-1.8 MPa) before drying, DT could be re-established in seedlings with a radicle length up to 2 mm. Flow cytometric analyses and MT visualisation in radicle cells of growing M. truncatula seedlings showed that up to a radicle length of 2 mm, the cell cycle had not been resumed, as shown by the absence of DNA synthesis and cell division, which were first detected in 3 mm long radicles. Therefore DT could be re-established only before the resumption of the cell cycle in the radicles. Dehydration of seedlings with a 2 mm protruded radicle, with or without previous PEG treatment, caused disassembly of MTs. Upon rehydration MTs were not reassembled in radicle cells of untreated seedlings, while PEG-treated seedlings were able to reconstitute the microtubular cytoskeleton and develop into normal seedlings. Dehydration of untreated seedlings with a 2 mm protruded radicle also led to an apoptotic-like DNA fragmentation in radicle cells, while in PEG-treated seedlings DNA integrity was maintained. The results showed that for different cellular components, desiccation-tolerant seedlings may apply distinct strategies to survive dehydration, either by further repair or avoidance of the damages. This thesis also investigated the changes in the expression of various genes related to seed development, DT, cell cycle and cytoskeleton during loss and re-establishment of DT in germinating seeds and in seedlings of M. truncatula. The transcript levels of the studied genes in radicle cells were relatively quantified by real time PCR, using specific primers for M. truncatula. Clear changes in transcript abundance were detected during and after germination and in response to osmotic treatment and dehydration. DT-related genes ( EM6 , PER1 and sHSP18.2 ) were down regulated during germination and up regulated by osmotic treatment, which correlated with the loss and re-acquisition of DT in the radicles. The expression pattern of the developmental gene ABI3 was similar to that of the stress related genes, corresponding with a possible control of the stress response by this gene. Abundance of LEC1 transcript correlated more with the germination process than with osmotic stress. The cytoskeleton genes ( ACT and TUB ) were up-regulated during germination, not affected substantially by osmotic treatment and down-regulated by subsequent dehydration, which was related with the massive breakdown of the cytoskeleton upon dehydration of seedlings. Expression of CDC2a , one of the key regulators of the G 1 -to-S transition, was clearly associated with the occurrence of the first cell cycle in the growing radicle. Radicles that have gone through the first cell cycle (3 mm long) may respond to osmoticum in a similar fashion as desiccation tolerant radicles, in terms of gene regulation. However, the resumption of the cell cycle appears to be an overriding factor that abolishes re-establishment of DT. Recalcitrant seeds are metabolically active in storage and this leads to a short longevity. I.vera embryos are among the worst storable species known, retaining viability for not much longer than one or two weeks if stored in semi-permeable bags at 20 or 5 o C, respectively. Thus, an attempt was made to slow down the metabolism of I.vera embryos and, consequently, prolong their short storability. For this, embryos were stored in an osmotic medium (PEG at -1.7 MPa) with or without the addition of ABA, a well known germination inhibitor. Besides slowing down the metabolism, osmotic stress and ABA can lead to the expression of genes involved in DT in plants or seeds. Storage in PEG was capable of keeping the germination rate of I.vera embryos at 100% until 30 days, either at 5 or 20 o C, thus causing a three-fold increase in storability, when compared to the embryos stored in semi-permeable bags. Starch content, as major food reserve, generally decreased with increasing storage duration. However, we found no direct relationship between starch content and viability/germination. The effect of ABA showed to be temperature-dependent, being positive at 20 o C and negative at 5 o C. The permanence of the embryos in PEG for 14 days did not render them more tolerant to desiccation. Another technique applied in order to prolong the storability was sealed storage of partially dried embryos. In this case, storability was better than the control, but not as long as in the PEG storage. Anyway, both approaches used in this thesis seem to be promising to prolong the naturally short storability of recalcitrant seeds. We hypothesize that mature recalcitrant seeds have completely lost unique seed traits such as DT and should therefore be regarded as vegetatively growing seedlings/plants rather than seeds and, hence, storage under controlled atmosphere may prove successful. This type of storage is common in long term conservation of fresh fruits and vegetables.