A seed after rain. The physiology of hydration memory

The first rain can leave a lasting effect

A desert cactus seed can take up water during brief wetting and then dry again. In some species, the next contact with water leads to faster germination than in seeds that previously remained dry. This lasting effect of hydration is called hydration memory. Memory here means a physiological state of the seed that survives the drying period. It can be detected by comparing germination patterns following the same final water supply. [López-Urrutia et al. 2014, pp. 91–93]

López-Urrutia and colleagues combined this test with RNA and protein analysis. They studied seeds of Ferocactus townsendianus (F. peninsulae var. townsendianus in the publication), now accepted by POWO as a separate species. This name change clarifies the biological scope of the experiment. The molecular results concern seeds of this plant collected in June 2012 at several locations in Baja California Sur. [López-Urrutia et al. 2014, pp. 91–93][2-WWW]

Wetting, drying and restarting germination

Control seeds were placed on moist filter paper, 25 per dish. Temperature remained constant at 30°C, with light and darkness alternating every 12 h. Germination was identified by radicle emergence. One preparation treatment involved 72 h under these conditions, followed by drying for four, fifteen or seventy days. Another treatment used one to three cycles, each comprising 24 h of hydration and four days of drying. Drying took place on a laboratory shelf at 23–27°C and 50–60% relative humidity. [López-Urrutia et al. 2014, pp. 92–94, 98]

After preparation, seeds returned to germination conditions. Mean radicle emergence time was about eight days in the control and about four days after a hydration and drying cycle. The shorter time also persisted after seventy days of dry conditions. For the seed, this meant earlier completion of the process when water became available again. This time was measured in the final germination stage. It did not include all the preceding days of preparation and drying. [López-Urrutia et al. 2014, pp. 92–94, 98]

The result is relevant in an environment where soil surface moisture is brief. Earlier completion of germination may leave more time for root development during the next wet episode. This is an ecological interpretation of the response obtained in the laboratory. The study discussed did not track subsequent seedling survival at a natural site. [López-Urrutia et al. 2014, pp. 92–94, 98]

What remains in the RNA

One cycle of 72 h of hydration and four days of drying was selected for molecular analysis. Prepared and control seeds were compared, along with two-day-old seedlings arising from both groups. Material was frozen in liquid nitrogen, then total RNA and proteins were isolated from the same samples. This separation allowed the response of the dry seed after treatment to be compared with the picture at the next developmental stage. [López-Urrutia et al. 2014, pp. 93–95]

The electrophoretic profile of total RNA in F. townsendianus retained a similar pattern after the cycle. The clearest bands corresponded to ribosomal RNA. The observation indicates that the overall integrity of this pool was maintained. Sequences and quantities of individual messenger RNAs, from which cells read instructions for protein synthesis, were not measured. The stable total-RNA pattern therefore remains one level of evidence, while selective preservation of particular transcripts is a hypothesis requiring more detailed measurements. [López-Urrutia et al. 2014, pp. 93–95]

Proteins separated by two properties

Proteins were separated by two-dimensional electrophoresis. In the first direction, they were separated by isoelectric point. This is the pH at which a protein's positive and negative charges balance. Separation in the second direction depended on molecular mass. In the resulting gel image, each detected spot represented material with similar properties in both dimensions. Software compared the positions and intensities of corresponding spots, accounting for background and differences in total gel intensity. [López-Urrutia et al. 2014, pp. 93–96]

A total of 321 spots were detected. Some differed in intensity after the cycle, and further changes accompanied the transition from seed to seedling. Protein profiles of prepared and control seeds therefore differed. The result supports the existence of a physiological state maintained after previous wetting. The term ‘expression’ in this paper refers mainly to the relative presence of proteins in the gel analysis. Their synthesis, degradation rates and distinct modifications would need to be separated using further methods. [López-Urrutia et al. 2014, pp. 93–96]

Hypotheses about reserves and ubiquitin

Possible proteins were proposed for 37 spots on the basis of mass and isoelectric point compared with databases of proteins from other plants. Candidates included storage proteins, inhibitors of enzymes that break down reserves, and components of the ubiquitination pathway. Ubiquitination involves attaching ubiquitin to other proteins. It can alter their subsequent fate and is one way of regulating the amount and activity of proteins in a cell. These candidates connect the observed memory with control over the use of stored substances and reorganisation of the cellular apparatus. [López-Urrutia et al. 2014, pp. 93, 96–98]

Spot identities were preliminary. Two distinct proteins can have similar masses and isoelectric points, and one spot can contain more than one component. No peptide identification by mass spectrometry was performed, nor an experiment in which removing a particular protein abolished accelerated germination. The relationship between previous wetting, shorter germination time and a different protein profile therefore remains the strongest documented finding. The detailed regulatory network is the next level of explanation. [López-Urrutia et al. 2014, pp. 93, 96–98]

A reference point for subsequent experiments

The experiment provides two linked findings. Under final germination conditions, prepared seeds responded faster, while molecular analysis retained traces of the previous treatment. The number of seeds per dish and preparation time are important for reproducing the result. The publication does not specify the number of independent seed batches for the entire molecular analysis as clearly. The conditions used describe a particular physiological test. Establishing a procedure for another population or species requires its own comparison with a control and observations of subsequent development. [López-Urrutia et al. 2014, pp. 96–98]

The plant's description and the names under which it appeared in the literature are given in the Ferocactus townsendianus profile. F. pilosus has a separate study of seed responses to wetting cycles. Keeping these materials separate allows both the repeatability of the phenomenon across species and the actual extent of knowledge of its mechanism to be traced. [2-WWW][Contreras-Quiroz et al. 2016, Table 2]