The olive tree is known for its irregular crop production from year to year . Although different plant species exhibit such phenomenon, the physiological processes involved are not universal, but rather species-specific . However, it has become clear that the genetic networks triggering the developmental phase-transitions share some common factors among species. Thus, Ulger et al.  reported that some endogenous plant growth hormones like the abscisic acid (ABA), gibberellins like the gibberellic acid (GA3) and auxins like the indole-3-acetic acid (IAA) have important roles on the alternate bearing. Additionally, the stored carbohydrate amounts in the plants differ between the on- and off-years, and consequently, an association between carbohydrate mobilization and biennial bearing was proposed .
The olive tree has a natural tendency to produce high number of flowers and fruits. Yet, the developing seeds inside the fruits produce molecular messengers (like the gibberellins) that inhibit the floral induction, arresting the buds and directing them towards shoots or leaves (vegetative buds) instead of flowers (reproductive buds). Likewise, the growing of a large number of fruits may produce the depletion of the carbohydrate reserves in the tree. Therefore, different strategies can be used to reduce or even eliminate the alternate bearing in the olive tree: i) reduction of the number of fruits, by means of a proper pruning the year before the expected high production; ii) reduction of the density of the fruits at a very early stage of development (very small fruits); iii) early harvest of the developed fruits (large green fruits; even though the flowering inhibition has started at such stage, this strategy has some influence); and iv) prevention of the depletion of the carbohydrate reserves in the tree (eg, supplying a convenient irrigation to favor the plant nourishment and reserve accumulation). These facts indicate that the periodicity in bearing is indeed an adaptation mechanism of the plant to avoid excessive depletion of its reserves, to assure that part of them are devoted to a significant vegetative growth (leaf and shoot buds), and a defensive mechanism to cope with restrictive environmental conditions like drought and macronutrient/micronutrient deficiencies .
So far, several miRNA have been identified and characterized in plants, and their roles in diverse biological processes have been documented in many instances. They are involved in many physiological processes, like development and stress responses [9, 16, 33]. The study of miRNA with traditional methods may be complicated, but the recent high-throughput sequencing methodologies have become an excellent approach to discover miRNA in different organisms [34–36].
In this study, we sequenced and assessed the sRNA of six olive libraries constructed from two organs, each at different developmental stages, using the Illumina high-throughput system to identify a comprehensive set of alternate bearing-associated miRNAs in O. europaea L. With a total of 93,526,915 sequence reads, these libraries represent, to our knowledge, the deepest olive tree miRNA sampling to date. Thus, it is about 550 times higher than the one produced by Donaire et al. , which is the only olive tree miRNA report previously published to date. In that study, two libraries were constructed (juvenile and mature shoots), and 18 families of known and five putative miRNA were identified in O. europaea L. In addition to those, we have discovered four more conserved miRNAs within 22 families.
The high-abundance miRNA (except the miR156a, miR156b, miR156c and miR156d) were generally ubiquitously expressed in the six libraries, suggesting a correlation between the abundance of miRNA and their expression levels. Supporting our data, evolutionarily conserved miRNA have been found often among the most abundant miRNA by other studies . The miR156 was previously identified in adult olive tree shoots . However, in that study, the miR156 had a very low count. Moreover, the miR156 regulates the expression of the Squamosa Promoter-binding protein-Like (spl) gene, which plays a role in the juvenile-to-adult and annual phase transitions [12, 13, 24]. Shalom et al.  analyzed the expression of the miR156 in "on" and "off" citrus trees, and no significant differences were found between samples. They also analyzed the expression of the spl gene, and detected upregulation in "off" year trees, as compared to "on" year trees. Supporting that, we found that the expression of the miR156 (which is a negative regulator of the spl gene) in "on" year juvenile olive tree sample pool (JON) was higher than in the "on" year mature one (NON).
The expression level of several miRNA varied between the six analyzed libraries, indicating a differential functional role of the involved genes in the development-associated regulation. The miR395 that we have found was also detected in the previous study on the olive tree , which targets transport and response-to-stimulus genes, being four-fold upregulated in the unripe fruit library (UF) as compared to the ripe fruit one (RF). Its expression was also higher in July (JON-JOFF) than in November leaves (NON-NOFF). The nutritional control has been considered as one of the principal regulatory mechanisms for alternate bearing . Yet, contradictory results have been reported on the involvement of the carbohydrate reserves on the reproductive development in the olive tree [30, 31, 39, 40]. Recently, Bustan et al.  showed that the demand on carbohydrates by the developing fruits is significant; yet the carbohydrate deficiency was not an essential controller for the alternate bearing. In addition, deficient boron transport was proposed as an important factor in the alternate bearing in avocado . Previously, it was reported that the miR395 might be involved in the salt-induced response pathways, such as the maintenance of the energy supply in maize . In the view of the previous reports and the present study, we conclude that the transport of nutrients (including ions) is involved in the development and ripening, with a significant role of the miR395 on the alternate bearing in such species.
The count of known miRNA in the leaf-derived libraries was higher than that of fruit-originated ones. Moreover, a higher diversification of miRNA was also found in the leaf libraries. These results indicate that more miRNA are involved in the leaf than in the fruit physiology. This is not surprising, taking into account that the latter represents a specific organ, whereas the former is a more general-purpose organ. Supporting our data, the contribution of leaves to the reproductive growth was found to be two-fold .
The miR156 and miR166 are the two most abundant conserved miRNA in our analyses, being also found in large amounts in other studies . The six libraries exhibited significant differential expression of the miR156 members. Their expression decreased approximately 2.5-fold in NON as compared to NOFF. The assessment of the “on-year” July (JON) and November (NON) leaf libraries showed that the miR156 expression was downregulated in the July (JON) leaf library, indicating the specificity of the miR156 for the November leaf stage. Previous studies also revealed that, besides the miR172, the miR156 also regulates the Squamosa Promoter-binding protein-Like (SPL) transcription factors, controlling the maintenance of the juvenile phase and the timing of the juvenile-to-adult phase transition in both Arabidopsis thaliana and olive tree [12, 13, 24]. The miR172 is considered one of the most ancient miRNA families in plants, being shown to function in regulating the transition between developmental stages [10, 14]. In our study, the expression of the miR172 members was clearly higher in the leaf libraries than in the fruit datasets, supporting their activity in the maintenance of juvenile stage. The higher expression of two miR172 members (miR172d and miR172e) in NOFF than in NON also supports this conclusion. Additionally, a similar pattern was observed for the miR172g and miR172h expression of July leaf libraries (JOFF and JON). Moreover, the July leaves (JON) expressed significantly more miR172d and miR172e than the November ones (NON). Except for miR172d, miR172e, miR172g and miR172h, the rest were ubiquitously expressed in the leaf libraries. Therefore, we conclude that the miR172 plays not the main, but a significant role in the onset of the developmental phase-transition in the olive tree.
The hormonal control has been proposed as one of the major regulatory mechanisms of alternate bearing . To give an example, members of the miR160 and miR319 (targeting the genes involved in the hormone-mediated signaling) were significantly regulated by bearing on the olive tree. The expression difference of the miR160 members between the on- and off-year varied from 4- to 6-fold, while its expression was slightly upregulated in juvenile leaves compared to mature leaves which was also detected in olive shoots . Whereas the difference between on- and off-years was about 1.8-fold for the miR319. Besides, significant fold changes were observed between the fruit libraries. Similarly, the fruit libraries showed considerable expression differences of the miR393 (which targets genes responsible for hormone-mediated signaling), indicating an important contribution of the hormone-mediated response on the olive tree development processes.
The analyses of the miRNA also revealed that a number of targets are related to functions which may be associated to alternate bearing, as in the case for floral development and organ morphogenesis. The comparison of the expression of the miR168 and miR396 between the on- and “off-year” libraries revealed small but significant differences. The effect of the miR396 on the leaf development via the targeting growth-regulating factors had been previously shown . Additionally, the miR166 and miR171 targeting genes involved in organ morphogenesis and developmental processes showed a fruit library-specific regulation. Thus, their expression was significantly upregulated in the unripe fruit (UF) library. It may be speculated that the suppression of these target genes may inhibit the maturation of fruits, albeit such hypothesis needs further investigation.
The KEGG analyses revealed that the genes targeted by the olive tree miRNA are largely involved in the carbohydrate metabolism and hormone signal-transduction pathways, followed by the brassinosteroid biosynthesis pathway. As discussed above, the nutrition and hormone control have been proposed as two principal factors involved in the alternate bearing. Besides, the brassinosteroids belong to the plant steroid hormone group that regulates the growth and development . Thus, our data further support that the nutritional and hormone control play essential roles in the alternate bearing for the olive tree.
In this study, in addition to the 135 conserved miRNA, we have also identified 38 putative novel miRNA in the olive tree, which exhibit the diversity of the miRNA expression in O. europaea L. and show the occurrence of more miRNA than previously known. This genome-wide survey of six olive tree miRNA libraries has allowed to discover the miRNA expression profiles associated to alternate bearing, and consequently can contribute to elucidate the processes involved in this relevant physiological phenomenon. These results allow to conclude that not just a few, but many genes, may be involved in the alternate bearing in the olive tree. Thus, the differentially-expressed miRNA identified in this study will be a valuable source for further assessments of alternate bearing-associated genes in such and other species.
A comprehensive study on the olive tree miRNA related to the alternate bearing was performed. The miRNA of six olive libraries constructed from fruits (ripe and unripe) and leaves ("on-year" and "off-year" in July and in November, respectively) were subjected to Illumina deep-sequencing. The bioinformatics analyses of 93,526,915 reads identified 135 conserved miRNA belonging to 22 families, with 38 putative novel miRNA for the olive tree datasets. The expression of the miRNA varied greatly between the six libraries, indicating the contribution of diverse miRNAs in the balancing between the reproductive and the vegetative developments. The predicted targets of the miRNAs were categorized into 108 process-ontology groups with significant abundance. Among those, some potential alternate bearing-associated processes were found, such as development, hormone-mediated signaling and organ morphogenesis. The KEGG analyses indicated that the nutritional and hormonal control play essential roles in the alternate bearing of the olive tree. The regulation of the miRNAs under different developmental stages indicated that the nutritional and hormonal controls, together with the ones for the flowering processes had noteworthy impacts on the alternate bearing of O. europaea L. Our results provide significant data on the miRNA-dependent developmental phase transition interaction and advance perspectives in the miRNA profiling of the olive tree that may be also useful for other species.