A number of plant regulators including auxin, ethylene, gibberellins and abscisic acid (ABA) have been proposed as implicated in the control of berry development and ripening. However, the role played by these plant hormones in the strawberry fruit receptacle development and ripening processes remains very poorly understood. Our microarrays analysis may shed light regarding the physiological role played by these hormones in these important fruit processes.
Auxins
In strawberry fruits, it was proposed that the expansion of the receptacle cells during the early stages of fruit development that promotes the growth of the receptacle is mediated by the auxins synthesized by the achenes (Perkins-Veazie, 1995). The auxins are exported from the achenes to the receptacle through the vascular bundles promoting the fruit growth and inhibiting the ripening process by activation of fruit growth related genes while repress ripening-related genes (Perkins-Veazie, 1995). The declining of receptacle auxin content during the fruit development trigger receptacle fruit ripening through the induction of the expression of ripening-related genes (Given et al., 1988b).
The IAA levels in plants are modulated by a specific group of amidohydrolases (ILL2 amidohydrolases) that release the active hormone from its auxin-amino acid conjugated storage forms (Bitto et al., 2009). We have detected that the expression of two genes coding iaa-amino acid hydrolases was up-regulated in ripen receptacle (UCOESTup1303 and
UCOESTup2085). Similarly, the expression of two different indole-3-acetic acid-amido synthetase (GH3) genes (UCOESTup831 and UCOESTup2520) were up-regulated in ripen receptacle (Table 4). GH3 genes code IAA-amido synthetase which conjugates IAA to amino acids regulating the free auxin content in plants (Staswick et al., 2005; Wang et al., 2008). Involvement of these genes with ripening-associated process has been proposed in non- climacteric fruits as pepper or grape (Liu et al., 2005; Böttcher et al., 2010). Also, it has been proposed that these enzymes could participate in IAA degradation through the biosynthesis of IAA-conjugates that may inactivate the IAA function which enables ripening (Staswick 2009; Böttcher et al., 2010). Auxin-response factors (ARFs) are transcription factors acting on the auxin signaling pathway as activator or repressor of the expression of auxin regulated genes (Tiwari et al., 2003; Benjamins and Scheres, 2008). They can heterodimerize with Aux/IAA proteins modulating the expression of auxin-responsive genes (Wang et al., 2011). Our study also shows the transcript level increase of a gene that code ARF regulators (UCOESTup1428) in red ripen receptacle (Table 4). Besides, genes presenting a high sequence homology with germin-like genes (UCOESTup727) exhibited higher levels of expression in ripen strawberry fruit receptacle (Table 4). The expression of germin-like genes has been related with the evolution of the auxin content during the fruit development in plum (El-Sharkawy et al., 2010). Similarly, in our transcriptomic analyses, we have found six
SAUR genes whose expression was strongly up-regulated in ripen fruits (UCOESTup173,
182, 206, 928, 977 and 1136) (Table 2). SAUR (small auxin-up RNA) genes constitute a large auxin-responsive gene family whose function is largely unknown.
related proteins whose expression was down-regulated in ripen fruit receptacle (data not shown). The decreased expression of many auxin-related genes in ripen receptacles showed by our transcriptomic studies supports the clear relationship, previously reported, between a declining in the auxins content of the fruit receptacle and the fruit ripening. However, the increased expression of a different set of auxin-related genes in ripen receptacles also indicates that these hormones could play and additional physiological role in the ripening process, probably related with the secondary expansion of the cell of the fruit receptacle along the ripening (Medina-Escobar et al., 1997b; Benítez-Burraco et al., 2003; Palomer et
al., 2006). The results indicate that auxins use apparently different signaling pathways along the growth and ripening processes of fruit receptacles and open new perspectives that suggest that, similarly to those previously demonstrated for climacteric fruits (El-Sharkawy et al., 2010), the auxins seem also to be involved in some of the mechanisms that control the physiological processes involved in receptacle fruit ripening of this non-climacteric fruits.
Gibberellins
In strawberry, there is very scarce information related with the functional physiological roles that play the gibberelins (GAs) during the strawberry fruit growth and ripening process. It was observed that GA3 did not stimulate growth when applied to fruits from which achenes
had been removed (Dreher and Povaiah, 1982). In addition, it was demonstrated that the exogenous application of giberellic acid (GA3) to strawberry fruits delayed the development
of red color (Martínez, 1996). Later, Bustamante et al. (2009) described a reduction of the protein and mRNA levels of the ripen-related and fruit-specific enzyme β-xylosidase after the application of GA3 to strawberry white fruits. Recently, a preliminary study has proposed
that gibberellins could be involved in the development and ripening of the fruit receptacle (Csukasi et al., 2011). These studies suggest that giberellins could play a dual regulatory physiological role, first promoting receptacle growth in combination with the auxins produced in the achenes, and afterwards playing a relevant role related with the receptacle ripening process. Supporting this proposal, our microarrays studies have shown a clear induction in ripe fruit receptacle of two genes that coding enzymes involved in the biosynthesis of gibberellins, such as a Giberellin 2 oxidase (UCOESTup266) and a Giberellin 3 beta-dioxygenases (UCOESTup1835) (Table 4).
The role played for GAs probably could be related with the differentiation of cambial meristem cells to xylem tissue and with an increase of xylem lignification in receptacle vascular tissue during the ripening process. Experiments in poplar, aspen and tobacco have demonstrated that transgenic plants which over-expression of these genes exhibit significantly increased levels of xylem lignification (Biemelt et al., 2004). In accordance with this suggestion, our microarray studies have also shown the up-regulation of genes coding key enzymes of the lignin biosynthesis pathway, as cinnamyl CoA reductase and cinnamyl alcohol dehydrogenase in ripen fruit receptacles (UCOESTup583, 1780, 60, 194,
397 and 1065) (Table 4).
Abscisic acid (ABA) metabolism and signaling
There are only scarce studies relating the abscisic acid (ABA) with the strawberry fruit ripening process. However, it has been reported that ABA is present in both receptacle and achene tissues, with amounts changing during the berry development (Lis et al., 1978; Archbold and Dennis, 1984). Besides, it has been indicated that ABA treatments through
It was proposed that the increase in the receptacle abscisic acid (ABA) content could promote the strawberry ripening process (Perkins-Veazie, 1995). In plants, ABA biosynthesis proceeds through the plastid-localized 2-C-methyl-D-erithritol 4-phosphate (MEP) pathway (Cazzonelli and Pogson, 2010). Our microarray analyses have shown the expression of genes that coding an enzyme involved in crucial steps of ABA biogenesis pathway. One of these genes coding a 9-cis-epoxycarotenoid dioxygenase 3 (UCOESTup1120) which was induced in ripen fruit receptacle (Table 4). This gene has large amount of expression in ripe fruit receptacle which suggests that it could be involved in the ripening processes (Table 4). It has been shown that the silencing of this gene avoid anthocyanin production in ripen strawberry fruits (Jia et al., 2011). These results indicate that this metabolic pathway is operative in fruit receptacle probably to produce ABA in ripen receptacles and support that this hormone could play important physiological role in the strawberry fruit ripening process. Besides, we observed the up-regulation in red ripen fruit receptacles of the FaPYR/RCAR/PYL (UCOESTup888 and UCOESTup1025) gene (Table 4) that presents a significant sequence homology with putative ABA receptors containing the START domain (Weiner et al., 2010). The presence of the signatures SGLP (´cap` loop) and HRL (´lock`loop), always conserved in these proteins cDNA (Weiner et al., 2010), in the deduced protein sequence derived of the
FaPYR supports even more that this gene codes an ABA receptor. Recently, studies showed
that the down-regulation a similar but not identical FaPYR1 gene delayed the fruit ripening and also decreased the anthocyanin content in receptacle of strawberry ripen fruits. This expression silencing also showed that a set of ABA-responsive gene transcripts, including
ABI1 and SnRK2, was altered (Chai et al. 2011). Furthermore, the loss of red colouring in
FaPYR1 RNAi fruits could not be rescued by exogenously applied ABA, which could promote the ripening of wild-type fruits. These results demonstrate that the putative ABA receptor FaPYR1 acts as a positive regulator in strawberry fruit ripening (Chai et al. 2011). Additionally, we have also observed an increased amount of transcripts corresponding to a
FaASR gene (UCOESTup262) encoding an ABA stress and ripening-induced protein (ASR)
in receptacle of ripen fruits. These results indicated that FaASR gene has an important role in ripening process of ripen fruits. The ASR proteins have been reported to act as a downstream component involving in ABA signal transduction in both grape berry and tomato (Cakir et al., 2003; Shkolnik and Bar-Zvi, 2008). The grape and tomato ASR proteins have been proposed to regulate the transcription of sugar- and abiotic stress-regulated genes in fruit and vegetative tissues (Cakir et al., 2003; Saumonneau et al., 2008; Shkolnik and Bar- Zvi, 2008). Recently, Chen et al. (2011) showed that FaASR may be involved in strawberry fruit ripening. They observed that an increase in the content of endogenous ABA might increase FaASR expression partially contributing to the acceleration of strawberry fruit ripening (Chen et al., 2011). However, the functional mechanism through ASR protein regulates the ABA responses are not fully understood. Taken together, these results show that both FaPYR2/FaASR genes probably regulate the gene expression of ABA-dependent genes whose responses are not related with the organoleptics properties of the ripen fruit.
The sequences of genes UCOESTup162 and 1400 present a high homology of sequence with those that code HVA22 proteins of higher plants. In our transcriptomic analysis, we have found that the expression of both genes was up-regulated in strawberry ripen fruits (Table 4). Homologs of HVA22 have been identified in diverse eukaryotes, including plants, fungi, mammals, flies and worms, but not in any prokaryotes (Guo and Ho, 2008). In several vegetative tissues of plants, the expression of HVA22 genes is dramatically up-regulated by abscisic acid and abiotic stress, as cold, drought, extreme temperatures and salt stresses (Shen
regulating GA-mediated vacuolation/programmed cell death in barley aleurone. These proteins could inhibit vesicular trafficking involved in nutrient mobilization to delay coalescence of protein storage vacuoles as part of its role in regulating seed germination and seedling growth (Guo et al., 2008). Recently, it has been proposed that HVA22 proteins play a physiological role as suppressors of autophagy in both plants and yeast (Chen et al., 2009). Curiously, several studies have shown that the HVA22 promoter could be regulated by WRKY transcriptional factors in response to those conditions where increases in the ABA production proceed (Zhang et al., 2009). In this sense, it is noteworthy that several genes coding WRKYs TFs increased its expression along the ripening process in strawberry fruit receptacle. The existence of a relationship between WRKY and HVA22 expression must be clarified through functional studies based in reverse genetic approaches.
In opposition to those found for other hormones, we have not observed genes related with the biogenesis of ABA whose expressions are up-regulated in immature green receptacles (data not shown). So, our results could be in accordance with the proposal that ABA plays a physiological role only in the ripening process.
Abscisic acid (ABA) and Isoprenoid metabolism
Isoprenoids are functionally and structurally the most diverse group of plant metabolites (Thulasiram et al., 2007).They can operate as primary metabolites, participating in essential plant cellular processes, and as secondary metabolites, of which many have substantial commercial, pharmacological, and agricultural value. Isoprenoid end products participate in a wide range of physiological processes in plants acting in them both synergistically, such as chlorophyll and carotenoids during photosynthesis, or antagonistically, such as gibberellic acid and abscisic acid during seed germination (Vranova et al., 2012).
The function of isoprenoid compounds during environmental challenge is diverse. Some of them are involved in protection of the photosynthetic apparatus, the amount and composition of carotenoid compounds, particularly xanthophylls, with roles in detoxification of the cell from free radicals and reactive oxygen species (ROS) caused by high light intensity during drought stress (Demming-Adams and Adams, 1996; Niyogi et al., 1997; Munne-Bosch et al., 1999; Munne-Bosch and Alegre, 2000a,b). Others, as α-tocopherol, are also important antioxidant agents (Abbasi et al., 2007). Sterols have been reported as protectors of biological membranes during drought and cold stress (Nakamura et al., 2001), while the isoprene seems to increase the thermotolerance in leaves of some plant species (Singsaas et
al., 1997). In our studies microarrays, we showed different genes involved in the biosynthesis of isoprenoid, which were up-regulated in strawberry ripen fruits (UCOESTup922, 1670,
854, 1727, 2192, 1610, 72, 2169 and 208) (Table 4).
The phytohormone abscisic acid (ABA) is involved in the response to cold, salt and drought stress (Loyola et al., 2012). Water deficit triggers the production of ABA, which in turn causes stomatal closure and induces the expression of drought stress-related genes, whose products allow temporary acclimatisation of diverse plant species (Seki et al., 2007). Our study detected high transcript levels of a Phytoene synthase (UCOESTup1573) in red-ripen receptacle (Table 4). This enzyme is directly involved in the ABA biosynthesis, so that high expression levels of UCOESTup1573 gene might be involved in the ripening processes of red fruit receptacle (Table 4).
Ethylene
AP2/ERF (Ethylene Response Element) gene family encodes putative transcription factors (Shigyo et al., 2006). These families of transcription factors are mostly associated with response to biotic and environmental stress or acts as key regulators in developmental process (Zhang et al., 2009). In our microarrays platform, we have detected 10 AP2/ERF genes whose expressions were up-regulated (Table 1). The UCOESTup889 and
UCOESTup752 exhibited large levels of expression in red fruit which suggest that it could be involved in ripening processes. Recently, an AP2/ERF gene (SIAP2a) has been identified and functionally characterized through transcriptional profiling of fruit maturation (Chung et al., 2010). This ripening related gene, in contrast to previously described transcription factors, is a negative regulator of ripening (Chung et al., 2010).
Methyl Jasmonate (MeJA)
MeJA and its free jasmonic acid (JA), both referred as jasmonates, are lipid-derived signals that mediate plant stress responses and development processes such as seed germination, flower and fruit development, leaf abscission and senescence (Avanci et al., 2010; Wasternack et al., 2010). In our study, we have identified an enzyme S-Adenosyl-L- methionine:jasmonic acid carboxyl methyltransfera (UCOESTup69) that catalyzes the methylation of JA to form MeJA (Table 4). The S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase (JMT) have been functionally characterized corresponding to an enzyme of Arabidopsis thaliana (Seo et al., 2001). These studies suggest that this enzyme is a key for the jasmonate-regulated plant responses (Seo et al., 2001). Activation of JMT expression leads to production of methyl jasmonate that could act as an intracellular regulator, a diffusible intercellular signal transducer, and an airborne signal mediating intra- and inter- plant communications (Seo et al., 2001).
3.3.7.2. Signaling
Receptors
Plants contain a large family of receptor-like kinases (RLKs) that regulate various growth and developmental processes, phytohormone perception and biotic and abiotic stress responses (Nurnberger et al., 2006; Chae et al., 2009; Lehti et al., 2009). The adaptive responses of plants to extracellular o intracellular ligands and stimuli are governed by a functional continuum between the plant cell wall (CW) and the plasma membrane (PM) in which RLKs, with CW-bound extracellular domains, probably function as cell wall integrity sensors (Humphrey et al., 2007; Chinchilla et al., 2009). As numerous RLKs share conserved structural features within the extracellular domains, they can be grouped into protein subfamilies (Shiu et al., 2004). A group of RLKs that are regarded as potential CW-PM linkers are the lectin receptor kinases. This receptor contains extracellular lectin motifs that are known to bind various carbohydrates. Based on the extracellular lectin motifs three types can be distinguished G, C, and L (Bouwmeester and Govers, 2009). We have observed an increased amount of transcripts corresponding to three G-type lectin receptor kinases (UCOESTup431, 1732 and 2230) in receptacle of strawberry ripen fruit when compared with immature green receptacle (Table 4). G-type lectin receptor kinases are also called S-locus receptor kinases, historically known as S-domain RLKs. In Brassicaceae, the S-locus receptor kinase SRK acts as the stigmatic determinant of the self-incompatibility response
type lectin receptor kinases were also shown to play roles in plant defence. The rice gene Pi-
d2, for example, confers resistance to the fungal pathogen Magnaporthe grisea whereas
NgRLK1 from Nicotiana glutinosa was selected in a yeast two-hybrid screen as a putative interactor with capsicein, an elicitin from Phytophthora capsici (Kim et al., 2010). Besides the G-type lectin motifs the extracellular domains of these proteins contain cysteine-rich EGF-like (epidermal growth factor) and PAN (plasminogen-applenematode) motifs that both function in protein homodimerization (Naithani et al., 2007). As yet, the role of the G-lectin motif is unknown and there is also no evidence for a function in ligand binding.
Besides, we observed the up-regulation in red ripen fruit receptacles of different legume-like or L-type lectin receptor kinases (LecRKs) genes (UCOESTup121, 292, 434, 482, 732, 760 and 1675) (Table 4). The LecRKs have extracellular domains resemble soluble legume lectins which are ubiquitous in leguminous seeds (Andre et al., 2005). Although LecRKs are implied to function in diverse biological processes, their exact biological role has not yet been clarified. LecRKs that have been described to function during plant development include the SGC lectin RLK of Arabidopsis, which is required for proper pollen development (Wan et al., 2008). More recently, Xin et al. (2009) showed that a specific subfamily of LecRKs is responsible for negatively regulating the abscisic acid (ABA) response during seed germination and hypothesized that these genes directly or indirectly affect defence. Lately, several reports have presented data linking ABA-signalling with defence responses (Asselbergh et al., 2008). NbLRK1, a legume-like lectin receptor kinase from Nicotiana
benthamiana was reported to interact intracellularly with the Phytophthora infestans elicitin INF1 and seems to be involved in the subsequent INF1-induced cell death (Kanzaki et al., 2008). Another LecRK with a potential link to plant defence and pathogen response is LecRK79 in Arabidopsis (Gouget et al., 2006). LecRK79 mediates CW-PM adhesions and hence the continuum between the cell wall and the plasma membrane (Gouget et al., 2006). Its role in plant defence is, furthermore, supported by the observation that LecRK79 expression is induced upon inoculation with several nonhost and avirulent pathogens (Bouwmeester et al., 2009). Taken together, these data suggests that LecRKs play crucial roles in both developmental and adaptive processes.
Other receptors are leucine-rich repeat receptor-like kinases (LRR-RLKs), which contain LRRs in their extracellular domain. These receptors are involved in many developmental processes and in host responses to biotic and abiotic stresses (Chinchilla et al., 2009; Dievart
et al., 2011). Our microarray analysis have shown in ripen receptacle the over expression of genes that coding different leucine rich repeat-receptor like kinase (LRR-RLKs) (UCOESTup196, 205, 286, 432, 456, 1435 and 2542) (Table 4). Among them,
UCOESTup432 showed higher level of expression so that this receptor could have an important role in the fruit ripening processes (Table 4). Recently, a vast number studies have demonstrated major roles played by LRR-RLKs in plants during almost all developmental processes and in defense/resistance against a large range of pathogens (Dievart et al., 2011). Forward and reverse genetic approaches have revealed various physiological functions of
Arabidopsis LRR-RLKs (Morillo and Tax, 2006). Brassinosteroid insensitive1 (BRI1), the receptor for the plant steroid hormone, brassinolide (BL), constitutes one of the best studied plant LRR-RLK, and was shown to regulate stem elongation, vascular differentiation, seed size, fertility, flowering time, and senescence (Li et al., 2002; Nam and Li, 2002; Wang et
al., 2005). Binding of brassinolide to an island domain that folds back between LRR repeat 21 and 22 was suggested to provide a docking platform for the formation of heteromeric complexes with another LRR-RLK, BAK1 (BRI1- associated receptor kinase1). Recently
(Kemmerling et al., 2011). Another subset of plant LRR-RLKs has been shown to function as pattern recognition receptors mediating there cognition of microbial surface structures (pathogen or microbe associated molecular patterns, PAMPs/MAMPs) and plant innate immunity to microbial infection (Nürnberger and Kemmerling, 2006). For example,
Arabidopsis FLS2 (Flagellin Sensing 2) and EFR (EF-Tu Receptor) sense bacterial flagellin and elongation factor EF-Tu, and thereby confer basal immunity to microbial pathogens displaying the respective cognate ligand (Zipfel et al., 2006; Chinchilla et al., 2007). The large number of LRR-RLK that are encoded by the A. thaliana genome and the proven role of FLS2 in plant immunity have prompted us to undertake a systematic survey for additional