Endocarp lignification plays critical roles in seed protection and dispersal in some fruits and yet it occurs sporadically throughout angiosperm lineages. This prompts the question of whether it is an ancestral state of angiosperms or a more recent adaptation. Among plants in the family Rosaceae, Prunus is one of two genera (the other being Rubus) that form a lignified endocarp layer which provides an excellent opportunity to address evolutionary questions. Here we sought to better characterize peach stone formation and define the molecular pathways that control it in order to gain an insight into how Prunus species evolved a lignified endocarp. Results show that the peach endocarp layer accumulates lignin 5-6 weeks after bloom. Lignin deposition proceeds from the blossom end and extends throughout the entire endocarp over a ten day time period (Figure 1). Recent biochemical studies have shown that peach stones accumulate extremely high lignin contents (≈ 50% lignin) relative to other woody tissues (≈ 25% lignin) (R Scorza, J Ralph and F Lu, unpublished data). Therefore, understanding how peach stones accumulate so much lignin could have important implications for forestry, forage and bioenergy crops in which lignin regulation is central to a number of critical agricultural traits.
Global gene expression analysis during peach fruit development revealed the up-regulation of a number of PP, lignin and flavonoid pathway genes concurrent with lignin deposition and stone hardening (Figure 3). Genes in these pathways made up over 20% (14/65) of annotated genes showing >3 Log2-fold expression. The concurrent induction of the lignin and flavonoid pathways is in sharp contrast since these are competitive pathways that presumably draw on the same precursors generated by the PP pathway. Expression studies in dissected fruit revealed that there is a distinct spatial separation of some components of the two pathways (Figure 5). The PP gene PAL, which catalyzes the first step in PP biosynthesis, and three lignin pathway genes (CCoAOMT, POX and LACC) were found to be largely endocarp specific while expression of the flavonoid genes (CHS, DFR, F3H and LDOX) and two lignin pathway genes (C3H and CCR) were predominately expressed in the mesocarp and exocarp. C4H, which catalyzes the second step in the PP pathway, showed expression throughout the fruit but transcripts predominated in the endocarp (Figure 5, Additional File 7). The overlap in expression of the known lignin pathway genes C3H and CCR with the flavonoid pathway implies that they may have flavonoid associated functions. In other plant species, CCR and C3H genes tend to be comprised by small gene families and have probably diverged . Our gene expression data suggests that that the identified C3H and CCR family members may not be rate limiting to lignin biosynthesis but may play important roles in flavonoid metabolism. While these inconsistencies have yet to be resolved, collectively, the expression data reveals intricate connections between lignin and flavonoid pathway regulation during peach fruit development.
The identified lignin and flavonoid regulons (1 and 2, respectively) reveal additional cellular changes associated with secondary metabolism in fruits (Additional File 9). Not surprisingly, regulon 1 includes a number of cell wall biosynthesis and secondary wall formation enzymes. Cell wall modifications are essential for proper lignin polymerization and hardening . The shift to increased secondary metabolism also appears to be associated with decreased protein synthesis and membrane transporter expression. These changes may reflect cellular metabolic rewiring necessary to enable extreme increases in secondary metabolism.
The observed spatial/temporal coordination between lignin and flavonoid expression supports the model that lignin and flavonoid biosynthesis are competitive. During times of peak lignin deposition genes in the lignin biosynthesis pathway were strongly induced while flavonoid pathway genes were repressed (Figure 5). Expression levels of CHS and DFR were lowest in lignifying stone tissue relative to other developmental times or during ripening. Conversely, high flavonoid gene expression was correlated with lower expression of genes involved in lignin biosynthesis. This interpretation is complicated by our finding that PP pathway genes, PAL and C4H, were disproportionally associated with lignin pathway induction. Little PAL expression was observed in the flesh or skin even when flavonoid gene expression was at its peak. In contrast, C4H showed substantial induction in the mesocarp and exocarp, though still to a slightly more limited extent than the endocarp. This discrepancy could be explained by the fact that PAL is typically encoded by two to four closely-related genes while C4H is often a single gene . An initial survey of lignin and flavonoid gene families in the draft peach genome suggests that there may only be two PAL genes and a single C4H, while other PP and lignin pathway gene families appear to be similar in size as Arabidopsis (data not shown). Thus, we interpret the data to mean that unidentified PP family members may function in the mesocarp and exocarp, that PP precursors for flavonoid biosynthesis are produced at sufficient but relatively lower PP gene expression levels and/or that the flavonoid pathway can be fed by an, as of yet unidentified, pathway in fruit tissues. In previous functional studies, silencing of individual PP genes in plants has shown marked decreases in lignin biosynthesis with more limited impacts on flavonoid production [35, 36]. As with the current study, these apparent inconsistencies have gone largely unexplained but collectively point to the conclusion that at least some enzymes in the PP pathway may not be rate limiting to flavonoid biosynthesis. Upon public release of the peach genome sequence (currently being assembled, D Main and B Sosinski, personal communication), it should be possible to differentiate each family member and confirm whether or not the PP pathway is substantially up-regulated during flavonoid biosynthesis.
Mining of gene expression databases for apple, tomato and pepper revealed that induction of the lignin pathway in young fruit is unique to Prunus, while flavonoid pathway induction may have a more ancient origin. The lack of obvious flavonoid induction in pepper and tomato is consistent with the lack of anthocyanins in these fruit which derive their red colour primarily from carotenoids. In contrast, the induction of the flavonoid pathway in anthocyanin rich fleshy fruits is supported by studies in both strawberry and grape [37, 38]. In addition to colour, the flavonoid pathway contributes to a number of important agricultural traits including flavour, nutritive properties and disease/stress resistance. The combined data from peach and apple fruit development studies indicates that the early induction of the flavonoid pathway is limited to genes encoding enzymes involved in the initial steps of flavonoid biosynthesis and proanthocyanidin production.
When placed in a physiological context, the expression patterns of lignin and flavoniod pathway genes are consistent with known aspects of peach fruit development. Peach fruit grow on a sigmoidal curve and show a growth plateau that coincides with the timing of stone hardening. Previous studies in plum fruit show that stones rapidly begin to accumulate dry weight during this time period . This slow down in fruit expansion could be attributed to the substantial energy resources which go in to endocarp lignification and hardening. Our data support this model as lignin gene expression is induced at extremely high levels immediately prior to the slow down in fruit growth. What is perhaps surprising is that expression of flavonoid biosynthesis genes in the flesh and skin appears to occur around the same time as the onset of lignification but diminish before the endocarp substantially hardens. Thus, energy resources in the fruit appear to be carefully partitioned to enable flavonoid accumulation before stone hardening depletes the necessary energy and metabolic resources. Here, the peach cultivar 'Suncrest' was used which is a yellow fleshed variety with red skin. This colour pattern mirrors the higher flavonoid gene expression that we observed in skin. Thus, other peach cultivars with different colour patterns, such as red flesh or yellow skin, may have different flavonoid gene expression patterns. However, flavonoid gene induction is not necessarily associated with anthocyanin production especially since 'Suncrest' has yellow and not red flesh. Rather, it seems likely that early flavonoid induction may also function to protect young fruit against disease and herbivory. Both the lignin and flavonoid pathways are induced during stress and pathogen attack and function to enhance tissue rigidity, decrease digestibility and produce anti-microbial compounds . Young fruit tend to be highly resistant to pathogens and are undesirable to herbivores, in part, due to the presence of flavonoid compounds [41, 42]. Prunus fruits tend to become more susceptible to pathogens after stone hardening and become attractive to herbivores during ripening [43–45]. Thus, the flavonoid pathway serves somewhat opposite functions in Prunus fruits; pathogen and herbivore resistance in young fruit and herbivore attraction when fruit are mature and seeds are ready for dispersal. Still, it is important to bear in mind that the roles of the lignin and flavonoid pathways in fruit do vary substantially, as highlighted by pepper where lignin pathway induction during later stages of ripening drives capsaicinoid production which confers herbivore specificity [31, 46].
Endocarp lignification occurs in a wide range of angiosperms, including both dry and fleshy fruits. This implies that it is either an adaptive process that occurs through relatively simple evolutionary changes or that it represents an ancestral state in which case fruits with non-lignifying endocarps would have intermittently lost this character. In order to address this question, we examined the expression patterns of peach homologues of Arabidopsis genes known to control dehiscence. In Arabidopsis, SHP1/2, STK, IND and ALC act together to define the enb layer boundary and are under negative regulation by FUL and RPL . A previous expression study of SHP, STK and FUL, in peach fruit dissected 30 days after full anthesis, found that SHP was endocarp specific, STK was higher in mesocarp and FUL was substantially expressed in both the endocarp and mesocarp. This indicates differences in the control of peach stone formation and Arabidopsis dehiscence . We found that both SHP and STK were endocarp specific and steadily declined from the earliest fruit stage analysed (29 DAB) while FUL was consistently lower in the endocarp than the mesocarp or exocarp (Figure 6). These patterns mirror those found for the Arabidopsis counterparts and are consistent with a putative role for FUL as a negative regulator of SHP and STK . It is worth noting that FUL expression did not increase in the endocarp as SHP and STK declined. Thus, it appears that SHP and STK are not actively regulated by dynamic FUL levels in the endocarp; rather, it is probably the relative ratio of FUL that enables SHP and STK to promote endocarp differentiation. Surprisingly, ALC and IND expression did not significantly vary with respect to tissue type or developmental time. However, we can not rule out an endocarp specific role as these genes potentially act much earlier in fruit development than analysed here. In Arabidopsis, NST1 promotes enb lignification after tissue identity has been established . The decline of SHP and STK expression just prior to the onset of lignin deposition, followed by subsequent induction of NST1, suggests this same regulatory process may occur in peach stones. Collectively, these data indicate that peach stone formation and Arabidopsis dehiscence appear to be controlled by a highly conserved pathway of positive and negative regulatory transcription factors that first establish tissue identity and then, subsequently, activates a common pathway in order to promote secondary wall formation and lignification. These close similarities imply that endocarp lignification is probably an ancestral state of angiosperm fruit development. It is an intriguing possibility that the concomitant flavonoid pathway induction observed in fleshy fruit mesocarp and exocarp layers may also be more widely conserved and is, likewise, an ancestral condition.