OMT3 was found to be the major genetic determinant for this trait in two independent studies. Nevertheless, it is possible that OMT1 may contribute to the IBMP concentration, because OMT1 can synthesize IBMP and it is located at the edge of a QTL significantly contributing to this trait. Furthermore, the majority of IBHP , the precursor for the OMT1 and OMT3 biosynthesis of IBMP, is produced in the pulp of the berry complicating the factors that influence IBMP concentration. Our results raise questions that require additional research to clarify this relationship of transcript abundance to IBMP concentration, including determination of the rates of biosynthesis and catabolism, enzyme activities, volatilization of IBMP from the berry, as well as the concentrations of substrates for the enzymes involved. There are a number of other transcriptomic ripening studies in grapes and other fruit species. Many of these have compared broad developmental stages with partial genome microarrays. One study compared transcriptomic responses of the lates stages of ripening of whole berries of Chardonnay. This study used a different microarray platform with only about half of the genome represented on the array. In this study, 12 genes were found to be differentially expressed in each of the 3 different stages investigated. There were approximately another 50 genes that were differentially expressed at one stage versus another. Several genes were proposed as good candidates for markers of ripeness and these were also examined in Cabernet Sauvignon berries using qPCR. Several of these candidate genes are consistent with our results in the present study. They include CCD4a , hydroponic nft channel a late embryogenesis abundant protein , a dirigent-like protein , and an S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase .
Of these, the transcript expression of SAMT was found to be temperature insensitive. Like the previous study, the present study focused on very close stages in the mature berry when fruit flavors are known to develop. In contrast to the previous study on Chardonnay, there were massive changes in the transcript abundance in hundreds of GO categories over this narrow window of ripening. This may in part be due to using six biological replicates rather than the standard three, which probably improved the detection of significantly changing transcripts. In addition, we used a different threshold level for statistical significance and an improved microarray platform, which was able to detect double the number of transcripts. In the present study, many differences were found between the skin and the pulp, °Brix levels and the interaction of tissue and °Brix. Important fruit ripening processes were affected including ethylene signaling, senescence, volatile aroma production, lipid metabolism and cell wall softening. These data indicate that fruit ripening in the late stages of maturity is a very dynamic and active process.Ethylene is involved in climacteric fruit ripening with a CO2 burst preceding the rise in ethylene. In tomato, this occurs at the time the seeds become mature in the mature green fruit stage. At this stage, tomato fruits become sensitive to ethyene and can continue through the ripening stage. Prior to the mature breaker stage, ethylene cannot promote tomato ripening to full ripeness. In non-climacteric fruit, there is no respiratory burst of CO2 and the ripening of most non-climacteric fruits was thought not to respond significantly to an extra application of ethylene. However, recently some non-climacteric fruit such as strawberry, bell pepper and grape have been found to produce a small amount of ethylene and appear to have responses to ethylene at certain stages.
In the study of grapes, this peak was observed just before the start of veraison, followed by decreases in ethylene concentrations for several weeks afterwards; the late mature stages of ripening were not examined. Ethylene action is dependent upon ethylene concentration and ethylene sensitivity or signaling. In this study, there were clear and significant changes in transcript abundance of genes involved in ethylene signaling and biosynthesis in the late stages of berry ripening. Seeds become fully mature at this time . Perhaps there is a signal from the seeds when they become mature that allows the fruit to ripen and senesce? Perhaps small amounts of ethylene are produced or there is a change in sensitivity to ethylene? Seymour et al. suggested the response of EIN3 might be a common signaling mechanism for both climacteric and non-climacteric fruit. The responses of VviEIN3 in this study and in a pepper fruit ripening study are consistent with this hypothesis. In addition, the transcript abundance of VviEIN3 in grape is very responsive to ethylene and the ethylene inhibitor, MCP. There are many other factors other than fruit development that can influence ethylene signaling. Could chilling of the fruit or other aspects of the processing of the grapes influence these responses? Could there be some influence of other abiotic or biotic stresses? These are questions that can only be addressed in future studies with additional experiments that are designed to answer these questions.Hydroxycinnamic acid amides are a group of plant secondary metabolites found in a wide range of plant species. Many studies have identified critical roles that HCAAs play in plant growth and developmental processes, including cell division, cytomorphogenesis, flowering, cell wall cross-linking, tuberization, and stress responses.
These compounds are antioxidants and effective free radical scavengers with anticarcinogenic, antihypertensive, antimicrobial, and other potentially therapeutic activity of significant benefit to human and animal health. Due to the diversity of carbon skeletons, HCAAs can be divided into many categories such as the polyamine conjugates hydroxycinnamoylspermidine , −spermine , and -putrescine . HCSpd and HCPut are predominant in the plant kingdom, while only a few plants are rich in HCSpm compounds . HCSpm, rare in nature, exhibit unique health benefits. For instance, N1 , N14-bis spermine is a major compound that confers the hypotensive and antiparasitic activities in fruit of the Chinese medicinal species Lycium chinense. Trypanothione reductase, an essential enzyme for survival of pathogenic protozoa such as Leishmania and other trypanosomes, is inhibited by afourfold lower concentration of Kukoamine A compared with its spermidine counterpart [N1 , N10-bis -spermidine]. Kukoamine A also shows anticancer activity and attenuates insulin resistance and fatty liver disease. In addition, N1 -coumaroylspermine, but not N1 -coumaroylspermidine, is found to efficiently inhibit mammalian and crayfish neuroreceptors in vitro, an ability of great interest for pest management as well as pain management.Like most solanaceous plants that have been phytochemically analyzed, Solanum fruit crops such as eggplant are rich in HCAAs with spermidine or putrescine, but not spermine, as the polyamine moiety. However, HCSpms possess medicinal properties distinct from those of the other, much more common HCAAs. Although HCSpms have been known for a few decades as rare plant HCAAs of potential therapeutic value, the committed enzyme catalyzing the condensation of hydroxycinnamoyl-CoA with spermine has not been identified so far. In this study, we identified and characterized SpmHT, a spermine exclusive HT from S. richardii, a wild eggplant relative found in Africa.Structurally SpmHT shares linear similarities to other polyamine hydroxycinnamoyl transferase family enzymes, such as spermidine HT and putrescine HT . However, SpmHT has two unique features compared with other HTs. First, SpmHT has the highest activity among all known polyamine HTs, nft growing system as its Vmax is more than 5, 7, 14, 21, and 84 times that of SbHCT, SrSHT, AtSDT, AtSCT, and CcHCT, respectively. Second, SpmHT only utilizes spermine as acyl acceptor. Several catalytic-activityrelated residues have been identified in HTs by mutagenesis, such as Thr-36 and Ser-38 in SbHCT. These two residues are specifically involved in hydroxycinnamoyl moiety binding. The positions corresponding to Thr-36 and Ser-38 are replaced by Leu in SpmHTs, and by Val and Thr in SHTs . Whether these two substitutions in SpmHT have an impact on the catalytic activity is worth further study. As far as the polyamine moiety binding, molecular docking indicates that SpmHT prefers spermine to spermidine based on FullFitness and cluster formation. Three residues involved in the formation of hydrogen bonds are highly conserved in all putative SpmHTs, whereas the corresponding sites in SrSHT are substituted by distinct residues in SrSHT. Further structural study is needed to address how SpmHT specifically selects the acceptor substrate and why it has high enzyme activity.
Our previous analyses show that HCSpms in S. richardii fruit are mainly di- or tri- hydrocaffeoyl acylated spermine . However in vitro studies exhibited a specific monohydrocaffeoyl acylation of spermine by SpmHT. It is not clear whether SpmHT can use di- or tri- hydrocaffeoyl CoA as the substrate in planta. Another possibility is that monosubstituted spermine conjugate may be an acyl acceptor for a second hydroxycinnamoyl transfer by another acyltransferase. In the case of two native tobacco HTs , NaDH29 mediates the initial acylation step specifically on Spd and is not able to perform the second acylation. Further acylation is committed by NaCV86 to act on monoacylated spermidines. Further elucidation requires structural or mutagenesis studies and in vivo functional analysis. SpmHT is highly expressed in fruit of S. richardii but barely in any tissue of S. melongena or other relatives we profiled. Therefore, SpmHT has been selectively silenced in eggplant and many other plants, suggesting that the reduced expression of SpmHT in eggplant fruit would represent an interesting model to investigate molecular mechanism of eggplant phytochemical evolution during the domestication process. High expression of SpmS ensures the sufficiency of Spm for HCSpm synthesis in S. richardii fruit. In contrast, there is almost no expression of SpmS in S. melongena fruits. Hence HCAA composition in S. richardii might be achieved through coordinated expression of HCAA structural genes SpmHT and SpmS. Their coordinated expression may be regulated by some master regulators. MYBs have been shown in other species to regulate HCT expression. For example, in Nicotiana attenuata, MYB8 controls phenolamide levels by directly activating the transcription of three polyamine hydroxycinnamic acid transferases. Since then, they have been similarly implicated in both monocots and eudicots such as strawberry. A recent study showed that a positive regulator ORA59 could bind to the promoter of an Arabidopsis agmatine coumaroyl transferase and enabled its expression and HCAAs biosynthesis to respond to simultaneous activation of the JA/ET signaling pathways. It would be interesting to identify the master regulators involved in SpmHT biosynthesis in order to activate the pathway and study the function of SpmHT. High levels of SpmS expression suggest that abundant Spm is synthesized in S. richardii fruit. This is unusual because Spm is synthesized at lower levels than Spd in most plants. Generally speaking, Spd is thought to contribute to higher vegetative growth, less shriveling , and longer life span in transgenic plants over expressing SpdS, while elevated Spm promotes abiotic and biotic stress tolerance by inducing the expression of defense genes in plants . Hence, it appears that Spd is largely implicated in developmental processes, whereas Spm is more likely involved in stress response. However, excessive Spm maycause abnormal development and consequently there must be tight regulation of polyamine homeostasis for normal growth of plants.The pineapple Merr is the 3rd most important fruit traded globally, but it is susceptible to chilling injury . Chilling injury is a complex physiological disorder that occurs in tropical and sub-tropical fruit, including pineapple, after exposure to low temperatures . Chilling may occur in the field in winter grown fruit causing pre-harvest chilling injury, or after harvest when fruit are stored at low temperatures, i.e., post harvest chilling injury; PCI . Losses of up to 80% have been attributed to PCI of pineapples after storage at 10 ◦C , which reduces wider market ability of the fruit. Because of the magnitude of the problem presented by PCI, the focus of this study was limited to this phenomenon and did not include pre-harvest chilling injury.The severity of PCI depends on the temperature to which fruit is exposed and the duration of exposure. PCI symptoms in pineapple are initiated after 1–3 weeks storage at temperatures below 12 ◦C followed by a transfer to room temperature . Symptoms mainly occur in the fleshy region adjacent to the core and the earliest manifestation is tissue translucency symptoms . As the symptoms intensify, browning or blackening of the tissue becomes noticeable. The resulting fruit damage, also called “internal browning” , “black heart,” or “endogenous brown spot,” occurs internally with no external signs of injury . IB-susceptible pineapple cultivars belonging to the ‘Queen’ group show symptoms within 1–2 weeks after storage at temperatures below 10–12 ◦C . The tissue first turns translucent, then brown, in both the core and the fleshy tissue of the fruit .