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Petal senescence, or programmed cell death (PCD) in petals, is a developmentally regulated and genetically programmed process. During petal senescence, petal cells show morphological changes associated with PCD: tonoplast rupture and rapid destruction of the cytoplasm. This type of PCD is classified as vacuolar cell death or autolytic PCD based on morphological criteria. In PCD of petal cells, characteristic morphological features including an autophagy-like process, chromatin condensation, and nuclear fragmentation are also observed. While the phytohormone ethylene is known to play a crucial role in petal senescence in some plant species, little is known about the early regulation of ethylene-independent petal senescence. Recently, a NAC (NAM/ATAF1,2/CUC2) transcription factor was reported to control the progression of PCD during petal senescence in Japanese morning glory, which shows ethylene-independent petal senescence. In ethylene-dependent petal senescence, functional analyses of transcription factor genes have revealed the involvement of a basic helix-loop-helix protein and a homeodomain-leucine zipper protein in the transcriptional regulation of the ethylene biosynthesis pathway. Here we review the recent advances in our knowledge of petal senescence, mostly focusing on the morphology of senescing petal cells and the regulatory mechanisms of PCD by senescence-associated transcription factors during petal senescence., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Main Conclusion: The role of mannitol differs from that of glucose, fructose and sucrose in sepal cell expansion associated with flower opening in Delphinium × belladonna. Sepals of Delphinium × belladonna are colored and much larger than the petals. To determine whether the role of mannitol in sepal growth associated with flower opening differs from those of ubiquitous metabolic sugars including glucose, fructose and sucrose, we investigated changes in cell number, subcellular concentrations of soluble carbohydrates, and osmotic potential in sepals during flower opening in Delphinium × belladonna cv. Bellamosum. The number of epidermal cells in the sepals did not increase from the stage when sepal pigmentation started, whereas the cell area increased during flower opening, indicating that petal growth during flower opening depends on cell expansion. Mannitol concentrations in the vacuole at three different stages were approximately 100 mM, which were much higher than the other carbohydrate concentrations, but they decreased slightly at open stage. In contrast, mannitol concentration in the cytoplasm was 56 mM at bud stage, but it increased to 104 mM at open stage. Glucose and fructose concentrations in the vacuole at open stage increased to 45 and 56 mM, respectively. Total osmotic potential in apoplast and symplast, which was partially due to soluble carbohydrates, was almost constant during flower opening. Therefore, mannitol may be acting constitutively as the main osmoticum in the vacuole where it may contribute to the maintenance of the osmotic balance between the cytoplasm and vacuole in open flowers. The role of mannitol differs from those of glucose, fructose, and sucrose in sepal cell expansion in Delphinium × belladonna.

In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals.

To clarify whether climacteric-like increases in ethylene production of senescing petals are also induced in the absence of the gynoecium in cut carnation (Dianthus caryophyllus cv. Barbara) flowers, we compared ethylene production and expression of ethylene-biosynthesis genes in detached petals and in petals, which remained on flowers (attached petals). No significant difference in longevity was observed between the attached and detached petals when held in distilled water, and both showed the inward rolling typical of senescing flowers. Treatment with silver thiosulfate complex (STS), an ethylene inhibitor, similarly delayed senescence of attached and detached petals. Climacteric-like increases in ethylene production of petals and gynoecium started on the same day, with similar bursts in attached and detached petals. Transcript levels of DcACS1 and DcACO1 were very low at harvest and increased similarly during senescence in both petal groups. Removal of the gynoecium did not significantly delay wilting of attached petals. In flowers with the gynoecium removed, the petals produced most of the ethylene while production by the other floral organs was very low, suggesting that wound-induced ethylene is not the reason for the ineffectiveness of gynoecium-removal in inhibiting flower senescence. These results indicate that ethylene biosynthesis is induced in carnation petals irrespective of the gynoecium., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Autophagy is one of the main mechanisms of degradation and remobilization of macromolecules, and it appears to play an important role in petal senescence. However, little is known about the regulatory mechanisms of autophagy in petal senescence. Autophagic processes were observed by electron microscopy and monodansylcadaverine staining of senescing petals of petunia (Petunia hybrida); autophagy-related gene 8 (ATG8) homologues were isolated from petunia and the regulation of expression was analysed. Nutrient remobilization was also examined during pollination-induced petal senescence. Active autophagic processes were observed in the mesophyll cells of senescing petunia petals. Pollination induced the expression of PhATG8 homologues and was accompanied by an increase in ethylene production. Ethylene inhibitor treatment in pollinated flowers delayed the induction of PhATG8 homologues, and ethylene treatment rapidly upregulated PhATG8 homologues in petunia petals. Dry weight and nitrogen content were decreased in the petals and increased in the ovaries after pollination in detached flowers. These results indicated that pollination induces autophagy and that ethylene is a key regulator of autophagy in petal senescence of petunia. The data also demonstrated the translocation of nutrients from the petals to the ovaries during pollination-induced petal senescence.

Anthocyanins are major flower pigments that can be affected by copigments, colorless compounds that can modify anthocyanin coloration to more intense and bluer. Thin-layer chromatography (TLC) is an available technique to separate and analyze anthocyanins and copigments. To easily and comprehensively detect copigments, we added function of mixture of compounds to TLC; by slantingly cross loading samples on TLC, compounds are symmetrically developed at various angle lines from the upper origin to individual R(f) values and cross each other in an orderly fashion, where mixture is simultaneously performed with separation. Occurrence of copigments can be detected as a coloration change on the developed line of anthocyanin. Pink sweet pea (Lathyrus odoratus L.) petals were analyzed by the cross-TLC and a more intense spot and a paler spot on the anthocyanin line were detected. As each spot overlapped with an ultraviolet absorbance line, each of these ultraviolet absorption compounds was purified and identified as kaempferol 3-rhamnoside and 2-cyanoethyl-isoxazolin-5-one, respectively. Whereas kaempferol 3-rhamnoside is a flavonoid and had a general copigment effect of more intense and bluer coloration change, 2-cyanoethyl-isoxazolin-5-one is a compound whose structure is outside of conventional categories of copigments and had a novel effect to change anthocyanin coloration paler while maintaining color tone. We determined that the search for copigments should be carried out without pre-existing prediction of structures and effects. We have shown that slantingly cross loading samples system on plate-type chromatography is an effective technique for such comprehensive analysis of molecular interaction., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Petal growth associated with flower opening depends on cell expansion. To understand the role of soluble carbohydrates in petal cell expansion during flower opening, changes in soluble carbohydrate concentrations in vacuole, cytoplasm and apoplast of petal cells during flower opening in rose (Rosa hybrida L.) were investigated. We determined the subcellular distribution of soluble carbohydrates by combining nonaqueous fractionation method and infiltration-centrifugation method. During petal growth, fructose and glucose rapidly accumulated in the vacuole, reaching a maximum when petals almost reflected. Transmission electron microscopy showed that the volume of vacuole and air space drastically increased with petal growth. Carbohydrate concentration was calculated for each compartment of the petal cells and in petals that almost reflected, glucose and fructose concentrations increased to higher than 100 mM in the vacuole. Osmotic pressure increased in apoplast and symplast during flower opening, and this increase was mainly attributed to increases in fructose and glucose concentrations. No large difference in osmotic pressure due to soluble carbohydrates was observed between the apoplast and symplast before flower opening, but total osmotic pressure was much higher in the symplast than in the apoplast, a difference that was partially attributed to inorganic ions. An increase in osmotic pressure due to the continued accumulation of glucose and fructose in the symplast may facilitate water influx into cells, contributing to cell expansion associated with flower opening under conditions where osmotic pressure is higher in the symplast than in the apoplast.

Petal senescence is a type of programmed cell death (PCD) that is tightly regulated by multiple genes. We recently reported that a putative membrane protein, InPSR26, regulates progression of PCD during petal senescence in Japanese morning glory (Ipomoea nil). Reduced InPSR26 expression in transgenic plants (PSR26r lines) resulted in accelerated petal senescence with hastened development of PCD symptoms, and transcript levels of autophagy-related genes were reduced in the petals. Autophagy visualized by monodansylcadaverine staining indicated reduced autophagic activity in the PSR26r plants. The results from our recent studies suggest that InPSR26 acts to delay the progression of PCD during petal senescence, possibly through regulation of the autophagic process. In this addendum, we discuss the role of autophagy in petal senescence as it relates to these findings.

In senescent petals of Ipomoea nil, we investigated the expression of genes showing homology to genes involved in animal programmed cell death (PCD). Three encoded proteins were homologous to apoptotic proteins in animals: Bax inhibitor-1 (BI-1), a vacuolar processing enzyme (VPE; homologous to caspases) and a monodehydroascorbate reductase [MDAR; homologous to apoptosis-inducing factor (AIF)]. AIFs harbor an oxidoreductase domain and an apoptotic domain. MDARs exhibit homology to the AIF oxidoreductase domain, not to the apoptotic domain. The three other genes studied relate to autophagy. They encode homologs to vacuolar protein sorting 34 (VPS34) and to the Arabidopsis autophagy-related proteins 4b and 8a (ATG4b and ATG8a). The transcript abundance of MDAR decreased continuously, whereas that of the other genes studies exhibited a transient increase, except ATG4b whose abundance stayed high after an increase. Treatment with ethylene advanced the time to visible petal senescence, and hastened the changes in expression of each of the genes studied. In order to assess the role of VPS34 in petal senescence, we studied the effect of its inhibitor 3-methyladenine (3-MA). 3-MA reduced the time to visible petal senescence, and also accelerated the time to DNA degradation. Remarkably, 3-MA increased the time to nuclear fragmentation, indicating that the time to visible petal senescence was independent of nuclear fragmentation. The data on 3-MA might suggest the idea that autophagy is not a cause of PCD, but part of the remobilization process.

The onset and progression of petal senescence, which is a type of programmed cell death (PCD), are highly regulated. Genes showing changes in expression during petal senescence in Japanese morning glory (Ipomoea nil) were isolated and examined to elucidate their function in PCD. We show here that a putative membrane protein, InPSR26, regulates progression of PCD during petal senescence in Japanese morning glory. InPSR26 is dominantly expressed in petal limbs and its transcript level increases prior to visible senescence symptoms. Transgenic plants with reduced InPSR26 expression (PSR26r lines) showed accelerated petal wilting, with PCD symptoms including cell collapse, ion and anthocyanin leakage, and DNA degradation accelerated in petals compared to wild-type plants. Transcript levels of autophagy- and PCD-related genes (InATG4, InATG8, InVPE, and InBI-1) were reduced in the petals of PSR26r plants. Autophagy visualized by monodansylcadaverine staining confirmed that autophagy is induced in senescing petal cells of wild-type plants and that the percentage of cells containing monodansylcadaverine-stained structures, most likely autophagosomes, was significantly lower in the petals of PSR26r plants, indicating reduced autophagic activity in the PSR26r plants. These results suggest that InPSR26 acts to delay the progression of PCD during petal senescence, possibly through regulation of the autophagic process. Our data also suggest that autophagy delays PCD in petal senescence.

Depending on the species, the end of flower life span is characterized by petal wilting or by abscission of petals that are still fully turgid. Wilting at the end of petal life is due to programmed cell death (PCD). It is not known whether the abscission of turgid petals is preceded by PCD. We studied some parameters that indicate PCD: chromatin condensation, a decrease in nuclear diameter, DNA fragmentation, and DNA content per nucleus, using Prunus yedoensis and Delphinium belladonna which both show abscission of turgid petals at the end of floral life. No DNA degradation, no chromatin condensation, and no change in nuclear volume was observed in P. yedoensis petals, prior to abscission. In abscising D. belladonna petals, in contrast, considerable DNA degradation was found, chromatin was condensed and the nuclear volume considerably reduced. Following abscission, the nuclear area in both species drastically increased, and the chromatin became unevenly distributed. Similar chromatin changes were observed after dehydration (24 h at 60 degrees C) of petals severed at the time of flower opening, and in dehydrated petals of Ipomoea nil and Petunia hybrida, severed at the time of flower opening. In these flowers the petal life span is terminated by wilting rather than abscission. It is concluded that the abscission of turgid petals in D. belladonna was preceded by a number of PCD indicators, whereas no such evidence for PCD was found at the time of P. yedoensis petal abscission. Dehydration of the petal cells, after abscission, was associated with a remarkable nuclear morphology which was also found in younger petals subjected to dehydration. This nuclear morphology has apparently not been described previously, for any organism.

We isolated several senescence-associated genes (SAGs) from the petals of morning glory (Ipomoea nil) flowers, with the aim of furthering our understanding of programmed cell death. Samples were taken from the closed bud stage to advanced visible senescence. Actinomycin D, an inhibitor of transcription, if given prior to 4 h after opening, suppressed the onset of visible senescence, which occurred at about 9 h after flower opening. The isolated genes all showed upregulation. Two cell-wall related genes were upregulated early, one encoding an extensin and one a caffeoyl-CoA-3-O-methyltransferase, involved in lignin production. A pectinacetylesterase was upregulated after flower opening and might be involved in cell-wall degradation. Some identified genes showed high homology with published SAGs possibly involved in remobilisation processes: an alcohol dehydrogenase and three cysteine proteases. One transcript encoded a leucine-rich repeat receptor protein kinase, putatively involved in signal transduction. Another transcript encoded a 14-3-3 protein, also a protein kinase. Two genes have apparently not been associated previously with senescence: the first encoded a putative SEC14, which is required for Golgi vesicle transport, the second was a putative ataxin-2, which has been related to RNA metabolism. Induction of the latter has been shown to result in cell death in yeast, due to defects in actin filament formation. The possible roles of these genes in programmed cell death are discussed.

To clarify the relationships of flower senescence, especially sepal abscission, and ethylene receptor gene expression in different flower parts, we isolated two cDNAs encoding ethylene receptors Dl-ERS1-3 and Dl-ERS2 from Delphinium flowers. Deduced polypeptides possessed no response regulator domain, indicating that they belong to a family of ethylene response sensor (ERS) ethylene receptors. Dl-ERS1-3 and Dl-ERS2 exhibited constitutive levels during flower senescence. Exogenous ethylene increased transcript levels in sepals, which are influenced by ethylene but not in gynoecia and receptacles, which produce ethylene. It was suggested that expression of ethylene receptor genes under ethylene exposure was differentially regulated in each organ of the flower.

Programmed cell death (PCD) was studied in the petals of Antirrhinum majus, Argyranthemum frutescens, and Petunia hybrida, using DNA degradation and changes in nuclear morphology as parameters. The petals exhibit loss of turgor (wilting) as a visible symptom of PCD. DNA degradation, as shown on agarose gels, occurred in all species studied, prior to visible wilting. The number of DNA masses in all the petals of a flower, determined by flow cytometry, markedly increased in Argyranthemum and Petunia, but decreased in Antirrhinum. Many small DNA masses were observed in Argyranthemum and Petunia. The surface of each small DNA mass stained with the lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide (DiOC6), indicating that these masses were surrounded by a membrane. In Antirrhinum, in contrast, the chromatin fragmented into several small spherical clumps that remained inside a large membranous structure. Nuclear fragmentation, therefore, did not occur in Antirrhinum, whereas nuclear fragmentation possibly was a cause of the small DNA masses in Argyranthemum and Petunia. It is concluded that at least two contrasting nuclear morphologies exist during PCD. In the first, the chromatin fragments inside the nucleus, not accompanied--or followed--by nuclear fragmentation. In the second, a large number of DNA masses were observed each enveloped by a membrane. The second type was probably due, at least partially, to nuclear fragmentation.

We isolated a homolog of the potential anti-apoptotic gene, defender against apoptotic death (DAD1) from gladiolus petals as full-length cDNA (GlDAD1), and investigated the relationship between its expression and the execution processes of programmed cell death (PCD) in senescing petals. RNA gel blotting showed that GlDAD1 expression in petals was drastically reduced, considerably before the first visible senescence symptom (petal wilting). A few days after down-regulation GlDAD1 expression, DNA and nuclear fragmentation were observed, both specific for the execution phase of PCD.

2-C-methyl-D-erythritol, a soluble carbohydrate that is not ubiquitously found in higher plants, was detected in the ethanol extract from Phlox subulata petals and isolated using HPLC. The isolated compound was identified by 1H-NMR, 13C-NMR and Cl-MS spectra. 2-C-methyl-D-erythritol was a major soluble carbohydrate in petals, leaves and stems. In petals, the concentration of 2-C-methyl-D-erythritol markedly increased during flower development and opening and was similar in concentration to glucose, a ubiquitous metabolic sugar. This suggests that 2-C-methyl-D-erythritol may contribute to flower opening in association with glucose in the P. subulata.

Methyl glucoside andmyo-inositol are present in all organs of rose (Rosa hybridaL.). To investigate the possible role of these carbohydrates in the opening of cut roses, flowers with a 10, 20 or 40-cm-long stem and a single flower bud (about 1.5 cm in diameter) were placed in water and flower opening and changes in sugar content in flowers and stems examined for 7 d. The longer the stem of the cut flower, the larger was the flower diameter. In stems, the concentration of carbohydrates, including methyl glucoside andmyo-inositol markedly decreased before flower opening. In petals, contents of glucose, methyl glucoside andmyo-inositol also decreased before flower opening, but those of fructose, sucrose and xylose did not. When glucose and methyl glucoside were added to the vase water (4%) flower opening was clearly promoted; this was accompanied by an increase in methyl glucoside and fructose concentrations in petals. On the contrary,myo-inositol inhibited flower opening, and this was accompanied by an increase inmyo-inositol and xylose concentrations in petals. These results suggest that methyl glucoside and/or its metabolites are transported into the petal cells, thereby lowering the osmotic water potential and promoting flower opening.Myo-inositol is not readily metabolized, and exogenousmyo-inositol given at a high concentration may act as an extracellular osmolyte, which inhibits water uptake and flower opening.Copyright 1999 Annals of Botany Company [ABSTRACT FROM AUTHOR]

• A homolog of ACO gene was cloned and designated it DcACO2. • The predicted protein of DcACO2 was highly homologous to DcACO1 protein. • The expression of DcACO2 was relatively high in style, but was low in petals. • Ethylene and ACC treatments increased DcACO1 expression, but not DcACO 2 expression. • The expression of DcACO1 and DcACO2 was differently regulated among floral organs. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) catalyzes the final step of ethylene biosynthesis. ACO proteins are also reportedly encoded by a multigene family. Although the presence of several ACO genes is suggested in carnation (Dianthus caryophyllus L.), DcACO1 is the only ACO gene in which full-length cDNA has been isolated. This study aimed to clone another ACO gene in carnation flowers and investigate changes the expression of the two ACO genes in floral organs during flower senescence. We cloned a homolog of the ACO gene from carnation gynoecium and designated it DcACO2. Transcript levels of DcACO2 were higher in the style and ovary than in the petals at harvest, although DcACO2 transcript levels in these organs increased during flower senescence. The ACO activity in the style was very high at harvest, suggesting that this high activity could be attributed to the translation of the DcACO2 transcript. However, DcACO2 transcript was only detected at very low levels in the petals of senesced flowers. Ethylene and ACC treatments accelerated petal wilting and increased ethylene production of the petals, style, and ovary. In the style and ovary, the DcACO1 transcript level was markedly higher than the DcACO2 transcript level as a result of ethylene and ACC treatments, and DcACO1 transcript levels in the petals were also increased by ethylene and ACC treatments. These results indicate that the expression of DcACO1 and DcACO2 are differently regulated among floral organs during senescence in carnation flowers. [ABSTRACT FROM AUTHOR]

To understand the biochemical mechanism underlying flower opening, the manner of cell expansion, soluble carbohydrate concentration, and expression of expansin and xyloglucan endotransglucosylase/hydrolase (XTH) genes were investigated in the petals of Oriental lily (Lilium 'Sorbonne'). Microscopic observation revealed that petal growth during flower opening mainly depended on cell expansion, which was accompanied by increases in glucose and fructose concentrations in the petals. The adaxial and abaxial sides of the petals grew at different rates during flower opening with petal reflection. To determine the concentration of soluble carbohydrates and the expression of expansin and XTH genes in adaxial and abaxial epidermal cells and parenchyma cells, these cells were separated using tweezers. We confirmed that these cells could be sufficiently separated. Glucose and fructose concentrations were higher in adaxial epidermal cells than in abaxial epidermal cells at the stage immediately preceding flower opening, but these differences diminished during flower opening. Three expansin genes, LhEXPA1 , LhEXPA2 , and LhEXPA3 , and two XTH genes, LhXTH1 and LhXTH2 were isolated. LhXTH1 transcript levels in the petals markedly increased during flower opening and were higher in adaxial epidermal cells than in other types of cells. Conversely, the levels of the three EXPA transcripts decreased during flower opening and there were slight differences in their levels among different cell types, with a few exceptions. In conclusion, differences in glucose and fructose concentrations between adaxial and abaxial epidermal cells, together with the expression of LhXTH1 , may contribute to cell expansion associated with flower opening. • Adaxial and abaxial epidermal cells and parenchyma cells were sufficiently separated. • Sugar concentrations were higher in adaxial than in abaxial epidermal cells. • Three expansin genes and two XTH genes were isolated from petals. • LhXTH1 transcript level was higher in adaxial cells than in the other types of cells. • There were slight differences in three EXPA expression among cell types. [ABSTRACT FROM AUTHOR]