Programmed Cell Death in Relation to Petal Senescence in Ornamental Plants

Received 14 Jul. 2004 Accepted 13 Nov. 2004Supported by the Hi-Tech Research and Development (863) Program of China (2001AA241201) and the Hubei Provincial Natural ScienceFoundation of China (99J112).*Author for correspondence. Tel: +86 (0)27 8728 2010; Fax: +86 (0)27 8728 2095; E-mail: <[email protected]>.

Journal of Integrative Plant BiologyFormerly Acta Botanica Sinica 2005, 47 (6): 641−650

http://www.blackwell-synergy.comhttp://www.chineseplantscience.com

Programmed Cell Death in Relation to Petal Senescence in Ornamental PlantsYuan ZHOU1, Cai-Yun WANG1, 2*, Hong GE3, Frank A. HOEBERICHTS4 and Peter B. VISSER5

(1. State Key Laboratory for Biology of Horticultural Plants, Huazhong Agricultural University, Wuhan 430070, China;2. College of Landscape Architecture, Beijing Forestry University, Beijing 100083, China;

3. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China;4. Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia – CSIC,

Avda de los Naranjos s/n 46022, Valencia, Spain;5. Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Camino de Vera s/n 46022, Valencia, Spain)

Abstract: Cell death is a common event in all types of plant organisms. Understanding the phenomenonof programmed cell death (PCD) is an important area of research for plant scientists because of its role insenescence and the post-harvest quality of ornamentals, fruits, and vegetables. In the present paper, PCDin relation to petal senescence in ornamental plants is reviewed. Morphological, anatomical, physiological,and biochemical changes that are related to PCD in petals, such as water content, sink-source relationships,hormones, genes, and signal transduction pathways, are discussed. Several approaches to improving thequality of post-harvest ornamentals are reviewed and some prospects for future research are given.Key words: ornamental plants; petal senescence; post-harvest quality; programmed cell death.

Cell death includes two different cellular processesthat result in the death of cells: (i) cell necrosis; and (ii)programmed cell death (PCD). Cell necrosis, as a re-sult of a sudden, traumatic event, is not regulated bygenes, whereas PCD, also known as apoptosis inanimals, is a common programmed process that is ge-netically defined and involving a number of regulatorygenes, stimulatory events, and signaling pathways(Danon et al. 2000). Several typical hallmarks of PCDhave been reported in past years. Morphological changesin cells that are observed during the process of PCDare nuclear condensation, plasma membrane blebbing,and budding of so-called apoptotic bodies (Faragher etal. 1987), which was described in cell culture inducedby heat shock (McCabe et al. 1997), as well as in thecells of senescing petals, such as four-o’clock flower(Mirabilis jalapa L.; Li et al. 1994). At the molecularlevel, RNA, proteins, and lipids are degraded as an-other feature of PCD, which was also investigated in

senescent petal tissue of four-o’clock flower (Li et al.1994). A breakdown pattern of DNA fragments, alsoknown as “DNA ladders”, is the simplest hallmark ofPCD that can be observed in both plants and animals.DNA degradation in plant cells has been investigated indifferent developmental cycles and in different organs(Cheah and Osborne 1978; Orzaez and Granell 1997;Danon et al. 2000; Xu and Hanson 2000).

The PCD plays an important role in plant develop-ment and stress responses. In xylogenesis and rootgrowth, the tracheary elements (TEs) and root cap cellshave been observed to undergo PCD (Wang et al. 1996b;Cao et al. 2003). PCD has been investigated in severalstages of reproduction, including somatic embryogen-esis (Havel and Durzan 1996), sex determination (deLong et al. 1993), the growth of pollen tubes (Wang etal. 1996c), the disappearance of the monocot aleuronelayer, and in the endosperm of seeds (Wang et al.1996d), and also occurs in different senescent plant

.Review.

Journal of Integrative Plant Biology (Formerly Acta Botanica Sinica) Vol. 47 No. 6 2005642

organs, involving leaves, stems, petals, and carpels, inwhich DNA fragmentation and morphological changeshave been detected. Furthermore, several PCD-relatedgenes have been cloned (Hoeberichts and Woltering2003). With regard to pathogenesis, the hypersensitiveresponse (HR) at the site of the infection is consideredto be a form of PCD in plants for which genetic con-trol is beginning to be elucidated (Greenberg 1996).

Petal senescence, an often rapid and synchronousprocess that marks the final stage of a flower’s lifespan,is the consequence of PCD occurring in individual cells.In flowering ornamentals, the condition of the petals isthe most important determinant of their freshness and,thus, their economic value. Understanding the geneticsand biochemistry of petal senescence triggered by PCDas a result of aging and biotic or abiotic stress is there-fore essential for improving the post-harvest quality ofornamentals. The purpose of the present review is tohighlight some PCD-related events that occur duringpetal senescence in cut flowers and to review the cur-rent knowledge on signals and signaling pathways in-volved in PCD. Finally, several practical approaches todelaying petal-associated PCD are discussed.

1 Methods to Study the Phenomenon of PCDin Flowers

Several approaches to investigating PCD in orna-mentals can be applied. Studying senescing petals un-der controlled conditions provides an easy system tofollow the process of PCD in cut flowers. The modelflower species currently used are characterized by anextreme rapid senescence, such as the daylily(Hemerocallis spp.; Guerrero et al. 1998; Panavas andRubenstein 1998; Panavas et al. 1998a), or by theirextreme sensitivity to ethylene, such as the carnation(Dianthus caryophyllus L.; van Altvorst and Bovy 1995;Ten Have and Woltering 1997). Generally, the PCD ofsenescing petals is characterized by changes that oc-cur in individual cells, such as plasma membraneblebbing, cellular and nuclear condensation andfragmentation, and the cleavage of DNA intooligonucleosomes (Rubinstein 2000). Morphological

changes in the nuclei of cells that undergo PCD can beobserved at the subcellular level using an electronmicroscope. DNA fragmentation can be visualized inagarose gels. Cells in the process of PCD can easily bedistinguished from healthy cells by in situ terminaldeoxyribonucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL; Mittler and Lam 1995) or in situ end-labeling (ISEL; Ning et al. 1999). Using cDNAmicroarray analysis, gene expression in cut flowers canbe evaluated at several stages of petal wilting and pu-tative transcription factors associated with PCD canbe identified (van Doorn et al. 2003). Identification,isolation, and cloning of these PCD-related genes mayprovide us with important tools to investigate the pre-cise mechanism of the PCD of flowers in ornamentals.

2 Environmental Factors Inducing PCD inFlowers

A very striking response of flowers to a biotic envi-ronmental factor is PCD caused by pollination (Stead1992; van Doorn 1997). Pollination generally initiatesPCD and promotes rapid petal senescence. The amountof pollen on the stigma is positively correlated withethylene production and induced senescence in Eustomaflowers (Ichimura et al. 1998). It was observed thatflowers tested from Phalaenopsis developed PCDwithin 1 d after pollination (Halevy 1998). In Petuniainflata L., PCD was initiated 24 h after compatible pol-lination (Xu and Hanson 2000).

Cut flowers are usually subjected to water loss af-ter harvest. In addition, the vascular system of cut flow-ers often gets plugged owing to microbial growth invase water, leading to water deficiency in the flowers(Wang et al. 1996a). This phenomenon is very com-mon in most cut flowers. Water deficiency shortensthe longevity of flowers, both directly and indirectlyvia PCD. Increased abscisic acid (ABA) and ethyleneare observed in carnations and are considered as im-portant signals in PCD (Nukui et al. 2004). Senescenceinduced by water deficiency can be delayed when pro-tein synthesis is inhibited by cycloheximide (Beja-Talet al. 1995).

Yuan ZHOU et al.: Programmed Cell Death in Relation to Petal Senescence in Ornamental Plants 643

The PCD of petals in post-harvest ornamentals isoften related to wounding of the plants. For example,in Portulaca flowers, wounding of the filaments, butnot the petals, halved the corolla lifetime. Senescenceas a consequence of PCD is associated with the re-lease of ethylene (Ichimura and Suto 1998).

Application of a heat shock is known to turn off thesynthesis of many proteins and to upregulate the pro-duction of heat shock proteins (Vierling 1991). A 24 hheat shock applied to carnations slowed petal senes-cence and delayed the evolution of ethylene, whereaselevated levels of both 1-aminocyclopropane-1-car-boxylate (ACC) synthase and ACC oxidase were ob-served (Rubinstein 2000; Verlinden and Woodson 1998).2.1 Changes in sink-source relationships

The PCD in the form of senescence is responsiblefor the recycling of nutrients and energy reserves inplants. Proteins and lipids are degraded and exported inthe form of amino acids, sugars etc. That is, the flow-ers switch from being a sink to a source during PCD.The metabolizing process, for example, is just the in-verse of the bud differentiation from being a source toa sink in sweet osmanthus (Wang et al. 2002; WangCY and Cheng ZW 2003, unpublished data). Many pa-rameters related to the sink-source, such as a declineof fresh weight in petals, dry weight, soluble (vacuolar)acid invertase (INV) activity, and soluble carbohydratecontent, are often linked to the onset of flower PCD inornamental plants. However, starch content does notchange significantly (Kapchina and Yakimova 1989; Reidet al. 2002). Ethylene may act as the key signal in thesephysiological changes. Changes in the expression ofINV genes correlate with the onset of rapid PCD dur-ing floral development of four-o’clock flowers(Mirabilis jalapa L.). It has been suggested that su-crose metabolism may be a key component of the sinkstrength in flowers during PCD (van Doorn 2004).2.2 Hormones

To ethylene-sensitive flowers, the importance ofethylene emissions in regulating the PCD process hasbeen stressed by several authors. As an example, gyno-ecium excision delays the onset of ethylene production

and greatly prolongs the vase life of flowers. Moreover,gynoecium excision diminishes the senescence-promot-ing effect of auxins, ABA, ACC, and benzyl adenine(BA) treatments in carnations (Jones 2003). In somecarnation cultivars that lack anthers, it was hypoth-esized that water stress-induced accumulation of ABAtriggers ethylene production in the gynoecium of flow-ers undergoing natural senescence instead of pollina-tion (Nukui et al. 2004). Inhibitors of ethylenesynthesis , such as amino oxyacet ic acid ornorbornadiene, or compounds that interfere with eth-ylene binding, such as CO2 or silver thiosulfate (STS),greatly prolong the life of ethylene-sensitive flowers(van Altvorst and Bovy 1995). The interconnectionbetween various hormones that play a role in the se-nescence of petals was elegantly shown by usingSAG12-ipt transformed petunias, which are charac-terized by an increased cytokinin production in theflowers. Flowers of these transgenic petunias were lesssensitive to ethylene and accumulated less ABA thanwild-type flowers, resulting in increased flower lon-gevity (Chang et al. 2003).

For ethylene-insensitive flowers, ABA and cytoki-nins are possibly the main signals for triggering PCD inpetals. Treatment with ABA hastens PCD-associatedevents, such as ion leakage, lipid peroxidation etc.(Panavas et al. 1998b). Generally, cytokinins appearto inhibit the PCD of petals. In the daylily (Hemerocallisfulva L.), the longevity of isolated flowers was signifi-cantly enhanced by cytokinins (Panavas et al. 1998a).Endogenous ABA levels in daylily petals increase slowlybefore flower opening and exogenous auxin delays petalPCD (Rubinstein 2000).2.3 Anatomical, biochemical and molecularchanges observed during PCD

The PCD morphology is recognized by the conden-sation of the cytoplasm and nucleus and shrinkage ofthe plasma membrane away from the cell wall (McCabeet al. 1997). Cells developing PCD can be detected usingTUNEL. Using this technique, cells commencing PCDwere detected in senescing gypsophila flower petals ata time when no other symptoms of senescence were


observed. Ethylene greatly stimulated, whereas STSdelayed, the appearance of TUNEL-positive nuclei(Hoeberichts et al. 2005). In Osmanthus fragrans Lour.,cells with TUNEL-positive nuclei were detected insenescing petals. Treatment with 200 mg/L ethephon(ETP) can increase the number of cells developing PCD,especially in senescing petals 48 h after treatment withETP (Wang CY and Cheng ZW 2003, unpublished data).

One hallmark feature of PCD is the observation ofso-called “DNA ladders”. The DNA in PCD nuclei isdegraded by various types of endonucleases. DNA frac-tionation occurs at internucleosomal sites, giving riseto DNA ladders of approximately 200 bp in agarosegels (Richberg et al. 1998). The DNA ladders werefound only in the advanced stage of petal senescence(36 h after pollination) in petunia flower petals. In thiscase, both single-stranded and double-stranded DNaseactivities were induced during petal cell death and wereenhanced by Ca2+ (Xu and Hanson 2000). InAlstroemeria, the timing of PCD is initiated at an ex-tremely early stage. Nuclear and cellular degradationhad already commenced before the flowers were fullyopen, whereas DNA laddering continued to increasethroughout petal development (Wagstaff et al. 2003).2.4 Genes related to PCD

The PCD is an active process that is regulated atboth the transcriptional and the translational level(Lawton et al . 1990; Nooden et al. 1997). InAlstroemeria, the expression of the partial cDNA of thesenescence-related gene Alstroemeria defender againstdeath-1 (ALSDAD-1) was downregulated after theflowers were fully open and before signs of petal in-rollment were present (Wagstaff et al. 2003). Manyother genes, with possible functional homology to PCD-related genes, have been discovered using microarrays(Swidzinski et al. 2002; van Doorn et al. 2003). Theexpression patterns of these genes will have to be con-firmed individually by quantitative reverse transcrip-tion-polymerase chain reaction (RT-PCR) or on North-ern blots. Sequencing the differentially expressed genesfrom aging petals revealed that a large proportion (upto 30%) of the transcripts represent metallothionin-like

proteins (Breeze et al. 2004). For iris and carnation,altered gene expression is already visible prior to petalwilting. It was found that 94% of more than 2 000transcripts were upregulated during carnation petalsenescence. The expression of all these transcripts wasinduced by treatment with ethylene and delayed aftertreatment with STS. Sucrose has a similar delayingeffect on the same genes as STS. This indicates thatsugar may be an early regulator of senescence(Hoeberichts et al. 2003; van Doorn et al. 2003). Incarnations, an ethylene insensitive-3 (EIN3)-like tran-scription factor and a zinc finger protein with a RING-domain present in many eukaryotic transcription fac-tors were found to be upregulated during senescence(Hoeberichts et al. 2003). A homeodomain protein, aclass of proteins generally also representing transcrip-tion factors, was found to be involved in senescence,as well as a casein kinase, probably one of the keyfactors in the PCD signal transduction pathway (Wakiet al. 2001). Three ethylene receptor genes, DC-ERS1,DC-ERS2, and DC-ETR1, were identified previouslyin carnation (Shibuya et al. 2002). The mRNA for DC-ERS2 and DC-ETR1 was present in considerableamounts in petals, ovaries, and styles of the flower atthe full-opening stage. In petals, the level of DC-ERS2mRNA showed a decreasing trend towards the late stageof flower PCD. However, DC-ETR1 mRNA showedlittle or no change in any of the tissues duringsenescence. Exogenous ethylene did not affect the lev-els of DC-ERS2 and DC-ETR1 mRNAs in petals(Shibuya et al. 2002). Two cDNA clones encoding pu-tative ethylene receptors were obtained from the gyno-ecium in Delphinium 4 d after anthesis and exhibitedhigh sequence similarity to Arabidopsis thaliana ERS1(Pun and Ichimura 2003). Many other genes with in-creasing transcript levels during petal senescence havebeen identified in other species, such as Phalaenopsis,daylily, petunia etc., of which an overview is given byRubinstein (2000).

Ethylene inhibitors are ineffective on ethylene-insen-sitive flowers (Wang et al. 2001a, 2001b), Here, pro-teolysis seems to be a dominant process in flower


senescence and is associated with increased activity ofserine, cysteine, and metallo-endoproteases. In theshort-lived cut flowers of Iris sect. oncocyclus, sev-eral petal senescence-inducing proteases were visual-ized and characterized, including the effect of specificinhibitors applied in vivo or in vitro (van Doorn et al.2004). Flower senescence was associated with a de-cline in petal fresh and dry weight and protein content,as well as an increase in cell sap pH, amino acid content,and ion leakage. The changes in protein content andpH were evident already at the closed-bud stage. De-tached petals senesced at the same rate and pattern asintact flowers, indicating that these petals undergo aPCD. Treatment with cycloheximide, an inhibitor ofde novo protein synthesis, inhibited flower openingwhen applied at an early stage (–2 d) and delayed se-nescence when applied at later stages (1 d or 2 d) offlowering. These observations support the theory thatfloral senescence is an active process involving theprotein synthesis of proteolytic enzymes. Separationof petal proteins isolated at the stage of closed buds byactivity gels revealed three distinct proteolytic activi-ties (van Doorn et al. 2004).2.5 Putative signal transduction pathways

The specific components involved in signal trans-duction leading to PCD and the senescence of flowersare hardly known. However, it is reasonable to assumethat, in general, signaling in flower PCD is governedby the same complex of factors that trigger plant stressresponses in other organs. Primary regulators of PCD,either induced by abiotic stress or by pathogen attack,seem to be the plant hormones salicylic acid, jasmonicacid, and ethylene. A specific PCD response in the plantmay depend on the availability of these hormones inthe affected organs, the biosynthesis of which, in turn,may depend on the activity of certain transcriptionfactors, changes in ion fluxes, Ca2+ channels, GTPases,mitogen-activated protein kinases (MAPKs) etc. (Raoet al. 2000). Borochov et al. (1994) described a modelof a pathway that is based on their observations in roseand petunia. They found that the activity of phospholi-pase A (PLA) and phospholipase C (PLC) increases in

the petals of these flowers before PCD commences.They deduced from other studies that PLC would hy-drolyze the membrane component phosphatidylinositoldiphosphate to yield inositol triphosphate anddiacylglycerol. Inositol triphosphate then acts as a sec-ond messenger to elevate calcium levels in the cytosol,which, in turn, gives rise to an ethylene burst.Interestingly, PLC activity is usually linked to activa-tion of a heterotrimeric G-protein. In the orchidPhalaenopsis, a 42-kDa peptide was detected onimmunoblots using an antiserum that reacts with thenucleotide-binding site of G-proteins in animals (Poratet al. 1994).

As referred to earlier in the present paper, anotherfactor of great importance to the PCD of plants iscalcium. Endogenous calcium increases flower sensi-tivity to applied ethylene in Phalaenopsis. Applicationof EGTA, a calcium chelator, decreases this sensitivity.Furthermore, calcium stimulates in vitro phosphoryla-tion of microsomal membranes (Porat et al. 1994).Thus, PCD seems to be controlled, in part, by increasesin cytosolic calcium, probably via activation of a cas-cade of events including the upregulation ofphospholipase, NADPH-oxidase, and GTPases, theformation of active oxygen species, such as superox-ide anion and hydrogen peroxide (Rao et al. 2000).

3 Approaches to Improving the Quality ofPost-harvest Ornamentals by Delaying PetalPCD

Ethylene production is the most important signal forthe onset of the PCD of flowers in ornamentals. In-hibitors of ethylene production may delay PCD andimprove the quality of ornamentals after harvest. Formany cut or potted flowers, 1-methylcyclopropene (1-MCP) and other cyclopropenes effectively block eth-ylene responses, providing possibilities to replace STS(Feng et al. 2004). Fumigation with 500 ng/L 1-MCPeffectively prolongs the vase life of cut Dendrobiumflowers (Ketsa et al. 2001a, 2001b).

Enhanced sink-strength contributes to the preven-tion of PCD in cut flowers. Sugar and nutrient salts in


the flower solution will help to delay PCD. Trehalosehas been reported as an inhibitor of PCD for ethylene-insensitive flowers, such as gladiolus and tulips (Yamadaet al. 2003). A concentration of 0.1 mol/L trehalose inthe vase solution delayed the floret senescence of cutgladiolus spikes, but tended to delay the unfolding offlorets. Pretreatment with sucrose improved vase lifeand increased the number of unfolded florets. The num-ber of fully unfolded florets was significantly lowerwhen the flowers were treated with sucrose and treha-lose on the cut gladiolus spikes (Yamada et al. 2003).The addition of inorganic nutrient salts (INS) to flower-opening solution (FOS) stimulated bud opening of cutcarnations. A combination of 150 µmol.m−2.s−1 photo-synthetic photon flux density (PPFD) and INS in FOSwas more effective in reducing the time required forbud opening at the early bud stage (Fujiwara et al. 2004).Mannitol plays a specific role in promoting spike elon-gation in cut snapdragons (Antirrhinum majus L.), aphenomenon that was not observed with glucose,sucrose, or sorbitol (Ichimura et al. 2000). A concen-tration of 5% sucrose was found to be the best forextending the flower vase life of cut carnation flowers,and was associated with a delayed ethylene peak. Su-crose reduces the sensitivity to ethylene when ethyleneconcentrations are still under 0.5 µL/L (Pun et al. 2001).Sucrose, in combination with thidiazuron (TDZ), provedvery effective in delaying the senescence of Lupinusdensiflorus Benth. flowers, suggesting that TDZ mayalso have some role in modulating the effects of ethyl-ene in cut inflorescences of L. densiflorus. It has alsobeen reported that sucrose and TDZ can greatly im-prove the post-harvest performance of phlox cut flowerheads (Mackay e t al . 2003) . At 1 mmol/L,cyclohexamide delayed wilting of tulips, ethylene-in-sensitive flowers (Jones and McConchie 1995).Acetylaldehyde and ethanol, when applied at lowconcentrations, extend the vase life of cut carnationflowers up to 50% (Podd and van Staden 1999, 2002;Pun et al. 2001), probably because of the inhibition ofovary development and subsequent decreased carbo-hydrate sink strength.

4 Prospects and Future Research

The PCD is an important process during petal se-nescence and is, therefore, associated with the post-harvest quality of ornamentals, especially the vase lifeof cut flowers. With an objective of producing flowerswith a longer vase life, to preserve their freshness dur-ing transport, or to design new vase water additivesthat extend flower longevity, we are challenged to studypetal PCD down to the molecular level. The knowledgeobtained may be used to improve flowers geneticallyby introducing target genes into the plant via geneticmodification. In recent years, several gene-transferprocedures have been developed for some major com-mercial cut flowers (for reviews, see Zuker et al. 1998;Vainstein 2002), but their application is still limited owingto the recalcitrant nature of established varieties of manyornamental species. However, in our laboratories, wehave made considerable progress in designing cultivar-independent transformation methods for roses,carnations, and chrysanthemums (Bovy et al. 1999;Visser et al. 2000; Condliffe et al. 2003a, 2003b; Visseret al. 2005). In the case of carnations, the transgenicapproach has already been shown to be successful. InArabidopsis, a mutant ethylene receptor ETR1 genewas found to be able to efficiently block ethylene sen-sitivity (Chang et al. 1993). When this gene wasoverexpressed in carnations using a flower-specificpromoter, vase life was extended threefold, from 8 dto 24 d (Bovy et al. 1999). However, transgenic cutroses transformed with the same gene construct didnot result in a significant prolonged vase life, probablyowing to the relatively limited sensitivity of cut rosesto ethylene (Visser PB 2004, unpublished data). Thisdemonstrates that specific solutions should be soughtfor different ornamental species.

Recently, various novel cell death-associated plantproteases have been identified (for reviews, seeWoltering et al. 2002; Woltering 2004). The elucida-tion of the role of these or similar proteases duringpetal senescence and the subsequent identification oftheir specific targets may result in novel inhibitors aimed


at prolonging vase life.Understanding the complex network of signals in-

volved in PCD will also help us design new strategiesfor the improvement of post-harvest flower quality.More effective and advanced techniques are now avail-able to obtain a holistic view of synchronous eventsthat take place during PCD. Microarray analysis of tran-scripts allows the evaluation of thousands of differentgenes at the same time in response to specific treat-ments (Hoeberichts et al. 2003; van Doorn et al. 2003;Breeze et al. 2004). This will provide us with a rangeof putative target genes to block or induce, either byexogenously administered components or by geneticinterference via the genetic modification of flowers. Inaddition, microarray analysis may serve to identify new,plant-specific genes involved in PCD, because manyof the well-characterized animal PCD genes do not haveobvious sequence homologs in plants.

Microarrays could also be used for diagnosticpurposes; for example, to determine the optimal har-vest time, for monitoring flower quality during trans-port and storage, or to identify new cultivars with im-proved post-harvest characteristics.

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Programmed Cell Death in Relation to Petal Senescence in Ornamental Plants