inhibited by the proteasome-specific inhibitor clasto-lactacystin-b-lactone. In addition, an unexpected shift inlocation for the sGFP-P fluorescence was observed uponprolonged viral infection. Over time, levels of fluorescentmaterial decreased in the ER and nuclear envelope while itincreased in the cytosol and also to a large extent in thenucleoplasm. FRAP experiments (fluorescence recoveryafter photobleaching) suggested that sGFP-P was activelytargeted to the nucleoplasm in a microtubule-dependentfashion. Although an increased degradation rate of sGFP-P compared with sGFP–HDEL and its partial occurrencein the cytoplasm might suggest a role for ER-associateddegradation in sGFP-P disposal, the failure to inhibit thisdegradation using proteasome inhibitors and its time-dependent distribution into the nucleoplasm questionsthis conclusion. However, the absence of sGFP-P transportthrough the secretory pathway seems to exclude ER-mediated vacuolar degradation as described for a mal-folded invertase fusion in yeast [6].

This study by Brandizzi et al. raises severalinteresting questions: is sGFP-P an unusual substratethat escapes the ‘normal’ ERAD pathway via transportto the nucleoplasm? Is transport to the nucleoplasmthe reason for the long half-life of sGFP-P comparedwith ricin A chains [4]? Does the slow degradation ofsGFP-P prevent a precise measurement of proteasomalinhibition by proteasome inhibitors and, therefore,exclude the proteasome as the degradation machinery?

And finally, is nucleoplasmic targeting for the disposalof malfolded proteins a novel mechanism specific toplants? If so, what is the biological purpose behindthis? Answers to these and probably many otherquestions will require future studies. Undoubtedly,only after extensive research using endogenous mal-folded plant proteins we will learn whether and howERAD operates in the plant kingdom.

References

1 Kostova, Z. and Wolf, D.H. (2003) For whom the bell tolls: proteinquality control of the endoplasmic reticulum and the ubiquitin–proteasome connection. EMBO J. 22, 2309–2317

2 Brodsky, J.L. and McCracken, A.A. (1999) ER protein quality controland proteasome-mediated protein degradation. Semin. Cell Dev Biol.10, 507–513

3 Ellgaard, L. and Helenius, A. (2003) Quality control in the endoplasmicreticulum. Nat. Rev. Mol. Cell Biol. 4, 181–191

4 Di Cola, A. et al. (2001) Ricin A chain without its partner B chain isdegraded after retrotranslocation from the endoplasmic reticulumto the cytosol in plant cells. Proc. Natl. Acad. Sci. U. S. A. 98,14726–14731

5 Brandizzi, F. et al. (2003) ER quality control can lead to retrogradetransport from the ER lumen to the cytosol and the nucleoplasm inplants. Plant J. 34, 269–281

6 Hong, E. et al. (1996) A pathway for targeting soluble misfolded proteinsto the yeast vacuole. J. Cell. Biol. 135, 623–633

1360-1385/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.tplants.2003.08.004

Role of death in providing lifeline to plants

Preeti Dahiya

Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich, UK NR4 7UH

As the major transporters and distributors of water and

minerals, xylem vessels and tracheids are the lifeline of

plants. Interestingly, the building blocks of these water

pipes are dead tracheary elements and vessel elements

that have the process of cell death integrated into their

differentiation programme. Using the Zinnia in vitro

model system for xylogenesis, a key nuclease that is

responsible for nuclear degradation during the terminal

stages of tracheary element differentiation has been

identified recently.

The evolution of the water-conducting system was one ofthe most significant developments in the long evolutionaryhistory of the land plants: one that subsequently played akey role in the occupation of dry land by plants. The xylemvessels of modern plants are perfectly adapted to carrylarge volumes of water and solutes to great heights underhuge negative pressure. During the course of theirdifferentiation, future vessels acquire lignified secondarywall thickenings and subsequently autolyse all their

cellular contents. A mature xylem vessel is, therefore, afunctional corpse, a tube or pipe composed of a series ofinter-connected cells known as tracheary elements (TE) [1](Figure 1). These cells have a unique cell death programmethat is activated as the final act of differentiation. Duringthis programme, the various organelles of the cell arecarefully disassembled, and the degradation of thechromosomal DNA in the nucleus is a characteristicfeature of this irreversible process [2]. Recent studies,using the Zinnia TE model system, have made significantprogress towards our understanding of the molecularmechanisms behind the process of nuclear degradationduring TE differentiation.

In vitro model system for tracheary element

differentiation

Sequential analysis of the developmental events duringtracheary element differentiation is difficult in intactplants because of their central position in the stem and thelimited numbers of vessels formed by the plant. The Zinniamodel system for in vitro xylogenesis (TE formation)provides a robust and amenable alternative. In thisCorresponding author: Preeti Dahiya (preeti.dahiya@bbsrc.ac.uk).

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system, mesophyll cells isolated from the leaves of Zinniaelegans trans-differentiate into TE in response to auxinand cytokinin [3] (Figure 1). Molecular and biochemicalanalysis of the Zinnia system has contributed significantlytowards our understanding of the mechanisms behind celldeath during TE differentiation. Using this system, JunIto and Hiroo Fukuda [4] have provided direct evidence forthe role of a particular nuclease, ZEN1, during nucleardegradation of TE.

ZEN1 is a vacuolar nuclease specific to the tracheary

elements

Ito and Fukuda [4] studied the functional role of a varietyof nucleases during the final stages of the differentiatingTE. The authors identified ZEN1 as the key nucleaseresponsible for nuclear DNA degradation. In addition they

also provided additional evidence of its role in TEdifferentiation: (i) ZEN1 is targeted to the central vacuole,(ii) ZEN1-like nucleases are likely to be plant specific, (iii)ZEN1 is exclusively expressed during the cell death phaseof TE differentiation.

Role of ZEN1 during tracheary element nuclear

degradation

ZEN1 is a Zn2þ-requiring nuclease that was initiallyisolated by Thelen and Northcote [5] using a Zinniaculture of differentiating TEs. Thelen and Northcoteidentified seven active nucleases; six of which, includingZEN1, are induced transiently in 60-h Zinnia culturewhile the differentiation of TE cells is underway. Extend-ing this work, Ito and Fukuda [4] demonstrated that thecell extract from this stage is capable of degrading isolatednuclei in the presence of Zn2þ; however, addition of theanti-ZEN1 antibody can suppress the nuclear degradation.Furthermore, antisense suppression of the ZEN1 retardsnuclear degradation in the differentiating TEs, even afterthe collapse of the central vacuole, but does not completelyabolish it. These results provided evidence that ZEN1plays an important role during TE nuclear degradation. Inaddition to ZEN1, five more nucleases were analysed by Itoand Fukuda and none of those had any direct effect on TEnuclear integrity.

Central vacuole of the tracheary element – the storage

house of lethal weapons

The rapid collapse of the central vacuole is the moststriking feature of the final stages of TE cell death [6]. Theautolysis of the cellular organelles, including nucleus,takes place soon after vacuole collapse, probably as a resultof the release of a range of hydrolytic enzymes. Interest-ingly, the nuclear degradation activity of ZEN1, whichcontains a vacuolar targeting sequence, also followsvacuolar disintegration. Moreover, the overexpression ofthe ZEN1 gene showed no increase in its enzymaticactivity towards the nucleus, suggesting that the activityof the nuclease is either insulated in the vacuole or is onlyactivated after vacuolar collapse [4]. The vacuole acts torestrain the premature release of hydrolases [4,7,8],thereby avoiding the premature initiation of programmedcell death (PCD).

However, not all known hydrolases are targeted to thevacuole. Many cell wall-degrading enzymes with neutralpH optima are probably active even before vacuolarcollapse [9] and several nucleases are likely to be targeteddirectly to the nucleus [4]. The combined activation andaction of all these hydrolases results in the controlledautolysis of the TE cell organelles but the molecularmechanisms behind their mutual interactions are not yetunderstood.

Have plants evolved their own way to execute death?

The direct correlation of TE nuclear degradation withZEN1 expression level suggests a central role for thisenzyme during TE cell death. Interestingly, the ZENfamily and their homologues in other plant species showsequence identity to the Nuclease I gene family (referred toas S1-type nucleases by Ito and Fukuda); which are all

Figure 1. The functioning corpse: water-conducting xylem vessels. (a) Scanning

electron micrograph showing xylem vessels in vivo. Spiral hoops of the secondary

wall deposition are marked by black arrows. White arrowheads indicate the cell

junction between two Zinnia tracheary elements that are connected via apical and

basal perforation in their end walls to form a continuous vessel or pipe [24,25]. (b)

and (c) Trans-differentiation in the Zinnia in vitro model system. (b) Mesophyll cell,

freshly isolated from the leaves of Zinnia elegans. Green chloroplast surrounds the

central vacuole of the cell; the nucleus is marked by an arrowhead. (c) Trans-differ-

entiated mesophyll cell showing morphological characteristics of the tracheary

elements. Arrows mark the spiral hoops of the secondary wall thickening, compar-

able to (a). The cell has undergone programmed cell death (PCD) to loose all its

cytoplasmic content. Scale bars ¼ 10 mm.

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activated during various types of cell death. A closehomologue of ZEN1 in Arabidopsis, BFN1, is inducedduring leaf and stem senescence [10]. Similarly, BEN1, thebarley homologue of ZEN1, is likely to be an importantcandidate during endosperm nuclear degradation [11,12].Almost all the other plant Nuclease I gene that have beenstudied in various plant species are also involved in celldeath [10,13]; with the exception of mung bean nuclease,which is reported to be associated with DNA synthesis andrepair [14].

Database searches have failed to find any animalhomologue for the ZEN family, which suggests theexistence of a plant-specific mechanism for nucleardegradation [4]. Moreover, continuing lack of evidencefor classical caspase-specific activity in plants [15] addsweight to the hypothesis that plants might have evolvedspecific regulators and independent mechanisms to exe-cute death.

An important point remains unanswered by this newwork – the mechanism of chromosomal DNA degradation.S1 nucleases are fungal enzymes that are highly specificfor single-stranded RNA and DNA, which they cleave inthe presence of Znþ2 to release mainly 50 mononucleotides[16]. Whether the ZEN family of enzymes can really bethought of as S1 enzymes is debatable because they appearto have double-stranded DNA activity as well. How theycan be the sole candidate enzymes for the chromosomaldisassembly is unclear and it will be important, throughgenetic and functional genomic approaches, to sort out thein vivo role of the other nucleases that might also beinvolved in the process.

Tracheary elements might have a special cell death

programme

Tracheary element (TE) differentiation has served asuseful model system for studying cell death duringdifferentiation in plants. Interestingly, several genes andenzymes, with crucial roles during TE cell death, show TE-specific expression patterns [17]. This specificity points tothe special cytological and molecular requirements duringTE differentiation. The activation ZEN1 nuclease, forexample, is limited to TE differentiation only and is notexpressed during leaf senescence or stress-induced death.However, unlike ZEN1, the expression of its familymembers ZEN2 and ZEN3 is not confined to the celldeath of TEs but is also seen during leaf senescence aswell [4,10]. Interestingly, as demonstrated by Ito andFukuda, suppression of ZEN2 and ZEN3 did notaffect the cell death process during TE differentiation,whereas the absence of ZEN1 activity represses nucleardegradation almost completely. In addition to thesenucleases, TE-specific serine and thiol proteinases werealso not activated in sucrose-starved Zinnia cells andcotyledons [18].

This specificity of the gene expression pattern alsoextends to other plant developmental events with inte-grated cell death programmes. The Arabidopsis BFN1gene, for example, is induced during leaf and stemsenescence, but its expression was not detected inseedlings grown in phosphate-deplete medium or duringgermination [10].

As indicated by the specific expression patterns of ZEN1and BFN1, it is likely that differentiating plant organsreceive a range of specific molecular signals and eachorgan has its own unique machinery to execute cell deathin accordance with its specific requirements. There mighteven be a case for rethinking the nature of the cell deathprocess in TE and PCD. It could be argued that twodifferent programmes are operating. The first, PCD, whichis a programme up and running in all living plant cells, canbe activated at any time by a variety of insults, for examplephoto-oxidative stress [19] or hypersensitive response.Here, a condensed cell corpse is formed and DNA ladderingcan be seen, as in animal paradigms. The second is celldeath as an integral part of the terminal cell differen-tiation, leading to a functional corpse. Examples are skinkeratinocytes in animals [20] and TE in plants. Becausethe signalling pathways leading to death are different ineach case, it is possible that although some of the PCDmachinery might be involved, other aspects of the processmight be TE specific. Thus, ZEN1 might be involved in TEdeath, but not in PCD, about which we still know little inplants.

Future perspectives

Cell death in multicellular organisms is aimed at theisolation and removal of damaged cells and is equallyessential to the development and maintenance of organ-isms in the animal and plant kingdoms. Although theprocess is better understood in animal system, there isincreasing evidence that plant cell death has adapted tothe specific features of plant development and defence,integrating plant-specific mediators or growth factors andplant-specific processes such as nutrient mobilization andsecondary cell wall synthesis [15,21]. This implies thatplant cell death regulators might not be readily identifiedby sequence comparisons with animal genes, and ‘novel’regulators such as ZEN1 might emerge from existing plantprotein families. Increasing numbers of cDNA AFLP [22],ESTs and microarrays [23], together with mutational andfunctional genomics approaches, should help to identifykey components of the TE cell death.

Acknowledgements

I thank Keith Roberts for helpful suggestions, discussion and criticalreading of the manuscript. I also acknowledge Kim Findlay for technicalhelp with the scanning electron microscope.

References

1 Ye, Z.H. (2002) Vascular tissue differentiation and pattern formation inplants. Annu. Rev. Plant Biol. 53, 183–202

2 Obara, K. et al. (2001) Direct evidence of active and rapid nucleardegradation triggered by vacuole rupture during programmed celldeath in Zinnia. Plant Physiol. 125, 615–626

3 Fukuda, H. and Komamine, A. (1980) Establishment of anexperimental system for the tracheary element differentiation fromsingle cells isolated from the mesophyll of Zinnia elegans. PlantPhysiol. 65, 57–60

4 Ito, J. and Fukuda, H. (2002) ZEN1 is a key enzyme in the degradationof nuclear DNA during programmed cell death of tracheary elements.Plant Cell 14, 3201–3211

5 Thelen, M.P. and Northcote, D.H. (1989) Identification and purificationof a nuclease from Zinnia elegans L.: a potential molecular marker forxylogenesis. Planta 179, 181–195

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6 Groover, A. et al. (1997) Programmed cell death of plant trachearyelements: differentiating in vitro. Protoplasma 196, 197–211

7 Kuriyama, H. (1999) Loss of tonoplast integrity programmed intracheary element differentiation. Plant Physiol. 121, 763–774

8 Funk, V. et al. (2002) The Arabidopsis xylem peptidase XCP1 is atracheary element vacuolar protein that may be a papain ortholog.Plant Physiol. 128, 84–94

9 Ohdaira, Y. et al. (2002) Activity of cell-wall degradation associatedwith differentiation of isolated mesophyll cells of Zinnia elegans intotracheary elements. Planta 215, 177–184

10 Perez-Amador, M.A. et al. (2000) Identification of BFN1, a bifunctionalnuclease induced during leaf and stem senescence in Arabidopsis.Plant Physiol. 122, 169–179

11 Aoyagi, S. et al. (1998) BEN1 and ZEN1 cDNAs encoding S1-typeDNases that are associated with programmed cell death in plants.FEBS Lett. 429, 134–138

12 Brown, P.H. and Ho, T.H.D. (1987) Biochemical-properties andhormonal-regulation of barley nuclease. Eur. J. Biochem. 168,357–364

13 Bariola, P. and Green, P.J. (1997) Plant ribonucleases. In Ribonu-cleases: Structure and Function (D’Alessio, G. and Riordan, J.F., eds),pp. 163–190, Academic Press

14 Grafi, G. and Larkins, B.A. (1995) Activity of single-stranded DNAendonucleases in mung bean is associated with cell division. Plant Mol.Biol. 29, 703–710

15 Hoeberichts, F.A. and Woltering, E.J. (2003) Multiple mediators ofplant programmed cell death: interplay of conserved cell deathmechanisms and plant-specific regulators. BioEssays 25, 47–57

16 Desai, N.A. and Shankar, V. (2003) Single-strand-specific nucleases.FEMS Microbiol. Rev. 26, 457–491

17 Kuriyama, H. and Fukuda, H. (2002) Developmental programmed celldeath in plants. Curr. Opin. Plant Biol. 5, 568–573

18 Beers, E.P. and Freeman, T.B. (1997) Proteinase activity duringtracheary element differentiation in Zinnia mesophyll cultures. PlantPhysiol. 113, 873–880

19 Tamagnone, L. et al. (1998) Inhibition of phenolic acid metabolismresults in precocious cell death and altered cell morphology in leaves oftransgenic tobacco plants. Plant Cell 10, 1801–1816

20 Jacobson, M.D. et al. (1997) Programmed cell death in animaldevelopment. Cell 88, 347–354

21 Roberts, K. and McCann, M.C. (2000) Xylogenesis: the birth of acorpse. Curr. Opin. Plant Biol. 3, 517–522

22 Milioni, D. et al. (2002) Early gene expression associated with thecommitment and differentiation of a plant tracheary element isrevealed by cDNA-amplified fragment length polymorphism analysis.Plant Cell 14, 2813–2824

23 Demura, T. et al. (2002) Visualization by comprehensive microarrayanalysis of gene expression programs during transdifferentiation ofmesophyll cells into xylem cells. Proc. Natl. Acad. Sci. U. S. A. 99,15794–15799

24 McCann, M.C. et al. (2000) Targeted cell death in xylogenesis.In Programmed Cell Death in Animals and Plants (Bryant, J.A.et al., eds), pp. 193–201, BIOS Scientific Publishers, Oxford,UK

25 Nakashima, J. et al. (2000) Autolysis during in vitro tracheary elementdifferentiation: formation and location of the perforation. Plant CellPhysiol. 41, 1267–1271

1360-1385/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.tplants.2003.08.003

Plant iNOS: conquest of the Holy Grail

David Wendehenne, Olivier Lamotte and Alain Pugin

UMR INRA 1088/CNRS 5184/Universite de Bourgogne Plante-Microbe-Environnement, 17 rue Sully, BP 86510, Dijon 21065 cedex,

France

In animals, nitric oxide (NO) is produced by a family of

enzymes named nitric oxide synthases (NOSs). Although

no NOS-like gene has been found in the Arabidopsis

thaliana genome, biochemical studies have suggested

that a NOS-like protein is likely to be activated in plants

resisting pathogens. This protein has been recently iden-

tified as a variant P protein of glycine decarboxylase. This

discovery means that studies of nitric oxide signalling

functions in plants are now entering a new phase.

Nitric oxide (NO) has been one of the most studiedmolecules in biological science over the past 15 years.NO exerts crucial regulatory functions in a wide range ofphysiological processes, including neurotransmission,vasodilatation, defence against invading microorganismsand apoptosis [1]. The biological reactivity of NO is dic-tated primarily by its interaction with transition metalcentres and cysteine thiols of numerous proteins. Thesemodifications modulate protein function to transduce apanoply of cellular-control signals [2].

In animals, nitric oxide synthases (NOSs) catalysethe production of L-citrulline and NO from L-arginine,NADPH and O2 [3,4]. Three NOS isoforms encoded by threedistinct genes were originally identified and named accord-ingtothetissuefromwhichtheywerefirst isolated:neuronalNOS (nNOS), endothelial NOS (eNOS) and immunologicNOS (iNOS), which was initially characterized in macro-phages. All isoforms share 50–60% identity and havecommon features of bound cofactors, substrate and co-substrates (Figure 1a). According to their main mode ofregulation and functions, these enzymes are grouped intotwo broad categories, constitutive and inducible [5]. ThenNOSandeNOSareconstitutiveenzymesstrictlycontrolledby Ca2þ-activated calmodulin (Figure 1b). This tightregulation makes constitutive NOSs ideal for generatingNO as a signalling molecule. The iNOS is regulatedprimarily at the transcriptional level by cytokines ormicrobial products and secondarily at the post-translationallevel by calmodulin, even at basal free Ca2þ concentration.Thus, iNOS is notably distinguished from the constitutiveisoforms by its prolonged production of large amounts ofNO. Consequently, iNOS is well suited as a mediator ofcellular defence against invading microorganisms.Corresponding author: David Wendehenne (wendehen@dijon.inra.fr).

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