Journal of Experimental Botany, Vol. 48, No. 307, pp. 181-199, February 1997
REVIEW ARTICLE
The molecular biology of leaf senescence
Vicky Buchanan-Wollaston1
Plant Molecular Biology Laboratory, Department of Biological Sciences, Wye College, University of London,Wye, Ashford, Kent TN255AH, UK
Received 27 March 1996; Accepted 30 August 1996
Abstract
Senescence is a complex, highly regulated, develop-mental phase in the life of a leaf that results in theco-ordinated degradation of macromolecules and thesubsequent mobilization of components to other partsof the plant. The application of molecular biology tech-niques to the study of leaf senescence has, in the lastfew years, enabled the isolation and characterizationof a large range of cDNA clones representing genesthat show increased expression in senescing leaves.The analysis of these genes and identification of thefunction of the encoded proteins will allow a pictureof the complex processes that take place during senes-cence to be assembled. To date, genes encodingdegradative enzymes such as proteases and nucle-ases, enzymes involved in lipid and carbohydratemetabolism and enzymes involved in nitrogen mobil-ization have all been identified as senescence-enhanced genes. A variety of other genes of no obvioussenescence-related function have also been identified;their role in senescence may be less predictable and,possibly, more interesting.
The combined action of several internal and externalsignals may be involved in the induction of senes-cence. Analysis of the regulatory mechanisms control-ling the expression of senescence-induced genes willallow the signalling pathways that are involved inthe regulation of senescence to be elucidated.Experiments with transgenic plants and mutants arealready shedding light on the role played by cytokininsand ethylene in regulating senescence in leaves.
The sight of falling red and gold autumn leaves or ofyellowing wheat fields brings to mind the end of a season
or the death of a plant. The leaves of both annual andperennial plants must eventually die. However, what maynot be immediately apparent is that the senescence ofleaves is not simply concerned with death, but that leafsenescence represents a key developmental phase in thelife of both annual and perennial plants which is asordered and complex as any other phase of development.Indeed, leaf senescence is as important a developmentalphase as those of growth and maturation. During its lifespan, a leaf undergoes at least three phases of develop-ment. Initially, it is expanding rapidly, importing carbonand nitrogen and undergoing rapid protein synthesis untilits full capacity for photosynthesis is reached. Then themature leaf becomes an asset to the plant, contributingto the supply of carbon, during which time, proteinturnover is at a consistently low level. This continuesuntil internal or external conditions initiate the onsetof senescence. Leaf senescence is a period of massivemobilization of nitrogen, carbon and minerals from themature leaf to other parts of the plant and is a highlyregulated, ordered series of events involving cessation ofphotosynthesis, disintegration of chloroplasts, breakdownof leaf proteins, loss of chlorophyll and removal of aminoacids. In annual plants, which include most agriculturalcrops and, in addition, the model plant species,Arabidopsis, the mobilizable nutrients from the entireplant are stored ultimately in the developed seeds. Sincea considerable percentage of nitrogen for grain filling isderived from vegetative plant parts, with only a minorportion being taken up from the soil during seeddevelopment, remobilization from senescing leaves iscentral for the nutrient budget in seed crops (Feller andKeist, 1986).
In addition to the programmed type of leaf senescencewhich takes place during plant development, the degrada-tion of macromolecules and mobilization of cellular com-ponent from leaves occurs as a response to external,environmental factors. Plants have to respond rapidlyto deteriorating environmental conditions since, unlike
animals, they cannot move in order to find a morefavourable situation. One response that plants can makeis to remove those parts of the plant that are not essential.For example, a diseased leaf will senesce, die and dropoff the plant, thus helping to prevent spread of diseaseand allowing the rest of the plant to continue in itsdevelopment. Similarly, nitrogen deficiency, light limita-tion and drought stress will initiate the onset of senescencewhich may result in early seed development and reducedplant life span. However, a difference from the naturaland programmed senescence is that, in these cases, theprocesses may be reversible if the stress conditions arerelieved before senescence has progressed beyond a certainpoint (Stoddart and Thomas, 1982).
In spite of the extensive biochemical and physiologicalresearch that has been applied to the study of leafsenescence there are still many unanswered questionsconcerning the process. Analysis of senescence in differentplant systems and experiments using different inductionmethods has often given conflicting results that are some-times difficult to interpret. Current understanding of theevents that occur during leaf senescence has been reviewedrecently by Smart (1994). This review will focus on recentstudies on the molecular analysis of genes that areinvolved in leaf senescence. The application of molecularbiology techniques to the study of leaf senescence shouldmake a major contribution to the previous biochemicalstudies since the isolation and characterization ofsenescence-related genes and mutants allows the analysisof the problem to be approached from a differentdirection.
The aims of molecular studies into leaf senescence are,firstly, to identify and clone genes encoding enzymesthat are involved in the senescence process, to character-ize these genes and to determine their function in orderto formulate an overall view of the enzymatic changesthat take place during senescence. Using biochemicalmethods, it has often been difficult or impossible toidentify senescence-specific enzymatic changes because ofthe high background of similar enzymes that are alreadypresent in the plant cell or, more probably, because theproteins are produced at low levels. If a gene can beidentified that is specifically expressed during senescencethen the product of that gene must have a function inthe senescence process. The cloning of a senescencespecific gene allows considerable information to beobtained about the timing of expression of the gene, thesite of activity, and the possible function of its proteinproduct. Secondly, once a senescence-enhanced gene hasbeen identified, the mode of regulation of this gene canbe studied. The upstream regulatory regions of the genescan be cloned and characterized and analysed bycomparison with other genes that show induced expres-sion during senescence. The isolation and characteriza-
tion of transcription factors, proteins that bind to theseupstream regions, will allow analysis of the signallingevents that control the senescence process. Thirdly, theidentification of mutants that are defective in a senes-cence function opens the way for an alternative approachto the analysis of senescence. Genes that are expressedat levels too low to be identified by other proceduresmay be identifiable by mutation; map based cloningtechniques can be applied to clone the genes involved.Furthermore, if a number of mutants defective in theregulation of senescence can be identified, the signallingpathways can be dissected by comparing gene expressionpatterns in these mutants to identify common signallingfunctions and branch points within the pathways thatare involved in controlling the complex interactions thatoccur during senescence.
Understanding the basis for the onset of senescence inplants and isolation of the genes involved in the senescenceprocess is of fundamental importance for future agro-nomic improvements. It has been known for many yearsthat the main diversity in crop yield is due to differencesin the duration of photosynthetic activity (Watson, 1952).Stay green variants of maize and sorghum have been usedin breeding programmes to enhance the yield of theseimportant grain crops and the stay green phenotype alsoappears to increase disease resistance (Thomas and Smart,1993). Stay green varieties of ornamental plants couldalso be very useful to extend their value. If senescencecould be delayed for a few days before harvest in foragecrops such as alfalfa, the yield and protein quality of thatcrop could be greatly improved. Also, the proteindegradation that occurs after harvest in forage cropsreduces the nutritive value of the crop and can result inthe production of detrimental nitrogenous compounds;preventing this decay would be very beneficial. Differentfunctions of senescence will need to be manipulatedto achieve these aims and therefore, a knowledge ofthe genes involved in the control and degradativeprocesses of senescence is essential before any molecularattempts can be made to modify the process in a dir-ected way.
Genes expressed during leaf senescence
Leaf senescence does not occur by a passive decay mech-anism, but, rather, is an actively regulated process thatinvolves co-ordinated expression of specific genes. Duringsenescence, the levels of total RNA decrease and theexpression of many genes is switched off (Bate et ah,1991; Hensel et ah, 1993; Lohman et al., 1994). It hasbeen postulated for some time that the senescence processmay depend on de novo transcription of nuclear genesand recent molecular studies have shown that this is
the case. New transcripts were originally detected asa changed pattern of products observed in in vitrotranslation experiments using mRNA from senescingleaves when compared to green leaves (Malik, 1987;Thomas et al., 1992). Recently, using differential screen-ing and subtractive hybridization techniques, many
Molecular biology of leaf senescence 183
cDNA clones representing senescence-enhanced geneshave been identified from a range of different plants andthese are summarized in Table 1. The identification ofgenes that are expressed specifically during senescence isthe best evidence that de novo transcription is requiredfor senescence to proceed.
Table 1. Cloned genes that show increased levels of transcription during senescence
Hensel et al., 1993Smart et al., 1995M Fife and VBW unpublishedSmart et al.. 1995
Lohman et al., 1994Buchanan-Wollaston and Ainsworth, 1997Buchanan-Wollaston and Ainsworth, 1997Genschik et al.. 1996Garbanno et al.. 1995Taylor et al.. 1993Graham et al., 1992McLaughhn and Smith, 1994Kim and Smith, 1994«
Kim and Smith, 19946
VBW. unpublished
VBW, unpublished
Smart et al.. 1995
VBW, unpublished
King ei al., 1995Ryu and Wang. 1995Bernhard and Mahle, 1994Kawakami and Watanabe, 1988Kamachi et al., 1992Buchanan-Wollaston and Ainsworth, 1997King etal., 1995VBW, 1994Coupe et al., 1995Buchanan-Wollaston and Ainsworth, 1997Hsieh et al., 1995Buchanan-Wollaston and Ainsworth, 1997Buchanan-Wollaston and Ainsworth, 1997Smart et al., 1995Buchanan-Wollaston and Ainsworth, 1997VBW. unpublishedVBW. unpublishedVBW, unpublishedHanfrey et al. 1996Hanfrey et al, 1996Buchanan-Wollaston and Ainsworth. 1997Davies and Grierson. 1989Fray et al.. 1994
T h e designation of the cDNA clone.'Deduced by comparison with sequences of previously characterized genes published in DNA and protein databases. In general, the functions of
these genes in senescence has not been proved.The class of gene is proposed,where enough information is available, as defined in Fig. 2.'Plant species from which the gene was isolated.'Indicates type of enzyme (proteases) or shows any other situations where induced expression of the gene has been detected.
The majority of the senescence-related genes isolated todate have been identified using methods aimed at theisolation of any cDNA clone that represents an mRNAspecies that occurs specifically in senescing leaves and notin green leaves or that has a significantly increased levelof expression relative to green leaves. cDNA librariesconstructed from mRNA isolated from senescing leaveshave been screened differentially using labelled cDNAfrom green or senescing leaves; those clones showinghybridization to the senescing leaf probe and not thegreen leaf probe have been selected for further analysis.This method of differential screening has been used suc-cessfully to identify senescence-related genes from plantssuch as Ambidopsis (Hensel et al., 1993; Lohman et al.,1994), Brassica napus (Buchanan-Wollaston, 1994) andmaize (Smart et al., 1995). However, differential screeningis useful only in the detection of cDNA clones thatrepresent genes that are expressed at fairly high levels inthe tissue of interest since the message has to be reason-ably abundant to be labelled sufficiently in the probe. Toidentify genes expressed at lower levels, a subtractivehybridization technique has been applied to identify senes-cence-related genes in B. napus which has enabled manysenescence-related genes to be identified (Buchanan-Wollaston and Ainsworth, 1997) (Table 1).
Analysis of senescence-related genes
Once a cDNA clone that detects an increased transcriptlevel in senescing tissue has been identified, Northernhybridization can be used to characterize the expressionof the gene in terms of steady-state mRNA levels. cDNAclones can be characterized by their patterns of temporalexpression during leaf development and also by the levelsof expression in other parts of the plant. However, inorder to identify the precise time during leaf senescenceat which the expression of a specific gene is induced, itis essential to characterize the biochemical and physiol-ogical changes that take place during leaf senescence.Physiological parameters such as chlorophyll content andphotosynthetic rate have been used to determine thetiming of senescence and to identify the different stagesof senescence (Smart et al., 1995). However, it is not easyto determine when senescence actually starts and, for thisreason, many senescence experiments have made use ofartificial methods of inducing senescence, such asdetaching leaves or dark treatment (Thimann, 1980).Although these induction methods may cause effectssimilar to those that occur during natural senescence,significant differences are found (Nooden, 1988), and thestudy of genes showing induced expression after suchtreatments is fraught with problems. This was shownconvincingly by Becker and Apel (1993) who found that,
of three cDNAs representing genes expressed during dark-induced senescence, only one showed induced expressionduring natural senescence; expression of the other twogenes was presumed to be stress related.
The most obvious sign of senescence is yellowing ofthe leaf; the progress of senescence can been divided intostages on the basis of the amount of yellowing that hasoccurred. However, by the time the leaf appears yellowto the eye the chlorophyll content has fallen to around50% of that in the mature green leaf (Hanfrey et al.,1996) and, therefore, senescence is well underway by thetime the leaf is senescing visibly. Chlorophyll measure-ments are simple to make, the amount of chlorophyll ina leaf giving a reasonable estimation of the stage ofsenescence. The timing of expression of many senescence-related genes can be defined according to the level ofchlorophyll present in the senescing leaves. However,some senescence-related genes identified in B. napus showinduced expression well before any reduction in chloro-phyll levels can be detected (VB-W, unpublished data).In spite of this, Northern analysis, using RNA isolatedfrom leaves at a range of developmental stages, definedby chlorophyll levels, is a useful tool to characterizesenescence-related cDNAs.
Patterns of gene expression during leafsenescence
On the basis of pattern of expression and the function ofthe protein products, six classes of genes that could beinvolved in leaf senescence were proposed previously(Smart, 1994). Class 1 contains 'housekeeping' genes thatcontrol essential metabolic activities of the cell and areexpressed at a constant level throughout the life of theleaf. These genes are not specific to senescence. Classes 2and 3 both consist of genes that are expressed in greenleaves, but whose expression may affect senescence at alater stage. These genes are active well before senescencestarts and are switched off before any signs of senescenceoccur. In Class 2, the encoded proteins become activatedduring senescence, whereas in Class 3, the encoded pro-teins may cause the initiation of senescence by theirabsence. Class 4 includes regulatory genes, which areexpressed immediately prior to or at the onset of senes-cence and which are expressed for a relatively short timeonly. Class 5 includes genes involved with the mobilizationprocesses that occur specifically during senescence. Class6 consists of genes involved with the mobilization ofstorage products that may also function during otherdevelopmental stages. Genes in the latter two classeswould be expressed from the onset of senescence until thedeath of the leaf.
From the expression analysis carried out with thesenescence-related cDNAs cloned from B. napus, awide variety of different temporal patterns of mRNA
expression is evident. A Northern blot showing hybridiza-tion of a range of different senescence-related cDNAs tototal RNA isolated from B. napus leaves at differentstages of development is shown in Fig. 1; transcriptionalactivation at every stage of senescence can be detected.Expression of the gene encoding the small subunit ofRUBISCO is shown, levels of transcript for this gene aremaintained until the SSI stage, i.e. the same stage atwhich chlorophyll breakdown is detectable, and then theystart to fall. Many of the senescence-related genes startto show increased transcript levels at the same stage(SSI). When expression patterns obtained by Northernanalysis are examined, it is clear that, whilst some of theB. napus genes fit into the scheme proposed by Smart,the situation is more complex than was first proposedand that, based on the expression patterns, four newclasses can be added (Fig. 2). The B. napus clones wereidentified by differential screening or subtractive hybrid-ization and identification of genes in the last three of theoriginal six classes (Classes 4, 5 and 6) would be expected.Genes in Class 1 would not be identified by these methodsbecause of the constitutive nature of their expression,whilst genes in Classes 2 and 3, with reduced expressionduring senescence, have not been the focus of the Brassicaresearch.
No genes of the Class 4 type have been identified fromBrassica. Genes of this type, which includes regulatorygenes, may be cloned infrequently since they are likely tobe expressed at low levels. Brassica clones representingClass 5 genes are typified by LSC54 and LSC222 whichshow increased levels of expression at the SSI stage,where chlorophyll levels are just starting to decrease (Figs1,2). LSC25 also falls into this class although its expres-sion is initiated much earlier (at the MG2 stage). Noclones representing Class 6 have been identified in Brassicaalthough clones of this type have been identified in otherplants. For example, a malate synthase gene from cucum-ber falls into Class 6 (Graham et al., 1992).
Four new classes of gene are proposed (Fig. 2). Brassicagenes were cloned which are expressed strongly early inleaf development and again during senescence (LSC8 andLSC101 are examples, Fig. 1), the difference from theClass 6 pattern being that there is a significant level ofexpression in the mature leaves. This class has beentermed Class 10. Another class of genes includes thosegenes that show an expression pattern in which mRNAlevels increase gradually over the period of leaf develop-ment from the youngest leaves and through the senescencestages. These Class 7 genes include the Brassica clonesLSC7, LSC210, LSC212, and LSC460 (Figs 1, 2). Class8 genes are similar, in terms of the period of expression,but differ in the respect that mRNA levels are low in theearly stages of leaf development, but increase dramaticallyat a particular stage of senescence. An example of thistype of gene is LSC94. The last class of genes, termed
fe*
Molecular biology of leaf senescence 185
YG MG1 MG2 SSI SS2 SS3
RUBISCO
LSCS4
LSC222
LSC25
LSC7
LSC210
LSC212
LSC460
LSC94
LSC680
LSC550
LSC101
LSC8
I I • •
Fig. 1. mRNA expression during leaf development. Northern blotscarrying RNA isolated from B. napus leaves at six different stages ofdevelopment were hybridized with 32P-labelled inserts from the differentcDNA clones. YG refers to RNA isolated from fully expanded greenleaves from young plants, MG1 refers to RNA isolated from maturegreen leaves isolated from plants which had just started flowering, MG2refers to RNA isolated from mature green leaves on plants that hadjust started silique development, SSI, SS2 and SS3 refer to RNAisolated from senescing leaves showing 98%, 60% and 35% of green leafchlorophyll levels, respectively. Only the leaves from which the SS3RNA was isolated showed visible yellowing or reduced total RNAlevels. 10 jig of total RNA was loaded in each track. As a control, anArabidopsis RUBISCO small subunit gene probe was hybridized to thesame RNA samples.
Fig. 2. Expression of genes during leaf development. Patterns of expression of senescence-related genes during leaf development can be used todivide the genes into classes. Class 1-6 are as proposed by Smart (1994). Classes 7-10 are proposed after analysis of different expression patternsobtained with a range of genes that show induced expression during leaf senescence in Brassica napus (Fig 1). The stages of development YG,MG1 etc. are the same stages as were used for RNA isolation (Fig 1, legend), levels of chlorophyll and RNA in each sample are indicated as thepercentage of the level in mature green leaves. The Brassica genes that represent each class are indicated.
Class 9, includes the Brassica genes LSC550 and LSC680,in which expression is induced specifically at some stageduring senescence but, in contrast to Class 5 genes,expression does not continue to the last stages of senes-cence. The period of high levels of mRNA is short forthese genes and is restricted to senescence stages SSI(LSC550) or SS2 (LSC680).
The range of different expression patterns identified bythe Northern analysis implies that the transcription ofthese senescence-related genes is not regulated by thesame mechanism and that a variety of regulatory path-ways are likely to be involved in controlling the sequentialenzymatic steps needed for the co-ordinated degradativeprocesses that take place. The regulatory proteins thatcontrol the expression of these genes must be present atvarious times during senescence. Therefore, regulatorygenes of Class 4 may be indistinguishable from Class 5genes on the basis of expression patterns.
Differential or subtractive screening as a method toidentify genes that are expressed during leaf senescence,
represents a powerful tool to aid in the unravelling of thecomplex processes that are taking place. The products ofgenes that show increased mRNA expression during leafsenescence are likely to be important for the process ofleaf senescence. However, it must be borne in mind, thatthe levels of mRNA detected in Northern analysis areusually, but not always, indicative of the transcriptionalactivity of the gene; the amount of hybridization obtainedalso depends on the relative stability of the message andthis may vary for different genes or in different circum-stances. Levels of a transcript for the gene represented byLSC7, for example, which shows increasing abundanceduring senescence as levels of total RNA are falling,could be accounted for by assuming that the stability ofthe mRNA from this gene is greater than that of theother RNA species as senescence progresses. However, itis a reasonable assumption that, if the stability of amRNA is high during leaf senescence when general RNAdegradation is increased, then the product of that RNAis important. For example, mitochondria must retain their
function during senescence, since respiration must con-tinue, whilst chloroplasts become inactive and are dis-mantled. The gene represented by LSC8 encodes amitochondrial enzyme NADH:ubiquinone oxidoreduc-tase, and shows elevated mRNA expression during senes-cence in B. napus and broccoli. This may be representativeof an essential message required for mitochondrial func-tion having enhanced stability over other members of thetotal RNA population (Pogson et al, 19956).
These methods will not successfully identify senescence-related genes of the Class 2 type whose products aresynthesized in green leaves and stored in an inactive formuntil the onset of senescence results in the activation ofthe enzymatic functions. It is not known whether thistype of control is important in the regulation or func-tioning of the senescence processes.
In summary, a large number of cDNA clones repres-enting genes that show induced or enhanced expressionduring leaf senescence have been identified by variousresearch groups. Having analysed the expression patternsshown by these genes the logical next step is to identifythe functions of the gene products in the senescenceprocess. This can be achieved, in some cases, by DNAsequence analysis followed by DNA and protein sequencedatabase searches for similar sequences that have identi-fiable functions. Table 1 shows a summary of the senes-cence-enhanced genes that have been isolated to date,together with the proposed function of the gene whichhas been determined after identifying genes of similarsequence. Where it has been possible to determine frompublished information, the expression class of each geneis also shown. This table does not show the manysenescence-enhanced genes that have been isolated thatshow no sequence similarities to previously sequencedgenes in the databases. The functions of these genesremain to be identified.
Genes involved in protein degradation
The degradation of proteins is probably the most signific-ant breakdown process that takes place during senescencesince the remobilization of amino acids is very importantto supply the developing organs elsewhere in the plant.Genes encoding several different types of protease havebeen identified in senescing leaves from various plants(Table 1). One of the most obvious enzymatic events thatoccurs in the senescence process is proteolysis and it istherefore not surprising that there is de novo transcriptionof protease genes and synthesis of proteins. However,increased levels of specific proteases in senescing leaveshave been hard to detect reproducibly by biochemicalmethods due to the high concentration of proteasesalready present in the vacuole of the developingleaf (Huffaker, 1990; Feller and Fischer, 1994). Theidentification of senescence-enhanced protease genes is,
Molecular biology of leaf senescence 187
therefore, significant. Much of the protein within aphotosynthesizing cell is located within the chloroplastand it is likely that the initial steps in protein degradationoccur within these organelles. Chloroplasts do containproteases; protein degradation can be detected in isolatedintact chloroplasts (Mitsuhashi and Feller, 1992) andClpP and ClpC protease subunits have been detected inthe chloroplasts of Arabidopsis (Shanklin et al., 1995).However, senescence-specific protease activity that isinvolved with the dismantling of the photosyntheticapparatus within the chloroplast, remains to bedemonstrated.
Three of the senescence-enhanced protease genes, isol-ated from maize, Arabidopsis and B. napus show sequencesimilarity to a class of cysteine protease represented byseed-specific proteases from cereals such as the oryzain yprotease from rice (Watanabe et al., 1991). These enzymesare produced in germinating seeds and, presumably, func-tion in the remobilization of storage proteins to supplydeveloping seedlings. They are likely to perform a similarfunction in senescing leaves. Three other types of cysteineprotease showing enhanced levels in senescing leaves havebeen identified. One of these, represented by the cDNAclone SAG12 appears similar in protein sequence topapain-like proteases (Lohman et al., 1994). This gene isexpressed specifically in senescing leaves and, as isdescribed below, work with the promoter of the gene hasshown it to be tightly regulated during leaf development(Gan and Amasino, 1995). In contrast, the third cysteineprotease that has been identified in B. napus, representedby cDNA clone LSC790, is expressed at all stages of leafdevelopment and also in flowers and germinating seeds(Buchanan-Wollaston and Ainsworth, 1997). The levelof transcripts of this gene is relatively high in young greenleaves, decreases in mature leaves and increases signific-antly during senescence. The most similar sequence to theprotein encoded by the LSC790 gene is a drought-inducedcysteine protease, RD19, that has been isolated fromArabidopsis (Koizumi et al., 1993). The B. napus generepresented by LSC760 which encodes an aspartic pro-tease showed a different pattern of expression; the genewas expressed similarly in young and mature green leavesbut expression increased during senescence (Buchanan-Wollaston and Ainsworth, 1997).
One of the genes shown to be senescence enhanced inmaize encodes a protein similar to a vacuolar processingenzyme from castor bean seeds, which is involved withthe conversion of vacuolar proteins to their mature forms(Smart et al., 1995). The expression of this gene duringsenescence may indicate that some of the senescence-related degradative enzymes are synthesized in the greenleaf, stored inactive in the vacuole, and activated by suchprocessing enzymes once senescence is in progress.
The location of the proteases that are synthesizedduring senescence remains to be determined. The protein
sequence deduced from the two cysteine proteases andthe aspartic protease clones identified in B. napus indicatesthat they all have similar hydrophobic N terminal regionswhich are likely to direct the protein into the endoplasmicreticulum. None have obvious chloroplast transit peptidesequences which might indicate their localization in thechloroplast. However, the protein sequence derived fromthe cloned gene can be used to identify antigenic peptidesequences; this has been done for the LSC7 protein andthe peptides synthesized have been used to raise antibodiesthat are specific for the LSC7 type of protease. Preliminaryimmunolocalization experiments with senescing B. napusleaf tissue has indicated that the protease encoded byLSC7 may be located within the chloroplast in senescingleaves (Fife and Buchanan-Wollaston, unpublishedresults). Further experiments such as this with othersenescence-related proteases will help to elucidate theproblem of how and where protein degradation takesplace during leaf senescence.
The ubiquitin pathway for targeted protein degradationis important in the elimination of abnormal cytosolicproteins and also in the rapid turnover of short-livedproteins. Ubiquitin is a highly conserved protein that isfound in all eukaryotes (Callis and Vierstra, 1989).Proteins that are destined for degradation are 'tagged' byubiquitination in a series of ordered events that require aseries of ubiquitin-activating enzymes, El and ubiquitin-conjugating enzymes, E2. Multi-ubiquitinated complexesare then recognized by an ATP-dependent protease com-plex which degrades the target protein (Rechsteiner,1991). It appears unlikely, from the study of the structureof genes from the ubiquitin pathway, that any of theproteins could be involved in degradation of chlorophyllproteins (Vierstra, 1993). Therefore, ubiquitin may notbe involved in the massive breakdown of proteins duringsenescence. However, the identification of an E2 typeubiquitin carrier protein gene that shows increased expres-sion during leaf senescence in TV. sylvestris (Genschiket al., 1994) and the observation of increased levels ofexpression of a polyubiquitin gene in senescing leaves ofpotato (Garbarino et al., 1995) may indicate that ubiqui-tin-dependent degradation of proteins does occur duringsenescence, possibly directed at specific cytosolic proteins.
Genes involved in nucleic acid breakdown
The nucleic acids, in particular the rRNA, within asenescing cell are an important source of carbon, nitrogenand especially phosphorus. The level of DNA in a senesc-ing cell remains relatively constant as senescence pro-gresses, but the levels of ribosomal RNA decrease(Makrides and Goldthwaite, 1981). RNase activity inleaves has been shown to increase during senescence(Green, 1994). Three RNase genes that show increasedexpression under phosphate-limited growth conditions
have been identified in Arabidopsis. One of these genes,RNS2, is also expressed at high levels during senescenceand is likely to be important for the degradation of RNAand remobilization of Pi in senescing leaves (Taylor et al.,1993; Bariola et al., 1994). The fate of the purines andpyrimidines resulting from nucleic acid degradation is notclear, but they are presumably broken down further, toconserve their nitrogen and carbon components forexport. The increased levels of uricase and xanthineoxidase observed in the peroxisomes of senescent leavesmay indicate that purine catabolism takes place in thisorganelle (Vicentini and Matile, 1993; Pastori and delRio, 1994).
Genes involved in lipid remobilization
The membranes of plant cells constitute a valuable storeof lipid molecules which can be mobilized and used bythe senescing leaf. The level of total lipids decreases insenescing leaves and it appears that the membranes ofthe cell including the thylakoid membranes are metab-olized to provide energy for the senescence process(Koiwai et al., 1981; Wanner et al., 1991). Respiratoryactivity in a senescing leaf is high and a continuous energysupply is necessary to allow degradation and mobilizationprocesses to take place. Meanwhile, the supply of photo-synthate to support respiration is decreasing due todismantling of the chloroplasts. There are several lines ofevidence that suggest that membrane components aremetabolized in senescing leaves to provide energy forrespiration. Firstly, the carbon component of 14C-labelledoleic acid, incorporated in galactolipids by feeding togreening etiolated barley, is released mostly as CO2 duringsenescence (Wanner et al., 1991). The respiratory quotient(RQ) of senescing leaves tends to be low, implying thatthe substrate for respiration is fatty acids rather thansucrose. Also, levels of key enzymes of the glyoxylatecycle, malate synthase and isocitrate lyase have beenshown to increase in senescing leaves of barley (Gut andMatile, 1988). The glyoxylate cycle, originally identifiedin micro-organisms as the pathway allowing growth onacetate, was shown to operate in higher plants in endo-sperm tissue of castor oil seeds (Kornberg and Beevers,1957). The cycle is involved with the conversion of acetylCo A, produced after lipid breakdown, to four carbonacids for subsequent conversion to carbohydrates, par-ticularly sucrose, by the reversal of glycolysis, or gluco-neogenesis (Fig. 3). The presence of the pathway ischaracterized by two key enzymes, malate synthase andisocitrate lyase and these enzymes are synthesized incotyledons during post germinative growth when storedlipid is being mobilized to provide energy for growth(Comai et al., 1989). The importance of the glyoxylatecycle in senescence has been confirmed by the detectionof increased expression of the genes encoding these two
enzymes during leaf senescence in cucumber (Grahamet al, 1992; McLaughlin and Smith, 1994).
Therefore, it appears that cellular lipids, such as thegalactolipids, which are the main component of thethylakoid membranes, or the endomembrane lipids, maybe metabolized during senescence via the glyoxylate cycle(Vicentini and Matile, 1993; McLaughlin and Smith,1995). Sucrose can be synthesized from the four carbonproducts of the glyoxylate cycle via the process of glucone-ogenesis, which converts TCA cycle components to suc-rose via a reversal of the glycolytic pathway. This sucrosecan be used for respiration or exported from the senescingleaf. The co-ordinate expression of the gene encodingphosphoenolpyruvate carboxykinase, a key enzymeinvolved in the conversion of lipids to sugars, with thegenes for malate synthase and isocitrate lyase during thenatural senescence of cucumber cotyledons, adds weightto the suggestion that the expression of these genes isrelated to gluconeogenesis (Kim and Smith, 1994a). PEPcarboxykinase catalyses the conversion of oxaloacetate toPEP, an essential step for gluconeogenesis to occur.Similarly, increased expression of peroxisomal NAD-malate dehydrogenase, one of the enzymes involved inthe glyoxylate cycle (Fig. 3), has been detected in senesc-ing cucumber cotyledons (Kim and Smith, 19946).
The senescence-related increase in expression of genesthat encode enzymes involved in the glycolytic pathwaymay also be indicative of the importance of gluconeogen-esis as a senescence-related process. Genes encodingenzymes such as fructose, 1,6, bisphosphate aldolase andglyceraldehyde-3-phosphate dehydrogenase are expressedin young B. napus leaves, expression is lower in maturegreen leaves and then increases again during senescence(Fig. 1). These enzymes may be involved in supportingthe increased respiration observed during senescence or,may be important for the synthesis of sucrose fromcomponents of degraded lipids and proteins, via glucone-ogenesis. Interestingly, another gene that is expressedduring senescence is that encoding pyruvate orthophosph-ate dikinase. Senescence-induced expression of this genehas been observed in maize (Smart et al., 1995) and alsoin B. napus (VBW, unpublished results). This enzyme isa key component of C4 photosynthesis where its role isto synthesize phosphoenolpyruvate (PEP) from pyruvateto restore PEP levels for CO2 fixation. The gene encodingthis enzyme is expressed at high levels in young developingleaves in maize (a C4 plant); expression is decreased inmature leaves and then increases again during senescence.In B. napus (a C3 plant) very low levels of expression aredetectable in young and mature green leaves and expres-sion increases during senescence. The implication of thisis that pyruvate orthophosphate dikinase enzyme, inaddition to its role in C4 photosynthesis, has an alternativerole to play during senescence in both C3 and C4 plants.The most likely role for this enzyme is in the gluconeo-
Molecular biology of leaf senescence 189
genesis pathway, where it may be involved in the conver-sion of pyruvate, produced from amino acid breakdown,into PEP.
Recently, senescence-related expression of a geneencoding phospholipase D, an enzyme that may beinvolved in the hydrolysis of membrane phospholipids,has been shown in dark-induced senescence of castorbean leaves (Ryu and Wang, 1995). This enzyme maycatalyse the first degradative step leading to the deteriora-tion of membrane integrity during senescence (Thompsonet al., 1987). In addition, the enhanced level of expressionof a j3-galactosidase gene that has been detected in senesc-ing asparagus ferns may be involved with the mobilizationof galactose during degradation of the galactolipids whichare the major components of the thylakoid membranes(King et al., 1995).
Genes involved in chlorophyll breakdown
Leaf yellowing, the visible sign of senescence, is due topreferential degradation of chlorophyll over carotenoids(Matile, 1992). The enzymatic steps by which chlorophyllis degraded have not been clearly defined; current know-ledge is reviewed by Smart (1994). It is likely that thecatabolism of chlorophyll starts with the chlorophyll stillbound to the membrane protein within the chloroplast,with the removal of the phytol tail by an enzyme such aschlorophyllase to yield chlorophyllide. The magnesiumatom is then removed by a Mg-dechelatase, the ring isopened by a dioxygenase and the binding protein releasedfor degradation. The remaining chlorophyll catabolite isthen transported to the vacuole where further metabolismtakes place. There is no evidence that any of the compon-ents of the chlorophyll molecules are exported from theleaf. This pathway has been proposed after the analysisof chlorophyll catabolites in senescing leaves using wildtype and stay green mutants that are defective in chloro-phyll breakdown (Matile, 1992; Smart, 1994; Vicentiniet al., 1995). However, none of the genes encodingenzymes involved in this process have been identified asyet, although a ferridoxin gene that shows persistentexpression during senescence, when photosynthesis-related ferridoxin genes would be switched off, may berequired by the dioxygenase step and thus may have arole in chlorophyll breakdown (Smart et al., 1995). It ispossible that some of the senescence-related cDNA clonesthat show no similarities to previously identified genes,may represent genes that encode enzymes involved inchlorophyll degradation.
Genes involved in nitrogen remobilization
Degradation of macromolecules during senescence is fol-lowed by remobilization of the components to developingareas of the plant. The nitrogen that is present in protein
and nucleic acid molecules is converted to transportableamino acids, in particular the amides, glutamine andasparagine, which are the predominant amino acids inthe phloem transported from senescing leaves (Kamachiet al, 1992; Feller and Fischer, 1994). It is likely that,during senescence, ammonia is released by deaminationof amino acids and catabolism of nucleic acids and isconverted into glutamine by the enzyme glutamine synthe-tase (GS). There are two distinct forms of GS found inplant leaves—GS1 which is located in the cytosol andGS2 which is plastidial (Kamachi et al., 1991). Duringsenescence, the activity of GS2 decreases while that ofGS1 tends to increase (Kamachi et al., 1992). The senes-cence-related increase of expression of genes encodingGS1 has been seen in several different plants (Table 1;Fig. 1) which demonstrates the importance of GS duringsenescence and confirms the prediction of its role in theconversion of the nitrogen from macromolecules intoglutamine. The expression of GS1 appears to be mainlyconfined to the vascular bundles where it is, presumably,involved in the generation of glutamine for transport,while GS2 is found in photosynthetic tissues where itsmain role is in the reassimilation of photorespiratoryammonia (Kamachi et al., 1992). Therefore, the increasein expression of the GS1 gene that is observed in senescingleaves is likely to be due to an increased requirement forglutamine synthetase to transport the mobilizable nitro-gen away from the senescing leaf. Similarly, the increasedexpression of the gene encoding asparagine synthetasethat has been observed in senescing asparagus ferns islikely be required for the synthesis of asparagine fortransport from the senescing leaf (King et al., 1995).
The source of the ammonia and glutamate for thesynthesis of glutamine by the action of glutamine synthe-tase is likely to be through transamination and deamin-ation reactions involving amino acids released from thedegraded proteins (Feller and Fischer, 1994). The amidegroup from most amino acids can be transferred toa-ketoglutarate by the action of a transaminase to produceglutamate, and glutamate dehydrogenase may then beinvolved in the production of ammonia from glutamate(Smart, 1994). Other sources of ammonia may be fromthe breakdown of other macromolecules such as nucleicacids. The source of the a-ketoglutarate, a component ofthe TCA cycle, may be maintained by substrates supplied
Molecular biology of leaf senescence 191
from the glyoxylate cycle or the transamination reactionswill liberate a variety of 2-oxo-acids that can enter theTCA cycle and be used either for energy production orto synthesize a-ketoglutarate for further transaminationreactions. Amino acids such as threonine and serinecannot normally be metabolized by transamination; asenescence-specific threonine dehydratase that has beenidentified in tomato may be involved in the release ofammonia from these amino acids during senescence(Szamosi et al., 1993).
A summary of the metabolic pathways that may beinvolved in the remobilization of macromolecules duringsenescence, leading to export of nitrogen as the amides,glutamine and asparagine, and sucrose, synthesized bygluconeogenesis, is depicted in Fig. 3. The expression ofgenes encoding enzymes of the glyoxylate cycle is rare inplant cells, occurring only in germinating seeds where oilstores are remobilized for respiration, and in senescingleaves where it is likely that it functions to mobilizemembrane lipids to use in respiration or to synthesizesucrose which is exported from the senescing cell. Thesuccinate produced from the conversion of acetylCoA inthe glyoxylate cycle can feed into the TCA cycle where itcan be converted to oxaloacetate for gluconeogenesis ora-ketoglutarate for transamination to glutamate. Thenitrogen component of the mobilized amino acids isprobably converted via glutamate to glutamine by glutam-ine synthetase and some may be converted to asparagineby asparagine synthetase for export. A proportion of theglutamate may be converted back to a-ketoglutarate byglutamate dehydrogenase to provide ammonia for theglutamine synthesis. The carbon skeletons from the aminoacids can enter the gluconeogenesis pathway as pyruvate,acetyl CoA or a-ketoglutarate. The senescence-relatedexpression of the pyruvate orthophosphate dikinase geneis probably involved with the conversion of the pyruvate,produced from amino acid breakdown, into PEP to enterinto the gluconeogenic pathway.
Senescence-related genes of unknown function
The senescence-enhanced genes discussed so far, encodeenzymes that appear to be involved in the degradativeand remobilization processes of senescence. In manycases, increased levels of the encoded enzymes have been
Fig. 3. Possible metabolic scheme controlling senescence-related mobilization of macromolecule components. 2-oxo-acids produced by transaminationof amino acids contribute to the pools of pyruvate, acetyl CoA or a-ketoglutarate. Glutamine and asparagine are transported from the senescingcell. Sucrose, synthesized via gluconeogenesis. is either used for respiration or is exported. The arrows indicate the direction which the processesare likely to take during senescence. Expression of genes encoding the following enzymes is elevated during senescence: (1) Fructose 1,6 bisphosphatealdolase and glyceraldehyde-3-phosphate dehydrogenase (required for both respiration and gluconeogenesis) (VB-W, unpublished). (2) Pyruvateorthophosphate dikinase (Smart et al., 1995; VB-W, unpublished). (3) PEP carboxykinase (Kim and Smith, 1994a). (4) Glutamine synthetase(Bemhard et al., 1994; Kawakami and Watanabe, 1988; Kamachi et al., 1992). (5) Asparagine synthetase (King et al., 1995). (6) Isocitratelyase (McLaughlin and Smith, 1994). (7) Malate synthase (Graham et al., 1992). (8) NAD malate dehydrogenase (Kim and Smith, 1994ft). (9)Various proteases (Hensel et al.. 1993; Smart et al., 1995; Lohman et al., 1994). (10) Various lipid-degrading enzymes such as phospholipase Dand delta 9 desaturase (Ryu and Wang. 1995; Fukuchi-Mizutani et al., 1995).
detected during senescence and the enhanced expressionof these genes could have been predicted. However, it isnot as easy to postulate a senescence-related function forsome of the other genes that have been identified.
Senescence-enhanced expression of metallothionein-likegenes has been detected in several plants (Table 1). In B.napus the expression of a gene encoding ferritin has beenfound to increase during senescence. Both metallothione-ins and ferritin function by binding metal ions—iron ionsin the case of ferritin and a range of divalent metal ionsin the case of metallothionein (Theil, 1987; Kagi, 1991).The possible functions of this type of protein in senescenceare not clear. It is possible, that, during leaf senescence, therelease of metal ions from the protein breakdown thatoccurs is sufficient to warrant a detoxification role.Alternatively, these metal ions may form a valuableresource to the future development of the plant and sothe presence of a metal binding protein may have afunction in the storage, and possibly transport, of metalions. There is some evidence that metallothioneins mayact in mammalian systems to protect DNA from oxidativedamage caused by free radicals (Chubatsu and Meneghini,1993). Leaf senescence is an oxidative process; breakdownof chlorophyll and membranes causes an increase in freeradical production. Therefore, the presence of metallo-thionein proteins may protect the nuclear DNA fromdamage, thus allowing expression of senescence-specificgenes required for the process to take place.
During senescence, it is essential that the senescing cellsremain viable for as long as possible to allow maximumexploitation of their cellular components. Therefore, it islikely that there will be mechanisms in place to protectthe cells from damage by internal or external factors.Levels of free radicals and reduced oxygen species suchas hydrogen peroxide increase in response to environ-mental stress and also during senescence (Merzlyak andHendry, 1994). The plant responds to the presence ofthese toxic compounds by the synthesis of a range ofantioxidative enzymes such as superoxide dismutase,ascorbate peroxidase and catalase (Foyer et al., 1994).Catalase is located in peroxisomes which become con-verted to glyoxosomes in senescing leaves (Vicentini andMatile, 1993). The function of catalase, acting in parallelwith ascorbate peroxidase located in the chloroplast, isto detoxify hydrogen peroxide that is produced duringphotorespiration in photosynthetic cells and also duringdegradation of macromolecules during senescence (Foyeret al., 1994). Catalases are present at all stages of leafdevelopment and three catalase genes showing differentialexpression have been identified in Nicotiana plumbagini-folia (Willekens et al., 1994). A catalase gene has beenidentified in B. napus that shows high levels of expressionin late senescence (Buchanan-Wollaston and Ainsworth,1997). The enzyme encoded by this gene may function toprotect the senescing cell from damage at later stages of
senescence, when, possibly, the chloroplast-locatedascorbate peroxidase system becomes less active.
Other genes showing senescence-related expression maybe involved in the metabolism of sulphur. A B.napus geneencoding ATP sulphurylase shows enhanced expressionduring senescence (Fig. 1; Buchanan-Wollaston andAinsworth, 1997). This enzyme activates sulphate in thepresence of ATP in the first step in the pathway tocysteine and methionine biosynthesis (Schmidt and Jager,1992). Free cysteine is converted to the tripeptide gluta-thione which appears to have an important role in theresponse of a plant to many external and internal stresses(Wingate et al., 1988; Foyer et al., 1994). Glutathionemay act as a storage molecule for sulphur and thisreaction might be important for the mobilization ofsulphur released during macromolecule degradationfrom the senescing leaf. The gene encoding glutathioneS-transferase also shows increased expression in senescingleaves (Smart et al., 1995). This enzyme is involved withdetoxification of herbicides by conjugation with gluta-thione and the senescence-related expression may berelated to stress. Alternatively, it could have some role insulphur metabolism during senescence.
Three different cytochrome P450 genes, that areexpressed in senescent and not green leaves, have beenidentified in B. napus (Table 1; VBW, unpublishedresults). In plants, cytochrome P450s are key enzymesinvolved in the synthesis of a large variety of secondaryplant metabolites including flavenoids, phytoalexins andlignin (Butt and Lamb, 1981). The role of these enzymesin leaf senescence is not understood. Another class ofgene that has been found to show increased expressionin B. napus senescence is that encoding proteins normallyassociated with the pathogen response (Hanfrey et al.,1996). These genes include PRla, a chitinase and a geneshowing similarity to an antifungal protein. It is possiblethat the presence of the PR proteins in senescing tissuesreflects the vulnerability of the tissues and performs anancillary function as part of the defence system againstinvading pathogens. Senescing leaves, containing a rela-tively high concentration of breakdown products andhaving a weakened physical structure, are extremely sus-ceptible to pathogen attack. Alternatively, these genesmay play fundamental roles in senescence since their rolein protection against pathogens is not clear. It is possiblethat the expression of some of the PR proteins could beconcerned with the induction and control of leaf senes-cence and only indirectly with defence against pathogens.
Therefore, the senescence-related function of a genecannot necessarily be determined by the DNA sequence,even if similar genes can be identified in the database. Inaddition, a certain number of senescence-enhanced genesthat have been identified in the screening show no similar-ity to sequences within the databases and hence theirfunctions cannot be speculated on. In these cases, expres-
sion of the genes as antisense in transgenic plants andanalysis of the effects of this on the senescence processcould help to determine the functions of these genes insenescence. Using this method, TOM 13, a gene involvedin tomato fruit ripening was identified as encoding ACCoxidase, an enzyme required for ethylene biosynthesis(Hamilton et al., 1990).
Genes expressed in other degradativephysiological processes
There are similarities between many of the events thattake place during leaf senescence and fruit ripening, forexample, chlorophyll degradation, increased activity ofhydrolytic enzymes and, in some cases, ethylene synthesis.The involvement of genes common to the two processesmight, therefore, be expected. Several cDNA clones rep-resenting genes that show increased expression duringleaf senescence were identified by screening clones origin-ally shown to be expressed during tomato fruit ripening(Davies and Grierson, 1989). One of these genes, TOM13,encodes the ethylene biosynthetic enzyme ACC oxidase(or EFE) and antisense expression of this gene was foundto delay leaf senescence in transgenic tomato plants(Picton et al., 1993). Another of the ripening-relatedgenes shown to be expressed in senescing leaves wasTOM75 which encodes a membrane channel protein thatmay be involved in the transport of small molecules fromsenescing cells (Fray et al., 1994).
Other physiological processes that involve metabolicevents similar to those that occur during leaf senescenceinclude petal senescence and post-harvest deterioration.Genes that have been identified showing induced expres-sion in these systems show common functions to thoseidentified in senescence. For example, genes involved inethylene biosynthesis are expressed in senescing carnationflowers (Park et al., 1992) and in broccoli after harvest(Pogson et al., 1995a). Protease genes are expressed insenescing ovaries in pea (Granell et al., 1992) and in thepetals of day lily and carnation (Jones et al., 1995;Valpuesta et al., 1995). A gene encoding a delta 9desaturase, possibly involved in the degradation of mem-brane fatty acids has been shown to be expressed insenescing rose petals (Fukuchi-Mizutani et al., 1995). Inmany cases, genes that have been identified as beinginvolved in one of these physiological processes have alsobeen found to be expressed in an alternative processes(e.g. fruit ripening and leaf senescence [Davies andGrierson, 1989]; post-harvest degradation and leafsenescence [King et al., 1995]).
Identification of genes that show induced expressionduring leaf senescence and analysis of possible functionsof the proteins encoded by these genes have not, so far,contributed very much to our current knowledge aboutthe processes that occur during senescence. The reasons
Molecular biology of leaf senescence 193
for this are that, in general, the cloned genes that caneasily be assigned functions have already been implicatedin senescence from enzyme studies. Transgenic plantanalysis using the genes that, from comparison withhomologous genes, have no obvious function in senes-cence or which have no similar sequences in the databases,will be more likely to generate new information on thesenescence processes that has not been accessible bybiochemical studies. Also, the identification of more senes-cence-related genes will help to fill in the picture of thesenescence process that is starting to emerge. The genesidentified so far, however, do open new ways for studyingthe regulation of senescence.
Regulation of leaf senescence
The events that take place during senescence appear tobe highly co-ordinated to allow the maximum use of thecellular components before necrosis and death occurs.Complex interactions involving the perception of specificsignals and the induction of cascades of gene expressionmust occur to regulate this process. Expression of thegenes coding for senescence-related proteins is likely beregulated via common activator proteins that are, in turn,activated directly or indirectly by hormonal signals. Littleis known about the mechanisms involved either in theinitial signalling or subsequent co-ordination of the pro-cess. However, the identification of senescence-relatedgenes provides the tools required for the molecularanalysis of the factors involved in the regulation ofsenescence.
There are two main approaches that may be taken forthe analysis of transcriptional regulation of gene expres-sion. One of these involves the identification and analysisof mutants that are defective in the process of interest,an approach which can be powerful if a large number ofmutants can be identified reasonably easily. For example,mutants defective in ethylene responses can be identifiedat the seedling level and dissection of the ethylene responsepathway using mutants in Ambidopsis is well underway(Zarembinski and Theologis, 1994; Ecker, 1995). Therole of ethylene in leaf senescence will be discussed later.Senescence is a complex process involving diverse signalsand the identification of regulatory mutants would requirethe characterization of a large number of potential senes-cence mutants. So far several groups, including ours,have expended considerable effort in trying to identifyAmbidopsis mutants defective in leaf senescence, withvery little success (Bernhard and Matile, 1994).
The alternative approach to analyse the regulation ofleaf senescence is to study the transcriptional activationof senescence-related genes. Cloning and characterizationof the promoters of genes that show induced expressionduring senescence will enable the critical elements thatare involved in regulating senescence to be defined. Even
if the genes under study are not the primary sites at whichsenescence regulation occurs, it should be possible totrace back the regulatory steps to the first step in thepathway. In this way, the initial signals that regulate theonset of senescence can be denned. Of the many senes-cence-related genes that have been identified, Northernanalysis has indicated that the transcription of the genesis not induced at the same stage of senescence (e.g. Fig. 1).Therefore, the regulation of gene expression during senes-cence may involve several different signalling pathways,which are themselves co-ordinately regulated, activatingtranscription at different senescence stages.
The onset of senescence
The onset of senescence can be induced by many differentfactors both internal and external. Environmental stressessuch as temperature, drought, poor light or nutrientsupply and pathogen attack will all result in prematureinitiation of senescence. However, leaf senescence is animportant part of the development of the plant and whena leaf reaches a certain age, or when the reproductivephase of the plant reaches a certain stage, senescence willbe initiated in the leaf even if the plant is growing underfavourable conditions. The original signals that act toregulate senescence, initiated under these different condi-tions may be different but, in general, the senescenceprocesses involved are likely to be the same.
All the major plant hormones have been implicated asbeing involved in the senescence process, but only cytoki-nins and ethylene have been shown definitively to have arole in the regulation of senescence (Smart, 1994). Theinitiation of leaf senescence caused by the developmentof ripening fruit has led to the proposal of a specific'death hormone' that is produced by developing fruit andtransported to the leaves (Engvild, 1989). Substancessuch as jasmonic acid have been proposed as having thistype of role but there is little evidence to support this andjasmonate appears more likely to be involved in stress-related responses which may result in senescence (Beckerand Apel, 1993; Reinbothe et cil., 1994).
Role of cytokinin in leaf senescence
Treatment with cytokinins has been shown to delay leafsenescence in many plants (van Staden et al., 1988).Cytokinins are synthesized mainly in the root and trans-ported to the rest of the plant in the xylem. It has beenshown that the level of cytokinins in xylem sap declineswhen senescence is initiated and it has been suggestedthat a reduced level of cytokinins in the leaf may causethe onset of senescence (Nooden et al., 1990).
Transgenic plants have been produced that have alteredlevels of cytokinins due to expression of an isopentenyl-transferase gene (IPT, the enzyme that catalyses the rate-
limiting step in cytokinin biosynthesis). Early experimentswhere this gene was expressed from a constitutive or heatshock promoter resulted in plants which did show delayedonset of leaf senescence, but the specific effects of thecytokinins on leaf senescence were hard to interpretbecause the plants grew abnormally (Smart et al., 1991).Recently, the IPT gene has been cloned under the regula-tion of the promoter for SAG 12—a senescence-specificArabidopsis gene, encoding a protease (Lohman et al.,1994). Transformed tobacco plants carrying this genefusion developed normally up to the senescence stage,whereupon senescence was significantly retarded in theleaves from the transformed plants (Gan and Amasino,1995). The leaves of the transgenic plants showed nosigns of senescence, the photosynthetic activity remainedhigh and more flowers were produced. From this resultit is clear that the levels of cytokinin within a leaf are akey factor in initiating the onset of senescence. The mostimportant component of this experiment was the availab-ility of the senescence-specific promoter for the SAG 12gene which is expressed only during leaf senescence and,therefore, has an autoregulatory role. Expression of thepromoter is induced as senescence starts, cytokinins aresynthesized, senescence is halted, and the SAG12 pro-moter switched off. Thus, there is no cytokinin overprod-uction to affect the normal development of the plant(Gan and Amasino, 1995).
The control by cytokinins is likely to be at the transcrip-tional level—the presence of cytokinins above a certainlevel inhibits the expression of senescence-related genes.A fusion between the SAG 12 promoter and the GUSreporter gene, when transformed into tobacco showedextremely high levels of the GUS prgtein in senescingleaves (Gan and Amasino, 1995). However, when thisfusion was present in the same plant as the SAG 12promoter: :IPT gene fusion, the levels of GUS seen weremuch lower, indicating that the cytokinins produced bythe IPT fusion were inhibiting the expression of GUSfrom the senescence-enhanced promoter. Two cytokinin-repressed genes have been identified in cucumber cotyle-dons (Teramoto et al., 1995). The expression of thesegenes in mature green leaves is repressed in the presenceof externally applied cytokinins. The level of transcriptfor both genes is very low in young leaves and increasessignificantly in mature and senescing tissue, presumablydue to reduced levels of cytokinins present in this tissue.Therefore, it is clear that cytokinins, either directly orindirectly via a signalling pathway, can inhibit the tran-scription of senescence-related genes.
Role of ethylene in the regulation of leafsenescence
Ethylene plays a regulatory role in many plant processessuch as seed germination, seedling development, fruit
ripening, and flower senescence. Ethylene is also involvedin plant responses to external signals, for example,responses to wounding, pathogens and stress caused byenvironmental pollutants such as ozone (Zarembinskiand Theologis, 1994). In climacteric fruits, ethylene pro-motes ripening by co-ordinately inducing the expressionof a large number of genes encoding enzymes requiredfor the ripening process (Theologis, 1993) and in thesenescence of some flowers, ethylene appears to have asimilar regulatory role (Borochov and Woodson, 1989).Ethylene has been implicated in the regulation of leafsenescence in certain plants but, as is described below,recent evidence with transgenic plants and ethyleneresponse mutants has indicated that, although ethylenehas an effect on senescence, it is not an essential regulatorof the process. In many plants, ethylene appears to haveno role in ripening or senescence; for example, in theripening of fruits of non-climacteric plants such as straw-berry, in the senescence of some flowers such as day liliesand in leaf senescence of some plants, in particular themonocots, there is no requirement for ethylene signalling(Smart, 1994; Valpuesta et al., 1995).
Ethylene biosynthesis from S-adenosyl methionineinvolves two enzymes, 1-amino-cyclopropane-l-carboxylic acid (ACC) synthase and ACC oxidase.Increased expression of the genes encoding these twoenzymes has been detected in ripening fruit, senescingpetals and during post-harvest degradation of broccoli(Hamilton et al, 1990; Park et al, 1992; Pogson et al.,19956). In senescing tomato leaves, increased expressionof the ACC oxidase gene was detectable early in senes-cence, before chlorophyll loss had commenced (Johnet al., 1995). Experiments with transgenic plants thatwere expressing the antisense of the ACC oxidase geneshowed that when the expression of this gene wasrepressed and ethylene production reduced there was amarked effect on the rate of fruit ripening, and a smallereffect on leaf senescence (Picton et al., 1993). Furtheranalysis of leaf senescence in these transgenic plantsshowed that senescence was delayed 10-14 d in thetransgenic plants and the leaves remained photo-synthetically active during this period. However, oncesenescence had started, the progression of senescence inthe leaves of the transgenic plants matched that takingplace in the wild-type leaves (John et al., 1995).
Mutants of tomato that are defective in the ethylenesignal transduction pathway have been used to study theeffects of ethylene insensitivity on fruit ripening andsenescence. For example, plants carrying the Never Ripemutation, which has been shown to block ethylene percep-tion, produce fruit that ripen extremely slowly. The effectsof the Never Ripe mutation have been studied in somedetail by Lanahan et al. (1994). The flowers produced bythese mutants do not senesce and show considerable delayin abscission. However, the leaves on the mutant plants,
Molecular biology of leaf senescence 195
apart from showing a small delay in the onset ofsenescence, senesced normally. In parallel experiments,transgenic tomato plants overproducing ACC synthasewere analysed; these plants produced high levels of ethy-lene which resulted in premature senescence of the flowers,but increased rates of leaf senescence were not observed.In fact, leaves on the ethylene overproducing plantsremained green longer than control leaves; this lack ofsenescence may be related to the lack of fruit set due toflower abortion.
Experiments with the ethylene-insensitive Arabidopsismutant, etr\-\, showed that leaf senescence was similarlydelayed; leaves on mutant plants had an approximately30% longer life span than the wild-type leaves althoughthe photosynthetic activity of these longer lived leavesdeclined (Grbic and Bleeker, 1995). Once initiated how-ever, the rate of senescence and the levels of expressionof senescence-induced genes (SAGs) was found to besimilar to that of the wild type. Treatment of wild-typeplants with exogenous ethylene resulted in prematuresenescence with earlier expression of the SAGs althoughthe increased expression was age related. Only the oldermature green leaves showed a response to ethylene byinducing expression of SAGs, the younger, but still maturegreen leaves showed only marginal increases in SAGexpression. This effect is comparable to results withtomato fruit where immature tomato fruit fail to ripen inresponse to ethylene and it appears that the tomato fruitshave to be of a certain age before the ripening processcan be induced by ethylene (Grierson and Kader, 1986).Also, ethylene could not induce the expression of ripeningspecific mRNAs in immature tomato fruit (Lincoln andBaker, 1987). These results have led to the conclusionthat other factors are involved in the regulation of geneexpression in both fruit ripening and leaf senescence(Grbic and Bleeker, 1995) and that these 'age-relatedfactors' are essential for both processes. The processesdiffer in that tomato fruit ripening requires both 'age-related factors' and ethylene while for leaf senescence tooccur, 'age-related factors' only are necessary. The roleof ethylene in leaf senescence appears to be as some kindof modulator in determining the timing of leaf senescence.
Ethylene treatment of young Arabidopsis leaves doesnot result in increased expression of senescence-relatedgenes (Grbic and Bleeker, 1995; VBW, unpublishedresults) although the expression of other ethylene-regulated genes unrelated to senescence is induced in thesame tissue (Chen and Bleeker, 1995). Thus, transcriptionof senescence-related genes is not activated by ethyleneunless the leaf is 'prepared' to senesce, in which casesenescence is enhanced and there is concomitant expres-sion of senescence-related genes. Therefore, the ethylenesignalling pathway is probably not involved directly inregulating the transcription of genes during leaf senes-cence. The mechanism(s) by which ethylene modulates
the rate of senescence is unclear, but it may be that thepresence of ethylene increases the sensitivity of an inter-action that takes place at some point in the signallingpathway that is controlled by the presence of 'age-relatedfactors'. In the absence of ethylene, transmission of thesignals occurs later, possibly due to the requirement fora higher concentration of 'age-related factors'.
As well as hormonal signals, alterations in levels ofmetabolites may be involved in regulating expression ofsome of the senescence-enhanced genes. For example,some of the genes involved in the glyoxylate pathwaythat show senescence-induced expression are also regu-lated by levels of carbon compounds (Graham et al.,1994; Kim and Smith, 1994a). It is possible that changesin levels of metabolites, that occur due to the reducedrate of photosynthesis, may have stimulatory effects onthe transcription of senescence-related genes (Henselet al., 1993).
Thus, there appear to be at least four signalling path-ways that can be involved in the regulation of genesinvolved in leaf senescence.(a) Cytokinin levels: Senescence can only be initiated whenthe cytokinins in the leaf fall below a threshold level.Maintaining the cytokinin levels inhibits transcriptionalregulation of senescence-enhanced genes and prevents theonset of senescence.(b) Signals from developing sinks: Leaf senescence isrepressed, or even reversed, when developing sinks areremoved (Crafts-Brandner, 1991). Tomato plants thatoverproduce ethylene produce no flowers or fruit and leafsenescence does not occur, possibly because of lack ofsignal from the developing fruit (Lanahan et al., 1994).This signal does not overcome the cytokinin thresholdsignal since flower and seed development did not induceleaf senescence in the transgenic tobacco plants expressingIPT in the leaves (Gan and Amasino, 1995). However,in normal plants cytokinin levels may be influenced bythe signal from developing sinks.(c) Ethylene: Antisense and mutant studies showingdelayed leaf senescence have indicated that ethylene altersthe timing of senescence. Increased levels of ethyleneenhance the rate of senescence progression, but only inleaves that are already programmed to start senescence.Ethylene does not appear to activate senescence-relatedgenes directly, but probably modulates the activation ofthe genes by other signals. Ethylene may have a role inrepressing the expression of genes involved in photosyn-thesis (Grbic and Bleeker, 1995).(d) Level of photosynthate and other metabolites: Theearliest sign of senescence initiation is the decline incarbon fixation rates which, in Arabidopsis and someother plants, commences as soon as full leaf expansion isreached (Hensel et al., 1993). The decline in photosyn-thesis, resulting in reduced fixed carbon availability, may
be a signal for the induction of expression of senescence-related genes.
These signals could act in concert to regulate theexpression of senescence-related genes. The studiesdescribed here involve Arabidopsis and tomato leaf senes-cence. However, it is possible that the regulation ofsenescence in these two plants is not identical. Forexample, Arabidopsis leaf senescence does not appear toinvolve a signal from developing fruit (Hensel et al.,1993) while that of tomato and other fruiting plantsobviously does. Therefore, although recent results haveshed some light on the regulation of leaf senescence, thedevelopmental and physiological differences betweendifferent plants add a new level of complexity.
The isolation and characterization of promoters thatcontrol gene expression during senescence and the analysisof the factors that bind to these promoters and affecttheir expression during different developmental and envir-onmental conditions may help to unravel the complexitiesof the regulation of leaf senescence as a whole. Thepromoter for the LSC54 gene, encoding a metallothion-ein-like protein, from B. napus (Buchanan-Wollaston,1994), has been cloned and fused to the GUS reportergene (Butt and Buchanan-Wollaston, unpublishedresults). Analysis of the expression of this promoter: :GUSfusion in transgenic Arabidopsis has indicated that thepromoter is highly induced during natural senescence inArabidopsis. The promoter also shows increased activityin response to wounding and during dark inducedsenescence.
Interestingly, it has recently been found in this laborat-ory that expression of this gene in Arabidopsis appears tobe linked to the disease resistance response to the patho-gen Peronospora parasitica. The disease resistanceresponse to pathogens in plants can often take the formof the hypersensitive response (HR) in which an infectedplant reacts to an invading pathogen by killing the cellsin the specific area of infection. This reaction of the planthas been likened to the programmed cell death phenom-enon that has been studied in animals (Bowen, 1993:Mittler and Lam, 1995). It has been shown that, in cellsthat are responding to pathogen invasion by induction ofthe hypersensitive response, the promoter for the LSC54gene is expressed very specifically, prior to cell death(Butt, Can, Holub, Beynon, and Buchanan-Wollaston,unpublished results). HR-associated cell death has beenshown to require active plant metabolism and be depend-ent on de novo transcription and translation of host genes(Yang et al., 1993). Therefore, this result, that may pointto HR-induced expression of the LSC54 gene, could implythat the signals involved in the disease resistance response,that allow the plant to respond to the presence of thepathogen and prevent its spread, have elements that arecommon to pathways that are controlling cellular senes-cence. Further analysis of this promoter will allow the
isolation of transcription factors and analysis of theseshould lead to the dissection of the regulatory steps thatare involved in the senescence-related expression of thisgene.
The application of molecular biology techniques tostudy the events that take place during leaf senescencehas allowed considerable progress to be made in the lastfew years. Many senescence-related genes have now beenidentified and, although the functions of some of theencoded proteins have not been unexpected, transgenicplant analysis to identify the functions of others hasbegun to open new perspectives on the processes thatoccur during senescence. The availability of genes, pro-moters and mutants has allowed a clearer picture to beobtained concerning the regulatory mechanisms that areinvolved in senescence.
Acknowledgements
I would like to thank Charles Ainsworth for his invaluableadvice on this manuscript and his help with the figures. I alsothank Dennis Baker for critically reading the manuscript.
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