Large-scale identi®cation of leaf senescence-associated
genes
Shimon Gepstein, Gazalah Sabehi, Marie-Jeanne Carp, Taleb Hajouj, Mizied Falah Orna Nesher, Inbal Yariv, Chen Dor and Michal Bassani
Faculty of Biology, Technion, Israel Institute of Technology, Haifa 32000, Israel
Received 1 July 2003; revised 19 August 2003; accepted 28 August 2003. For correspondence (fax972 4 8225153; e-mail gepstein@tx.technion.ac.il).
Summary
Leaf senescence is a form of programmed cell death, and is believed to involve preferential expression of a speci®c set of `senescence-associated genes' (SAGs). To decipher the molecular mechanisms and the pre-dicted complex network of regulatory pathways involved in the senescence program, we have carried out a large-scale gene identi®cation study in a reference plant,Arabidopsis thaliana. Using suppression subtrac-tive hybridization, we isolated approximately 800 cDNA clones representing SAGs expressed in senescing leaves. Differential expression was con®rmed by Northern blot analysis for 130 non-redundant genes. Over 70 of the identi®ed genes have not previously been shown to participate in the senescence process. SAG-encoded proteins are likely to participate in macromolecule degradation, detoxi®cation of oxidative meta-bolites, induction of defense mechanisms, and signaling and regulatory events. Temporal expression pro-®les of selected genes displayed several distinct patterns, from expression at a very early stage, to the terminal phase of the senescence syndrome. Expression of some of the novel SAGs, in response to age, leaf detachment, darkness, and ethylene and cytokinin treatment was compared. The large repertoire of SAGs identi®ed here provides global insights about regulatory, biochemical and cellular events occurring during leaf senescence.
Keywords:Arabidopsis, genomics, leaf senescence, senescence-associated genes, subtraction suppression hybridization.
Introduction
Leaf senescence, the last stage of development that pre-cedes death, is a genetically programmed process. Senes-cence program is thus believed to be regulated by speci®c genes (Buchanan-Wollaston, 1997; Buchanan-Wollaston et al., 2003; Dangl et al., 2000; Gan and Amasino, 1997; Quirinoet al., 2000; Smart, 1994; Yoshida, 2003). Expres-sion of many genes associated with photosynthetic activity and other anabolic processes is downregulated during leaf senescence (Gepstein, 1988), whereas others are upregu-lated. Indeed, several dozen genes upregulated during senescence, designated as senescence-associated genes (SAGs) have been identi®ed in various species (Buchanan-Wollaston and Ainsworth, 1997; Dangl et al., 2000; Lee et al., 2001; Lohmanet al., 1994; Nam, 1997). The function of the predicted SAGs' products may give a clue to the biochemical, regulatory, and cellular pathways of senes-cence. The downregulation of a certain set of genes on the
Robatzek and Somssich, 2002), and components of intra-cellular protein traf®cking (Gutermanet al., 2003). Among the genes that are upregulated during leaf senescence are several whose transcript levels accumulate also under abiotic and biotic stresses (Binyaminet al., 2000; Hanfrey et al., 1996; Johnet al., 1997; Quirinoet al., 1999; Weaver et al., 1998).
While these genes offer insights into the molecular basis of senescence, the number of SAGs identi®ed so far cannot account for the myriad biochemical and cellular events involved in responses to exogenous and endogenous senescence-affecting agents, the operation of multiple signaling cascades, and the execution of the senescence syndrome. Furthermore, the available list of SAGs repre-sents genes identi®ed in a wide range of monocot and dicot species. These species may not share identical regu-latory pathways. For example, while leaf senescence of several monocarpic species is controlled by ¯ower and fruit development, such linkage was not found in Arabidopsis thalianaecotype Landsberg erecta (Nooden and Penney, 2001). Thus, the information accumulated from different unrelated species may not allow the predic-tion of an integrated network operating during leaf senes-cence. A global genomic study in a single reference species is therefore required and should provide this information.
Most known SAGs have abundant products and are read-ily detected and repeatedly reported in the literature. To identify also the non-abundant genes, we used suppression subtractive hybridization (SSH), which was speci®cally designed for comparing gene expression in different tis-sues or at different developmental stages (Diatchenko et al., 1996). The SSH method is based on generation of libraries of differentially expressed clones by subtraction of tester cDNA (in our case, senescing leaf) with an excess of driver cDNA (prepared from mature leaf). This PCR-based method includes also a normalization step, which equalizes the abundance of cDNA within the tester popula-tion, and a subtraction step that excludes the common sequences. The major advantage of the SSH technology is the enrichment of rarely transcribed clones, and therefore higher sensitivity is attained than by other methods of differential screening.
We present here the most comprehensive gene identi®-cation study made, so far, of the monocarpic senescence program in a single reference plant. A large fraction of the identi®ed genes has not yet been studied in the context of senescence, thereby contributing to the unraveling of mole-cular events regulating this important developmental stage. A more complete inventory of genes in a single reference may also enable the integration of the regulatory pathways of senescence. Furthermore, this information is crucial for identifying targets for the manipulation of leaf senescence.
Results and discussion
The SSH approach and its advantages in identifying senescence-related genes
The driver cDNA for the construction of the SSH library was synthesized from mRNA isolated from fully expanded mature green rosette leaves, harvested from plants that had not yet commenced ¯owering and contained the max-imal levels of chlorophyll (Figure 1a,b). The tester cDNA was produced from mRNA isolated from senescent leaves whose chlorophyll levels were around 60% of the initial values. Leaves at this stage contain heterogeneous cell
populations, with the oldest cells found at the leaf margins and mature green cells in the central region of the leaf (Figure 1a,b). Thus, a pool of leaves at this stage is a good source for isolating SAGs acting throughout the senes-cence process.
A global study of genes whose expression is upregulated during natural senescence ofArabidopsisleaves was car-ried out by analysis of clones obtained from the SSH libraries. Out of 800 isolated cDNA clones, 350 were ana-lyzed for their sequence and 175 non-redundant sequences were con®rmed by Northern blot. Table 1 summarizes Northern blot analyses for 127cDNA clones. This study revealed about 70 new SAGs induced inArabidopsis, add-ing signi®cantly (doubladd-ing) to the number of known SAGs. The list also contains around 50 genes that had already
been reported, although in various species, as related to senescence. Some of the listed SAGs with predicted func-tions similar to previously reported SAGs have different Accession numbers. Identi®cation of these known SAGs not only supports previous senescence studies but also con®rms the enrichment of senescence-speci®c clones by SSH. Abundant genes that function in the execution phase of senescence, such as hydrolases, have been repeatedly reported in many species. In our set of SAGs, the proportion of these abundant clones is not higher than that of other genes. These results are not surprising because the SSH method enriches for the non-abundant genes that other-wise would not have been detected. It has been calculated from a large population of enhancer trap lines that about 10% of the total number of genes in Arabidopsis are
Table 1Differential display of leaf senescence clones
Annotation Accession M O References
Macromolecular degradation and recycling
Cysteine protease component At1g47128 1, 2, 3, 4, 20, 21 Cysteine protease At4g16190 1, 2, 3, 4, 20, 21 Cathepsin B-like cysteine protease At4g01610 1, 2, 3, 4, 20, 21 Gamma vacuolar processing enzyme At4g32940 7 , 19
AALP At5g60360 1, 2, 3, 4, 20, 21
APG8 At3g15580 29, 30
Ubiquitin carrier protein At1g14400 6, 28
26 s Proteasome ATPase subunit At1g53750 28
SKP1 interacting partner 6 At2g21950 7
ATP-dependent Clp protease subunit At5g53350 5 Endonuclease-putative At1g35160
Amino acid transport
Amino acid permease At1g58360 Cationic amino acid transporter putative At3g03720
Aspartate amino transferase At5g11520 10
Vegetative storage protein 2 At5g24770 11 Vegetative storage protein 1 At5g24780 11 Lipid metabolism
Lipase-putative At2g42690 12
Omega-6 fatty acid desaturase At3g12120
Phospholipase D putative At3g15730 NA 22, 23 Sugar metabolism
Beta amylase putative At3g23920 Cell wall
Glycine rich protein At1g30460
Beta-1,3 glucanase like protein At3g55430 18 Glycine-rich cell wall protein-like At4g18280
Beta glucosidase-like protein At4g27830 9, 10 Nucleotide sugar epimerase At4g30440
Xyloglucan endo-1,4 beta-D-glucanase At4g30270 6 Endo-polygalacturonase At4g57510
Xylose isomerase At5g57655 Metabolism
Lipoic acid synthase At5g08410 Lipoic acid synthase At2g20860 Acid phosphatase-like protein At5g34850
Acid phosphatase type 5 At3g17790 24
Table 1continued
Annotation Accession M O References
Detoxification
Metallothionein-like protein At1g07600 1, 4, 16 Metallothionein-like protein At3g09390 1, 4, 16
Metallothionein 2b At5g02380 1, 4, 16
Dehydroascorbate reductase At1g19570
Gluthatione S transferase At1g78380 4, 25, 26 Glutathione S transferase At5g17220 4, 25, 26 Phospholipid hydroperoxide gluthatione transferase At4g11600
Cu/Zn superoxide dismutase-like protein At5g18100 26
Glyoxalase II At1g53580 NA 8
Copper Amine Oxidase At4g12290 31
Ferittin 1 precursor At5g01600 4
Dioxygenase putative At2g25450
Catalase 3 At1g20620 4
Stress, pathogenicity and secondary metabolites
Myrosinase-binding protein At1g52040
SAG29 At5g13170 NA 8
Aldehyde dehydrogenase precursor At3g48000 8
Cinnamyl alcohol dehydrogenase At1g09500 8
Cinnamyl alcohol dehydrogenase ELI3 At4g37990 NA 8
Allene oxide cyclase At3g25760 NA 13
Drought induced cysteine proteinase At4g39090 Wound-responsive protein At1g75380 Patatin-like protein At2g26560
Harpin putative At5g53730 10
Nitrilase II At3g44300 8
Protein induced upon wounding At4g24220 Limonene cyclase-like protein At4g16740 Squalene epoxidase At5g24160
nsLipid transfer protein precursor At5g59310 10 NADPH oxidoreductase putative At1g75280
Trypsin inhibitor At1g73260 Regulatory genes
TonB-dependent receptor At1g05340 myo-inositol 1-phosphate synthase At2g22240 Receptor-like protein kinase At5g48380
Calcium-binding protein At1g18210 15
Cyclic nucleotide-regulated ion channel At2g23980 15, 17
Calcium-binding protein At2g43290 15,17
Calmodulin regulated ion channel At5g54250 15, 17 Apoptosis inhibitor-putative At1g68820
RAP2.4-AP2 domain TF At1g78080 Lim domain protein At2g39900 Auxin responsive GH3-like protein At4g37390 Auxin-regulated protein putative At2g45210 G protein beta subunit-like protein At2g43770 Ras-related small GTP-binding At5g47201 Zinc finger-like protein At3g52800 Ring H2 finger protein At4g11360 Zinc finger-like protein At5g10650 Small nuclear ribonuclear protein At3g07590 SSH clones involved in various processes
Dioxygenase putative At2g25450
No Apical Meristem (NAM) At1g52880 6
speci®cally expressed during senescence so that reported SAGs represent only a limited sample (Heet al., 2001). The 127SAGs (Table 1) represent one-third of the available SSH clones; thus, this library, composed of many yet unidenti-®ed genes, is an excellent source for large-scale functional genomics studies.
Most of the cDNA sequences are readily aligned to DNA sequences found in the Arabidopsisdatabase. Less than 50% of the sequences are annotated as unknown or hypothetical. The remaining clones have signi®cant homol-ogy to sequences with known or predicted function, allow-ing us to assign, for each clone, a putative role in the regulation or execution of senescence.
SAGs have been grouped into several categories, based on their predicted function. Examples are: degradation of macromolecules and recycling of metabolites, oxidative metabolism and detoxi®cation of oxygen reactive species, responses to pathogens, biosynthesis of secondary
meta-bolites, and regulation of the initiation and progression of senescence. In the following sections, we outline some of the classes of SAGs.
Macromolecule breakdown: protein degradation and recycling
Senescence-associated genes isolated in our study, which participate in cellular protein degradation processes, repre-sent components of the various known proteolytic systems acting in most subcellular compartments. This group of senescence-associated proteins includes: cysteine pro-teases, aspartic propro-teases, and components of the ubiqui-tin/proteosome system and of the novel autophagic (APG) pathway.
The isolated cDNA clones encoding cysteine proteases found in our study represent different groups. The Arabidopsisaleurain-like protein (AALP) that shares 70% Table 1continued
Annotation Accession M O References
NADPH-fettihmprotein reductase At4g30210
SAG21 At4g03280
LSG clones with unknown functions
At1g13990 At1g21670 At1g30420 At1g51200 At1g59870 At1g63010 At1g67840 At1g70900 At1g71950 At1g73325 At1g73750 At3g02040 At3g15580 At3g22600 At3g25480 At3g26100 At3g44100 At3g51130 At3g51730 At3g62550 At4g11910 At4g13250 At4g27020 At4g35750 At4g39670 At5g10860 At5g19540 At5g43850
homology with the barley enzyme aleurain and with g oryzain, both involved in germination, is upregulated also during senescence. Of special interest is our ®nding of the senescence-related increased expression of a gene encod-ing a cathepsin B-like cysteine proteinase (At4g01610). Cathepsin B, the animal counterpart, is a major lysosomal cysteine peptidase that has been reported to accumulate during liver aging (Keppler et al., 2000). The observed elevated expression of the vacuolar processing enzyme gene indicates that, in addition to de novo synthesis of proteases during leaf senescence, some of the senes-cence-related proteases are stored in the vacuole, and are then activated by processing enzymes once senescence is in progress (Kinoshitaet al., 1999).
The involvement of ubiquitin-proteasome-pendent pro-teolysis during leaf senescence is re¯ected by an increase in the expression of the ubiquitin genes encoding enzymes associated with the ubiquitination cascade (ubiquitin con-jugating enzymes). Expression of some of the genes encod-ing proteasome constituent proteins (At1g53750) also increased during the advance of leaf senescence. This ®nding suggests that the whole ubiquitin-proteasome path-way is active, at least inArabidopsis, and contributes to intensive protein degradation (Ingvardsen and Veierskov, 2001). Furthermore, a regulatory role for ubiquitin-depen-dent proteolysis during senescence has been proposed. A mutation in the gene encoding the ORE9 F-box protein caused a delay in the initiation of leaf rather on the pro-gression of senescence (Wooet al., 2001). The ORE9 F-box protein interacts with a component of the plant SCF com-plex that controls selective ubiquitination and subsequent proteolysis of targeted proteins. Thus, ORE9 may function in the initiation of senescence and is involved in the degra-dation, through ubiquitin pathway, of a putative key reg-ulatory repressor of senescence. Another mutant (dLs1), defective in a gene encoding an arginyl-tRNA; proteinargi-nyl transferase (R-transferase) and involved in the N-end rule proteolytic pathway in yeast and mammals, exhibited delayed senescence. This observation suggests that the N-end rule pathway plays an important role in the progress of leaf senescence (Yoshidaet al., 2002).
Our ®nding that the senescence-related cDNA clone At3g15580 is identical to the APG8 gene suggests its asso-ciation to a novel autophagic pathway in plants (Doelling et al., 2002). During autophagy, bulk cytosolic constituents and organelles are sequestered in specialized autophagic vesicles and are delivered into the vacuoles for their degra-dation. This pathway could provide an additional route to protein degradation during senescence. The APG8 gene encodes a component of this autophagic pathway in yeast. An Arabidopsis mutant altered in another component (APG7) of the autophagic pathway displayed premature senescence under nutrient-limiting conditions (Doelling et al., 2002). Recently, a novel autophage gene (AtAPG9)
related to senescence has been identi®ed (Hanaokaet al., 2002) but has not yet been found in our subtraction library. Taken together, the results suggest active autophagic recycling during leaf senescence.
Senescence promotes movement of nutrients from the vegetative parts to the fruits or to the seeds. Vegetative storage proteins (VSPs) were suggested to serve as a storage buffer between N losses from senescing leaves and grain ®lling later in the growth cycle (Rossato et al., 2002). We have observed an increase in the steady-state levels of the VSP1 and VSP2 transcripts during senescence, supporting their postulated function. Before being loaded into the vascular system, amino acids are modi®ed into organic nitrogen compounds, such as the amides gluta-mine and asparagine. The upregulation of genes encoding glutamine synthase (At1g55090) and aspartate amino transferase (At5g11520) during Arabidopsis leaf senes-cence is consistent with this notion.
Carbohydrate and lipid metabolism
The upregulation in the levels of b-amylase transcripts (At3g23920) in senescing leaves ofArabidopsissupports previous studies describing the intensive polysaccharide breakdown during senescence. It has been hypothesized that sugars, in addition to their important role in energy supply, might act as signal molecules triggering the senes-cence program (Yoshida, 2003). The hexokinase-mediated sugar signaling pathway has been suggested to regulate the initiation of senescence (Daiet al., 1999). Lipid degra-dation also accompanies the other characteristic catabolic processes of leaf senescence (Thompsonet al., 1998), and is re¯ected by an increase in the expression of a lipase (At2g42690) and Phospholipase Da(Fanet al., 1997). It is most likely that lipid degradation is not just a symptom of the senescence process but may also act in regulating the progression of age-dependent senescence (He and Gan, 2002). They identi®ed an SAG encoding an acyl hydrolase that catalyzes the release of oleic acid from triolein. Over-expression of this gene accelerated senescence whereas antisense suppression inhibited the normal progression of senescence. The release ofa-linoleic acid may provide a precursor for the synthesis of jasmonic acid, a senescence-promoting hormone (Heet al., 2002). A gene whose pro-duct is involved in jasmonic acid biosynthesis and was identi®ed in our SSH library is the allen oxide cyclase (At3g25760) that was also found by Heet al. (2002).
Pathogenesis-related proteins and biosynthesis of secondary metabolites
The non-speci®c lipid transfer protein represented by one of the SSH clones might also be involved in the pathogen defense mechanism. Recent studies indicate that lipid transfer proteins are not involved in intracellular lipid traf-®cking ± the role they were initially proposed to have ± but rather in plant resistance to biotic and abiotic stresses (Blein et al., 2002). Their extracellular localization may re¯ect a need for lipid transfer in the formation of hydrophobic protective layers (cutin and suberin) and in the inhibition of fungal growth. One of the well-characterized defense mechanisms in plants is the hypersensitive response (HR) that is commonly triggered by pathogen attack, causing rapid cell death in the region around the infection site. Our ®nding of new senescence-related genes that have been shown to participate in the HR con®rms previous assump-tions (Quirino et al., 1999, 2000) that there is an overlap between pathways leading to cell death in both leaf senes-cence and HR. The extent and signi®cance of this overlap are, however, not yet clear. The temporal expression pat-tern of the newly identi®ed HR-related gene, the lethal leaf spot homolog, clearly indicates its exclusive participation in the late stages of the senescence syndrome (Figure 2). The gene encoding myrosinase-binding protein (At1g52040), whose transcript is upregulated during senescence, has been implicated in defense responses (Eriksson et al., 2002). The enzyme myrosinase degrades the secondary compounds glucosinolates upon wounding and their pro-ducts actively involved in defense against pests. Although, the senescence-associated expression of this gene has not been demonstrated, pathogen-independent induction of several defense genes during senescence has been reported (Quirino et al., 1999) and indicates the involve-ment of defense genes in the senescence program.
Oxidation and detoxification mechanisms
Senescence in general is a regulated oxidative process that involves enhancement in the generation of several reactive oxygen species (ROS; Dangl et al., 2000). Although ROS may have a role in the terminal irreversible phase of senes-cence, an excess of reactive oxygen might be catastrophic and has to be removed to allow essential biochemical processes, nutrient recycling and transport. The increase in the expression of genes encoding antioxidative defense enzymes prevents possible irreversible damage by ROS in the pre-terminal stages of senescence (Danglet al., 2000). Cu/Zn superoxide dismutase (At5g17220) is one of the oxygen radical scavenging enzymes that were differentially expressed in senescing stages. The major antioxidants ascorbate and glutathione (Danglet al., 2000) are produced via the ascorbate±glutathione cycle driven by four enzymes; one of them is the dehydroascorbate reductase whose transcripts are accumulated during senescence (Table 1). Other genes with products involved in
detoxi®ca-tion processes are ferritin and metallothionein. The precise role of these two proteins is not clear, but they may have dual roles: detoxi®cation of metal ions released during protein breakdown and/or to function as metal-binding proteins for storage or transport into developing organs (Buchanan-Wollaston and Ainsworth, 1997). Evidence from mammalian cells suggests that metallothioneins protect DNA from damage (Chubatsu and Meneghini, 1993).
Regulatory genes
It is expected that a set of genes may regulate the initiation and/or the rate of progress of the senescence syndrome, and these are major targets for `gene hunting' studies. The initiation and progression of leaf senescence is likely to be Figure 2.Temporal expression pro®les of various SAGs.
RNA gel blots analyses displaying various kinetic patterns.
(a) SAGs with basal expression in pre-senescence stages: (1) Cationic amino acid transporter; (2) amino acid permease; (3) methalothionein. (b) Early expressed genes: (4) xylose isomerase; (5) RING H2 ®nger protein. (c) SAGs displaying transient expression: (6) lipid transfer protein and; (7) calmodulin-like protein.
regulated primarily at the transcriptional level (Heet al., 2001). The SSH method, which has proved to be a powerful tool for the ampli®cation of non-abundant transcripts in other systems (Diatchenkoet al., 1999), allowed us to iden-tify a number of new SAGs encoding potential regulators. In addition to the two reported senescence-related recep-tors, senescence-associated receptor kinase (SARK; Hajouj et al., 2000) and senescence-induced receptor kinase (SIRK; Robatzek and Somssich, 2002), we revealed in our screen a cDNA clone that represents a receptor-like protein kinase (At5g48380). Other mRNAs for signal transduction compo-nents preferentially accumulated as leaves senesce are represented by the clones encoding a small GTP-binding protein (At5g47201), related to the oncogene RAS and a G protein beta subunit-like protein (At2g43770).
Another SSH-derived clone encodes an AP2 domain transcription factor, which is identical to RAP2.4 and belongs to the EREBP (ethylene responsive element-bind-ing protein) subfamily. These features make it an attractive candidate for a transcription factor functioning in the leaf senescence program. The gene encoding a putative Lim domain protein (At2g3990). This protein carries a zinc motif called LIM domain and belongs to a family known to participate in transcription and cytoskeleton organization. A study carried out in sun¯owers suggested that Lim pro-teins participate in gene transcription, possibly by assem-bling and stabilizing transcription complexes (Mundel et al., 2000). A tobacco Lim protein acts as a potential transcription factor in lignin biosynthesis (Kawaoka, 2001). Furthermore, suppression of this Lim protein caused simultaneous reduction in the transcript levels of some phenylpropanoid pathway genes, resulting in low lignin content in transgenic plants.
Other genes whose transcript levels increased during senescence belong to known transcription factor families including a zinc ®nger-like protein and a RING-H2 ®nger protein. The present research also demonstrates senes-cence-induced expression of the genes encoding a calmo-dulin-regulated ion channel (At5g54250) and calcium-binding protein (At1g18210). Together with our recent observation concerning the involvement of a speci®c Ca2-dependent protein kinase in the regulation of leaf senescence (Gutermanet al., 2003), these results suggest that Ca2may serve as a second messenger in the regula-tion of leaf senescence. Another interesting SAG repre-sented by clone At1g68820 shows sequence homology to the animal apoptosis inhibitor (IAP) that regulates apopto-sis by binding and inhibiting capsases (Lotocki and Keane, 2002). The sequence of the plant gene contains the follow-ing:N-glycosylation site, a protein kinase C phosphoryla-tion site,N-myristolation site, and a RING H2 zinc ®nger. This multidomain sequence and the putative role of the human homolog (IAP) in apoptosis support its possible role as a regulatory gene related to leaf senescence.
An interesting observation is the upregulation of At1g05340, which is also analyzed and annotated by the MIPSA. thalianadatabase and indicates its association to Ton B-dependent receptor protein (IPR000531). In bacteria, the Ton B protein interacts with outer membrane receptor proteins that carry high-af®nity binding and energy-depen-dent uptake of speci®c substrates. An example is the active iron transport system involved in iron uptake by bacteria (Braun and Braun, 2002). Although neither the gene nor the protein has been isolated or characterized in plants, they may be implicated in ion transport processes acting mainly during the late developmental stages of leaf senescence.
Temporal patterns of gene expression during leaf senescence
Temporal expression patterns may indicate the role of each gene during the various senescence steps from the initia-tion signal to the terminal phase of cell death. The kinetics of SAGs may be divided into different general categories (Buchanan-Wollaston, 1997; Danglet al., 2000), and several of them are considered here (Figures 2 and 3). The ®rst category includes genes that have some degree of basal expression in non-senescent leaves, but their mRNA levels increase markedly during the successive stages of senes-cence. Among this group are genes encoding cationic amino acid transporters, amino acid permease and metal-lothionein (Figure 2a). Genes belonging to a second cate-gory are expressed during the emergence of the in¯orescence stem just prior to or at the onset of senes-cence and may suggest their participation in the initiation
Figure 3.Temporal expression of senescence-associated regulatory genes. RNA gel blots analyses displaying various kinetic patterns of the following regulatory genes.
phase of senescence. Among this group are the RING-H2 ®nger protein and xylose isomerase (Figure 2b). Genes whose products are involved in the terminal phase of leaf senescence during which irreversible loss of cell integrity and viability occurs would display a characteristic pro®le of late expression (Figure 2d). An example is the lethal leaf spot 1 (lls1) gene, known to be involved in the HR in corn, which is preferentially expressed at the very late phase of the senescence syndrome (Figure 2d, 9). The product of this gene is probably responsible for the irreversible necrosis typically involved in this stage of senescence (Danglet al., 2000). In contrast to the senescence-enhanced genes, chlorophylla/b-binding (cab) protein expression displayed the typical temporal pattern of a senescence-downregu-lated gene re¯ecting the decline of photosynthesis during senescence (Figure 2).
Regulatory genes whose expression is associated with leaf senescence not only are considered to play a crucial role primarily in triggering the onset of senescence but may also have a role in the regulation of various stages through-out the senescence syndrome. Indeed, expression of selected regulatory genes, isolated in the present study, was analyzed and showed different temporal patterns (Figure 3). There are representatives of early genes whose expression is induced at the very early stages of senes-cence, indicating a regulatory role during the initiation phase. There are genes whose expression is induced at early stages, but thereafter their transcript levels drop. The gene encoding Ras-related small GTP-binding protein dis-played such temporal pro®le (Figure 3). Others (putative apoptosis inhibitor and putative receptor-like protein kinase) are also induced at early stages, but their transcript levels stay high throughout senescence (Figure 3). Genes whose expression is induced very late such as the zinc ®nger protein may participate in the late senescence stages known to be associated with irreversible necrosis. A bipha-sic time course was found for the expression of the putative Lim domain protein, suggesting a function both during maturation and later only at the terminal stages of the senescence program (Figure 3).
The expression patterns of senescence-enhanced genes may provide valuable information concerning the sequence of events of the senescence program.
Leaf detachment and hormonal regulation of gene expression during senescence
The fact that leaf senescence is induced and in¯uenced by many different exogenous and endogenous factors implies that there are multiple pathways in the regulation of this process (He et al., 2001). Leaf detachment, for example, causes considerable stress and consequently induces pre-mature senescence. Similar prepre-mature senescence is re¯ected also in the short shelf life of harvested vegetables.
Many of the physiological changes occurring during post-harvest senescence, such as chlorophyll loss, deterioration of cellular structures and ®nally cell necrosis, show simi-larity to developmental leaf senescence (Figure 1c). To determine the relevance of SAGs identi®ed in attached leaves (Table 1) to post-harvest senescence, the expression pattern of some of the genes was determined in detached leaves and compared with that of attached ones (Figure 4). Three of the tested genes behaved similarly in detached and attached leaves, while NADPH ferrihemoprotein reduc-tase had a different expression pro®le in the two senescing systems (Figure 4). As there is also a clear distinction in the initiation and progression rate of leaf yellowing under light or in darkness, comparison of gene expression was made with detached leaves incubated either in light or in dark-ness. Dark treatment that hastened leaf yellowing was as effective in the induction of three of the selected genes NADPH ferrihemoprotein reductase, Gluthatione S-trans-ferase, and a putative protein (clone #636, At4g13250), whereas expression of the lls1 gene was not in¯uenced.
not surprising to ®nd that the expression of a common subset of senescence-associated genes is dependent on interaction of two or more different hormones (Heet al., 2001). Hormones represent only part of a complex of multi-ple factors involved in the network of pathways of the senescence program (Heet al., 2001). Better understanding of individual pathways and the identi®cation of subgroups of genes that require operation of each speci®c pathway for expression will allow us to identify common senescence promoter sequences.
Senescence-enhanced gene expression in monocarpic plants as compared to autumn leaves
A recent genomic study aimed at understanding the mole-cular program of leaf senescence in autumn leaves of the aspen tree (Populus) has yielded large-scale sequencing and analysis of expressed sequence tags (ESTs) obtained by comparison of two cDNA libraries prepared from autumn and young leaves (Bhaleraoet al., 2003). The infor-mation obtained from this study allows us to compare and estimate the degree of similarity between gene expressions
in the two extreme variations of leaf senescence systems; monocarpic senescence as represented byArabidopsisand autumn leaves of perennial trees. Although no subtractive steps were carried out during the construction of the libraries of aspen, as performed in our present study, the relative EST abundance in the senescing library may pro-vide an approximate indication of transcript levels of indi-vidual genes.
The four most abundant ESTs found in aspen autumn leaves are also predominant among the cDNA clones of the Arabidopsissenescing subtraction library. Similar to our ®nding inArabidopsis, the most redundant expressed gene is the metallothionein whose transcript was 13 times more abundant in the autumn leaves as compared to young leaves (Bhaleraoet al., 2003). In addition to the metallothio-nein, genes encoding early-light-inducible proteins (ELIP), proteases and components of the ubiquitin degradation pathway are found at the top of the list as the most abun-dant expressed genes in the aspen autumn leaf. In general, stress-related proteins are preferentially expressed and abundant inArabidopsisand other monocarpic senescing leaf (Quirinoet al., 1999) and are similarly highly expressed Figure 4.Effects of age, leaf detachment, darkness, and exogenous application of ethylene and cytokinins on the expression of various senescence-associated genes.
in the autumn leaf of aspen (Bhaleraoet al., 2003). One example is the ELIP, which is known to accumulate in thylakoids during not only early stages of light-induced greening but also stress (Binyaminet al., 2001). Although ELIP has not as yet been found in ourArabidopsis senes-cing subtraction library, this gene was shown to be pre-ferentially expressed in senescing leaves of tobacco, a monocarpic plant (Binyaminet al., 2001). Thus, ELIP may prove to play an additional and important role in the senes-cence program of both perennial trees and monocarpic plants.
Intensive proteolytic degradation is a universal process characteristic of all senescing plant organs and occurs in wide spectrum of species including monocots, dicots, and autumn leaves of perennials (Danglet al., 2000). The cel-lular proteolysis in all studied senescing systems is carried out by similar mechanisms including aspartic, Cys pro-teases and the ubiquitin degradation pathway. The degree of similarity between the senescing systems of monocarpic and autumn leaves is especially high in the family of Cys proteases. Out of the 10 reported genes encoding Cys proteases in aspen (Table 4 in Bhaleraoet al., 2003), eight Arabidopsisorthologs have been identi®ed in our study as senescence-regulated genes. Among the common genes are those encoding Cys protease-like protein (At4g16190), Cys protease SAG12 (At5g45890), Protease RD19A (At4g39090), Oryzain (At3g45310), Cathepsin B-like Cys proteinase (At4g01610, At1g02300), Cys Protease RD21A (At1g47128), and AALP (At5g60360). Senescence-enhanced expression of the vacuolar processing enzyme (At4g32940) was also detected in ourArabidopsisstudy and is not listed as senescence-regulated gene in autumn leaves of aspen (Bhaleraoet al., 2003). In general, vacuolar cys proteases are the most predominant proteases in senescing leaves. As the vacuole plays a signi®cant role in defense against pathogens and pests, a defense rather than re-mobilization role has been suggested for several of the senescence-related proteases (Thomaset al., 2003). Vacuolar proteases may not have even a role in the process of chloroplast (and/or other organelle) protein degradation until the very late lytic stages of the vacuole following tonoplast disin-tegration.
The ubiquitin/proteosome pathway for targeted protein degradation is essential for the control of protein turnover throughout leaf development. The increase in the expres-sion of the ubiquitin-related genes in both monocarpic senescing leaves (Table 1) and autumn leaves of aspen may indicate involvement of ubiquitin in the process of massive protein degradation during senescence. However, in contrast toArabidopsis, transcript levels of components of the proteosome were not upregulated in autumn aspen leaves (Bhaleraoet al., 2003). This result re¯ects differences between the two senescing systems and may indicate an increase in the proteosome activity exclusively in senescing
Arabidopsisleaf and probably in other monocarpic plants, but not in autumn leaves. However, it may also be possible that the accumulated levels of the proteosome components in autumn leaves of aspen are suf®cient to breakdown the presumably high levels of ubiquitinated proteins during senescence.
The overall gene expression patterns and the extensive similarity and overlapping in the listed genes of autumn leaves (Bhaleraoet al., 2003), ofArabidopsis(Table 1) and other annual senescing leaves (Buchanan-Wollastonet al., 2003) strongly support the notion that the general pattern of leaf senescence metabolism is, to a large extent, similar in a wide range of senescing systems. However, as the senes-cence syndrome is triggered in perennial autumn leaves by day length shortening, rather than by the development of reproductive organs, it is not surprising thatArabidopsis orthologs of some of the identi®ed aspen senescence-regulated genes are either unknown or not related to senescence (Bhaleraoet al., 2003). Similar differences in expression of some of the senescence-enhanced genes are expected even between related monocarpic plants whose senescence is triggered by different signals. For example, the Columbia ecotype (Col-0) ofArabidopsis, which was also studied in the present research, displays a phenom-enon characteristic of many monocarpic plants; removal of ¯ower or bolt increases the longevity of the whole plant (Nooden and Penney, 2001). This correlation suggests a link between reproductive organ development and induction of senescence. However, unlike soybean plants whose leaf senescence is controlled by reproductive structures, male and female sterile mutants and surgical removal of in¯or-escence bolts inArabidopsisdid not increase the longevity of the individual leaves (Nooden and Penney, 2001). Some differences in gene activation related to the induction phase of senescence in various monocarpic species are expected, and comparative genomic studies are required to identify genes related to the induction phase.
or even higher order mutants of different SAGs could be generated, and their phenotypes, related to senescence, may reveal redundant regulatory pathways. The availability of this collection of SAGs would also allow computer analysis of theArabidopsisgenomic sequences in search of common regulatory motifs in the SAG's promoters.
Experimental procedures
Plant material
Following 2±3 days of cold strati®cation, seeds ofA. thalianaCol-0 were germinated and grown on peat pellets (Jiffy 7, Kappa Fore-nade Well) in a temperature-regulated growth room at 2318C with 14-h day/10-h night cycles. For the experiment of the attached leaf senescence, rosette leaves, in position ®fth and sixth of each plant, were harvested just before the emergence of the in¯ores-cence stem and were designated as fully expanded mature leaves (FX). Leaves representing various progressive senescence stages were harvested after the in¯orescence transition and are presented in Figure 1. For the experiment of detached leaf senescence and hormonal regulation, mature leaves were excised before the emer-gence of the in¯orescence stem and incubated in Petri dishes in darkness or under continuous light at 2318C for various periods as indicated.
Leaves were ¯oated either on distilled water or aqueous solu-tions of 1 mMACC or benzyl adenine (0.001 mM).
Isolation of RNA and RNA gel blot analysis
Total RNA was prepared according to Hajoujet al. (2000) from rosette leaves. RNA was separated on 1.0% (w/v) agarose formal-dehyde gels and blotted to nylon ®lters (NytranN, Schleicher and Schuell, Dassel, Germany). For RNA blots, universal primers (M13 forward and reverse) were used for PCR ampli®cation and synth-esis of the probes. However, if redundancy of sequences has been found in theArabidopsisdatabases, speci®c probes were pro-duced by PCR ampli®cation using speci®cally designed primers for each of the genes presented in experiments described in Figures 3 and 4. Labeling was performed using the kit rediprime DNA labeling system (RPN1633/1634, Amersham, Uppsala, Swe-den). After an overnight hybridization, the membrane was washed, visualized after 2 h with phosphor imager, and exposed to X-ray ®lm.
Suppression subtractive hybridization
Suppression subtractive hybridization was performed with the PCR-Select cDNA Subtraction kit (Clontech Laboratories Inc., Palo Alto, CA, USA) as described by the manufacturer. Two micrograms of senescent leaf mRNA (tester) and 2mg of fully expanded mature green leaf mRNA (driver) were used. The PCR products generated by the SSH were cloned into the PUC57vector using the T-cloning kit (MBI-Fermentas, St Leon-Rot, Germany).
Sequencing and homology analysis
Nucleotide sequence of each insert was determined at the Sequen-cing Services, Technion, Haifa using Dye-deoxy terminators. Sequence homology was analyzed using theBLASTprogram.
Acknowledgement
We thank Dr B. Horwitz for helpful discussions and for critically reading the manuscript.
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