Even though cells may be able to compensate for p16INK4a loss and still undergo a growth arrest characterised by most if not all senescent features, the importance of the tumor sup‐ pressor p16INK4a in senescence is clear as p16-null tumor cells can be driven into senescence by the sole re-expression of p16INK4a: Induced p16INK4a expression in glioma cells caused a typical senescent phenotype [41], reversing promoter hypermethylation allowed for the reexpression of endogenous p16INK4a in oral squamous cell carcinoma cells leading to senes‐ cence [42], inducible p16INK4a expression in osteosarcoma cells induced senescence after 3-6 days, potentially irreversible after 6 days [43] and inducible p16INK4a in human melanoma cells caused a senescent phenotype after 3-5 days in the absence of p53 [44, 45]. Moreover, even in normal early passage human fibroblasts the ectopic introduction of p16INK4a or func‐ tional peptides thereof initiated cell cycle arrest and senescent features [46, 47]. In line with this, melanoma associated germline mutations of p16INK4a are impaired in inducing a cellular senescence program in melanoma cells and this disability to promote senescence may con‐ tribute to the melanoma-risk of p16INK4a linked melanoma-prone families [45].

#### **5. Timing of Senescence by repression and activation of p16INK4a**

#### **5.1. p16INK4a repression**

**Figure 2.** The Cell Cycle

28 Senescence and Senescence-Related Disorders

This simplified model, focusing on early cell cycle entry, illustrates that hypophosphorylat‐ ed, active pRb represses E2F-mediated transcription. The action of CDKs, exemplified by the cyclin D dependent CDK4 and 6, phosphorylate pRb and thus release E2F to activate tran‐ scription of early DNA replication genes. p16INK4a and p21Waf1, the latter usually activated by

With regard to senescence, it is long known that p16INK4a levels accumulate and cause growth arrest and senescence when cells approach their replicative life span [16-23]. More‐ over, in long term tissue culture studies cells that were able to overcome senescence com‐ monly had lost p16INK4a and p53 expression [24]. Increased p16INK4a expression is also linked

Interestingly, despite this clear correlation of p16INK4a up-regulation with senescence there is some evidence from p16INK4a is not strictly required for senescence to occur. Evidence form mouse models show that mouse embryonic fibroblasts (MEFs) of p16-null mice undergo a comparable number of cell divisions as wild type MEFs before entering senescence [34] [35]*,* while in primary melanocytes, which were lentivirally transduced to express oncogenic

p53, inhibit CDKs and retain pRb in its active cell cycle inhibitory state.

to oncogene induced and other forms of premature senescence [25-33].

**4. The role of the tumor suppressor p16INK4a in senescence**

In fact, the ability to induce senescence in response to accumulated or sudden genomic stress is probably the most important tumor suppressive function of p16INK4a. In line with this consideration it is not surprising that p16INK4a expression is tightly repressed at the chro‐ matin and transcriptional level in "young" proliferating cells and in cells with extensive re‐ newal capacities, such as stem cells. The polycomb protein Bmi1, which is also known as "stem cell factor" is facilitating repression of the INK4a locus at the chromatin level [48, 49]. Intriguingly, in a functional feedback loop, in human fibroblasts the Bmi1-mediated repres‐ sion of p16INK4a requires active pRb and also H3K27 (histone 3/lysine 27] trimethylation fa‐ cilitated by the histone methyltransferase EZH2 in concert with a second polycomb protein, SUZ12 [50]. Crucially, Bmi1 chromatin binding can be inhibited by its phosphorylation through the MAPK and p38 signalling pathways [51]. Hence these pathways are able to di‐ rectly oppose p16INK4a repression and lead to its transcriptional activation via Ets and Sp-1 during oncogene-induced senescence.

ent on the SWI/SNF subunits BRG1 and hSnf5, and the relaxed chromatin structure allows tran‐ scription factor access [71] (Figure 3a). Alterations of hSnf5 are associated with early childhood rhabdoid cancer and re-expression of hSnf5 in rhabdoid cancer cells leads to p16INK4a accumula‐

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The best understood transcription factors driving p16INK4a expression and thereby growth ar‐ rest and senescence are Ets1 and Ets2. They are effectors of MAPK signalling, often in re‐ sponse to oncogenic stress, such as activating N-RAS or B-RAF mutations, which induce an increase in p16INK4a levels [25, 29, 37, 52]. In line with this, human fibroblasts with biallelic mutations in p16INK4a did increase mutant p16INK4a expression in response to RAS signaling or expression of ectopic Ets, but failed to arrest or undergo senescence [74]. Interestingly in‐ creased p16INK4a expression has also been linked to loss of p53 and this appears to be corre‐ lated with increased Ets protein half-life [75]. Another member of the Ets transcription factor family, ESE-3, was independently identified as a down stream target of p38 signalling and caused senescence via p16INK4a up-regulation [76]. p38 signalling has been linked to en‐ hanced p16INK4a expression before and this involved the downstream transcription factor Sp-1, which was proposed to be required for p16INK4a up-regulation during senescence in hu‐ man fibroblasts [77]. Sp-1 was reported to engage the p300 enhancer leading to further

(A) During the proliferative cellular life-time, EZH2 in cooperation with SUZ12 trimethy‐ lates H3K27 at the genomic INK4a locus and these histone modifications attract the poly‐ comb repressor BMI1, which maintains the p16INK4a promoter region inaccessible for

tion, growth arrest and senescence [71, 72] and this requires functional p16INK4a [73].

p16INK4a upregulation [78] (Figure 3b).

**Figure 3.** Schematic presentation of p16INK4a regulation

In concert with Bmi-linked chromatin-remodelling events a number of transcription factors fa‐ cilitate p16INK4a repression during the proliferative life-time of cells. The perhaps most impor‐ tant transcriptional repressors of p16INK4a are Id proteins, with the main representative Id1. Id proteins function by binding to E-box DNA sequences to repress the INK4a promoter and im‐ portantly by interfering with Ets transcriptional complex formation and thereby inhibiting the main INK4a transcriptional activator, Ets, in two ways [52, 53]. In line with this, high Id1 levels were associated with early stage melanoma whereas premalignant and interestingly also more advanced melanoma showed limited Id1 expression [54]. This suggests a role of the Id1/ p16INK4a regulative connection during melanomagenesis and Id1 may be dispensable in later melanoma stages once p16INK4a is either more tightly repressed by engaging repressive histone modifications or inactivated by other mechanisms. Id1 down-regulation on the other hand is usually associated with cell differentiation and senescence [55]. Consequently, ectopic expres‐ sion of Id1 delayed senescence in melanocytes [56] and keratinocytes [57], while MEFs lacking Id1 prematurely senesced due to increased p16INK4a levels [58, 59]. Another way to oppose Ets driven p16INK4a transcription was identified, when Cdh1, an adaptor protein of the anaphase promoting complex, was shown to bind to and promote degradation of Ets2 and thereby in‐ creased the replicative life span of MEFs [60], while the Epstein-Barr virus protein LMP1 re‐ presses p16INK4a by promoting the nuclear export of Ets2 [61, 62]

Interestingly, p16INK4a may also be repressed by the oncogene β-catenin, which has been linked to melanoma. β-catenin binds the INK4a promoter at a conservative β-catenin/Lef/Tcf binding site and thereby directly represses its transcription. Consequently β-catenin silenc‐ ing increased p16INK4a levels in A375P human melanoma cells, while stabilization of β-cate‐ nin together with oncogenic N-RAS led to prevention of senescence and thus, immortalization [63]. Importantly, a role of β-catenin in melanocyte senescence is controver‐ sial as nuclear β-catenin was commonly found in benign melanocytic nevi [64-66] and these lesions were proposed to be senescent by some investigators [29, 67], this again is controver‐ sial, as benign nevi are not be distinguished from normal melanocytes or primary melano‐ mas using a range of common senescence markers [68]. It would clearly be interesting to test whether nuclear β-catenin does overlap with the expression of p16INK4a in benign nevi as the latter is mosaic and not found in all cells [29] and co-localisation or lack of it could help clar‐ ify this debate. Another repressor of p16INK4a is the "T-box transcription factor" Tbx2, this transcription factor binds to corresponding T-box DNA sequence elements [69]. Tbx2 overexpression was identified in melanomas and associated with melanoma progression [70].

#### **5.2. p16INK4a expression**

When cells reach their finite life span the pendulum at the INK4a promoter swings from repres‐ sion to activation and the SWI/SNF chromatin remodeling complex, replaces Bmi1 repressors and relaxes chromatin structures around the INK4 promoter region, which is strictly depend‐ ent on the SWI/SNF subunits BRG1 and hSnf5, and the relaxed chromatin structure allows tran‐ scription factor access [71] (Figure 3a). Alterations of hSnf5 are associated with early childhood rhabdoid cancer and re-expression of hSnf5 in rhabdoid cancer cells leads to p16INK4a accumula‐ tion, growth arrest and senescence [71, 72] and this requires functional p16INK4a [73].

The best understood transcription factors driving p16INK4a expression and thereby growth ar‐ rest and senescence are Ets1 and Ets2. They are effectors of MAPK signalling, often in re‐ sponse to oncogenic stress, such as activating N-RAS or B-RAF mutations, which induce an increase in p16INK4a levels [25, 29, 37, 52]. In line with this, human fibroblasts with biallelic mutations in p16INK4a did increase mutant p16INK4a expression in response to RAS signaling or expression of ectopic Ets, but failed to arrest or undergo senescence [74]. Interestingly in‐ creased p16INK4a expression has also been linked to loss of p53 and this appears to be corre‐ lated with increased Ets protein half-life [75]. Another member of the Ets transcription factor family, ESE-3, was independently identified as a down stream target of p38 signalling and caused senescence via p16INK4a up-regulation [76]. p38 signalling has been linked to en‐ hanced p16INK4a expression before and this involved the downstream transcription factor Sp-1, which was proposed to be required for p16INK4a up-regulation during senescence in hu‐ man fibroblasts [77]. Sp-1 was reported to engage the p300 enhancer leading to further p16INK4a upregulation [78] (Figure 3b).

**Figure 3.** Schematic presentation of p16INK4a regulation

SUZ12 [50]. Crucially, Bmi1 chromatin binding can be inhibited by its phosphorylation through the MAPK and p38 signalling pathways [51]. Hence these pathways are able to di‐ rectly oppose p16INK4a repression and lead to its transcriptional activation via Ets and Sp-1

In concert with Bmi-linked chromatin-remodelling events a number of transcription factors fa‐ cilitate p16INK4a repression during the proliferative life-time of cells. The perhaps most impor‐ tant transcriptional repressors of p16INK4a are Id proteins, with the main representative Id1. Id proteins function by binding to E-box DNA sequences to repress the INK4a promoter and im‐ portantly by interfering with Ets transcriptional complex formation and thereby inhibiting the main INK4a transcriptional activator, Ets, in two ways [52, 53]. In line with this, high Id1 levels were associated with early stage melanoma whereas premalignant and interestingly also more advanced melanoma showed limited Id1 expression [54]. This suggests a role of the Id1/ p16INK4a regulative connection during melanomagenesis and Id1 may be dispensable in later melanoma stages once p16INK4a is either more tightly repressed by engaging repressive histone modifications or inactivated by other mechanisms. Id1 down-regulation on the other hand is usually associated with cell differentiation and senescence [55]. Consequently, ectopic expres‐ sion of Id1 delayed senescence in melanocytes [56] and keratinocytes [57], while MEFs lacking Id1 prematurely senesced due to increased p16INK4a levels [58, 59]. Another way to oppose Ets driven p16INK4a transcription was identified, when Cdh1, an adaptor protein of the anaphase promoting complex, was shown to bind to and promote degradation of Ets2 and thereby in‐ creased the replicative life span of MEFs [60], while the Epstein-Barr virus protein LMP1 re‐

Interestingly, p16INK4a may also be repressed by the oncogene β-catenin, which has been linked to melanoma. β-catenin binds the INK4a promoter at a conservative β-catenin/Lef/Tcf binding site and thereby directly represses its transcription. Consequently β-catenin silenc‐ ing increased p16INK4a levels in A375P human melanoma cells, while stabilization of β-cate‐ nin together with oncogenic N-RAS led to prevention of senescence and thus, immortalization [63]. Importantly, a role of β-catenin in melanocyte senescence is controver‐ sial as nuclear β-catenin was commonly found in benign melanocytic nevi [64-66] and these lesions were proposed to be senescent by some investigators [29, 67], this again is controver‐ sial, as benign nevi are not be distinguished from normal melanocytes or primary melano‐ mas using a range of common senescence markers [68]. It would clearly be interesting to test whether nuclear β-catenin does overlap with the expression of p16INK4a in benign nevi as the latter is mosaic and not found in all cells [29] and co-localisation or lack of it could help clar‐ ify this debate. Another repressor of p16INK4a is the "T-box transcription factor" Tbx2, this transcription factor binds to corresponding T-box DNA sequence elements [69]. Tbx2 overexpression was identified in melanomas and associated with melanoma progression [70].

When cells reach their finite life span the pendulum at the INK4a promoter swings from repres‐ sion to activation and the SWI/SNF chromatin remodeling complex, replaces Bmi1 repressors and relaxes chromatin structures around the INK4 promoter region, which is strictly depend‐

during oncogene-induced senescence.

30 Senescence and Senescence-Related Disorders

**5.2. p16INK4a expression**

presses p16INK4a by promoting the nuclear export of Ets2 [61, 62]

(A) During the proliferative cellular life-time, EZH2 in cooperation with SUZ12 trimethy‐ lates H3K27 at the genomic INK4a locus and these histone modifications attract the poly‐ comb repressor BMI1, which maintains the p16INK4a promoter region inaccessible for transcriptional activation. Once cells reach their finite life span or during premature senes‐ cence the histone modifications at the INK4a locus change from repressive methylations to activating acetylations, which attract the SWI/SNF complex. The SWI/SNF subunits hSnf5 and BRG1 are instrumental in opening the chromatin structure and allowing access of the transcriptional machinery to the p16INK4a promoter. (B) Ets transcription factors are down stream targets of MAPK signalling, exemplified here by RAS, and bind to E-box p16INK4 pro‐ moter motifs to activate gene expression. Id transcription factors can compete with Ets for DNA binding and oppose transcriptional expression. The levels of Id proteins decline with onset of senescence and Ets are able to promote p16INK4a expression.

**6.1. DNA damage and senescence**

**6.2. Drug induced senescence**

level of the stress signal.

**6.3. Oncogene-induced senescence and DNA damage**

The human genome is constantly exposed to genotoxic stress such as ultraviolet light, reactive oxygen species (ROS) as well as chemical and biological mutagens. To ensure the integrity of the genome cells have evolved a sophisticated safety system that can implement a cell cycle ar‐ rest to allow the repair of the incurred damage. The initial step in the complicated DNA dam‐ age repair process is the detection of DNA damage. The Mre11/Rad50/NBS1 (*MRN*) complex function as a DNA damage sensor is localized in nuclear foci at the sites of double strand breaks (DSBs) [102-104]. Here, the complex tethers DNA ends, processes and repairs free strands via endonuclease and exonuclease activities [105-107]. The MRN complex is also involved in the recruitment and activation of ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3 related) protein kinases, which in turn activate checkpoint-1/2 (CHK1/2) kinases leading to phosphorylation and thereby stabilization of a variety of target genes including p53 [108]. In the case of irreparable damage, the cell will be permanently retracted from the pool of dividing

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Several sources of genotoxic stress including cisplatin, cyclophosphamide, doxorubicine, taxol, vincristine, cytarabine, etoposide, hydroxyurea, bromodeoxyuridine, adriamycin, bleomycin mitomycin D, interferon beta, radiation, ROS, H2O2 have been used to trigger senescence-like growth arrest in vitro and in vivo allowing a detailed analysis of the under‐ lying signaling events [28, 111-122]. The crosslining agent cisplatin, for example, induced senescence in primary human fibroblasts and HCT116 colon carcinoma cells in a p53 and dose dependent manner [123, 124]). Interestingly, treatment of HCT116 cells with low cispla‐ tin concentrations lead to DNA damage and senescence whereas high drug concentrations induced apoptosis via superoxide production. Furthermore, in human lung cancer cells in‐ duced p53 expression enhanced the cytotoxic effect of cisplatin whereas p21waf1 overexpres‐ sion surprisingly lead to increased drug resistance [82]. Similarly, cisplatin-resistant human non-small cell lung cancer (NSCLC) cells could be sensitized to drug-induced senescence by re-expressing p16Ink4a [118]. Duale and co-workers compared changes in the gene expression profiles of a cisplatin treated human colon carcinoma cells (HCT116) and cell lines derived from testicular germ cell tumors (TGCTs). The systematic approach combining the acquired gene expression data and other publicly available microarray data identified 1794 genes that were differentially expressed including 29 senescence-related genes such as IGFBP 7, inter‐ leukin 1 and MAPK8 [125]. These findings highlight the notion that cellular response to che‐ motherapeutic agents is generally depending on the cellular context as well as the type and

In contrast to chemotherapeutic agents that directly damage the DNA, activated oncogenes do the harm by forcing the cell in uncontrolled, constitute replication cycles which leads to DNA replication stress and subsequent DNA damage [126, 127]. During this process DNA replication forks stall making the DNA more susceptible to single or double strand breaks.

cells by the induction of apoptosis or senescence (Figure 4] [109, 110].

#### **6. The role of the p53/p21 pathway in senescence**

The transcription factor and tumor suppressor p53 is often referred to as "guardian of the genome" and inactivating mutations in p53 are observed in about half of all human cancer cases. The p53 protein is a critical regulator of cell survival in response to cellular stress sig‐ nals including DNA damage, oncogene activation, hypoxia and viral infection (reviewed in [79]. In the absence of stress stimuli p53 gets rapidly ubiquitinated by one of several E3 li‐ gases including MDM2, MDM4, TOPORS, COP1, and ARF-BP1 and subsequently degraded in the proteasome [80, 81]. Stress signals, on the other hand, induce covalent modification usually by disrupting the interaction between p53 and the E3 ubiquitin ligases which pre‐ vents its degradation. Oncogenic stress, for example, activates the alternative reading frame product of the INK4a locus (p14ARF) that stabilizes p53 by binding and thereby inhibiting its negative regulator MDM2.

Several lines of evidence show convincingly that the p53 and its downstream effector p21Waf1 play a crucial role in the regulation of cell cycle arrest and senescence*.* Overexpression of p53 [82] and p21Waf1 [83-86] autonomously induced senescence in human cells and activation of p53 by either overexpression of p14ARF [87] or nutlin-3 treatment [88] induced senes‐ cence in a p21Waf1 dependent manner in human diploid fibroblasts (HDF) and human glio‐ blastoma cells respectively. Furthermore, inactivation of p53 or p19ARF (mouse homologue of human p14ARF) prevents senescence in mouse embryonic fibroblasts (MEF) [89-91] and human fibroblast lacking p21Waf1 can bypass the senescence growth arrest [83]. Further supporting evidence comes from studies that show that, inactivation of p53 using viral on‐ coproteins, anti-p53 antibodies or anti-sense oligonucleotides can extend the replicative life‐ span or even reverse the senescence growth arrest in human cells [38, 92-94]. However, it should be noted that although inactivation of the p53 pathway can weaken or even reverse the senescence arrest in some cells, there is emerging evidence that it fails to do so in cells with an activated p16INK4a/pRb pathway [38, 95-97]. Despite the clear evidence of p53's role in promoting senescence a study conducted by Demidenko and co-workers suggested that p53 can also act as an inhibitor of senescence [98]. Surprisingly, p53 was able to reverse a p16INK4a and p21Waf1 driven senescence response in human fibroblasts. Although the underly‐ ing mechanisms are not fully understood, inhibition of the mTor (mammalian target of rapa‐ mycin) pathway seems to be involved in the repression of cellular senescence [98-101].

#### **6.1. DNA damage and senescence**

transcriptional activation. Once cells reach their finite life span or during premature senes‐ cence the histone modifications at the INK4a locus change from repressive methylations to activating acetylations, which attract the SWI/SNF complex. The SWI/SNF subunits hSnf5 and BRG1 are instrumental in opening the chromatin structure and allowing access of the transcriptional machinery to the p16INK4a promoter. (B) Ets transcription factors are down stream targets of MAPK signalling, exemplified here by RAS, and bind to E-box p16INK4 pro‐ moter motifs to activate gene expression. Id transcription factors can compete with Ets for DNA binding and oppose transcriptional expression. The levels of Id proteins decline with

The transcription factor and tumor suppressor p53 is often referred to as "guardian of the genome" and inactivating mutations in p53 are observed in about half of all human cancer cases. The p53 protein is a critical regulator of cell survival in response to cellular stress sig‐ nals including DNA damage, oncogene activation, hypoxia and viral infection (reviewed in [79]. In the absence of stress stimuli p53 gets rapidly ubiquitinated by one of several E3 li‐ gases including MDM2, MDM4, TOPORS, COP1, and ARF-BP1 and subsequently degraded in the proteasome [80, 81]. Stress signals, on the other hand, induce covalent modification usually by disrupting the interaction between p53 and the E3 ubiquitin ligases which pre‐ vents its degradation. Oncogenic stress, for example, activates the alternative reading frame product of the INK4a locus (p14ARF) that stabilizes p53 by binding and thereby inhibiting

Several lines of evidence show convincingly that the p53 and its downstream effector p21Waf1 play a crucial role in the regulation of cell cycle arrest and senescence*.* Overexpression of p53 [82] and p21Waf1 [83-86] autonomously induced senescence in human cells and activation of p53 by either overexpression of p14ARF [87] or nutlin-3 treatment [88] induced senes‐ cence in a p21Waf1 dependent manner in human diploid fibroblasts (HDF) and human glio‐ blastoma cells respectively. Furthermore, inactivation of p53 or p19ARF (mouse homologue of human p14ARF) prevents senescence in mouse embryonic fibroblasts (MEF) [89-91] and human fibroblast lacking p21Waf1 can bypass the senescence growth arrest [83]. Further supporting evidence comes from studies that show that, inactivation of p53 using viral on‐ coproteins, anti-p53 antibodies or anti-sense oligonucleotides can extend the replicative life‐ span or even reverse the senescence growth arrest in human cells [38, 92-94]. However, it should be noted that although inactivation of the p53 pathway can weaken or even reverse the senescence arrest in some cells, there is emerging evidence that it fails to do so in cells with an activated p16INK4a/pRb pathway [38, 95-97]. Despite the clear evidence of p53's role in promoting senescence a study conducted by Demidenko and co-workers suggested that p53 can also act as an inhibitor of senescence [98]. Surprisingly, p53 was able to reverse a p16INK4a and p21Waf1 driven senescence response in human fibroblasts. Although the underly‐ ing mechanisms are not fully understood, inhibition of the mTor (mammalian target of rapa‐ mycin) pathway seems to be involved in the repression of cellular senescence [98-101].

onset of senescence and Ets are able to promote p16INK4a expression.

**6. The role of the p53/p21 pathway in senescence**

its negative regulator MDM2.

32 Senescence and Senescence-Related Disorders

The human genome is constantly exposed to genotoxic stress such as ultraviolet light, reactive oxygen species (ROS) as well as chemical and biological mutagens. To ensure the integrity of the genome cells have evolved a sophisticated safety system that can implement a cell cycle ar‐ rest to allow the repair of the incurred damage. The initial step in the complicated DNA dam‐ age repair process is the detection of DNA damage. The Mre11/Rad50/NBS1 (*MRN*) complex function as a DNA damage sensor is localized in nuclear foci at the sites of double strand breaks (DSBs) [102-104]. Here, the complex tethers DNA ends, processes and repairs free strands via endonuclease and exonuclease activities [105-107]. The MRN complex is also involved in the recruitment and activation of ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3 related) protein kinases, which in turn activate checkpoint-1/2 (CHK1/2) kinases leading to phosphorylation and thereby stabilization of a variety of target genes including p53 [108]. In the case of irreparable damage, the cell will be permanently retracted from the pool of dividing cells by the induction of apoptosis or senescence (Figure 4] [109, 110].

#### **6.2. Drug induced senescence**

Several sources of genotoxic stress including cisplatin, cyclophosphamide, doxorubicine, taxol, vincristine, cytarabine, etoposide, hydroxyurea, bromodeoxyuridine, adriamycin, bleomycin mitomycin D, interferon beta, radiation, ROS, H2O2 have been used to trigger senescence-like growth arrest in vitro and in vivo allowing a detailed analysis of the under‐ lying signaling events [28, 111-122]. The crosslining agent cisplatin, for example, induced senescence in primary human fibroblasts and HCT116 colon carcinoma cells in a p53 and dose dependent manner [123, 124]). Interestingly, treatment of HCT116 cells with low cispla‐ tin concentrations lead to DNA damage and senescence whereas high drug concentrations induced apoptosis via superoxide production. Furthermore, in human lung cancer cells in‐ duced p53 expression enhanced the cytotoxic effect of cisplatin whereas p21waf1 overexpres‐ sion surprisingly lead to increased drug resistance [82]. Similarly, cisplatin-resistant human non-small cell lung cancer (NSCLC) cells could be sensitized to drug-induced senescence by re-expressing p16Ink4a [118]. Duale and co-workers compared changes in the gene expression profiles of a cisplatin treated human colon carcinoma cells (HCT116) and cell lines derived from testicular germ cell tumors (TGCTs). The systematic approach combining the acquired gene expression data and other publicly available microarray data identified 1794 genes that were differentially expressed including 29 senescence-related genes such as IGFBP 7, inter‐ leukin 1 and MAPK8 [125]. These findings highlight the notion that cellular response to che‐ motherapeutic agents is generally depending on the cellular context as well as the type and level of the stress signal.

#### **6.3. Oncogene-induced senescence and DNA damage**

In contrast to chemotherapeutic agents that directly damage the DNA, activated oncogenes do the harm by forcing the cell in uncontrolled, constitute replication cycles which leads to DNA replication stress and subsequent DNA damage [126, 127]. During this process DNA replication forks stall making the DNA more susceptible to single or double strand breaks. The subsequent activation of the DNA damage response (DDR) machinery delays cell-cycle progression by initiation of the ATM/ATR-CHK1/2 pathway and stable knockdown of any one of these DDR genes was sufficient to bypass RAS induced senescence in human fibro‐ blasts. Specifically, in the absence of ATM, ATR, Chk1 or Chk2, human fibroblasts continued to proliferate despite Ras expression, and BrdU incorporation increased from approximately 5% in the control sample to 15% in cells deficient for a DNA damage protein. Interestingly, the inhibition of both the p16INK4a and DNA damage pathway enhanced the effect and en‐ abled over 60% of the cells to replicate DNA [126]. In a similar study, Bartkova and co-work‐ ers showed that the suppression of ATM or p53 allowed the MRC5 and BJ human fibroblasts to bypass oncogene-induced senescence using overexpressed Mos (an activator of the MAPK pathway) or Cdc6 (a DNA replication licensing factor). However, in this report p16INK4a depletion alone did not weaken the senescence response [127].

To protect the integrity of their DNA, cells need to be able to sense DNA damage and acti‐ vate response pathways that coordinate cell cycle progression and DNA repair. Ataxia telan‐ giectasia mutated (ATM) and ATM-Rad3-related (ATR) kinases are important DNA damage checkpoints proteins that transduce signals from the DNA damage sensors to the effector proteins that control cell cycle progression, chromatin restructuring, and DNA repair. ATM and ATR activate other kinases such as Chkl (activated by ATR) and Chk2 (activated by ATM) that phosphorylate and activate the tumour suppressor protein p53. ATM and ATR can additionally enhance p53 activity by directing p53 phosphorylation on Ser15. Activated p53 can halt progression of the cell cycle in the G1 phase, allowing DNA repair to occur and

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preventing the transmission of damaged DNA to the daughter cells.

**7. pRb in senescence: maintaining a secure proliferative arrest**

beyond the antiproliferative functions described in the cell cycle section 3.

development of senescence [9].

**7.1. pRb and SAHFs**

In the previous sections we have reviewed the pivotal role of the cell cycle inhibitors p16INK4a and p21Waf1 in keeping pRb in a hypophosphorylated, active state and explored the mecha‐ nisms of timing the expression of these tumor suppressors with the onset and maintenance of senescence. Here we explore the roles of active pRb in the senescence program, which go

There is longstanding evidence that pRb is required for an intact senescence program, for instance re-introduction of pRb into SAOS osteosarcoma cells that have lost its expression can cause senescence [128]. On the other hand inactivation of pRb with the viral oncoprotein E1A prevents senescence, while a mutant form of E1A that is impaired in pRb binding is un‐ able to prevent senescence [9]. Interestingly, another mutant E1A, that is able to bind pRb but is unable to interact with the histone deacetylases p300 and p400 leads to less efficient

These data highlight that chromatin remodeling plays an important role in the cellular sen‐ escence program. Indeed, during senescence E2F responsive genomic promoter regions are stably repressed from transcription by the establishment of heterochromatin regions. As mentioned above (section 3) these regions are visible as microscopical "senescence associat‐ ed heterochromatin foci" (SAHF) when cells are stained with certain DNA intercalating dyes such as DAPI. The term heterochromatin refers to a highly condensed protein-DNA structure, which is facilitated by modifications of the histone core molecules of chromatin and suppresses DNA access by the transcriptional machinery and thus gene expression of these regions is tightly suppressed (reviewed in [129]). The investigation of SAHF remains a progressing research field and is developing forward with improved microscopic imaging technology and the ability to isolate these complex structures and analyse them more pre‐ cisely and we can expect to understand even more about their role and configuration in the future. To date we know that SAHF formation coincides with the recruitment of heterochro‐ matin proteins by pRb when interacting with the histone deacetylase 1 (HDAC1] at E2F-re‐

**Figure 4.** DNA damage pathway in senescence

To protect the integrity of their DNA, cells need to be able to sense DNA damage and acti‐ vate response pathways that coordinate cell cycle progression and DNA repair. Ataxia telan‐ giectasia mutated (ATM) and ATM-Rad3-related (ATR) kinases are important DNA damage checkpoints proteins that transduce signals from the DNA damage sensors to the effector proteins that control cell cycle progression, chromatin restructuring, and DNA repair. ATM and ATR activate other kinases such as Chkl (activated by ATR) and Chk2 (activated by ATM) that phosphorylate and activate the tumour suppressor protein p53. ATM and ATR can additionally enhance p53 activity by directing p53 phosphorylation on Ser15. Activated p53 can halt progression of the cell cycle in the G1 phase, allowing DNA repair to occur and preventing the transmission of damaged DNA to the daughter cells.

#### **7. pRb in senescence: maintaining a secure proliferative arrest**

In the previous sections we have reviewed the pivotal role of the cell cycle inhibitors p16INK4a and p21Waf1 in keeping pRb in a hypophosphorylated, active state and explored the mecha‐ nisms of timing the expression of these tumor suppressors with the onset and maintenance of senescence. Here we explore the roles of active pRb in the senescence program, which go beyond the antiproliferative functions described in the cell cycle section 3.

There is longstanding evidence that pRb is required for an intact senescence program, for instance re-introduction of pRb into SAOS osteosarcoma cells that have lost its expression can cause senescence [128]. On the other hand inactivation of pRb with the viral oncoprotein E1A prevents senescence, while a mutant form of E1A that is impaired in pRb binding is un‐ able to prevent senescence [9]. Interestingly, another mutant E1A, that is able to bind pRb but is unable to interact with the histone deacetylases p300 and p400 leads to less efficient development of senescence [9].

#### **7.1. pRb and SAHFs**

The subsequent activation of the DNA damage response (DDR) machinery delays cell-cycle progression by initiation of the ATM/ATR-CHK1/2 pathway and stable knockdown of any one of these DDR genes was sufficient to bypass RAS induced senescence in human fibro‐ blasts. Specifically, in the absence of ATM, ATR, Chk1 or Chk2, human fibroblasts continued to proliferate despite Ras expression, and BrdU incorporation increased from approximately 5% in the control sample to 15% in cells deficient for a DNA damage protein. Interestingly, the inhibition of both the p16INK4a and DNA damage pathway enhanced the effect and en‐ abled over 60% of the cells to replicate DNA [126]. In a similar study, Bartkova and co-work‐ ers showed that the suppression of ATM or p53 allowed the MRC5 and BJ human fibroblasts to bypass oncogene-induced senescence using overexpressed Mos (an activator of the MAPK pathway) or Cdc6 (a DNA replication licensing factor). However, in this report

p16INK4a depletion alone did not weaken the senescence response [127].

34 Senescence and Senescence-Related Disorders

**Figure 4.** DNA damage pathway in senescence

These data highlight that chromatin remodeling plays an important role in the cellular sen‐ escence program. Indeed, during senescence E2F responsive genomic promoter regions are stably repressed from transcription by the establishment of heterochromatin regions. As mentioned above (section 3) these regions are visible as microscopical "senescence associat‐ ed heterochromatin foci" (SAHF) when cells are stained with certain DNA intercalating dyes such as DAPI. The term heterochromatin refers to a highly condensed protein-DNA structure, which is facilitated by modifications of the histone core molecules of chromatin and suppresses DNA access by the transcriptional machinery and thus gene expression of these regions is tightly suppressed (reviewed in [129]). The investigation of SAHF remains a progressing research field and is developing forward with improved microscopic imaging technology and the ability to isolate these complex structures and analyse them more pre‐ cisely and we can expect to understand even more about their role and configuration in the future. To date we know that SAHF formation coincides with the recruitment of heterochro‐ matin proteins by pRb when interacting with the histone deacetylase 1 (HDAC1] at E2F-re‐ sponsive promoters [130]. A number of heterochromatin-associated histone modifications were characteristically found associated with SAHF. These include the reversion of histone 3/lysine 9 acetylation (H3K9Ac) and histone 3/lysine 4 trimethylation (H3K4me3) and signif‐ icantly, since it is often used as a marker of senescence, the promotion of histone 3/lysine 9 trimethylation (H3K9me3) and H3K27me3. H3K9 trimethylation is a fundamental step dur‐ ing heterochromatin formation as it attracts HP1 proteins, which are pivotal for heterochro‐ matin assembly [9]. It is important to highlight that senescence does not always lead to a net increase in overall H3K9me3, suggesting that SAHF formation is local and indeed targeted via pRb to E2F promoters [131]. H3K9me3 is thought to be very stable and is able to prevent histone acetylases (HATs) to catalyze histone acetylations. Histone acetylations are com‐ monly associated with "euchromatin", which describes actively transcribed chromatin re‐ gions. Accordingly, SAHF formation is thought to "lock" chromatin regions that encode proliferation genes and thus contributes to the very secure silencing of E2F responsive pro‐ liferation genes, which is not reversible by physiological stimuli [9]. H3K9 trimethylation as‐ sociated with SAHF formation is thought to be catalyzed by either the SUV39H1 methyltransferase, which together with HP1 interacts with pRb during E2F promoter silenc‐ ing in senescence [132] or the RIZ1/PRDM2 H3K9 methyltransferase, which was also shown to co-operate in pRb gene repression and is inactivated in colon, breast and gastric carcino‐ ma [133]. Furthermore, a search for H3K9me3 interacting proteins to identify proteins in‐ volved in the senescence program identified JMJD2C, which is a H3K9 specific demethylase. Thereby it is the direct antagonist of the SUV39H1 H3K9 methyltransferase and its over-ex‐ pression is not surprisingly associated with several forms of cancer [134].

overexpressing the H3K9me3 favoring histone demethlyase JMJD2D or by silencing SUZ12, a polycomb protein involved in H3K9 and K27 methylation. Importantly, even without these methylated histone markers RAS induction still led to senescence with the occurrence of DAPI dense SAHF structures. The investigators concluded that SAHF formation is medi‐ ated through spatial rearrangement of pre-existing H3K9me3 and H3K27me3 regions but that these histone marks are not a prerequisite for the SAHF formation process [135]. Clear‐ ly, further studies are needed to fully define the combined roles of histone modifications,

Molecular Mechanisms of Cellular Senescence

http://dx.doi.org/10.5772/54120

37

Consistent with gene silencing and decreased acetylation during senescence is the downregulation of the histone acetylases p300/CBP, which was observed in melanocytes reaching

With regard to the role of chromatin remodelling in senescence, there is also evidence that ATP dependent chromatin remodeling complexes, such as SWI/SNF are required for senes‐ cence onset in roles other than the timely expression of p16INK4a as described in section 5.1.2: The introduction of the ATPase active component of the SWI/SNF complex, BRG1, into various cell lines induced senescent features. This is attributed to the BRG1 ability to interact with pRb and participate in E2F target promopter repression [137]. However development of senescence in response to BRG1 introduction was only convincing in cells that also lack the BRG1 homologue ATPase BRM [138]. Interestingly we found that BRG1 is able to inter‐ act with p16INK4a, and since p16INK4a is required for SAHF formation it might directly be in‐ volved in this process in co-operation with BRG1. However, the absence of BRG1, by specific silencing, does neither prevent p16INK4a induced growth arrest nor senescence associ‐ ated SAHF formation. It should be noted however, that the WMM1175 melanoma cells used in these experiments also express the BRG1 homologue BRM and therefore they still have functional SWI/SNF complexes even in the absence of BRG1. [139]. It still needs to be tested whether BRM also interacts with p16INK4a and whether the BRM-p16INK4a complex is involved in SAHF formation. This idea is in line with a suggested role for BRM in melanocyte senes‐ cence as it was demonstrated that BRM was recruited and required, albeit transiently, by the

Importantly, although the p53-p21 pathway was demonstrated to be capable of inducing a number of senescent features and does so in response to DNA damage, telomere dysfunc‐ tion and oncogene induced senescence [38, 97], E2F promoter silencing via SAHF formation requires an intact p16-pRb pathway [9, 141]. As expected, SAHF formation is prevented by cancer associated p16INK4a mutations [45]. Since p21Waf1 is capable of inhibiting all CDKs it remains unclear why it appears less effective in promoting a secure senescence program in

The current view is that pRb binds HDAC1 and attracts H3K9 and H3K27 methylating com‐ plexes (exemplified by SUV39H1 and EZH2/SUZ12) to promoters of E2F responsive prolif‐ eration genes leading to histone trimethylation and heterochromatin and thereby DAPI dense microscopic structures. Recent work indicates that H3K9 and H3K27, although usual‐ ly associated with SAHF, may not be a strict requirement. Instead the formation of DAPI dense structures is linked to the timing of presenescent replication events [135]. This view

heterochromatin and SAHF in senescence.

pRb-HDAC1 complex during the initiation of SAHF [140].

involving heterochromatin formation (Figure5).

in their finitive lifespan [136].

Lately there is a different view about the importance of SAHF emerging and SAHF forma‐ tion, may according to a recent study only be of major relevance in the oncogene induced senescence process that crucially involves DDR and the corresponding ATR signalling, as knock-down of ATR prevents SAHF formation during RAS induced senescence. The same study also found that SAHF resembling structures containing H3K9me3 do independently occur in proliferating oncogene expressing cells, however the investigators data show that these structures are not associated with E2F target promoters, which remain active. The au‐ thors concluded that SAHF structures might not be an essential feature of senescence [40].

More recently a groundbreaking study by the Narita group has combined microscopic, elec‐ tron microscopic and genomic data analysis to shed more light on SAHF formation and structure. Their significant findings are that H3K9me3 and H3K27me3 may in fact not di‐ rectly be involved in SAHF. Although these markers are usually associated with SAHF, the overall methylation status of histone tail lysines does not change within the genomic DNA during senescence. Moreover, the investigators found that SAHF formation was linked to pre-senescent replication timing and specific histone modifications were characteristic for early-, mid- and late replications genes. Significantly though, when they prevented SAHF formation by silencing of either pRb or the high mobility group AT-hook 1 (HMGA-1), which they and others had previously linked to SAHF formation, there was no change to H3K9me3 in proliferating cells [135] and this may explain the above-mentioned data by Mic‐ co et al [40]. Moreover, Chandra et al. reversed H3K9 and H3K27 trimethylation by either overexpressing the H3K9me3 favoring histone demethlyase JMJD2D or by silencing SUZ12, a polycomb protein involved in H3K9 and K27 methylation. Importantly, even without these methylated histone markers RAS induction still led to senescence with the occurrence of DAPI dense SAHF structures. The investigators concluded that SAHF formation is medi‐ ated through spatial rearrangement of pre-existing H3K9me3 and H3K27me3 regions but that these histone marks are not a prerequisite for the SAHF formation process [135]. Clear‐ ly, further studies are needed to fully define the combined roles of histone modifications, heterochromatin and SAHF in senescence.

sponsive promoters [130]. A number of heterochromatin-associated histone modifications were characteristically found associated with SAHF. These include the reversion of histone 3/lysine 9 acetylation (H3K9Ac) and histone 3/lysine 4 trimethylation (H3K4me3) and signif‐ icantly, since it is often used as a marker of senescence, the promotion of histone 3/lysine 9 trimethylation (H3K9me3) and H3K27me3. H3K9 trimethylation is a fundamental step dur‐ ing heterochromatin formation as it attracts HP1 proteins, which are pivotal for heterochro‐ matin assembly [9]. It is important to highlight that senescence does not always lead to a net increase in overall H3K9me3, suggesting that SAHF formation is local and indeed targeted via pRb to E2F promoters [131]. H3K9me3 is thought to be very stable and is able to prevent histone acetylases (HATs) to catalyze histone acetylations. Histone acetylations are com‐ monly associated with "euchromatin", which describes actively transcribed chromatin re‐ gions. Accordingly, SAHF formation is thought to "lock" chromatin regions that encode proliferation genes and thus contributes to the very secure silencing of E2F responsive pro‐ liferation genes, which is not reversible by physiological stimuli [9]. H3K9 trimethylation as‐ sociated with SAHF formation is thought to be catalyzed by either the SUV39H1 methyltransferase, which together with HP1 interacts with pRb during E2F promoter silenc‐ ing in senescence [132] or the RIZ1/PRDM2 H3K9 methyltransferase, which was also shown to co-operate in pRb gene repression and is inactivated in colon, breast and gastric carcino‐ ma [133]. Furthermore, a search for H3K9me3 interacting proteins to identify proteins in‐ volved in the senescence program identified JMJD2C, which is a H3K9 specific demethylase. Thereby it is the direct antagonist of the SUV39H1 H3K9 methyltransferase and its over-ex‐

36 Senescence and Senescence-Related Disorders

pression is not surprisingly associated with several forms of cancer [134].

Lately there is a different view about the importance of SAHF emerging and SAHF forma‐ tion, may according to a recent study only be of major relevance in the oncogene induced senescence process that crucially involves DDR and the corresponding ATR signalling, as knock-down of ATR prevents SAHF formation during RAS induced senescence. The same study also found that SAHF resembling structures containing H3K9me3 do independently occur in proliferating oncogene expressing cells, however the investigators data show that these structures are not associated with E2F target promoters, which remain active. The au‐ thors concluded that SAHF structures might not be an essential feature of senescence [40].

More recently a groundbreaking study by the Narita group has combined microscopic, elec‐ tron microscopic and genomic data analysis to shed more light on SAHF formation and structure. Their significant findings are that H3K9me3 and H3K27me3 may in fact not di‐ rectly be involved in SAHF. Although these markers are usually associated with SAHF, the overall methylation status of histone tail lysines does not change within the genomic DNA during senescence. Moreover, the investigators found that SAHF formation was linked to pre-senescent replication timing and specific histone modifications were characteristic for early-, mid- and late replications genes. Significantly though, when they prevented SAHF formation by silencing of either pRb or the high mobility group AT-hook 1 (HMGA-1), which they and others had previously linked to SAHF formation, there was no change to H3K9me3 in proliferating cells [135] and this may explain the above-mentioned data by Mic‐ co et al [40]. Moreover, Chandra et al. reversed H3K9 and H3K27 trimethylation by either

Consistent with gene silencing and decreased acetylation during senescence is the downregulation of the histone acetylases p300/CBP, which was observed in melanocytes reaching in their finitive lifespan [136].

With regard to the role of chromatin remodelling in senescence, there is also evidence that ATP dependent chromatin remodeling complexes, such as SWI/SNF are required for senes‐ cence onset in roles other than the timely expression of p16INK4a as described in section 5.1.2: The introduction of the ATPase active component of the SWI/SNF complex, BRG1, into various cell lines induced senescent features. This is attributed to the BRG1 ability to interact with pRb and participate in E2F target promopter repression [137]. However development of senescence in response to BRG1 introduction was only convincing in cells that also lack the BRG1 homologue ATPase BRM [138]. Interestingly we found that BRG1 is able to inter‐ act with p16INK4a, and since p16INK4a is required for SAHF formation it might directly be in‐ volved in this process in co-operation with BRG1. However, the absence of BRG1, by specific silencing, does neither prevent p16INK4a induced growth arrest nor senescence associ‐ ated SAHF formation. It should be noted however, that the WMM1175 melanoma cells used in these experiments also express the BRG1 homologue BRM and therefore they still have functional SWI/SNF complexes even in the absence of BRG1. [139]. It still needs to be tested whether BRM also interacts with p16INK4a and whether the BRM-p16INK4a complex is involved in SAHF formation. This idea is in line with a suggested role for BRM in melanocyte senes‐ cence as it was demonstrated that BRM was recruited and required, albeit transiently, by the pRb-HDAC1 complex during the initiation of SAHF [140].

Importantly, although the p53-p21 pathway was demonstrated to be capable of inducing a number of senescent features and does so in response to DNA damage, telomere dysfunc‐ tion and oncogene induced senescence [38, 97], E2F promoter silencing via SAHF formation requires an intact p16-pRb pathway [9, 141]. As expected, SAHF formation is prevented by cancer associated p16INK4a mutations [45]. Since p21Waf1 is capable of inhibiting all CDKs it remains unclear why it appears less effective in promoting a secure senescence program in involving heterochromatin formation (Figure5).

The current view is that pRb binds HDAC1 and attracts H3K9 and H3K27 methylating com‐ plexes (exemplified by SUV39H1 and EZH2/SUZ12) to promoters of E2F responsive prolif‐ eration genes leading to histone trimethylation and heterochromatin and thereby DAPI dense microscopic structures. Recent work indicates that H3K9 and H3K27, although usual‐ ly associated with SAHF, may not be a strict requirement. Instead the formation of DAPI dense structures is linked to the timing of presenescent replication events [135]. This view would explain the fact that SWI/SNF including BRM has been associated in a transient man‐ ner with the build up of SAHF structures; BRM can also interact with pRb but strictly is as‐ sociated with acetylated histones and replicating chromatin is acetylated [140]. The complete understanding of SAHF formation is still an evolving field. Regardless, it is widely accepted that functional p16INK4a and pRb as well as the HMGA, which accumulate at E2F target pro‐ moters during senescence, are critically required for SAHF arrangement. In regards to p16INK4a it is tempting to speculate that its specific role in SAHF formation goes beyond CDK inhibition and thus maintaining pRb in an active state.

**Author details**

Therese Becker1

burg, Germany

**References**

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and Sebastian Haferkamp2

Institute, Westmead Hospital, Westmead, New South Wales, Australia

1 Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium

Molecular Mechanisms of Cellular Senescence

http://dx.doi.org/10.5772/54120

39

2 Department of Dermatology, Venereology und Allergology, University Hospital Würz‐

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**Figure 5.** Chromatin remodeling and *SAHF formation*

#### **8. Conclusion**

Senescence is a potent means of tumor suppression. The mechanisms of senescence in‐ volve cell cycle regulatory protein functions in concert with the chromatin remodeling machinery to maintain a complex and secure withdrawal from proliferation. The under‐ standing of these mechanisms is still evolving and is predicted to identify novel targets for cancer therapy.

#### **Acknowledgements**

We thank Dr Kavitha Gowrishankar for her assistance with the illustrations and Dr Roland Houben for the critical review of the manuscript.

#### **Author details**

would explain the fact that SWI/SNF including BRM has been associated in a transient man‐ ner with the build up of SAHF structures; BRM can also interact with pRb but strictly is as‐ sociated with acetylated histones and replicating chromatin is acetylated [140]. The complete understanding of SAHF formation is still an evolving field. Regardless, it is widely accepted that functional p16INK4a and pRb as well as the HMGA, which accumulate at E2F target pro‐ moters during senescence, are critically required for SAHF arrangement. In regards to p16INK4a it is tempting to speculate that its specific role in SAHF formation goes beyond CDK

Senescence is a potent means of tumor suppression. The mechanisms of senescence in‐ volve cell cycle regulatory protein functions in concert with the chromatin remodeling machinery to maintain a complex and secure withdrawal from proliferation. The under‐ standing of these mechanisms is still evolving and is predicted to identify novel targets

We thank Dr Kavitha Gowrishankar for her assistance with the illustrations and Dr Roland

inhibition and thus maintaining pRb in an active state.

38 Senescence and Senescence-Related Disorders

**Figure 5.** Chromatin remodeling and *SAHF formation*

**8. Conclusion**

for cancer therapy.

**Acknowledgements**

Houben for the critical review of the manuscript.

Therese Becker1 and Sebastian Haferkamp2

1 Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Westmead Hospital, Westmead, New South Wales, Australia

2 Department of Dermatology, Venereology und Allergology, University Hospital Würz‐ burg, Germany

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**Section 3**

**Plant Senescence**

**Section 3**

**Plant Senescence**

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50 Senescence and Senescence-Related Disorders

duced senescence does not require the p16(INK4a) or p14ARF melanoma tumor sup‐

**Chapter 3**

**Plant Senescence and Nitrogen**

Additional information is available at the end of the chapter

Very early during their reproductive phase, annual plants initiate the process of senescence. Monocarpic senescence describes the last steps in these plants' development; senescence on organ level starts shortly after entering reproductive phase while after anthesis the whole

In the following we will focus on leaf senescence. Two different processes can be distin‐ guished in annual plants relying on different genetic programs. Before anthesis, sequential leaf senescence recycles nutrients from old to developing leaves which is mainly under the control of the growing apex and is arrested when no more new leaves develop and when the plant starts to flower and sets fruit. Monocarpic leaf senescence recovers valuable nutrients from the leaves during flower induction and anthesis to provide these to the developing re‐ productive organs [1, 2]. The latter is crucial for fruit and seed development and has a major impact on yield quantity and quality. In wheat salvaged nitrogen (N) from the leaves ac‐ counts for up to 90% of the total grain N content [3]. A complex regulation of many different metabolic pathways and expression of numerous genes underlies this process. How coordi‐ nation and interplay of many controlling factors, like hormones, genetic reprogramming, bi‐ otic and abiotic stresses are achieved is far from being understood, but it is already clear that

Thousands of genes are differentially regulated during senescence induction and progres‐ sion. To date forward and reverse genetic approaches as well as large-scale transcript profil‐ ing have identified almost 6.500 genes being differentially expressed during the course of leaf senescence including up-regulated as well as down-regulated genes [4]. The high num‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Bieker and Zentgraf; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Mobilization and Signaling**

Stefan Bieker and Ulrike Zentgraf

http://dx.doi.org/10.5772/54392

plant undergoes senescence and dies.

this regulatory network is highly complex and dynamic.

**1. Introduction**

**1.1. Senescence**

**Chapter 3**

## **Plant Senescence and Nitrogen Mobilization and Signaling**

Stefan Bieker and Ulrike Zentgraf

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54392

### **1. Introduction**

#### **1.1. Senescence**

Very early during their reproductive phase, annual plants initiate the process of senescence. Monocarpic senescence describes the last steps in these plants' development; senescence on organ level starts shortly after entering reproductive phase while after anthesis the whole plant undergoes senescence and dies.

In the following we will focus on leaf senescence. Two different processes can be distin‐ guished in annual plants relying on different genetic programs. Before anthesis, sequential leaf senescence recycles nutrients from old to developing leaves which is mainly under the control of the growing apex and is arrested when no more new leaves develop and when the plant starts to flower and sets fruit. Monocarpic leaf senescence recovers valuable nutrients from the leaves during flower induction and anthesis to provide these to the developing re‐ productive organs [1, 2]. The latter is crucial for fruit and seed development and has a major impact on yield quantity and quality. In wheat salvaged nitrogen (N) from the leaves ac‐ counts for up to 90% of the total grain N content [3]. A complex regulation of many different metabolic pathways and expression of numerous genes underlies this process. How coordi‐ nation and interplay of many controlling factors, like hormones, genetic reprogramming, bi‐ otic and abiotic stresses are achieved is far from being understood, but it is already clear that this regulatory network is highly complex and dynamic.

Thousands of genes are differentially regulated during senescence induction and progres‐ sion. To date forward and reverse genetic approaches as well as large-scale transcript profil‐ ing have identified almost 6.500 genes being differentially expressed during the course of leaf senescence including up-regulated as well as down-regulated genes [4]. The high num‐

© 2013 Bieker and Zentgraf; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ber of differentially regulated senescence-associated transcription factors (TF) demonstrates the dimensions of genetic reprogramming taking place. These TFs include 20 distinct fami‐ lies of which NAC-, WRKY-, C2H2-type zinc finger, EREBP- and MYB-families are most abundant [5]. Recently, Breeze et al. (2011) [4] published extremely important results of a high-resolution temporal transcript profiling of senescing Arabidopsis leaves giving insight into the temporal order of genetic events. One of the first steps at the onset of senescence is a shift from anabolic to catabolic processes. Amino acid metabolism and protein synthesis are down-regulated while expression of autophagy- and reactive oxygen species-related, and water-response genes is enhanced. In contrast to the following elevation of abscisic acid (ABA) and jasmonic acid (JA) signaling-related gene expression, cytokinin-mediated signal‐ ing is lowered just as chlorophyll and carotenoid biosynthesis. The next phases include down-regulation of carbon utilization and enhanced expression of cystein-aspartat proteas‐ es, carotene metabolism-associated genes and pectinesterases which is then followed by the reduction of photosynthetic activity and degradation of the photosynthetic apparatus coin‐ ciding with the increased activity of lipid catabolism, ethylene signaling and higher abun‐ dance of cytoskeletal elements [4].

are applied annually worldwide [13-15]. However, 50-70% of these nitrogenous fertilizer are lost to the environment [16], mostly due to volatilization of N2O, NO, N2 and NH3 and leach‐

trients to provide, but it also has a strong detrimental impact on the environment. Since surrounding ecosystems and potable water supply are endangered by oversaturation with nitrogenous compounds, it is necessary to improve application techniques and plant's nitro‐

Several different definitions of nitrogen and nutrient use efficiencies are on hand. The most common is the *nitrogen use efficiency* (NUE), which is defined as shoot dry weight divided by total nitrogen content of the shoots. The *usage index* (UI) takes absolute biomass into account as it is denoted by the shoot fresh weight times the NUE. Likewise, the *N uptake efficiency* (NUpE) takes into account the whole N content of the plant and the N supplied by fertiliza‐ tion per plant. The fraction of the N taken up, which is then distributed to the grain, can be obtained by calculating the *N utilization efficiency* (NUtE) (Grain weight per total N content). Other efficiencies, which seem to be more suitable for the use in applied sciences, are the *agronomic efficiency* (AE), *apparent nitrogen recovery* (AR) and the *physiological efficiency* (PE). AE, AR and PE do require an unfertilized control to be calculated. While AE measures the efficiency to redirect applied nitrogen to the grains, AR defines the efficiency to capture N from the soil. PE puts the N uptake into relationship with the outcome of grain (reviewed in [15]). *Nitrogen remobilization efficiency* (NRE) describes the plant's capacity to translocate al‐ ready assimilated nitrogen to developing organs. Finally, the *harvest index* (HI) and the *nitro‐ gen harvest index* (NHI) are often used terms. HI is the total yield weight per plant mass,

Emission of nitrogen to the environment could be strongly reduced by application of 'best management techniques' in agricultural practice like e.g. rectifying the rate of appliance by accounting for all other possible sources of nitrogen influx (carryover from previous crops, atmospheric deposits etc.), ameliorating the timing and also changing the method of appli‐ ance to reduce atmospheric losses [13]. Food production has doubled in the last 40 years. Most of this increase could be achieved by selection of new strains, breeding and application of greater amounts of fertilizer and pesticides and other techniques [12]. Amending of nu‐ trient use efficiencies of the crop plants was mostly accomplished via breeding programs by now. QTL selection for higher yields, increased oil or protein content has been pursued for decades. In wheat for example, increasing yield and grain protein content has been exten‐ sively studied, but improving both is restrained by the negative genetic relationship be‐

Nitrogen sources vary extremely encompassing organic and inorganic forms, small peptides and single amino acids, thus uptake systems need to be adjusted and well regulated in spa‐ tial and temporal activity. Although the predominant form in which N is taken up mainly

while NHI states the grain N content per whole plant N content.

**2. Nitrogen uptake, assimilation and distribution**

into the water. Thus nitrogen is not only one of the most expensive nu‐

Plant Senescence and Nitrogen Mobilization and Signaling

http://dx.doi.org/10.5772/54392

55

ing of soluble NO3

gen use efficiencies.

tween these traits [17, 18].

Hormonal control of senescence is conveyed especially by ethylene, jasmonic and salicylic acid, cytokinin and auxin. Many mutants with a delayed senescence phenotype could be traced back to impaired or up-regulated ethylene or cytokinin signaling, respectively [6, 7]. In adition, ABA acts as a positive regulator of leaf senescence. Recently a membrane-bound, leucine rich repeat containing receptor kinase (RPK1) has been identified to play an impor‐ tant role in ABA-mediated senescence induction in an age-dependent manner. Strikingly, *rpk1* mutant lines did not show significant alterations in developmental processes, which have been reported for numerous other ABA signaling defective mutant lines [5], except slightly shorter growth [8]. This kinase has been indentified to integrate ABA signals during seed germination, plant growth, stomatal closure and stress responses. Overexpression lines showed enhanced expression of several stress and H2O2-responsive genes [9, 10]. Mutant lines showed a delayed senescence phenotype with slower progression of chlorophyll deg‐ radation and cell death.

Induction and progression of leaf senescence demands a tight regulation of numerous proc‐ esses. Integration of nutritional cues, biotic and abiotic influences, plant development and age has to take place for the correct timing of onset and temporal advance of this complex developmental process. Despite the enormous efforts and achievements in this field, many of the regulatory mechanisms remain elusive.

#### **1.2. Nitrogen and agriculture**

The nowadays growth of population and thus increasing demand for food and oil crops forces agricultural industry to increase quantitative as well as qualitative yields. Until 2050 world's population is predicted to be as high as 9-10 billion people [11] and grain require‐ ment is projected to be doubled, mostly resulting from a higher demand for wheat fed meat [12]. As most of the cropping systems are naturally deficient in nitrogen, there is a funda‐ mental dependency on inorganic nitrogen fertilizers. 85-90 million tons of these fertilizers are applied annually worldwide [13-15]. However, 50-70% of these nitrogenous fertilizer are lost to the environment [16], mostly due to volatilization of N2O, NO, N2 and NH3 and leach‐ ing of soluble NO3 into the water. Thus nitrogen is not only one of the most expensive nu‐ trients to provide, but it also has a strong detrimental impact on the environment. Since surrounding ecosystems and potable water supply are endangered by oversaturation with nitrogenous compounds, it is necessary to improve application techniques and plant's nitro‐ gen use efficiencies.

ber of differentially regulated senescence-associated transcription factors (TF) demonstrates the dimensions of genetic reprogramming taking place. These TFs include 20 distinct fami‐ lies of which NAC-, WRKY-, C2H2-type zinc finger, EREBP- and MYB-families are most abundant [5]. Recently, Breeze et al. (2011) [4] published extremely important results of a high-resolution temporal transcript profiling of senescing Arabidopsis leaves giving insight into the temporal order of genetic events. One of the first steps at the onset of senescence is a shift from anabolic to catabolic processes. Amino acid metabolism and protein synthesis are down-regulated while expression of autophagy- and reactive oxygen species-related, and water-response genes is enhanced. In contrast to the following elevation of abscisic acid (ABA) and jasmonic acid (JA) signaling-related gene expression, cytokinin-mediated signal‐ ing is lowered just as chlorophyll and carotenoid biosynthesis. The next phases include down-regulation of carbon utilization and enhanced expression of cystein-aspartat proteas‐ es, carotene metabolism-associated genes and pectinesterases which is then followed by the reduction of photosynthetic activity and degradation of the photosynthetic apparatus coin‐ ciding with the increased activity of lipid catabolism, ethylene signaling and higher abun‐

Hormonal control of senescence is conveyed especially by ethylene, jasmonic and salicylic acid, cytokinin and auxin. Many mutants with a delayed senescence phenotype could be traced back to impaired or up-regulated ethylene or cytokinin signaling, respectively [6, 7]. In adition, ABA acts as a positive regulator of leaf senescence. Recently a membrane-bound, leucine rich repeat containing receptor kinase (RPK1) has been identified to play an impor‐ tant role in ABA-mediated senescence induction in an age-dependent manner. Strikingly, *rpk1* mutant lines did not show significant alterations in developmental processes, which have been reported for numerous other ABA signaling defective mutant lines [5], except slightly shorter growth [8]. This kinase has been indentified to integrate ABA signals during seed germination, plant growth, stomatal closure and stress responses. Overexpression lines showed enhanced expression of several stress and H2O2-responsive genes [9, 10]. Mutant lines showed a delayed senescence phenotype with slower progression of chlorophyll deg‐

Induction and progression of leaf senescence demands a tight regulation of numerous proc‐ esses. Integration of nutritional cues, biotic and abiotic influences, plant development and age has to take place for the correct timing of onset and temporal advance of this complex developmental process. Despite the enormous efforts and achievements in this field, many

The nowadays growth of population and thus increasing demand for food and oil crops forces agricultural industry to increase quantitative as well as qualitative yields. Until 2050 world's population is predicted to be as high as 9-10 billion people [11] and grain require‐ ment is projected to be doubled, mostly resulting from a higher demand for wheat fed meat [12]. As most of the cropping systems are naturally deficient in nitrogen, there is a funda‐ mental dependency on inorganic nitrogen fertilizers. 85-90 million tons of these fertilizers

dance of cytoskeletal elements [4].

54 Senescence and Senescence-Related Disorders

radation and cell death.

**1.2. Nitrogen and agriculture**

of the regulatory mechanisms remain elusive.

Several different definitions of nitrogen and nutrient use efficiencies are on hand. The most common is the *nitrogen use efficiency* (NUE), which is defined as shoot dry weight divided by total nitrogen content of the shoots. The *usage index* (UI) takes absolute biomass into account as it is denoted by the shoot fresh weight times the NUE. Likewise, the *N uptake efficiency* (NUpE) takes into account the whole N content of the plant and the N supplied by fertiliza‐ tion per plant. The fraction of the N taken up, which is then distributed to the grain, can be obtained by calculating the *N utilization efficiency* (NUtE) (Grain weight per total N content). Other efficiencies, which seem to be more suitable for the use in applied sciences, are the *agronomic efficiency* (AE), *apparent nitrogen recovery* (AR) and the *physiological efficiency* (PE). AE, AR and PE do require an unfertilized control to be calculated. While AE measures the efficiency to redirect applied nitrogen to the grains, AR defines the efficiency to capture N from the soil. PE puts the N uptake into relationship with the outcome of grain (reviewed in [15]). *Nitrogen remobilization efficiency* (NRE) describes the plant's capacity to translocate al‐ ready assimilated nitrogen to developing organs. Finally, the *harvest index* (HI) and the *nitro‐ gen harvest index* (NHI) are often used terms. HI is the total yield weight per plant mass, while NHI states the grain N content per whole plant N content.

Emission of nitrogen to the environment could be strongly reduced by application of 'best management techniques' in agricultural practice like e.g. rectifying the rate of appliance by accounting for all other possible sources of nitrogen influx (carryover from previous crops, atmospheric deposits etc.), ameliorating the timing and also changing the method of appli‐ ance to reduce atmospheric losses [13]. Food production has doubled in the last 40 years. Most of this increase could be achieved by selection of new strains, breeding and application of greater amounts of fertilizer and pesticides and other techniques [12]. Amending of nu‐ trient use efficiencies of the crop plants was mostly accomplished via breeding programs by now. QTL selection for higher yields, increased oil or protein content has been pursued for decades. In wheat for example, increasing yield and grain protein content has been exten‐ sively studied, but improving both is restrained by the negative genetic relationship be‐ tween these traits [17, 18].

#### **2. Nitrogen uptake, assimilation and distribution**

Nitrogen sources vary extremely encompassing organic and inorganic forms, small peptides and single amino acids, thus uptake systems need to be adjusted and well regulated in spa‐ tial and temporal activity. Although the predominant form in which N is taken up mainly depends on the plants adaption to the given environment and influences like fertilization, soil pH, temperature, precipitation and others [14, 19], most plants cover their N demand primarily through soil nitrate being provided by fertilization, bacterial nitrification and oth‐ er processes [15]. However, a wide range of different uptake system has evolved in plants. For example, oligopeptides can be taken up via OPT-proteins (oligopeptide transporters), ammonium via ammonium transporters (AMTs) and amino acids via amino acid transport‐ ers and amino acid permeases. Besides the *AtCLC* (ChLoride Channel) gene family, compris‐ ing 7 members of which two (*AtCLCa* and *AtCLCb*) have been shown to encode tonoplast located NO3 - /H+ antiporters [20, 21], two families of nitrate transporters have been identified in higher plants (NRT1 and NRT2), representing low- and high affinity transporter systems, respectively. Moreover the *NRT1*-family has been shown to comprise also di-/tripeptide transporters (PTR) [22].

low. Additionally, *AtNRT1-4* is predominantly expressed in the leaf petiole and the *atnrt1.4* mutant shows a nitrate content decreased by half in the petiole [22, 31]. *AtNRT1-6* is ex‐ pressed in the silique's and funiculus' vascular tissue and thought to play a nitrate provid‐ ing role in early embryonic development [32]. AtNRT1-8 functions in nitrate unloading from the xylem sap and is mainly located in xylem parenchyma cells within the vasculature [33], whereas AtNRT1-9 facilitates nitrate loading into the root phloem from root phloem com‐

High affinity nitrate uptake is conducted by members of the *NRT2*-family, comprising 7 genes in Arabidopsis. AtNRT2-1 has been shown to be one of the main components of the HATs. Mutant *atnrt2-1* plants displayed a loss of nitrate uptake capacity up to 75% at HAT-

nitrate combined with high sucrose supply, where NRT2-1 acts either as sensor or transduc‐ er [36]. Experiments with *Xenopus* oocytes revealed the requirement of a AtNAR2 protein for AtNRT2-1 function [37]. Mutants of either of these two components showed impaired ni‐ trate uptake at HAT-specific concentrations and hampered growth with display of N-starva‐ tion symptoms, in which, remarkably, the *atnar2* mutant phenotype appeared to be more pronounced [38]. The phenotype of the *atnrt2-7* mutant is similar to the phenotype of the

cumulation of nitrate in the vacuole. Mutation of either of these resulted in lower nitrate content. Ectopic overexpression of *AtNRT2-7* led to higher nitrate contents in dried seeds, where the gene is highly expressed under wild type conditions, and an increase in the ni‐ trate HATs uptake capacity by 2-fold. However, normal development was not impaired in the mutants as well as overexpressor plants [14, 20, 39]. Despite its high homology to *AtNRT2-1, AtNRT2-4* is not dependent on the function of *AtNAR2*. *AtNRT2-4* expression is highly induced upon nitrogen starvation in the outermost layer of young lateral roots [40].

Members of the *AMT1*- and *AMT2*-subfamilies are thought to be the main high affinity am‐ monium transporters in plants. Due to ammonium's toxic nature and convertibility from

(AA). The most abundant transport forms are glutamine, glutamate, asparagine and as‐

lesser extend [46]. Nitrate assimilation thus requires reduction to ammonium. Nitrate re‐

the cytoplasm, while the reduction of nitrite to ammonium is carried out in the plastids.

 to NH4

and NH4

 to NO2 -

activity is regulated posttranscriptionally via the availability of nitrate [42].

ductase (NR) realizes the first step by reducing NO3

partate [45] although direct transport of NO3

Here, nitrite reductase (NiR) converts NO2

 to NH3 and the thus varying membrane permeability, its uptake and transport needs to be tightly regulated [41, 42]. AMTs are regulated transcriptionally by N-supply, sugar and daytime and provide an additive contribution to ammonium transport [41]. AtAMT1-1 contributes 30-35 % as does AtAMT1-3, while AtAMT1-2 provides only 18-25% [43, 44]. AtAMT1-1 transports ammonium as well as its analog methyl-ammonium. Additionally, its

*atclca* mutant. The *AtCLCa* gene has been shown to encode a NO3

concentrations [35]. Furthermore, lateral root growth is repressed under low

Plant Senescence and Nitrogen Mobilization and Signaling

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57

+ almost always includes incorporation into amino acids

+ also takes place but to a much

<sup>+</sup> making it readily available for the in‐

. This reaction takes place in

/H+ antiporter enabling ac‐

panion cells [34].

specific NO3

NH4 +

**2.2. Nitrogen assimilation**

Assimilation of NO3

#### **2.1. Nitrogen transporter systems**

Four constituents of nitrate uptake are known, constitutive (c) and inducible (i) high- (HAT) and low-affinity (LAT) transporters, respectively. The high-affinity system's KM ranges from ~ 5-100 µM, varying with plant species, and a maximal influx via this system of 1 µMol \* g-1 \* h-1 has been determined [23, 24]. At nitrate concentrations of 10 mM the influx rate via the LATs can reach up to ~24 µMol \* g-1 \* h-1 [24].

The *NRT1*-family comprises 53 genes in Arabidopsis which are classified as LATs. *AtNRT1-1* (*CHL1*) was the first member to be identified in 1993 and has been shown to en‐ code a proton-coupled nitrate transporter [25]. Studies with *Xenopus* oocytes have shown that this transporter protein possesses two different states, one serving low-affinity and the other one high-affinity nitrate uptake [26, 27], thus the distinction between high and lowaffinity nitrate transporters is overridden in this case. Switching between these two modes of action is conferred by phosphorylation of threonin at position 101 [28]. *AtNRT1-1* is ex‐ pressed in the cortex and endodermis of mature roots and in the epidermis of root tips. Ad‐ ditionally, a nitrate sensing function regulating the plants primary nitrate response has been strongly indicated for the AtNRT1-1 protein by several lines of evidence. The *chl1-5 (atnrt1-1-5)* mutant, a deletion mutant with no detectable *CHL1* transcript, is deficient in ni‐ trate uptake and initiation of the primary nitrate response. The *chl1-9* mutant is defective in nitrate uptake but not in the primary nitrate response. The *chl1-*9 mutant has a point muta‐ tion between two transmembrane domains. When threonine 101 was mutated to mimic or repress phosphorylation and transformed into the *chl1-5* background, primary nitrate re‐ sponse could be repressed or enhanced [29]. Constitutive expression identifies AtNRT1-2 as part of the Arabidopsis cLATs. Its transcript is only found in root epidermal cells [22]. Ex‐ pression of *AtNRT1-5* is nitrate inducible; however, the response to nitrate is much slower than for *AtNRT1-1*. AtNRT1-5 has been shown to be a pH-dependent, bidirectional nitrate transporter, with subcellular localization in the plasma membrane of root pericycle cells near the xylem implicating an involvement in long-distance nitrate transport [30]. Experi‐ mental evidence suggests nitrate storage in leaf petioles to be associated with the function of AtNRT1-4. Here, nitrate content is relatively high, while nitrate reductase (NR) activity is low. Additionally, *AtNRT1-4* is predominantly expressed in the leaf petiole and the *atnrt1.4* mutant shows a nitrate content decreased by half in the petiole [22, 31]. *AtNRT1-6* is ex‐ pressed in the silique's and funiculus' vascular tissue and thought to play a nitrate provid‐ ing role in early embryonic development [32]. AtNRT1-8 functions in nitrate unloading from the xylem sap and is mainly located in xylem parenchyma cells within the vasculature [33], whereas AtNRT1-9 facilitates nitrate loading into the root phloem from root phloem com‐ panion cells [34].

High affinity nitrate uptake is conducted by members of the *NRT2*-family, comprising 7 genes in Arabidopsis. AtNRT2-1 has been shown to be one of the main components of the HATs. Mutant *atnrt2-1* plants displayed a loss of nitrate uptake capacity up to 75% at HATspecific NO3 concentrations [35]. Furthermore, lateral root growth is repressed under low nitrate combined with high sucrose supply, where NRT2-1 acts either as sensor or transduc‐ er [36]. Experiments with *Xenopus* oocytes revealed the requirement of a AtNAR2 protein for AtNRT2-1 function [37]. Mutants of either of these two components showed impaired ni‐ trate uptake at HAT-specific concentrations and hampered growth with display of N-starva‐ tion symptoms, in which, remarkably, the *atnar2* mutant phenotype appeared to be more pronounced [38]. The phenotype of the *atnrt2-7* mutant is similar to the phenotype of the *atclca* mutant. The *AtCLCa* gene has been shown to encode a NO3 - /H+ antiporter enabling ac‐ cumulation of nitrate in the vacuole. Mutation of either of these resulted in lower nitrate content. Ectopic overexpression of *AtNRT2-7* led to higher nitrate contents in dried seeds, where the gene is highly expressed under wild type conditions, and an increase in the ni‐ trate HATs uptake capacity by 2-fold. However, normal development was not impaired in the mutants as well as overexpressor plants [14, 20, 39]. Despite its high homology to *AtNRT2-1, AtNRT2-4* is not dependent on the function of *AtNAR2*. *AtNRT2-4* expression is highly induced upon nitrogen starvation in the outermost layer of young lateral roots [40].

Members of the *AMT1*- and *AMT2*-subfamilies are thought to be the main high affinity am‐ monium transporters in plants. Due to ammonium's toxic nature and convertibility from NH4 + to NH3 and the thus varying membrane permeability, its uptake and transport needs to be tightly regulated [41, 42]. AMTs are regulated transcriptionally by N-supply, sugar and daytime and provide an additive contribution to ammonium transport [41]. AtAMT1-1 contributes 30-35 % as does AtAMT1-3, while AtAMT1-2 provides only 18-25% [43, 44]. AtAMT1-1 transports ammonium as well as its analog methyl-ammonium. Additionally, its activity is regulated posttranscriptionally via the availability of nitrate [42].

#### **2.2. Nitrogen assimilation**

depends on the plants adaption to the given environment and influences like fertilization, soil pH, temperature, precipitation and others [14, 19], most plants cover their N demand primarily through soil nitrate being provided by fertilization, bacterial nitrification and oth‐ er processes [15]. However, a wide range of different uptake system has evolved in plants. For example, oligopeptides can be taken up via OPT-proteins (oligopeptide transporters), ammonium via ammonium transporters (AMTs) and amino acids via amino acid transport‐ ers and amino acid permeases. Besides the *AtCLC* (ChLoride Channel) gene family, compris‐ ing 7 members of which two (*AtCLCa* and *AtCLCb*) have been shown to encode tonoplast

in higher plants (NRT1 and NRT2), representing low- and high affinity transporter systems, respectively. Moreover the *NRT1*-family has been shown to comprise also di-/tripeptide

Four constituents of nitrate uptake are known, constitutive (c) and inducible (i) high- (HAT) and low-affinity (LAT) transporters, respectively. The high-affinity system's KM ranges from ~ 5-100 µM, varying with plant species, and a maximal influx via this system of 1 µMol \* g-1 \* h-1 has been determined [23, 24]. At nitrate concentrations of 10 mM the influx rate via the

The *NRT1*-family comprises 53 genes in Arabidopsis which are classified as LATs. *AtNRT1-1* (*CHL1*) was the first member to be identified in 1993 and has been shown to en‐ code a proton-coupled nitrate transporter [25]. Studies with *Xenopus* oocytes have shown that this transporter protein possesses two different states, one serving low-affinity and the other one high-affinity nitrate uptake [26, 27], thus the distinction between high and lowaffinity nitrate transporters is overridden in this case. Switching between these two modes of action is conferred by phosphorylation of threonin at position 101 [28]. *AtNRT1-1* is ex‐ pressed in the cortex and endodermis of mature roots and in the epidermis of root tips. Ad‐ ditionally, a nitrate sensing function regulating the plants primary nitrate response has been strongly indicated for the AtNRT1-1 protein by several lines of evidence. The *chl1-5 (atnrt1-1-5)* mutant, a deletion mutant with no detectable *CHL1* transcript, is deficient in ni‐ trate uptake and initiation of the primary nitrate response. The *chl1-9* mutant is defective in nitrate uptake but not in the primary nitrate response. The *chl1-*9 mutant has a point muta‐ tion between two transmembrane domains. When threonine 101 was mutated to mimic or repress phosphorylation and transformed into the *chl1-5* background, primary nitrate re‐ sponse could be repressed or enhanced [29]. Constitutive expression identifies AtNRT1-2 as part of the Arabidopsis cLATs. Its transcript is only found in root epidermal cells [22]. Ex‐ pression of *AtNRT1-5* is nitrate inducible; however, the response to nitrate is much slower than for *AtNRT1-1*. AtNRT1-5 has been shown to be a pH-dependent, bidirectional nitrate transporter, with subcellular localization in the plasma membrane of root pericycle cells near the xylem implicating an involvement in long-distance nitrate transport [30]. Experi‐ mental evidence suggests nitrate storage in leaf petioles to be associated with the function of AtNRT1-4. Here, nitrate content is relatively high, while nitrate reductase (NR) activity is

antiporters [20, 21], two families of nitrate transporters have been identified

located NO3

56 Senescence and Senescence-Related Disorders

transporters (PTR) [22].

**2.1. Nitrogen transporter systems**

LATs can reach up to ~24 µMol \* g-1 \* h-1 [24].

Assimilation of NO3 - and NH4 + almost always includes incorporation into amino acids (AA). The most abundant transport forms are glutamine, glutamate, asparagine and as‐ partate [45] although direct transport of NO3 and NH4 + also takes place but to a much lesser extend [46]. Nitrate assimilation thus requires reduction to ammonium. Nitrate re‐ ductase (NR) realizes the first step by reducing NO3 to NO2 - . This reaction takes place in the cytoplasm, while the reduction of nitrite to ammonium is carried out in the plastids. Here, nitrite reductase (NiR) converts NO2 to NH4 <sup>+</sup> making it readily available for the in‐ corporation into AAs in a NADH-dependent manner. Assimilation of ammonium into AAs involves chloroplastic glutamine synthetase 2 (GS2) and glutamate synthase (Fd-GO‐ GAT), which generates glutamine and glutamate (for detailed review see [14]). Glutamine as well as glutamate serve as ammonium donor for the synthesis of all other amino acids including aspartate and asparagine, which in turn function as active NH4 + donor or as long-range nitrogen transport and storage form, respectively [47]. Alternatively carba‐ moylphosphate synthase can be involved in ammonium assimilation by producing carba‐ moylphosphate and successively citrulline and arginine. Assimilation in non-green tissues is achieved in plastids in a similar manner, although here GOGAT depends on NADH in‐ stead of ferredoxin. Carbon skeletons are essential for the acquisition of inorganic nitro‐ gen in AAs. Especially the demand for keto-acids has to be met (see [14] and references within). These are predominantly obtained from the TCA-Cycle in the form of 2-oxogluta‐ rate (2OG) [47, 48]. 2OG is used for incorporation of photorespiratory ammonium, result‐ ing in the production of glutamate, which in turn can be utilized by GS1 and GS2 to produce glutamine. This displays the intricate interconnection between carbon and nitro‐ gen metabolism, in which N uptake and assimilation is also influenced via photosynthet‐ ic rates [47]. Besides direct assimilation into AAs, nitrate can also be stored in the vacuole and in the chloroplast. Vacuolar nitrate concentrations can vary enormously, as vacuolar nitrate also contributes to turgor maintenance and might have a nitrate storage function to maintain the cytosolic nitrate concentrations which are more constant [49].

ply showed a stronger decline of photosynthetic activity and more pronounced senescence symptoms than plants sufficiently supplied with nitrogen [51]. Furthermore these plants showed a more pronounced and earlier drop in (Glu+Asp)/(Gln+Asn) ratio during the pro‐ gression of senescence indicating an additional adaption to low nitrogen conditions through enhanced nitrogen remobilization. In this experiment, also a significant increase in the ratio of hexose to sucrose was observed at the beginning of senescence which was higher in Nstarved plants. This indicates that sugar-related senescence induction is dependent on the availability of nitrogen [51]. However, high sugar contents repress photosynthesis and can induce early SAG expression while late SAG expression is repressed. Diaz et al. (2005) [52] showed sugar accumulation to be lower in some recombinant inbred lines which display early leaf yellowing, thus pointing out a mayor function for sugar accumulation alone but the regulating function of the C/N balance during induction of monocarpic senescence is widely discussed. Recently, trehalose-6-phosphate (T6P) was identified as a main signaling component in this pathway. T6P inhibits the activity of Snf1-related protein kinase (SnRK1). Zhang et al. (2009) [53] showed the T6P/SnRK1 interaction in Arabidopsis seedling extracts and other young tissues treated with T6P. Additionally, Delatte et al. (2011) [54] confirmed the inhibition of SnRK1 by T6P with plants overexpressing the SnRK1 catalytic subunit gene *KIN10.* These plants were insensitive to trehalose treatments. Further verification of T6P as signaling molecule was provided by several studies. In wheat the interaction of T6P and SnRK1 has been suggested to play a role during grain filling [55]. Wingler et al. (2012) [56] conducted a study with *otsA* and *otsB* expressing Arabidopsis plants *otsA* encodes the bacte‐ rial T6P synthase gene, *otsB* the T6P phosphatase gene; therefore, overexpression leads to in‐ creasing or decreasing T6P contents, respectively. A significantly higher accumulation of glucose, fructose and sucrose was observed in *otsB* expressing plants and these plants dis‐ played a delayed senescence phenotype. But most interestingly, these plants were rendered less susceptible to the induction of senescence-associated genes by sugar feeding in combi‐ nation with low nitrogen supply, whereas *otsA* plants induced senescence and anthocyanin

Plant Senescence and Nitrogen Mobilization and Signaling

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59

Another signaling component involved in senescence induction is light quantity and quali‐ ty. Senescence can be induced by the darkening of individual leaves. However, darkening of the whole plant resulted in delay rather than in induction of leaf senescence in Arabidopsis and sunflower plants [57, 58]. Brouwer et al. (2012) [59] recently revealed the involvement of photoreceptors in dark and shading induced leaf responses. They applied different shading conditions to single leaves of Arabidopsis plants. Depending on the amount of light per‐ ceived, different biological programs were induced, leading to either acclimation to the new light conditions or leaf senescence. Furthermore, *phytochrome A* mutant lines displayed ac‐ celerated chlorophyll degradation under all shading conditions except darkness, displaying

its involvement in the perception of and adaption to changing light conditions [59].

A tight linkage between stress response and leaf senescence is demonstrated by the function of several members of the NAC- and WRKY-family [60]. For example, *At NTL9 (NAC TRANSCRIPTION FACTOR LIKE 9)* mediates osmotic stress signaling during leaf senes‐

synthesis upon external supply of 2% glucose.

#### **3. Senescence induction and nitrogen mobilization**

As mentioned above, induction of senescence is a highly complex regulated and dynamic process. Besides developmental cues, there are numerous other possible impacts. Nutrition‐ al starvation, photosynthetic activity, pathogen infections, carbon accumulation, carbon to nitrogen ratio, photoperiod and various other cues can lead to senescence induction on ei‐ ther organ or whole plant level. Both natural and stress induced senescence are accompa‐ nied with the remobilization of valuable nutrients from various organs of the plant. In the following we will again focus on the situation in leaves.

#### **3.1. Senescence induction**