- Email: [email protected]
Senescence-Associated Markers
C H A P T E R
17 Senescence-Associated Markers Jyoti Bala⁎,†, Anupam J. Das†, Hoshang Unwalla⁎ ⁎
Department of Immunology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, United States †Molsys Scientific, Bangalore, India
1 INTRODUCTION 1.1 Senescence Senescence is a process related to biological aging that is characterized by dysfunction and deterioration of vital functioning, ultimately leading to mortality (Watanabe et al., 2013; Jeyapalan and Sedivy, 2008; Leshem, 1988; Lim et al., 2007; Jansson and Thomas, 2008). It is an active and regulated mechanism that dramatically changes the physiological and morphological condition of a biological organism. It is also tightly regulated at the metabolism and gene levels. There are several factors that affect the process of senescence, including various internal and environmental factors. In addition, oxidative stress and reactive oxygen species (ROS) play a significant role in senescence. Plant senescence has been widely studied (e.g., Guo and Gan, 2005; Bleecker, 1998; Thomas and Donnison, 2000), and the role of senescence in plant growth, development, and death processes are well known. There are several factors that play crucial roles in the processes of plant senescence. Molecular mechanisms like signaling, hormonal regulation, and epigenetic involvements regulate transitory changes in plant senescence that subsequently lead plants from juvenility to maturity and ultimately to death (Uauy et al., 2006; Guo and Gan, 2006; Umehara et al., 2008; Ougham et al., 2007; Brusslan et al., 2012). Evidently, leaf possess distinctive and reliable genetic systems which provide insights about senescence. Moreover, leaf senescence offers an alternative way to explore the plant senescence and biogenesis processes. It has been well established that leaf senescence is regulated by a compactly precise genetic regulatory mechanism at multiple cellular and gene levels (Fraga and Esteller, 2007; Miller et al., 1999; Oh et al., 1997; Suzuki and Makino, 2013; Hinderhofer and Zentgraf, 2001). Still, the molecular processes of fundamental leaf senescence and plant senescence are not yet completely explored.
Senescence Signalling and Control in Plants https://doi.org/10.1016/B978-0-12-813187-9.00017-2
273
© 2019 Elsevier Inc. All rights reserved.
274
17. Senescence-Associated Markers
Markers specific to plant senescence hold great potential for studying the fate of plant death and crop productivity. All living organisms have a certain definite life span. Plants, whether annual or perennial, have a vegetative growth phase postgermination. The formation of flowers or development of reproductive structure indicates the occurrence of the reproductive phase. As a plant becomes mature, and with the further passage of time, its metabolic activity deteriorates. The functional and molecular mechanisms at the cellular level decelerates, and the plant becomes old and eventually influences the senescence phases that in due course lead to the plant's death. Leaves and other organs fall from plants during the course of senescence in a process called abscission (Williams and Whitham, 1986; Van Doorn and Stead, 1997; Taesakul et al., 2015; McFadyen et al., 2012).
2 CHARACTERISTICS OF SENESCENCE It is quite evident that senescence is a tightly regulated degenerative process that eventually terminates the functional life of an organ or organism. Senescence in plants is manifested in various forms, such as whole-plant senescence, shoot senescence, and organ senescence (Fig. 1; Keskitalo et al., 2005; Hopkins et al., 2007; Bassham, 2007; Thomas et al., 2003; Fukuda, 1996). Whole-plant senescence begins with reproductive maturity, and ultimately the whole plant dies after seed production. It is typical of monocarpic plant species, which flower and fruit only once in their life cycle. This is also seen in annual and biennial plants. Meanwhile, shoot senescence mostly represents senescence of aerial shoots, and shoots subsequently die every year after flowering. Shoot senescence is seen in many perennial plants. Finally, organ senescence represents lateral organs such as leaves and fruits senescence and they die prior to the death of the whole plant. Senescence plays vital role not only in cell death, but in others mechanism such as by providing nutrient back to plants thereby crucial for well being for plant growth too. When senescence occurs, new, functionally efficient organs or organisms are created and old, functionally inefficient ones are discarded. Senescence ensures the retrieval and reutilization of mineral elements and organic nutrients from older, senescing organs to newly formed, growing organs (Matile et al., 1999; Thomas and Sadras, 2001; Hortensteiner, 2004;
Drought Flooding Whole plant senescence Deciduous senescence Progressive senescence
Senescence Pathogen Stress
Factors of senescence
Types of senescence
Leaf senescence
Aging
FIG. 1 Representation of the types of plant senescence and the factors responsible for plant senescence.
3 Senescence-Associated Markers in Plants
275
Zimmermann and Zentgraf, 2005; Giovannoni, 2007; Feller and Fischer, 1994). Additionally, synchronous (or seasonal) senescence avoids water loss due to transcription during critical seasonal changes, thus supporting the existence of the plant in an adverse environment.
3 SENESCENCE-ASSOCIATED MARKERS IN PLANTS 3.1 Cytosolic Glutamine Synthetase and Glutamate Dehydrogenase Pageau et al. (2006) reported on the role of stress and its effects on nitrogen management in Nicotiana tabacum leaves. They found two senescence-related markers, cytosolic glutamine synthetase (GS1) and glutamate dehydrogenase (GDH), which were mainly involved in the homeostasis of nitrogen status via nitrogen mobilization. They have observed differential regulation of nitrogen cell status upon pathogen attack, elicitors and by stress hormones. Their data validate GDH and GS1 association in leaf senescence and plant defense response upon pathogen interaction. Interestingly, they have seen that during natural senescence, the expression of GS2 and Nia decreased under given stress conditions. Conversely, GS1 and GDH messenger RNA (mRNA) was found to be enhanced. Differential expression of GS1 and GDH was observed depending on the given stress, which suggests that GS1 appeared to be more selective than GDH. Natural plant senescence and stress- induced senescence links were conferred, showing the prominence of GS1 and GDH in plant defense (Pageau et al., 2006).
3.2 Leaf Senescence-Associated Gene Homolog Markers Senescence is the ultimate phase of development in plant tissue. During this process, several senescence-associated genes (SAGs) have been validated that showed transcriptional upregulation. Kajimura et al. (2010) reported the isolation of nine crucial wheat SAG complementary deoxyribonucleic acid (cDNA) clones named TaSAG1-TaSAG9, and also assessed the feasibility of the SAG homologs as molecular markers based on their expression patterns. They have demonstrated the utilization of leaf SAG homologs as developmental markers in common wheat. These nine wheat SAGs were identified, validated, and established in wheat expressed sequence tag (EST) libraries based on homology to rice SAGs. Interestingly, they found that these wheat SAG transcripts were enhanced during natural senescence, as well as dark-induced senescence, in seedling leaves. However, the nine wheat SAGs also displayed variable expression patterns in developing and ripening seeds. They also showed the significance of transcript accumulation patterns of TaSAG5 and TaSAG6 that were enhanced linearly during the course of their studies on the flag leaf and seed. These could be utilized as molecular markers to assess the fate of wheat flag leaf senescence and seed maturation. Additionally, the transcript accumulation levels of the other six SAGs were found to be enhanced before the obvious necrotic cell death of seeding leaves displaying wheat hybrid necrosis (Kajimura et al., 2010). Their data validate that necrotic cell death in wheat hybrid necrosis is related to senescence and defense.
276
17. Senescence-Associated Markers
3.3 Characterization of Leaf Senescence Markers in Arabidopsis Using Metabolic Profiling A number of metabolic markers that differentiate Arabidopsis lines on the basis of senescence progress are crucial. Diaz et al. (2005) have characterized and determined markers for the variability of leaf senescence in Arabidopsis using metabolic profiling. Briefly, five recombinant inbred lines (RILs) of the Bay-0 × Shahdara differential leaf senescence phenotypes populations (from early senescing to late senescing) were studied to investigate the metabolic markers based on senescence-dependent metabolic changes. Studies have compared the extent of leaf senescence based on the genetic context of the diverse RILs of Arabidopsis (e.g., Arabidopsis thaliana; Diaz et al., 2005). They have characterized markers to decide the degree and variability of leaf senescence. Diaz et al. (2005) have shown differential leaf senescence phenotypes. Further, they have investigated metabolic markers that could differentiate Arabidopsis lines on the basis of senescence-dependent metabolism changes. Interestingly, the levels of gamma-aminobutyric acid, aspartate, leucine, isoleucine, and glutamate were estimated and correlated with parameters of age and the senescence phenotype of the RILs. Additionally, early variances were observed in the glycine/serine ratio. Their data suggest that these markers could be studied as predictive indicator for plant senescence behavior. Prognostic indicators for plant senescence behavior were established (Diaz et al., 2005). Amazingly, they also have noticed differences in the mobilization efficiency of asparagine, glutamine, and sulfate in late-senescing lines than early-senescing lines.
3.4 SSR Markers for Leaf Chlorophyll Content, Flag Leaf Senescence, and Cell Membrane Stability Elshafei et al. (2013) reported identification of novel markers linked to leaf senescence (i.e., leaf chlorophyll content, flag leaf senescence) and cell membrane stability traits in wheat plants under stress conditions. They have mapped the quantitative trait locus (QTL) for the three physiological traits. To identify the simple sequence repeat (SSR) markers, researchers have made drought-sensitive (Yecora Roja) and drought-tolerant (Pavon 76) genotypes. The SSR markers for physiological traits had genetic distances that have potential and could be used in breeding programs for drought tolerance in wheat (Elshafei et al., 2013).
3.5 Delayed Senescence Markers in Transgenic Tobacco Phytohormones such as cytokinins (CKs) are involved in vital functioning of plants growth and development. CKs are involved in cellular mechanisms at the cellular level by affecting cell division. Their roles in regulating the expression of photosynthetic-associated genes are well established. CKs hold great significance for its antisenescing action and have been utilized to delay the onset of plant senescence, efficient output, and postharvest storage. Skowron et al. (2016) have reported molecular markers related to delayed senescence in transgenic tobacco with higher CK levels and investigated the effect of enhanced CK biosynthesis on the onset of leaf senescence. Tobacco plants with genetically engineered gene for CK biosynthesis were designed and expressed in a senescence-related manner. These were further used for
3 Senescence-Associated Markers in Plants
277
analyzing the physiological and molecular markers. The authors have designed transgenic tobacco plants with an inserted gene pathway, ipt, fused with a senescence-specific promoter, PSAG12. Further, the effects of elevated hormone level in transgenic tobacco resulted in decreased chlorophyll, soluble proteins, and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) degradation in plants exposed to 9 days of light withdrawal. They have explored the conceivable association between the enhanced CK synthesis and antioxidant properties of PSAG12-IPT plants by considering three isoforms of superoxide dismutase (SOD; Skowron et al., 2016). The data signify the importance of molecular and physiological basis of the role of CKs in inhibiting leaf aging.
3.6 Telomere Shortening as a Marker of Cellular Senescence Senescent cells comprising replicative senescence and stress-induced premature senescence of all the tissues are alike and metabolically active. Although there are certain in vitro and in vivo characteristics that are known as biomarkers of aging and cellular senescence. Bernadotte et al. (2016) reported on markers of cellular senescence and demonstrated telomere shortening as a marker of cellular senescence. The authors have described the mechanisms of aging and elaborated the role of telomerase in this process. Telomeres have been established as an indicative of aging, as well as a possible factor in determining life expectancy. The telomere is a promising marker of cellular senescence; telomere theory is based on the telomere-shortening mechanism. Many researchers believe that the chief marker (telomere-shortening) of aging is actually the cause of aging. This is usually used to indicate cellular senescence. During the course of replication, telomeres get shorter with every cell division. Rationality of telomere shortening as a marker for cellular senescence was established on the basis of theoretical and experimental research.
3.7 Transcriptomic and Metabolomic Data to Characterize Leaf Senescence Markers Moschen et al. (2016) have validated the network and bio-signature analysis for the integration of transcriptomic and metabolomic data to illustrate leaf senescence in sunflowers. Weighted gene correlation network analysis and bio-signature discovery were performed to integrate transcriptomic and metabolomic data. Interestingly, 11 metabolites and 19 transcription factors (TFs) were found to be potential biomarkers. Moschen et al. (2016) have used an efficient tool for characterizing the chosen genes and metabolites that may be involved during the triggering and development of the leaf senescence process. The methodology offered great assistance in interpreting and foreseeing novel potential biomarkers of leaf senescence and aging in sunflower.
3.8 RAPD and ISSR Markers Associated With Leaf Senescence Milad et al. (2011) have reported random amplification of polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers associated with flag wheat leaf senescence during water stress. Flag leaf senescence is an imperative cause that reduced the crop yield under
278
17. Senescence-Associated Markers
stress. Studies have identified molecular markers associated with the flag leaf senescence gene in drought-sensitive genotype (variant-1) and drought-tolerant genotype (Veery) wheat under water-stressed conditions. Several RAPD and ISSR primers were established for polymorphism amid parental genotypes and F2 populations. Remarkably, their data validate four RAPD and two ISSR markers linked to the flag leaf senescence gene in wheat. Additionally, these four RAPD and two ISSR markers were related to QTL for the flag leaf senescence gene as an indicator of drought tolerance. These markers have potential and can be applied in wheat-breeding programs that would assist in selection of early generations.
3.9 Redox Markers for Drought-Induced Senescence Marquez-Garcia et al. (2015) have studied redox markers for drought-induced nodule senescence in soybean (Glycine max) leaves. Drought and water scarcity affect crop productivity and have a huge impact on the agricultural economy. With changing climate, drought stress-tolerant crops hold a prodigious amount of potential to enhance crop productivity. In this study, the authors compared the effects of drought on soybean leaves and nodules to describe phenotypic markers and changes in the cellular redox state, as well as the associations between leaf and nodule senescence during drought. They have observed that the nodule ascorbate pool was reduced considerably less during drought. Moreover, greater levels of transcripts encoding two peroxiredoxins were identified in nodules under drought stress; however, senescence-associated transcripts encoding redox-related proteins were comparable under both conditions.
3.10 REVOLUTA and WRKY53 Developmental Markers in Arabidopsis Xie et al. (2014) reported REVOLUTA and WRKY53 markers for leaf development in Arabidopsis. The growth and development of plants usually change with external environmental changes. During these changes, plants persuade leaf senescence to reallocate nutrients and from the leaves to the maturing seeds. The authors showed that class III homeodomain leucine zipper (HD-ZIPIII) TFs play a role in controlling the onset of leaf senescence in Arabidopsis. Additionally, other downstream genes, such as the HD-ZIPIII protein REVOLUTA (REV), play crucial roles in environment-controlled physiological processes. Studies have reported that REV acts as a redox-sensitive TF that positively regulates the expression of WRKY53, a chief regulator of age-induced leaf senescence. HD-ZIPIII proteins are vital for inducing WRKY53 in response to oxidative stress, and mutations in HD-ZIPIII genes strongly delay the onset of senescence. This work proposes cross-talk between early and late stages of leaf development that could significantly contribute to plant developmental studies and ultimately crop productivity.
4 SUMMARY Physiological and biochemical studies have contributed to the present understanding of plant senescence. In this chapter, we have discussed the history and current state if molecular investigations, molecular markers for senescence, and their involvement in senescence regulation mechanisms. Additionally, advance molecular approaches to the manipulation of
279
4 Summary
SSR marker
Telomere
Senescence-associated genes (SAGs)
RAPD and ISSR markers
Glutamate dehydrogenase
Markers Cytosolic glutamine synthetase
WRKY53 marker
Redox marker
Multiple omic data analysis (organelle, cell, organization) • Metabolomics • Proteomics • Transcriptomics • Genomics • Epigenomics • Phenomics
Tools to studies and analysis of senescence mechanism
FIG. 2 Plant senescence markers that have been utilized to study the process of senescence.
plant senescence were focused. Senescence has a huge impact on agricultural economy and crop productivity, which gives senescence studies a great impact. Therefore, studying the senescence process and senescence markers will contribute in-depth knowledge about plant development and thereby helps to manipulate and design strategies to delay senescence for higher crop yields, as well as for potential agricultural applications. Several advanced techniques and methods have been utilized to explore the senescence process and mechanisms, combining metabolic profiling and transcriptome analysis to achieve a comprehensive understanding about molecular regulation that would provide a smarter way to study plant metabolism and regulation during plant senescence (Fig. 2). Controlling senescence through breeding or genetic engineering might work to improve crop yield. It has been reported that delayed leaf senescence was caused by autoregulatory production of the CK hormone. In addition, studies have shown in maize plants, the knitted-I senescence specific gene was responsible for delayed senescence. These studies provide information that could help to delay the senescence and enhance the shelf life of plants. Development of advanced reliable and novel biomarker for senescence could help to predict early signs of senescence during cellular and stress conditions.
Acknowledgments Financial support from National Institute of Health (NIH) Grants R21-HL128141-01A1 and partial financial support from the Department of Immunology, Herbert Wertheim College of Medicine, Florida International University are greatly acknowledged.
280
17. Senescence-Associated Markers
Conflict of Interest The authors have declared no conflict of interest.
References Bassham, D.C., 2007. Plant autophagy–more than a starvation response. Curr. Opin. Plant Biol. 10, 587–593. Bernadotte, A., Mikhelson, V.M., Spivak, I.M., 2016. Markers of cellular senescence. Telomere shortening as a marker of cellular senescence. Aging (Albany NY) 8 (1), 3–11. Bleecker, A.B., 1998. The evolutionary basis of leaf senescence: method to the madness? Curr. Opin. Plant Biol. 1, 73–78. Brusslan, J.A., Alvarez-Canterbury, A.M.R., Nair, N.U., Rice, J.C., Hitchler, M.J., Pellegrini, M., 2012. Genome-wide evaluation of histone methylation changes associated with leaf senescence in Arabidopsis. PLoS ONE 7, e33151. Diaz, C., Purdy, S., Christ, A., Morot-Gaudry, J.F., Wingler, A., Masclaux-Daubresse, C., 2005. Characterization of markers to determine the extent and variability of leaf senescence in Arabidopsis. A metabolic profiling approach. Plant Physiol. 138 (2), 898–908. Elshafei, A.A., Mohamed, S., Al-Doss, A.A., Moustafa, K.A., Al-Qurainy, F.H., Barakat, M.N., 2013. Identification of new SRAP markers linked to leaf chlorophyll content, flag leaf senescence and cell membrane stability traits in wheat under water-stressed condition. Aust. J. Crop. Sci. 7 (6), 887–893. Feller, U., Fischer, A., 1994. Nitrogen metabolism in senescing leaves. Crit. Rev. Plant Sci. 13, 241–273. Fraga, M.F., Esteller, M., 2007. Epigenetics and aging: the targets and the marks. Trends Genet. 23, 413–418. Fukuda, H., 1996. Xylogenesis: initiation, progression, and cell death. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 299–325. Giovannoni, J.J., 2007. Fruit ripening mutants yield insights into ripening control. Curr. Opin. Plant Biol. 10, 283–289. Guo, Y., Gan, S., 2005. Leaf senescence: signals, execution, and regulation. Curr. Top. Dev. Biol. 71, 83–112. Guo, Y., Gan, S., 2006. AtNAP, a NAC family transcription factor, has an important role in leaf senescence. Plant J. 46, 601–612. Hinderhofer, K., Zentgraf, U., 2001. Identification of a transcription factor specifically expressed at the onset of leaf senescence. Planta 213, 469–473. Hopkins, M., Taylor, C., Liu, Z., Ma, F., McNamara, L., Wang, T., Thompson, J.E., 2007. Regulation and execution of molecular disassembly and catabolism during senescence. New Phytol. 175, 201–214. Hortensteiner, S., 2004. The loss of green color during chlorophyll degradation—a prerequisite to prevent cell death? Planta 219, 191–194. Jansson, S., Thomas, H., 2008. Senescence: developmental program or timetable? New Phytol. 179, 575–579. Jeyapalan, J.C., Sedivy, J.M., 2008. Cellular senescence and organismal aging. Mech. Ageing Dev. 129, 467–474. Kajimura, T., Mizuno, N., Takumi, S., 2010. Utility of leaf senescence-associated gene homologs as developmental markers in common wheat. Plant Physiol. Biochem. 48 (10–11), 851–859. Keskitalo, J., Bergquist, G., Gardeström, P., Jansson, S., 2005. A cellular timetable of autumn senescence. Plant Physiol. 139, 1635–1648. Leshem, Y.Y., 1988. Plant senescence processes and free radicals. Free Radic. Biol. Med. 5, 39–49. Lim, P.O., Kim, H.J., Nam, H.G., 2007. Leaf senescence. Annu. Rev. Plant Biol. 58, 115–136. Marquez-Garcia, B., Shaw, D., Cooper, J.W., Karpinska, B., Quain, M.D., Makgopa, E.M., Kunert, K., Foyer, C.H., 2015. Redox markers for drought-induced nodule senescence, a process occurring after drought-induced senescence of the lowest leaves in soybean (Glycine max). Ann. Bot. 116 (4), 497–510. Matile, P., Hörtensteiner, S., Thomas, H., 1999. Chlorophyll degradation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 67–95. McFadyen, L., Robertson, D., Sedgley, M., Kristiansen, P., Olesen, T., 2012. Time of pruning affects fruit abscission, stem carbohydrates and yield of macadamia. Funct. Plant Biol. 39, 481–492. Milad, S.I., Wahba, L.E., Barakat, M.N., 2011. Identification of RAPD and ISSR markers associated with flag leaf senescence under water-stressed conditions in wheat (Triticum aestivum L.). Aust. J. Crop. Sci. 5 (3), 334–340. Miller, J.D., Arteca, R.N., Pell, E.J., 1999. Senescence-associated gene expression during ozone-induced leaf senescence in Arabidopsis. Plant Physiol. 120, 1015–1024.
REFERENCES 281
Moschen, S., Higgins, J., Di Rienzo, J.A., Heinz, R.A., Paniego, N., Fernandez, P., 2016. Network and biosignature analysis for the integration of transcriptomic and metabolomic data to characterize leaf senescence process in sunflower. BMC Bioinform. 17 (Suppl. 5), 174. Oh, S.A., Park, J.H., Lee, G.I., Paek, K.H., Park, S.K., Nam, H.G., 1997. Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana. Plant J. 12, 527–535. Ougham, H., Armstead, I., Howarth, C., Galyuon, I., Donnison, I., Thomas, H., 2007. The genetic control of senescence revealed by mapping quantitative trait loci. Annu. Plant Rev. 26, 171–201. Pageau, K., Reisdorf-Cren, M., Morot-Gaudry, J.F., Masclaux-Daubresse, C., 2006. The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves. J. Exp. Bot. 57 (3), 547–557. Skowron, E., Trojak, M., Sobala, T., 2016. Molecular markers of delayed senescence in transgenic tobacco with enhanced cytokinin level. World Sci. News 51, 13–25. Suzuki, Y., Makino, A., 2013. Translational downregulation of RBCL is operative in the coordinated expression of Rubisco genes in senescent leaves in rice. J. Exp. Bot. 64, 1145–1152. Taesakul, P., Siriphanich, J., Van Doorn, W.G., 2015. Two abscission zones proximal to Lansium domesticum fruit: one more sensitive to exogenous ethylene than the other. Front. Plant Sci. 6, 264. Thomas, H., Donnison, I., 2000. Back from the brink: plant senescence and its reversibility. In: Programmed Cell Death in Animals and Plants. Bios, Oxford, pp. 149–162. Thomas, H., Sadras, V.O., 2001. The capture and gratuitous disposal of resources by plants. Funct. Ecol. 15, 3–12. Thomas, H., Ougham, H., Wagstaff, C., Stead, A.J., 2003. Defining senescence and death. J. Exp. Bot. 54, 1127–1132. Uauy, C., Distelfeld, A., Fahima, T., Blechl, A., Dubcovsky, J., 2006. A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314, 1298–1301. Umehara, M., Hanada, A., Yoshida, S., Akiyama, K., Arite, T., Takeda-Kamiya, N., Magome, H., Kamiya, Y., Shirasu, K., Yoneyama, K., 2008. Inhibition of shoot branching by new terpenoid plant hormones. Nature 455, 195. Van Doorn, W.G., Stead, A.D., 1997. Abscission of flowers and floral parts. J. Exp. Bot. 48, 821–837. Watanabe, M., Balazadeh, S., Tohge, T., Erban, A., Giavalisco, P., Kopka, J., Mueller-Roeber, B., Fernie, A.R., Hoefgen, R., 2013. Comprehensive dissection of spatiotemporal metabolic shifts in primary, secondary, and lipid metabolism during developmental senescence in Arabidopsis. Plant Physiol. 162, 1290–1310. Williams, A.G., Whitham, T.G., 1986. Premature leaf abscission: an induced plant defense against gall aphids. Ecology 67 (6), 1619–1627. Xie, Y., Huhn, K., Brandt, R., et al., 2014. REVOLUTA and WRKY53 connect early and late leaf development in Arabidopsis. Development (Cambridge, England) 141 (24), 4772–4783. Zimmermann, P., Zentgraf, U., 2005. The correlation between oxidative stress and leaf senescence during plant development. Cell. Mol. Biol. Lett. 10, 515–534.
17 Senescence-Associated Markers Jyoti Bala⁎,†, Anupam J. Das†, Hoshang Unwalla⁎ ⁎
Department of Immunology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, United States †Molsys Scientific, Bangalore, India
1 INTRODUCTION 1.1 Senescence Senescence is a process related to biological aging that is characterized by dysfunction and deterioration of vital functioning, ultimately leading to mortality (Watanabe et al., 2013; Jeyapalan and Sedivy, 2008; Leshem, 1988; Lim et al., 2007; Jansson and Thomas, 2008). It is an active and regulated mechanism that dramatically changes the physiological and morphological condition of a biological organism. It is also tightly regulated at the metabolism and gene levels. There are several factors that affect the process of senescence, including various internal and environmental factors. In addition, oxidative stress and reactive oxygen species (ROS) play a significant role in senescence. Plant senescence has been widely studied (e.g., Guo and Gan, 2005; Bleecker, 1998; Thomas and Donnison, 2000), and the role of senescence in plant growth, development, and death processes are well known. There are several factors that play crucial roles in the processes of plant senescence. Molecular mechanisms like signaling, hormonal regulation, and epigenetic involvements regulate transitory changes in plant senescence that subsequently lead plants from juvenility to maturity and ultimately to death (Uauy et al., 2006; Guo and Gan, 2006; Umehara et al., 2008; Ougham et al., 2007; Brusslan et al., 2012). Evidently, leaf possess distinctive and reliable genetic systems which provide insights about senescence. Moreover, leaf senescence offers an alternative way to explore the plant senescence and biogenesis processes. It has been well established that leaf senescence is regulated by a compactly precise genetic regulatory mechanism at multiple cellular and gene levels (Fraga and Esteller, 2007; Miller et al., 1999; Oh et al., 1997; Suzuki and Makino, 2013; Hinderhofer and Zentgraf, 2001). Still, the molecular processes of fundamental leaf senescence and plant senescence are not yet completely explored.
Senescence Signalling and Control in Plants https://doi.org/10.1016/B978-0-12-813187-9.00017-2
273
© 2019 Elsevier Inc. All rights reserved.
274
17. Senescence-Associated Markers
Markers specific to plant senescence hold great potential for studying the fate of plant death and crop productivity. All living organisms have a certain definite life span. Plants, whether annual or perennial, have a vegetative growth phase postgermination. The formation of flowers or development of reproductive structure indicates the occurrence of the reproductive phase. As a plant becomes mature, and with the further passage of time, its metabolic activity deteriorates. The functional and molecular mechanisms at the cellular level decelerates, and the plant becomes old and eventually influences the senescence phases that in due course lead to the plant's death. Leaves and other organs fall from plants during the course of senescence in a process called abscission (Williams and Whitham, 1986; Van Doorn and Stead, 1997; Taesakul et al., 2015; McFadyen et al., 2012).
2 CHARACTERISTICS OF SENESCENCE It is quite evident that senescence is a tightly regulated degenerative process that eventually terminates the functional life of an organ or organism. Senescence in plants is manifested in various forms, such as whole-plant senescence, shoot senescence, and organ senescence (Fig. 1; Keskitalo et al., 2005; Hopkins et al., 2007; Bassham, 2007; Thomas et al., 2003; Fukuda, 1996). Whole-plant senescence begins with reproductive maturity, and ultimately the whole plant dies after seed production. It is typical of monocarpic plant species, which flower and fruit only once in their life cycle. This is also seen in annual and biennial plants. Meanwhile, shoot senescence mostly represents senescence of aerial shoots, and shoots subsequently die every year after flowering. Shoot senescence is seen in many perennial plants. Finally, organ senescence represents lateral organs such as leaves and fruits senescence and they die prior to the death of the whole plant. Senescence plays vital role not only in cell death, but in others mechanism such as by providing nutrient back to plants thereby crucial for well being for plant growth too. When senescence occurs, new, functionally efficient organs or organisms are created and old, functionally inefficient ones are discarded. Senescence ensures the retrieval and reutilization of mineral elements and organic nutrients from older, senescing organs to newly formed, growing organs (Matile et al., 1999; Thomas and Sadras, 2001; Hortensteiner, 2004;
Drought Flooding Whole plant senescence Deciduous senescence Progressive senescence
Senescence Pathogen Stress
Factors of senescence
Types of senescence
Leaf senescence
Aging
FIG. 1 Representation of the types of plant senescence and the factors responsible for plant senescence.
3 Senescence-Associated Markers in Plants
275
Zimmermann and Zentgraf, 2005; Giovannoni, 2007; Feller and Fischer, 1994). Additionally, synchronous (or seasonal) senescence avoids water loss due to transcription during critical seasonal changes, thus supporting the existence of the plant in an adverse environment.
3 SENESCENCE-ASSOCIATED MARKERS IN PLANTS 3.1 Cytosolic Glutamine Synthetase and Glutamate Dehydrogenase Pageau et al. (2006) reported on the role of stress and its effects on nitrogen management in Nicotiana tabacum leaves. They found two senescence-related markers, cytosolic glutamine synthetase (GS1) and glutamate dehydrogenase (GDH), which were mainly involved in the homeostasis of nitrogen status via nitrogen mobilization. They have observed differential regulation of nitrogen cell status upon pathogen attack, elicitors and by stress hormones. Their data validate GDH and GS1 association in leaf senescence and plant defense response upon pathogen interaction. Interestingly, they have seen that during natural senescence, the expression of GS2 and Nia decreased under given stress conditions. Conversely, GS1 and GDH messenger RNA (mRNA) was found to be enhanced. Differential expression of GS1 and GDH was observed depending on the given stress, which suggests that GS1 appeared to be more selective than GDH. Natural plant senescence and stress- induced senescence links were conferred, showing the prominence of GS1 and GDH in plant defense (Pageau et al., 2006).
3.2 Leaf Senescence-Associated Gene Homolog Markers Senescence is the ultimate phase of development in plant tissue. During this process, several senescence-associated genes (SAGs) have been validated that showed transcriptional upregulation. Kajimura et al. (2010) reported the isolation of nine crucial wheat SAG complementary deoxyribonucleic acid (cDNA) clones named TaSAG1-TaSAG9, and also assessed the feasibility of the SAG homologs as molecular markers based on their expression patterns. They have demonstrated the utilization of leaf SAG homologs as developmental markers in common wheat. These nine wheat SAGs were identified, validated, and established in wheat expressed sequence tag (EST) libraries based on homology to rice SAGs. Interestingly, they found that these wheat SAG transcripts were enhanced during natural senescence, as well as dark-induced senescence, in seedling leaves. However, the nine wheat SAGs also displayed variable expression patterns in developing and ripening seeds. They also showed the significance of transcript accumulation patterns of TaSAG5 and TaSAG6 that were enhanced linearly during the course of their studies on the flag leaf and seed. These could be utilized as molecular markers to assess the fate of wheat flag leaf senescence and seed maturation. Additionally, the transcript accumulation levels of the other six SAGs were found to be enhanced before the obvious necrotic cell death of seeding leaves displaying wheat hybrid necrosis (Kajimura et al., 2010). Their data validate that necrotic cell death in wheat hybrid necrosis is related to senescence and defense.
276
17. Senescence-Associated Markers
3.3 Characterization of Leaf Senescence Markers in Arabidopsis Using Metabolic Profiling A number of metabolic markers that differentiate Arabidopsis lines on the basis of senescence progress are crucial. Diaz et al. (2005) have characterized and determined markers for the variability of leaf senescence in Arabidopsis using metabolic profiling. Briefly, five recombinant inbred lines (RILs) of the Bay-0 × Shahdara differential leaf senescence phenotypes populations (from early senescing to late senescing) were studied to investigate the metabolic markers based on senescence-dependent metabolic changes. Studies have compared the extent of leaf senescence based on the genetic context of the diverse RILs of Arabidopsis (e.g., Arabidopsis thaliana; Diaz et al., 2005). They have characterized markers to decide the degree and variability of leaf senescence. Diaz et al. (2005) have shown differential leaf senescence phenotypes. Further, they have investigated metabolic markers that could differentiate Arabidopsis lines on the basis of senescence-dependent metabolism changes. Interestingly, the levels of gamma-aminobutyric acid, aspartate, leucine, isoleucine, and glutamate were estimated and correlated with parameters of age and the senescence phenotype of the RILs. Additionally, early variances were observed in the glycine/serine ratio. Their data suggest that these markers could be studied as predictive indicator for plant senescence behavior. Prognostic indicators for plant senescence behavior were established (Diaz et al., 2005). Amazingly, they also have noticed differences in the mobilization efficiency of asparagine, glutamine, and sulfate in late-senescing lines than early-senescing lines.
3.4 SSR Markers for Leaf Chlorophyll Content, Flag Leaf Senescence, and Cell Membrane Stability Elshafei et al. (2013) reported identification of novel markers linked to leaf senescence (i.e., leaf chlorophyll content, flag leaf senescence) and cell membrane stability traits in wheat plants under stress conditions. They have mapped the quantitative trait locus (QTL) for the three physiological traits. To identify the simple sequence repeat (SSR) markers, researchers have made drought-sensitive (Yecora Roja) and drought-tolerant (Pavon 76) genotypes. The SSR markers for physiological traits had genetic distances that have potential and could be used in breeding programs for drought tolerance in wheat (Elshafei et al., 2013).
3.5 Delayed Senescence Markers in Transgenic Tobacco Phytohormones such as cytokinins (CKs) are involved in vital functioning of plants growth and development. CKs are involved in cellular mechanisms at the cellular level by affecting cell division. Their roles in regulating the expression of photosynthetic-associated genes are well established. CKs hold great significance for its antisenescing action and have been utilized to delay the onset of plant senescence, efficient output, and postharvest storage. Skowron et al. (2016) have reported molecular markers related to delayed senescence in transgenic tobacco with higher CK levels and investigated the effect of enhanced CK biosynthesis on the onset of leaf senescence. Tobacco plants with genetically engineered gene for CK biosynthesis were designed and expressed in a senescence-related manner. These were further used for
3 Senescence-Associated Markers in Plants
277
analyzing the physiological and molecular markers. The authors have designed transgenic tobacco plants with an inserted gene pathway, ipt, fused with a senescence-specific promoter, PSAG12. Further, the effects of elevated hormone level in transgenic tobacco resulted in decreased chlorophyll, soluble proteins, and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) degradation in plants exposed to 9 days of light withdrawal. They have explored the conceivable association between the enhanced CK synthesis and antioxidant properties of PSAG12-IPT plants by considering three isoforms of superoxide dismutase (SOD; Skowron et al., 2016). The data signify the importance of molecular and physiological basis of the role of CKs in inhibiting leaf aging.
3.6 Telomere Shortening as a Marker of Cellular Senescence Senescent cells comprising replicative senescence and stress-induced premature senescence of all the tissues are alike and metabolically active. Although there are certain in vitro and in vivo characteristics that are known as biomarkers of aging and cellular senescence. Bernadotte et al. (2016) reported on markers of cellular senescence and demonstrated telomere shortening as a marker of cellular senescence. The authors have described the mechanisms of aging and elaborated the role of telomerase in this process. Telomeres have been established as an indicative of aging, as well as a possible factor in determining life expectancy. The telomere is a promising marker of cellular senescence; telomere theory is based on the telomere-shortening mechanism. Many researchers believe that the chief marker (telomere-shortening) of aging is actually the cause of aging. This is usually used to indicate cellular senescence. During the course of replication, telomeres get shorter with every cell division. Rationality of telomere shortening as a marker for cellular senescence was established on the basis of theoretical and experimental research.
3.7 Transcriptomic and Metabolomic Data to Characterize Leaf Senescence Markers Moschen et al. (2016) have validated the network and bio-signature analysis for the integration of transcriptomic and metabolomic data to illustrate leaf senescence in sunflowers. Weighted gene correlation network analysis and bio-signature discovery were performed to integrate transcriptomic and metabolomic data. Interestingly, 11 metabolites and 19 transcription factors (TFs) were found to be potential biomarkers. Moschen et al. (2016) have used an efficient tool for characterizing the chosen genes and metabolites that may be involved during the triggering and development of the leaf senescence process. The methodology offered great assistance in interpreting and foreseeing novel potential biomarkers of leaf senescence and aging in sunflower.
3.8 RAPD and ISSR Markers Associated With Leaf Senescence Milad et al. (2011) have reported random amplification of polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers associated with flag wheat leaf senescence during water stress. Flag leaf senescence is an imperative cause that reduced the crop yield under
278
17. Senescence-Associated Markers
stress. Studies have identified molecular markers associated with the flag leaf senescence gene in drought-sensitive genotype (variant-1) and drought-tolerant genotype (Veery) wheat under water-stressed conditions. Several RAPD and ISSR primers were established for polymorphism amid parental genotypes and F2 populations. Remarkably, their data validate four RAPD and two ISSR markers linked to the flag leaf senescence gene in wheat. Additionally, these four RAPD and two ISSR markers were related to QTL for the flag leaf senescence gene as an indicator of drought tolerance. These markers have potential and can be applied in wheat-breeding programs that would assist in selection of early generations.
3.9 Redox Markers for Drought-Induced Senescence Marquez-Garcia et al. (2015) have studied redox markers for drought-induced nodule senescence in soybean (Glycine max) leaves. Drought and water scarcity affect crop productivity and have a huge impact on the agricultural economy. With changing climate, drought stress-tolerant crops hold a prodigious amount of potential to enhance crop productivity. In this study, the authors compared the effects of drought on soybean leaves and nodules to describe phenotypic markers and changes in the cellular redox state, as well as the associations between leaf and nodule senescence during drought. They have observed that the nodule ascorbate pool was reduced considerably less during drought. Moreover, greater levels of transcripts encoding two peroxiredoxins were identified in nodules under drought stress; however, senescence-associated transcripts encoding redox-related proteins were comparable under both conditions.
3.10 REVOLUTA and WRKY53 Developmental Markers in Arabidopsis Xie et al. (2014) reported REVOLUTA and WRKY53 markers for leaf development in Arabidopsis. The growth and development of plants usually change with external environmental changes. During these changes, plants persuade leaf senescence to reallocate nutrients and from the leaves to the maturing seeds. The authors showed that class III homeodomain leucine zipper (HD-ZIPIII) TFs play a role in controlling the onset of leaf senescence in Arabidopsis. Additionally, other downstream genes, such as the HD-ZIPIII protein REVOLUTA (REV), play crucial roles in environment-controlled physiological processes. Studies have reported that REV acts as a redox-sensitive TF that positively regulates the expression of WRKY53, a chief regulator of age-induced leaf senescence. HD-ZIPIII proteins are vital for inducing WRKY53 in response to oxidative stress, and mutations in HD-ZIPIII genes strongly delay the onset of senescence. This work proposes cross-talk between early and late stages of leaf development that could significantly contribute to plant developmental studies and ultimately crop productivity.
4 SUMMARY Physiological and biochemical studies have contributed to the present understanding of plant senescence. In this chapter, we have discussed the history and current state if molecular investigations, molecular markers for senescence, and their involvement in senescence regulation mechanisms. Additionally, advance molecular approaches to the manipulation of
279
4 Summary
SSR marker
Telomere
Senescence-associated genes (SAGs)
RAPD and ISSR markers
Glutamate dehydrogenase
Markers Cytosolic glutamine synthetase
WRKY53 marker
Redox marker
Multiple omic data analysis (organelle, cell, organization) • Metabolomics • Proteomics • Transcriptomics • Genomics • Epigenomics • Phenomics
Tools to studies and analysis of senescence mechanism
FIG. 2 Plant senescence markers that have been utilized to study the process of senescence.
plant senescence were focused. Senescence has a huge impact on agricultural economy and crop productivity, which gives senescence studies a great impact. Therefore, studying the senescence process and senescence markers will contribute in-depth knowledge about plant development and thereby helps to manipulate and design strategies to delay senescence for higher crop yields, as well as for potential agricultural applications. Several advanced techniques and methods have been utilized to explore the senescence process and mechanisms, combining metabolic profiling and transcriptome analysis to achieve a comprehensive understanding about molecular regulation that would provide a smarter way to study plant metabolism and regulation during plant senescence (Fig. 2). Controlling senescence through breeding or genetic engineering might work to improve crop yield. It has been reported that delayed leaf senescence was caused by autoregulatory production of the CK hormone. In addition, studies have shown in maize plants, the knitted-I senescence specific gene was responsible for delayed senescence. These studies provide information that could help to delay the senescence and enhance the shelf life of plants. Development of advanced reliable and novel biomarker for senescence could help to predict early signs of senescence during cellular and stress conditions.
Acknowledgments Financial support from National Institute of Health (NIH) Grants R21-HL128141-01A1 and partial financial support from the Department of Immunology, Herbert Wertheim College of Medicine, Florida International University are greatly acknowledged.
280
17. Senescence-Associated Markers
Conflict of Interest The authors have declared no conflict of interest.
References Bassham, D.C., 2007. Plant autophagy–more than a starvation response. Curr. Opin. Plant Biol. 10, 587–593. Bernadotte, A., Mikhelson, V.M., Spivak, I.M., 2016. Markers of cellular senescence. Telomere shortening as a marker of cellular senescence. Aging (Albany NY) 8 (1), 3–11. Bleecker, A.B., 1998. The evolutionary basis of leaf senescence: method to the madness? Curr. Opin. Plant Biol. 1, 73–78. Brusslan, J.A., Alvarez-Canterbury, A.M.R., Nair, N.U., Rice, J.C., Hitchler, M.J., Pellegrini, M., 2012. Genome-wide evaluation of histone methylation changes associated with leaf senescence in Arabidopsis. PLoS ONE 7, e33151. Diaz, C., Purdy, S., Christ, A., Morot-Gaudry, J.F., Wingler, A., Masclaux-Daubresse, C., 2005. Characterization of markers to determine the extent and variability of leaf senescence in Arabidopsis. A metabolic profiling approach. Plant Physiol. 138 (2), 898–908. Elshafei, A.A., Mohamed, S., Al-Doss, A.A., Moustafa, K.A., Al-Qurainy, F.H., Barakat, M.N., 2013. Identification of new SRAP markers linked to leaf chlorophyll content, flag leaf senescence and cell membrane stability traits in wheat under water-stressed condition. Aust. J. Crop. Sci. 7 (6), 887–893. Feller, U., Fischer, A., 1994. Nitrogen metabolism in senescing leaves. Crit. Rev. Plant Sci. 13, 241–273. Fraga, M.F., Esteller, M., 2007. Epigenetics and aging: the targets and the marks. Trends Genet. 23, 413–418. Fukuda, H., 1996. Xylogenesis: initiation, progression, and cell death. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 299–325. Giovannoni, J.J., 2007. Fruit ripening mutants yield insights into ripening control. Curr. Opin. Plant Biol. 10, 283–289. Guo, Y., Gan, S., 2005. Leaf senescence: signals, execution, and regulation. Curr. Top. Dev. Biol. 71, 83–112. Guo, Y., Gan, S., 2006. AtNAP, a NAC family transcription factor, has an important role in leaf senescence. Plant J. 46, 601–612. Hinderhofer, K., Zentgraf, U., 2001. Identification of a transcription factor specifically expressed at the onset of leaf senescence. Planta 213, 469–473. Hopkins, M., Taylor, C., Liu, Z., Ma, F., McNamara, L., Wang, T., Thompson, J.E., 2007. Regulation and execution of molecular disassembly and catabolism during senescence. New Phytol. 175, 201–214. Hortensteiner, S., 2004. The loss of green color during chlorophyll degradation—a prerequisite to prevent cell death? Planta 219, 191–194. Jansson, S., Thomas, H., 2008. Senescence: developmental program or timetable? New Phytol. 179, 575–579. Jeyapalan, J.C., Sedivy, J.M., 2008. Cellular senescence and organismal aging. Mech. Ageing Dev. 129, 467–474. Kajimura, T., Mizuno, N., Takumi, S., 2010. Utility of leaf senescence-associated gene homologs as developmental markers in common wheat. Plant Physiol. Biochem. 48 (10–11), 851–859. Keskitalo, J., Bergquist, G., Gardeström, P., Jansson, S., 2005. A cellular timetable of autumn senescence. Plant Physiol. 139, 1635–1648. Leshem, Y.Y., 1988. Plant senescence processes and free radicals. Free Radic. Biol. Med. 5, 39–49. Lim, P.O., Kim, H.J., Nam, H.G., 2007. Leaf senescence. Annu. Rev. Plant Biol. 58, 115–136. Marquez-Garcia, B., Shaw, D., Cooper, J.W., Karpinska, B., Quain, M.D., Makgopa, E.M., Kunert, K., Foyer, C.H., 2015. Redox markers for drought-induced nodule senescence, a process occurring after drought-induced senescence of the lowest leaves in soybean (Glycine max). Ann. Bot. 116 (4), 497–510. Matile, P., Hörtensteiner, S., Thomas, H., 1999. Chlorophyll degradation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 67–95. McFadyen, L., Robertson, D., Sedgley, M., Kristiansen, P., Olesen, T., 2012. Time of pruning affects fruit abscission, stem carbohydrates and yield of macadamia. Funct. Plant Biol. 39, 481–492. Milad, S.I., Wahba, L.E., Barakat, M.N., 2011. Identification of RAPD and ISSR markers associated with flag leaf senescence under water-stressed conditions in wheat (Triticum aestivum L.). Aust. J. Crop. Sci. 5 (3), 334–340. Miller, J.D., Arteca, R.N., Pell, E.J., 1999. Senescence-associated gene expression during ozone-induced leaf senescence in Arabidopsis. Plant Physiol. 120, 1015–1024.
REFERENCES 281
Moschen, S., Higgins, J., Di Rienzo, J.A., Heinz, R.A., Paniego, N., Fernandez, P., 2016. Network and biosignature analysis for the integration of transcriptomic and metabolomic data to characterize leaf senescence process in sunflower. BMC Bioinform. 17 (Suppl. 5), 174. Oh, S.A., Park, J.H., Lee, G.I., Paek, K.H., Park, S.K., Nam, H.G., 1997. Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana. Plant J. 12, 527–535. Ougham, H., Armstead, I., Howarth, C., Galyuon, I., Donnison, I., Thomas, H., 2007. The genetic control of senescence revealed by mapping quantitative trait loci. Annu. Plant Rev. 26, 171–201. Pageau, K., Reisdorf-Cren, M., Morot-Gaudry, J.F., Masclaux-Daubresse, C., 2006. The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves. J. Exp. Bot. 57 (3), 547–557. Skowron, E., Trojak, M., Sobala, T., 2016. Molecular markers of delayed senescence in transgenic tobacco with enhanced cytokinin level. World Sci. News 51, 13–25. Suzuki, Y., Makino, A., 2013. Translational downregulation of RBCL is operative in the coordinated expression of Rubisco genes in senescent leaves in rice. J. Exp. Bot. 64, 1145–1152. Taesakul, P., Siriphanich, J., Van Doorn, W.G., 2015. Two abscission zones proximal to Lansium domesticum fruit: one more sensitive to exogenous ethylene than the other. Front. Plant Sci. 6, 264. Thomas, H., Donnison, I., 2000. Back from the brink: plant senescence and its reversibility. In: Programmed Cell Death in Animals and Plants. Bios, Oxford, pp. 149–162. Thomas, H., Sadras, V.O., 2001. The capture and gratuitous disposal of resources by plants. Funct. Ecol. 15, 3–12. Thomas, H., Ougham, H., Wagstaff, C., Stead, A.J., 2003. Defining senescence and death. J. Exp. Bot. 54, 1127–1132. Uauy, C., Distelfeld, A., Fahima, T., Blechl, A., Dubcovsky, J., 2006. A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314, 1298–1301. Umehara, M., Hanada, A., Yoshida, S., Akiyama, K., Arite, T., Takeda-Kamiya, N., Magome, H., Kamiya, Y., Shirasu, K., Yoneyama, K., 2008. Inhibition of shoot branching by new terpenoid plant hormones. Nature 455, 195. Van Doorn, W.G., Stead, A.D., 1997. Abscission of flowers and floral parts. J. Exp. Bot. 48, 821–837. Watanabe, M., Balazadeh, S., Tohge, T., Erban, A., Giavalisco, P., Kopka, J., Mueller-Roeber, B., Fernie, A.R., Hoefgen, R., 2013. Comprehensive dissection of spatiotemporal metabolic shifts in primary, secondary, and lipid metabolism during developmental senescence in Arabidopsis. Plant Physiol. 162, 1290–1310. Williams, A.G., Whitham, T.G., 1986. Premature leaf abscission: an induced plant defense against gall aphids. Ecology 67 (6), 1619–1627. Xie, Y., Huhn, K., Brandt, R., et al., 2014. REVOLUTA and WRKY53 connect early and late leaf development in Arabidopsis. Development (Cambridge, England) 141 (24), 4772–4783. Zimmermann, P., Zentgraf, U., 2005. The correlation between oxidative stress and leaf senescence during plant development. Cell. Mol. Biol. Lett. 10, 515–534.