1. Introduction
1.3. The Molecular Process of Senescence
1.3.4. Whole transcriptome changes during leaf senescence
The onset and progress of senescence is accompanied by large changes in the plant transcriptome. Many hundreds of genes increase or decrease in expression level to facilitate the cellular changes that occur during senescence (Buchanan-Wollaston
et al., 2005; Breeze et al., 2011). These large scale transcription changes need to be
carefully controlled to ensure they occur in the correct spatial and temporal patterns. While there are many methods of regulation, control of transcription is arguably the most important since it is the first step to producing an active molecule, i.e., without transcription no functional product can be produced.
Since the process of senescence involves large scale transcriptome changes in a precise and defined order, the process of transcription can be monitored through
whole-genome expression changes (Breezeet al., 2011). In leaf 7 of Arabidopsis Col
0, grown in 16 hour day conditions, the major switch from a mature plant organ to a senescing leaf occurs at approximately 29 to 33 days after sowing (DAS, Breeze
et al., 2011). At around this time point, there is a clear and discernible decrease in
expression of genes relating to photosynthesis and cell maintenance and a concurrent increase in expression of genes relating to hormone biosynthesis, degradation and
transport (Breeze et al., 2011). However, the temporal transcriptome changes can
be explored further, showing distinct phases of gene expression over the course of a senescence in a leaf.
1.3.4.1. Decrease in expression of photosynthetic machinery related genes
The process of senescence can begin prior to leaves reaching full expansion. In Ara- bidopsis grown in 16 hour days, visible symptoms of senescence appear approximately 35 days after sowing, however, decrease in the expression of translation machinery
related genes occurs at approximately 19 DAS (Breezeet al., 2011). This includes
expression of genes involved in amino acid biosynthesis, tRNA aminoacylation and ribosomal protein synthesis. This indicates that retardation of the ribosomal expan-
sion is occurring, causing de novo protein synthesis to decrease as the leaf reaches
full maturity.
At around 23 days after sowing, genes involved in redirecting the tetrapyrrole syn- thesis pathway to chlorophyll biosynthesis are decreased in expression, suggesting synthesis of chlorophyll halts. Similarly, the expression of enzymes involved in the
carotenoid synthesis pathway (LUTEIN DEFICIENT 1, 2 and 5) decreases, thus
the production of carotenoid for the light harvesting complex II structure decreases alongside chlorophyll biosynthesis. This suggests the continuous production of pho- tosynthetic components halts at around 23 days after sowing, which leads to a decline in photosynthesis shortly after.
Concurrently with the decline in expression of genes relating to photosynthesis, expression of genes relating to regulatory pathways increases. Expression of JA and ABA biosynthesis increases at 23 DAS which is shortly followed by an increase in JA and ABA levels at approximately 25 DAS, alongside expression of JA and
ABA responsive genes (Breeze et al., 2011). Similarly, at this point a number of
transcription factors from the NAC, WRKY, NFY and Zinc-finger protein families are increased in expression, to modulate the downstream transcriptome changes. The increase in expression genes relating to hormone biosynthesis and transcription factor is brief and appears to reduce shortly after, suggesting there is a ‘burst’ of signalling, which triggers the ensuing molecular changes.
tional change occurs. At approximately 25 DAS, expression of genes relating to the carbon fixation decreases, including two members of the Rubisco small subunit. This reduction correlates with a drop in total protein content of the leaf, suggesting photosynthetic activity reduces and halts.
Consistent with this, over the next 5 - 10 days expression of many genes relating
to photosynthesis declines, includingLIGHT HARVESTING COMPLEX BINDING
PROTEINS (LHCB), which coordinate the photosystems of plant chloroplasts (Jans-
son, 1994) and the transcription factorGOLDEN2-LIKE (GLK2), which promotes
expression of photosynthesis apparatus (Waters et al., 2008). The decrease in ex-
pression of these genes appears to occur gradually over the course of senescence and some expression is detectable late into the process, perhaps indicating that some photosynthesis needs to occur to provide sufficient energy for metabolism and remo- bilisation.
1.3.4.2. Genes increased in expression during leaf senescence
Like the decrease in expression of photosynthetic genes, expression of genes related to degradation and recycling begin before the leaf has fully expanded. Expression of autophagy related genes are high at 21 DAS, indicating autophagic mechanisms
may be active prior to the onset of senescence (Breeze et al., 2011). Autophagy, or
‘self-eating’, is a form of internal recycling conserved across all eukaryotic organisms (Hughes & Rusten, 2007), involving the transport of organelles and/or cytoplasm
into the vacuole for degradation (Avila-Ospina et al., 2014). The prevailing theory
on autophagy is that it exists to clear unwanted cell components and maintains homeostasis. However, it differs from other degradatory mechanisms in that it is more involved with degradation of long-lived proteins and organelles, as opposed to the rapid breakdown of short lived molecules. Autophagy activity appears to increase
during senescence and nutrient deprivation or dark-treatment (Hanaokaet al., 2002;
Doelling et al., 2002; Xiong et al., 2005), suggesting it may have a role in clearing
and recycling molecules during stress.
If autophagy was a component of senescence, it may be expected that autophagy mutants would exhibit delayed senescence. Instead, it has been repeatedly observed
that autophagy mutants show accelerated senescence (Hanaokaet al., 2002; Doelling
et al., 2002; Xiong et al., 2005; Phillips et al., 2008). This possibly indicates that
autophagy is involved in the clearance of damaged organelles and oxidative species, thus maintaining homeostasis and allowing the cell to continue. In the absence of autophagy, the cell loses viability prematurely compared to the wild type equivalent, therefore senescence is initiated early and uses other mechanisms to clear proteins.
By 27 DAS, expression of metabolism related genes begins to increase. In partic-
ular, ß-carotene hydroxylases and the carotenoid cleavage enzymes CAROTENOID
catabolise carotenoid compounds in chloroplasts (Biswal, 1995). At a similar time,
expression of two metacaspases,METACASPASE 1 and 2, increase. Metacaspases
have been implicated in promoting programmed cell death (Collet al., 2010), there-
fore they may induce the final step of cell death during senescence. However no functional link between metacaspase expression and senescence has been observed.
Shortly after this, cell wall degradation related enzymes begin to increase in expres- sion levels. Pectinesterases, xylosidases, glucosyl hydrolases, ß-glucosidases, pectate lyases and pectin methylesterase inhibitors are expressed to degrade the cell wall and
perhaps release sugars for use in respiration (Leeet al., 2007). Expression of many
other catabolic enzymes is then induced, including many carbohydrate-degrading en- zymes such as pectinesterases, glycosyl transferases, glucosyl transferases and poly- galacturonases. This represents the catabolic phase of senescence, where intracellular components are degraded for recycling.
Alongside the increase in expression of enzymes for carbon based molecule degrada- tion, expression of many protease enzymes increases. Proteolytic activity increases
dramatically during senescence (Roberts et al., 2012), while treatment of tobacco
with protease inhibitors blocks the degradation of rubisco and retards senescence
(Carrionet al., 2013).
In particular, cysteine proteases are frequently associated with senescence, includ-
ing the senescence specific gene SAG12 (Grbic, 2003). SAG12 encodes a cysteine
protease that is uniquely expressed towards the final stages of senescence, alongside the yellowing observed as chlorophyll is degraded (Grbic, 2003). However, mutants of SAG12 do not show any phenotype, suggesting functional redundancy in the
proteolytic mechanisms (Oteguiet al., 2005).
In addition to SAG12, a number of serine- aspartic and metalloproteases are also
expressed in senescing tissue and appear to play important roles (Roberts et al.,
2012). This is not a disorganised burst of proteolytic activity, instead each protease appears to have highly restricted targets and functions, defined by its subcellular localisation and expression profile. Genomic analysis reveals a number of proteases include plastid targeting peptides (Adam & Clarke, 2002), suggesting a subset of proteases are targeted to the plastids and conduct protein degradation there. In
particular, the Zn2+ dependent metalloprotease FtsH6 has been implicated in de-
grading the LHCB3 protein of photosystem II during dark-induced senescence, high-
light treatment andin vitro (Żeliskoet al., 2005). However, mutants deficient in the
FtsH6 gene did not show altered rate of degradation of photosystem II during senes-
cence, suggesting functional redundancy between FtsH metalloproteases (Wagner
et al., 2011).
No proteases that are targeted to chloroplasts appear to breakdown Rubisco. In- stead, during senescence Rubisco is exported to the chloroplast membrane and ex- pelled by budding to form double membrane bound vacuoles known as Rubisco con-
a manner similar to autophagy (Avila-Ospina et al., 2014).
Perhaps the best understood degradation organelle formed during senescence are senescence associated vacuoles (SAVs). These small bodies accumulate in chloroplast
containing cells during senescence (Otegui et al., 2005). They are characterised
by high proteolytic activity and a pH approximately 0.8 lower then the cytosol,
possibly to facilitate protease activity (Otegui et al., 2005). The number of SAVs
directly correlates with the rate of chloroplast degradation and the majority of the
protease activity in senescing tissue (Martínez et al., 2008; Carrion et al., 2013).
Plastid targeted GFP relocates to SAVs during senescence (Otegui et al., 2005),
while chlorophyll A and rubisco were identified in SAVs using antibodies and HPLC
respectively (Martínezet al., 2008).
1.3.4.3. Control of gene expression through regulation of transcription
The coordinated gene expression of many thousands of genes brings about the ap- propriate and controlled decrease in photosynthetic maintenance followed by cellular degradation observed in senescing tissue. It is the correct spatial and temporal pat- terns that allow nutrients to be remobilised efficiently and effectively for continued growth of the plant. Control of this process begins at the transcriptional level.
Within the nucleus, genomic DNA is tightly packed around histone proteins, which are themselves condensed to form nucleosomes. DNA within this tightly coiled bun- dle is inaccessible to RNA polymerase and therefore genes within this region are unable to be transcribed and their expression is repressed. This level of packaging is dependent on factors such as histone modifications and DNA methylation which are highly adaptive to external stimuli (reviewed in Chinnusamy & Zhu, 2009). Modifi- cations to histone proteins such as acetylation will disrupt the protein-interactions that cause DNA to be condensed, therefore opening the DNA which can then be tran- scribed. Techniques such as DNase-seq have begun to elucidate the nature of DNA
packaging in plant development (Zhang et al., 2012b), but have yet to be applied to
plant stress responses.
After DNA is unpacked and exposed to cellular machinery, genes encoded along the newly accessible DNA are then available for transcription to RNA. Transcrip- tion of DNA to mRNA in Arabidopsis is primarily catalysed by the enzyme RNA POLYMERASE II, which recognises recognition and assembly of basal transcription apparatus at the core promoter, located on average 70 base pairs upstream of the mRNA coding region of a gene (Smale & Kadonaga, 2003). The best characterised core promoter region is the TATA box, a DNA motif rich in T/As located upstream of the transcription start site in 30-40% of eukaryotic genes, including Arabidopsis (Smale & Kadonaga, 2003; Molina & Grotewold, 2005). The TATA box is recognised by the TATA-recognition protein (TBP) and is usually located 25-35 base pairs up- stream from the transcription start site. The binding of the TBP and associated
proteins form the pre-initiation complex which facilitates the binding of RNA Poly- merase II, which acts as the first step to transcription.
The formation of the pre-initiation complex is promoted or repressed by auxiliary proteins known as transcription factors (TFs). The binding of one or more tran- scription factors to the DNA upstream of a coding region facilitates or prevents the formation of the pre-initiation complex and therefore promotes or represses transcrip- tion. Transcription factors form transient non-covalent interactions with sequences
of DNA 5 - 31bp long (Stewart et al., 2012), known as DNA motifs or recognition
sequences. The protein:DNA bonds are inherently weak to allow them be dynamic and respond to stimuli. Indeed, predictions from yeast indicate a higher number of low-specificity DNA-binding motifs are preferable to a lower number of more specific DNA binding motifs (Bilu & Barkai, 2005; Geisel & Gerland, 2011). This is because the higher number of motifs allows modulation of target gene expression through the binding of multiple transcription factors in different combinations. In addition, the higher number of motifs allows greater flexibility in individual recognition sites (Stewart & Plotkin, 2013). Therefore transcription factors offer one of the major sources of dynamic regulation in eukaryotic species.
Arabidopsis contains a disproportionately large number of transcription factors, with approximately 1716 coding sequences representing ~5% of the total number of
genes (Jin et al., 2014). Each of these transcription factors will regulate a set of
targets, which include, but are not exclusive to, other regulatory proteins. Research into the developmental biology of Arabidopsis has highlighted that small numbers of transcription factors regulate a large number of other transcription factors, therefore acting as a ‘master regulator’, coregulating large numbers of genes (Meyerowitz, 2002;
Kaufmannet al., 2010). The regulatory genes targeted by this master regulator will
in turn target ‘workhorse’ genes, which perform the functional changes in the cell
(Kaufmann et al., 2010). Manipulation of the function of these master regulators
has dramatic phenotypic effects (Meyerowitz, 2002; Kaufmannet al., 2010).
Multiple transcription factors have been identified as master regulators of senes- cence. The NAC family transcription factor ANAC092 has been studied in particular detail.
1.3.4.4. ANAC092/ORE1/AtNAC2
The NAC family of transcription factors
The NAC proteins are a large family of plant specific transcription factors char-
acterised by a unique NAC domain (Riechmann et al., 2000; Olsen et al., 2005b).
NAC transcription factors were originally identified as two independent proteins,
namedNAM (NO APICAL MERISTEM) from Petunia andCUC2 (CUP-SHAPED
COTYLEDON 2) from Arabidopsis, so named for the developmental defects induced
parison between the two coding sequences revealed a conserved domain that was
shared with two other proteins in Arabidopsis, subsequently named ARABIDOP-
SIS TRANSCRIPTION ACTIVATOR (ATAF) 1 and 2 (Aida et al., 1997). The
conserved domain was subsequently named the NAC domain, forNAM, ATAF and
CUC2. Whole genome sequencing and phylogenetic analysis has now revealed this
same domain conserved in 110 genes in Arabidopsis (Jensen et al., 2010), as well as
in many closely related genes of rice (Nuruzzaman et al., 2010), soybean (Leet al.,
2011), potato (Singh et al., 2013), barley (Christiansen et al., 2011), Populus (Hu
et al., 2010) and Physchomitrella (Rensinget al., 2008). No NAC proteins have been
identified in non-plant eukaryotes. Indeed, NAC proteins have been implicated in the development of specialised water transporting plant tissue, suggesting NAC pro-
teins are partly responsible for adaptation of plants to life on land (Xu et al., 2014),
therefore one of the key family of genes separating plants from other eukaryotic species.
The conserved NAC domain originally identified through NAM and CUC2 remains the defining feature of NAC proteins. It is always located at the N-terminus and
primarily involved with DNA-binding and dimerisation (Olsen et al., 2005a). The
C-terminal region of NAC proteins is more diverse and involved in transcriptional
activation and binding to auxiliary proteins (Jensen et al., 2010). The structure of
the NAC domain has been analysed in detail through crystallography of ANAC019,
which has been used as a model for NAC protein structure (Olsen et al., 2003;
Ernst et al., 2004; Welner et al., 2012). The 168 amino-acid NAC domain present
in ANAC019 shows a largely beta-sheet structure composed of 5 subdomains, whose
sequence does not conform to other identified DNA-binding structures (Ernstet al.,
2004). Studies of the crystal structure and in-solution protein revealed that the NAC domain interacts with DNA by inserting the conserved amino acid sequence
WKATGTD into the major groove of DNA (Welner et al., 2012). The WRKY
transcription factors from plants and GCM transcription factors from Drosophila
interact with DNA in a similar manner (Cohen et al., 2003; Yamasaki et al., 2012),
but using different sequences (Welner et al., 2012). This could indicate a highly
ancestral common ancestor, or alternatively it could be an example of convergent evolution.
The identification of the NAC consensus recognition sequence, i.e. the sequence of DNA that is recognised by all of the NAC proteins has been the subject of much study (Jensen & Skriver, 2014). The first recognition sequence for a NAC protein was identified by yeast 1-hybrid for ANAC019. The protein was identified to recognise a
core CGTG sequence in theEARLY RESPONSIVE TO DEHYDRATION STRESS
1 (ERD1) promoter region (Tran et al., 2004), which was confirmed to be the recog-
nition sequence using thein vitro technique EMSA and SELEX (Olsenet al., 2005a;
Buet al., 2008) . Crystallographic studies indicated ANAC019 dimers bound to two
base pairs (Welner et al., 2012). In many subsequent studies, the core CGT[G/A]
motif or similar has been identified as critical to NAC-DNA interactions (reviewed in Jensen & Skriver 2014), however a limited number have identified alternative se- quences. For example, ATAF2 has been identified to bind to a low specificity 35 base pair motif (Wang & Culver, 2012) and CBNAC was shown to bind to a GCTT
sequence in thePR1 promoter region (Kimet al., 2012). A protein-binding microar-
rray experiment for 12 Arabidopsis NAC proteins revealed differences between many of the NAC recognition sequence for different transcription factors, showing some
consistency within particular clades of NAC proteins (Lindemose et al., 2014). As
such, the CGT[G/A] appears to be a core sequence of NAC recognition, but this may vary based on the particular NAC gene in question.
The more variable C-terminal domain of NAC proteins has not been identified to have DNA-binding properties, but has been shown to act as a transcriptional acti-
vator (Tran et al., 2004; Taoka et al., 2004; Jensen et al., 2010) or repressor (Kim
et al., 2007a; Yamaguchi et al., 2010). Ten Arabidopsis NAC transcription factors
representing a wide spread of NAC phylogenetics and functions have been analysed for transactivation capability in yeast. In 9 cases, the transactivation capability was encoded by the C-terminal domain. The C-terminal domain of NAC proteins does not conform to a particular tertiary structure, instead it has been shown to remain flexible and form transient structures in some cases, a property known as ‘protein
intrinsic disorder’ (Kjaersgaard et al., 2011). Phylogenetic analysis has indicated
that the C-terminal domains of NAC domains are mostly non-conserved and instead
contain conserved short motifs (Jensenet al., 2010). Since C-terminal domains pro-
vide the transcriptional function of NAC proteins, their low conservation implies that NAC proteins are capable of a wide range of transcriptional activity.
NAC transcription factors were originally shown to regulate developmental signals,
where mutants exhibited severe developmental defects (Soueret al., 1996; Aidaet al.,
1997). Many NAC proteins are known to regulate particular developmental signals
such as lateral root formation (Xie et al., 2002; He et al., 2005), organ boundary
formation (Weiret al., 2004), secondary wall biosynthesis (Zhonget al., 2006, 2007b),
xylem cell speciation (Yamaguchiet al., 2010) and many more. Many NAC proteins
function in stress-responsive signals (Puranik et al., 2012; Nakashima et al., 2012).
NAC proteins have been shown to be involved in response to dehydration (Fujita
et al., 2004; Lu et al., 2006), salt-stress (He et al., 2005; Balazadeh et al., 2010b),
pathogen response (Bu et al., 2008; Wu et al., 2009; Wang & Culver, 2012; Kim
et al., 2012; Hickman et al., 2013), low oxygen levels (Christianson et al., 2009),
oxidative stress (Woo et al., 2004; Balazadeh et al., 2011; Wu et al., 2012b), age-
related resistance (Al-Daoud & Cameron, 2011) and heat stress (Shahnejat-Bushehri
et al., 2012). A small number, including NAP (Guo & Gan, 2006), JUB1 (Wuet al.,
2012a), NAC016 (Kimet al., 2013) and ANAC092 (Ohet al., 1997) have been shown
ANAC092 is a major positive promoter of leaf senescence
ANAC092 was originally identified by Ohet al.(1997). A population of Arabidopsis
were mutagenised using EMS, followed by studying them for a delayed senescence phenotype. Redundant mutations were removed which left four distinct, delayed