Developmental and Age-Related Processes That Influence the Longevity and Senescence of

Photosynthetic Tissues in Arabidopsis

Author(s): Linda L. Hensel, Vojislava Grbić, David A. Baumgarten and Anthony B. Bleecker
Source: The Plant Cell, Vol. 5, No. 5 (May, 1993), pp. 553-564
Published by: Oxford University Press
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The Plant Cell, Vol. 5, 553-564, May 1993 ? 1993 American Society of Plant Physiologists

Developmental and Age-Related Processes That Influence

the Longevity and Senescence of Photosynthetic Tissues
in Arabidopsis
Linda L. Hensel, Vojislava Grbic, David A. Baumgarten, and Anthony B. Bleecker1
Botany Department, Birge Hall, University of Wisconsin, Madison, Wisconsin 53706

Factors that influence the longevity and senescence of photosynthetic tissues of Arabidopsis were investigated. To de?
termine the influence of reproductive development on the timing of somatic tissue senescence, the longevity of rosette
leaves of the Landsberg erecta strain and of isogenic mutant lines in which flowering is delayed (co-2) or sterile flowers
are produced (ms1-Y) were compared. No difference in the timing of senescence of individual leaves was observed be?
tween these lines, indicating that somatic tissue longevity is not governed by reproductive development in this species.
To examine the role of differential gene expression in the process of leaf senescence, cDNA clones representing genes
that are differentially expressed in senescing tissues were isolated. Sequence analysis of one such clone indicated ho?
mology to previously cloned cysteine proteinases, which is consistent with a role for the product of this gene in nitrogen
salvage. RNA gel blot analysis revealed that increased expression of senescence-associated genes is preceded by declines
in photosynthesis and in the expression of photosynthesis-associated genes. A model is presented in which it is postu-
lated that leaf senescence is triggered by age-related declines in photosynthetic processes.

INTRODUCTION

The coupling of whole-plant senescence to reproduction

concomitant
is a cessation of vegetative meristem activity t
prevents
life history trait that is common to many annual and some the development of new photosynthetic tiss
perennial plant species and is referred to as the monocarpic
(Woolhouse, 1983; Nooden, 1988b). The two processes app
habit (Hildebrand, 1881). In nature, monocarpy is most char-
to be coordinately regulated in some cases but independen
controlled
acteristic of plant species classified as ruderals (Stebbins, 1950; in other species (see Nooden, 1988a, for a
Grimes, 1979). This ecological classification refers todiscussion).
plants,
such as Arabidopsis, that are adapted to growth in disturbed The timing of both leaf senescence and apical arrest is
environments. According to theory, ruderals have evolved lifeto involve interacting signals between the affected tis?
thought
sues risks
history traits that are compatible with the high mortality and the developing fruit. These interactions between
from the environment: a propensity for early reproductive de? and reproductive structures are generally referred
vegetative
to as correlative controls. A number of hypotheses concern-
velopment and high fecundity are two particularly noteworthy
traits. The early diversion of resources from vegetative
ing the to
nature of these correlative control signals have been
reproductive development in ruderals is thought to havepresented
con- (reviewed in Woolhouse, 1983; Kelly and Davies,
1988; Nooden, 1988b), but the biochemical nature of the sig?
tributed to the evolution of the monocarpic habit characteristic
of these species (Molisch, 1928). naling mechanisms has not been unequivocally established
The partitioning of resources between vegetative and re?higher plant.
for any
productive development in monocarpic plants involves a the regulatory mechanisms that govern the tim?
Although
ing of leaf senescence remain elusive, the actual processes
complex interplay of generative and degenerative processes
associated
that are thought to be under developmental control. The phys? with senescence of photosynthetic tissue have been
iological basis for these processes has been extensively
extensively characterized in a number of plant species. Com?
studied in a few annual crop species, such as soybean,
mon cot-
features observed across a range of species have led
ton, and maize (Eaton, 1955; Wittenbach, 1982; Crafts-
to the concept of the senescence syndrome: an orderly se?
quence
Brandner et al., 1984a; Crafts-Brandner and Egli, 1987; Fordof events involving the turnover of macromolecules
and Shibles, 1988). In general, monocarpic plant senescence
and lipids and the transport of mobilized nutrients out of the
senescing
involves a degeneration of existing vegetative tissues and a tissue. The most widely used biomarker for the
senescence syndrome is the rapid loss of chlorophyll as?
1 To whom correspondence should be addressed. sociated with the degeneration of chloroplast internal structure

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554 The Plant Cell

(Thomson and Plat-Aloia, 1987; see also Woolhouse, 1982).

Experimental evidence indicates that the senescence syn?
drome is under genetic control by the nucleus (Yoshida, 1961;
Ness and Woolhouse, 1980; Thomas et al., 1992). Recent
studies indicate that differential expression of specific genes
is associated with the senescence syndrome (Davies and
Grierson, 1989; Graham et al., 1992).
The monocarpic habit is exemplified by Arabidopsis, which
may undergo its entire life cycle in 8 to 10 weeks. Sexual
reproduction in Arabidopsis involves the generation of thou-
sands of offspring. Associated with this massive reproductive
effort, the leaves, stems, and fruits of the adult plant undergo
progressive senescence that ultimately results in the death
of the plant. We are interested in determining the mechanisms
involved in the coordination of these processes. In this report,
we make use of single-gene mutations that affect reproduc?
tive development to evaluate the role of reproduction in the
patterns of growth, development, and senescence of the Ara?
bidopsis rosette leaf. In addition, we examine the differential
expression of genes in the leaf associated with the senescence
syndrome.

RESULTS

Experimental Set Up

Environmental conditions such as light quality and quantity,

nutrient and water availability, temperature, and humidity have
a strong influence on the course of development in Arabidop?
sis. For this reason, we established culture conditions that
minimized environmental fluctuations. Although these condi?
tions of constant light, temperature, humidity, and soil moisture
are not necessarily optimum for growth and development,
plants were healthy and uniform in appearance, and the Lands-
berg erecta (Ler) strain produced more than 10,000 seeds within
the 50-day life cycle. Under these same conditions, delayed
flowering varieties grew vigorously for several months. In some
experiments, plants were grown in a 16-hr-light/8-hr-dark pho?
toperiod. Plants of the Ler strain grown under these conditions
were indistinguishable from plants grown under constant light.

Figure 1. Stages in the Life Cycle of the Landsberg erecta Strain

Life History Traits in Arabidopsis
Arabidopsis and Age-Related Changes in Rosette Leaf Number Fi

(A) 14 days after planting.

Figures 1A through 1D depict the life history of an Arabidop?
(B) 21 days after planting.
sis plant. Under the above constant environmental conditions,
(C) 37 days after planting.
the Ler strain of Arabidopsis developed initially as a rosette
(D) 53 days after planting.
of seven to eight leaves (Figure 1A). At ~12 days postgermi-
(E) 0 days after full leaf expansion.
nation, the transition from vegetative to inflorescence meristem
(F) 3 days after full leaf expansion.
occurred at the apex, resulting in the production of the highly
(G) 5 days after full leaf expansion.
branched inflorescence (Figures 1B and 1C) (Vaughan, (H) 1955;7 days after full leaf expansion.
Bowman etal., 1989; Schultzand Haughn, 1991; Shannon (I) and
9 days after full leaf expansion.
Meeks-Wagner, 1991). The most active period of flowering (J) and
11 days after full leaf expansion.

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 Longevity and Senescence in Arabidopsis

fruit development is accompanied by the sequential senes? somatic tissue senescence was first compared betw
cence of the original leaves of the rosette, as indicated by an wild-type Ler strain and ms1-1, a male-sterile mutant
initial stage of chlorophyll loss, followed by the complete dis- the Ler background (Koornneef et al., 1983). As shown
integration of the leaf tissue (Figure 1C). Developmental arrest ure 2, the absence of fruit development in the ms1-1 l
of the main inflorescence stem, characterized by a cessation not appreciably affect the timing of senescence of eith
of proliferative activity at the apex and a degeneration of the or inflorescence stem tissue. A delay in reproductive de
youngest flower buds, occurred at approximately day 40 ment also failed to have an effect on the longevity of
(Vaughan, 1955; Shannon and Meeks-Wagner, 1991; Alvarez leaves. Leaves of a late-flowering mutant line, co-2 (Rede
et al., 1992). During this later phase, the stem, cauline leaves, Koornneef et al., 1991), senesce over the same period
and seed pods became senescent and the seeds of the next as leaves of wild-type plants, as shown in the mortality
generation reached maturity (Figure 1D). of Figure 3A. A second late-flowering line, fca (Martinez-Z
Based on these general observations, we considered two and Somerville, 1990; Koornneef et al., 1991), was also
distinct processes that govern the course of monocarpic senes? ined with similar results (data not shown).
cence in Arabidopsis: (1) the senescence of the developed An alternative hypothesis for correlative control is the
organ systems, such as leaves, stems, and siliques, which we ent drain hypothesis in which it is postulated that chan
define as somatic senescence and (2) the cessation of gener- source-sink relationships between tissues may govern t
ative activity at the inflorescence meristems, which we refer ing of leaf senescence (Molisch, 1928). One could argu
to as apical arrest. In this study, we focused on the senescence sink demand resulting from inflorescence development
of somatic tissues using primarily the fifth and sixth leaves male-sterile line or from the development of additiona
of the rosette as our model. in the delayed flowering lines was governing leaf sen
The fifth and sixth leaves of the rosette represent the first
To test this possibility, the longevity of rosette leaves w
consistently adult leaves of the Ler strain (as defined for sured
the in the tf/7-2 (terminal flower) mutant line (Shan
Wassilewskija strain by Medford et al., 1992). Under the cul-
ture conditions used, individual leaves progressed through full
expansion to maturity followed by a period of progressive leaf 25 -i ?
yellowing and, finally, a degeneration and complete desicca?
tion of the tissue. This developmental sequence is shown for
leaf 5 in Figures 1E through U. Toobtain data from large popu-
lations of plants in a noninvasive way, we performed our initial
experiments to assess tissue longevity by simply recording
the time from visible emergence (1 to 2 mm) of an individual
leaf or stem internode to the time at which more than 50%
visible degreening of that tissue had occurred. For leaves, the
latter stage was invariably followed by a complete degenera?
tion and death of the tissue within 2 days. In the case of stem
tissue, senescence refers only to the degreening of the pho?
tosynthetic cortical tissues because stems may continue to
function in nutrient transport beyond this stage. Preliminary
ultrastructural analysis of the leaf and stem indicated that
degreening of both mesophyll cells of the leaf and cortical cells
of the stem is associated with the loss of thylakoid structure
and increases in plastoglobuli characteristic of the senescence
6J |1 11 211
syndrome (data not shown) (Thomson and Platt-Aloia, 1987).
Leaf number Internode
Figure 2. Longevity of Individual Leaves and Stem Segments of Wild-
Relationship between Reproduction and Somatic
Type (Ler) and Male-Sterile (ms1-1) Lines.
Tissue Senescence
Plants were grown in 16-hr-light/8-hr-dark cycles. Mean tissue longevity
(? se) is
The sequential senescence of the leaves of the rosette for rosette leaves 1 through 6 (numbered by order of emer?
tem-
porally correlated with inflorescence developmentgence from the meristem) is shown. Data for the first two pairs of leaves
(Figure 1).
are averaged because the time of emergence is often indistinguish-
In correlative control models for monocarpic senescence, it
able. Internode 1 is defined as the internode between the first and the
is hypothesized that signals or processes associated with in?
second flower produced on the primary inflorescence meristem. Tis?
florescence and/or fruit development are responsible for the
sue longevity was measured as the time from visual emergence (1
induction of the senescence syndrome in leaves (reviewed
to 2 mm) to inmore than or equal to 50% degreening. For leaf Ler,
Nooden, 1988b). To investigate the relationship between re? ms1-1, n = 45 plants. For internode Ler, n = 19; ms1-1,
n = 137 plants;
productive development and senescence in Arabidopsis,
n = 11.

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556 The Plant Cell

1251 Rosette Leaf Senescence as an

Results of the above experiments i

100 H leaf and stem senescence is not c
velopment in Arabidopsis. Rather
appears to be an intrinsic age-rel

| 75 H system.
To investigate the role of processes intrinsic to the leaf in
?
the timing of leaf senescence, we examined a number of cel?

?s 5o^
co
D Ler
? '?
lular parameters for age-related changes. Because the primary
0) function of the mature leaf is photosynthesis, we focused on
-J
? ms1-1 biomarkers associated with photosynthetic function. Results
25 co-2 shown in Figure 4 indicate that C02 fixation rates, while ini-
? ??
tially variable, decline with leaf age almost from the time of
full leaf expansion (Figure 4A). Extractable ribulose bisphos-
phate carboxylase (Rubisco) activity and soluble protein
showed similar declines associated with age (Figures 4B and
B
4C). The slower decline in Rubisco activity relative to C02 fix?
ation is due in part to the fact that fixation rates were measured
100 H
co
"% on the whole leaf, and Rubisco activity was determined from
leaf discs taken from the center of the leaf that senesces last
>
(Figure 1). For leaf discs, the decline in Rubisco preceded the
75 H
3 rapid loss in chlorophyll that is characteristic of the senescence
?*- syndrome (Figure 4D).
co
0)
50 H
o Ler Isolation of cDNA Clones Representing Genes That
Are Differentially Expressed during Senescence
25-^ ? ff/7-2

Whereas biological markers for photosynthetic function were

known, no such markers were known for the senescence
"t-1-1-1-r phase of leaf development. As mentioned in the Introduction,
5 10 15 20 25 30 several lines of evidence support the view that leaf senes?
cence is a developmentally controlled process that requires
Days from leaf expression of nuclear genes. To identify senescence-related
emergence
biomarkers, we screened a cDNA library made from mRNA
Figure 3. Survival Curves of Adult of
Leaves for using
senescing leaves Wild-Type (Ler), (Sambrook
differential hybridization Late-
Flowering (co-2), Male-Sterile (ms1-1),etand Terminal
al., 1989). Flower
Nine cDNA families that (tfH-2) Lines.
do not cross-hybridize
were identified
Data from leaves 5 and 6 were combined forand further
thischaracterized.
analysis. Data for two of these
Leaf lon?
cDNA clones2.
gevity was measured as given in Figure are shown here. These two senescence-
associated
(A) Ler, n = 322; co-2, n = 54; ms1-1, n =(SAG) 74. gene clones were designated SAG2
(B) Ler, n = 25; tfl1-2, n = 23. SAG4. As shown in Figure 5A, RNA gel blot analysis ind
that mRNA levels for both genes are elevated in sen
leaves and inflorescence stems. DNA gel blot analysis,

Meeks-Wagner, 1991; Alvarez et al., 1992). The tfl1-2 line pro-

duces a normal rosette and initiates flower development at the
same time as the isogenic Ler wild type. However, the devel?
opment of aberrant flower structures at the inflorescence
meristem causes early termination of inflorescence develop?
ment resulting in a 10-fold decrease in biomass accumulation
relative to wild type, as shown in Table 1. Results shown in
Figure 3B indicate that leaf longevity was not appreciably al?
tered by the diminished sink demand in tfl1-2.

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 Longevity and Senescence in Arabidopsis 557

in Figure 5B, indicated that both SAG2 and SAG4 are sing
copy genes within the Arabidopsis genome. Partial sequen
analysis (Figure 5C) indicated that SAG2 contains an open rea
ing frame that shares a high degree of derived amino aci
sequence similarity with a family of cysteine proteinases iden
fied in plants and animals (Fuchs and Gassen, 1989; Watan
et al., 1991). The identification of the product of SAG2 as a p
teinase is consistent with a role for this protein in th
mobilization of nitrogen within the leaf during the senescen
phase (Thayer et al., 1987).

Changing Patterns of Gene Expression Associated

with Senescence

The process of leaf senescence involves a shift in leaf f

tion from that of a photosynthetic organ to that of a stor
organ. To examine the role of differential gene express
during the progression from a photosynthetic function
remobilization, the expression patterns of marker ge
representing each stage in leaf development were used f
RNA gel blot analysis. Cloned probes representing chloroph
a/b binding protein (CAB) and ribulose bisphosphate carb
ylase small subunit (rbcS) were used as markers for
photosynthetic stage of leaf development. An elongation
tor (EF-1 a) gene (Curie et al., 1991) probe was used to repre
cytoplasmic housekeeping functions (protein synthesis).
two SAG cDNA clones described in the previous section ser
as markers for the senescence phase of leaf developm
To determine changes in steady state mRNA levels for
marker genes, total RNA was extracted from leaves (lea
7 and 8, Columbia strain) of increasing age. Representa
autoradiographs are shown and quantitative analysis of mR
levels for all marker genes are depicted in Figure 6. Res
indicated that transcript levels of nuclear-encoded, photos
thesis-associated (PAG) genes were highest in expand
leaves, dropped precipitously as the leaf reached full ex
sion, and then declined slowly with age relative to total RN
On the other hand, transcript levels for EF-1a remained f
constant relative to total RNA after full leaf expansion.
sages for the two SAG genes were expressed at lowest l
in the young fully expanded leaf and increased 10- to 20-f
in the senescing leaves. It should be noted that RNA le
depicted in Figure 6 are based on equal total RNA loads. H
ever, the amount of extractable rRNA per leaf declined as
leaf senesced, as shown in Figure 7. Two factors may contr
ute to the observed decline in rRNA: (1) decreases in rR
-2 0 2 4 6 8 10
in senescing cells and (2) decreases in the number of live
per leaf as necrotic areas developed. It was not possibl
Days from full expansion
differentiate between these possibilities from the experim
described. Correcting
Figure 4. Age-Related Changes in Photosynthetic for the changes
Parameters ofinAdult
rRNA, it was c
Rosette Leaves. lated that SAG2 and SAG4 increased fourfold and threef
(A) C02 assimilation rates. respectively, on a per leaf basis during senescence. By
(B) Extractable Rubisco activity. same analysis, PAG gene expression was 16-fold lowe
(C) Soluble protein. senescing leaves than in fully expanded leaves on a per
(D) Chlorophyll content. basis.

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558 The Plant Cell

B
SAG2 SAG4 SAG2 SAG4
STEM LEAF STEM LEAF E H E H
SAG 2 TEAAL P ET KDWR EDG -1 VS PV KD Q
Y 0 Y 0 Y 0 Y 0 ORYZAIN V DAP************-********17g
ALEURAIN
12.2 DA*************-******n*162
CATHEPSIN H GTGPY*PSV***KK*NF*****N*14 6
7.1 ?
SAG 2
4.1 ? GGCGSF^rFSTTGALEAAYHQAFG
ORYZAIN Y p**?**s***R*T**T<.2c2
3.1 - ALEURAIN *****?******T**T*lg6

2.3- CATHEPSIN H *********S*IAI*T*17 0

2.0.
SAG 2 KGIS LS EQQLVDCAGAFNNYGSN3
1.5- ORYZAIN V PPV*******A***TRY**F**S*226
ALEURAIN *N*************G***F*C**2I0
1.0.
CATHEPSIN H *ML * * A* ******* QD* * * * * CQ*1

0.2- 0.5- SAG 2 GLPSQAFEYIKSNGGLDTEKAYRY

ORYZAIN V ***********Y*******?**p*250
-ALEURAIN ***********Y***I***ES*P*234
CATHEPSIN H **********LY*K*IMG*DT*P*218

Figure 5. SAG Gene Analysis.

(A) Age-related expression in leaves and inflorescence stem. RNA gel blot analysis of young, fully ex
degreening tissue (O).
(B) DNA gel blot analysis of genomic DNA digested with EcoRI (E) or Hindlll (H) and probed with eith
size markers are given at the left in kilobases.
(C) DNA sequence analysis of the SAG2 cDNA open reading frame. Comparison of the predicted SAG2
teine proteinases: oryzain y from rice (Watanabe et al., 1991); aleurain from barley (Rogers et al., 19
and Gassen, 1989). Gaps are indicated by dashes and sequence identities by asterisks. The arrow m
to produce the N terminus of the mature protein.

DISCUSSION to the observed age-related senescence. Analysis of ph

thetic processes revealed that carbon fixation rates d
progressively from the time of full leaf expansion (Fi
The sequential senescence of Arabidopsis rosetteThis
leaves is
age-related, progressive loss of photosynthetic fu
associated with the time of maximum inflorescence develop?
from the time of leaf maturity may be a general feature
ment. We were surprised to discover that althoughnual
theseplants
two because this phenomenon has been obser
processes are normally juxtaposed in time, they are not cou- (Callow, 1974), Perilla (Batt and Woolhouse,
cucumber
pled in a causal way. Under the growth conditionsbarley
used, the
(Friedrich and Huffaker, 1980), wheat (Peoples
rosette leaves appear to have an intrinsic maximum1980),
life span
maize (Crafts-Brandner et al., 1984a), and so
that is independent of the reproductive status of the (Wittenbach,
plant. This 1982; Ford and Shibles, 1988). Wittenb
is clearly not the case with other monocarpic plants. In the
ferred to the photosynthetic decline as "functional sene
legumes, for instance, monocarpic senescence oftoleaves is
distinguish this process from the subsequent rapid
dependent on fruit development and affects both younger and
chlorophyll and macromolecular turnover associated w
older leaves (Nooden, 1988b). In some pea varieties, senes? syndrome (Wittenbach, 1983). The physio
senescence
cence may even progress from youngest to oldest leaves
basis for functional senescence is not known, altho
(Proebsting et al., 1976). The age-related nature of number
senescence of explanations have been suggested (Callow
in Arabidopsis is not restricted to rosette leaves. The Woolhouse,
photosyn? 1982; Nooden, 1988b). Perhaps, the simp
thetic tissues of the inflorescence stem also show age-related
planation for the observed functional decline in photosy
patterns of senescence. Based on our observations, mayallbepho?
that maintenance and repair of the photosynthe
tosynthetic tissues of the plant exhibit equally short paratus
life spans.
does not keep pace with the damaging effects
Therefore, the age of specific tissues appears to be the best
free-radical byproducts of photosynthesis in the leaf
predictor of when senescence will occur. Whether et vascular
al., 1992). In this sense, functional senescence of
tissues are also subject to age-related declines in function is
may conform to free-radical theories of aging applied
a question that can only be answered with a detailed anatomi-
mal systems (Harman, 1981; Bolli et al., 1989).
cal analysis. Whatever the mechanism, the consequence of age-r
The lack of evidence for extrinsic, correlative controls on leaf
decline in photosynthesis is that the leaf will ultimately
senescence in Arabidopsis led to a more detailed the examina-
compensation point at which carbon assimilation and
tion of intrinsic processes within the leaf that might ration
contribute
are equal. At this point, the leaf will no longer con

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 Longevity and Senescence in Arabidopsis 559

as a photosynthetic organ to the assimilatory needs of the rest

of the plant. However, the functionally senescent leaf contains
a significant pool of nutrients in the form of lipids and macro-
molecules. The chloroplasts of a mesophyll cell are of particular
importance in this regard because it has been estimated that
these organelles may contain over 50% of the protein and 75%
of the lipid of the cell (Forde and Steer, 1976; Dean and Leech,
1982). The mobilization of these nutrients is thought to occur
via salvage pathways that increase in activity at the terminal
stages of leaf development (Woolhouse, 1984; Nooden, 1988a).
As outlined below, we postulate that in Arabidopsis the acti?
vation of salvage pathways associated with senescence is
directly coupled to photosynthetic decline. This regulatory fea?
ture may be the critical one that distinguishes Arabidopsis from
other monocarpic plants such as soybean, in which the senes?
cence syndrome is correlatively controlled by reproductive
development. Interestingly, depodding of soybean plants will
delay the final stages of senescence in the leaf but does not
prevent photosynthetic decline (Wittenbach, 1982; Crafts-
Brandner et al., 1984b). In fact, the Rubisco enzyme may be
0)
o
degraded in leaves of depodded plants, but the mobilized N
c
CO
is apparently refixed in the paraveinal mesophyll as storage
c
protein (Franceschi et al., 1983; Wittenbach, 1983). Thus, in
3 soybean, depodding appears to result in uncoupling of photo?
n
co synthetic decline from activation of the senescence syndrome.
< ^ In this case, full activation of salvage pathways presumably
z
DC
E
0

0)
rx 100

75 H

co
E 50 H
o
co
o

? 25H
-30369

Days from full expa

Figure 6. Age-Related Changes

Genes.

Equal loads of total RNA were loaded in each lane and blots were hy?
bridized with 32P-labeled probes. Days from full expansion
(A) Representative autoradiographs of (B), (C), (D), (E), and (F).
Figure 7. Age-Related Changes in Extractable Ribosomal RNA per
(B) CAB. Leaf.
(C) rbcS.
(D) EF-1a. RNA quantities were evaluated by slot-blot analysis of RNA obtained
(E) SAG2. on a per leaf basis. Values were corrected for RNA losses during
(F) SAG4. purification.

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560 The Plant Cell

requires some form of signal from the developing fruit. In con?

trast, we have not yet found a similar situation in Arabidopsis
leaves. In addition to the analysis of the Arabidopsis mutants
affecting reproduction reported in this paper, we have exam?
ined existing hormone-deficient and insensitive mutants as well
as newly isolated mutants with delayed leaf senescence. In
no case have we found a condition in which the senescence
syndrome has been uncoupled from photosynthetic decline
(L.L. Hensel, V. Grbic, D.A. Baumgarten, and A.B. Bleecker,
unpublished results).
To consider the possible mechanisms by which the activa?
tion of salvage pathways may be coupled to photosynthetic
decline, we turn to the paradigm of differential gene expres?
sion. Based on our analysis of PAG and SAG gene expression,
we consider these biomarkers as representatives of two com? Senescence

peting developmental programs within the leaf. PAG genes

code for components of the photosynthetic apparatus and are
consequently expressed at highest levels during leaf expan?
Export Export
sion when chloroplast biogenesis is occurring (Figure 6; see
also Mullet, 1988). The lower PAG transcript levels observed
Figure 8. Schematic of Regulatory Pathways That May Influence Leaf
after full leaf expansion presumably represent messages uti?Longevity and Senescence.
lized in the maintenance and repair of existing chloroplasts
The emphasis of this schematic is on the transcriptional control of PAG
because chloroplast fission and biogenesis are thought to
and SAG genes. The darker lines outline a hypothetical model in which
cease at full leaf expansion (Pyke and Leech, 1992). The SAG
the following sequence of events is proposed: (A) maturation signals
genes appear to code for salvage-related functions that are
suppress PAG gene expression at full leaf expansion; (B) photosyn?
antagonistic to photosynthetic maintenance. The observed age-
thetic function declines as a result of inadequate maintenance; (C)
related expression patterns of SAG genes are consistent withmetabolites of photosynthesis, which act to repress SAG gene expres?
this concept: SAG transcripts are expressed at low levels insion in the young leaf, decline in concentration, resulting in derepression
young leaf tissues and increase in expression as photosynthe?
of SAG genes; (D) SAG gene products act to mobilize nutrients and,
sis declines (Figure 6). as a consequence, promote the senescence of the leaf tissues.
Potential regulatory processes that drive the expression of
PAG and SAG gene transcripts are presented diagrammatically
in Figure 8. These possible regulatory pathways can form the tissues (Crafts-Brandner et al., 1984a). The source of carbon
basis for mechanistic models that simulate the regulation of for sucrose synthesis in this case is unlikely to be photosynthe?
sis, but is rather through the p-oxidation of lipids because
these two sets of genes. As indicated in the diagram, the regu?
lation of the genes under consideration is complex. As one glyoxysomal pathways are known to increase during senes?
working model from which to generate and test specific hy- cence (Gut and Matile, 1988; DeBellis et al., 1990). In this
potheses, we suggest a two-step process in which (1) regard, the metabolite repression model has previously been
age-related declines in photosynthesis trigger the activation suggested for the senescence-associated expression of ma?
of SAG gene expression and (2) the products of the SAG genes late synthase in cucumber cotyledons (Graham et al., 1992).
act to mobilize nutrients and, as a consequence, reduce the The limited longevity of Arabidopsis somatic tissues is con?
viability of the tissues to the point of a loss of homeostasis sistent with the early reproductive development and high
and death. According to this model, PAG transcript levels, which fecundity of this species. In terms of evolutionary theory, the
influence the rate of damage repair in the mature leaf, may disposable soma hypothesis (Kirkwood and Cremer, 1982;
be determinants in the rate of photosynthetic decline and, thus, Kirkwood and Rose, 1991) predicts that species adapted to early
contribute indirectly to the timing of leaf senescence. An addi? reproduction will tend to invest less energy in somatic main?
tional key feature of this model is the speculation that SAG gene tenance because such maintenance requires resources that
expression is repressed in younger leaves by metabolites of are then unavailable for reproductive effort. In specific terms,
photosynthesis, and it is the derepression of these genes result? the dynamics of the Arabidopsis leaf represent a trade-off be?
ing from photosynthetic decline that initiates the senescence tween high photosynthetic output, which supports new growth,
syndrome. According to this model, the rate of decline in pho? particularly of reproductive structures, and maintenance and
tosynthesis after full leaf expansion will determine the timing repair, which contribute to longevity of somatic tissues once
of SAG gene expression and, therefore, the longevity of the they develop. The measure of this evolutionary trade-off for
leaf. The chemical identity of the metabolite(s) responsible is photosynthetic tissues may be in the age-related rate of pho?
not known, but it is unlikely to be sucrose because this trans- tosynthetic decline characteristic of a particular species. At
portable form of carbon may occur at high levels in senescent the opposite end of the continuum from Arabidopsis would be

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 Longevity and Senescence in Arabidopsis 56

the needles of some conifers, which are purported to main- all monocarpic plants conform to the predictions of this m
tain photosynthetic competence for years but are characterized As discussed above, the senescence of leaves can be del
by relatively low photosynthetic output (Oren et al., 1986). by removal of fruit in a number of species (Nooden, 19
The processes we have detailed also conform to the genetic even though this treatment does not prevent age-rel
theory of life span evolution known as the antagonistic pleio- declines in photosynthesis. In these systems, the activa
tropy hypothesis first elaborated by Williams (1957) and more of SAG gene expression is apparently not coupled to photo
recently discussed by Rose (Kirkwood and Rose, 1991; Rose, thetic decline but rather depends on other or additio
1991). According to this hypothesis, individual genes that have developmental signals for activation. This possibility i
a positive effect on reproductive fecundity but a negative ef? counted for in our model by the "developmental and age-re
fect on postreproductive survival of the parent will be selected signals" box in Figure 8. The developing Arabidopsis plant
for in evolution. The accumulation of these genes over evolu? parently either does not require these additional regula
tionary time is thought to determine the maximum life span systems or cannot afford them. In any case, the timing of
for a given species. Although this theory is generally applied senescence in Arabidopsis is adapted to the reproductiv
to animals, specific genes and associated mechanisms for an? quirements of the plant; the sequential senescence of
tagonistic pleiotropy have not been well characterized in animal rosette leaves coincides with the period of maximum prim
systems (Finch, 1990). On the other hand, the salvage path? inflorescence development and seed fill (Figure 1).
ways associated with the senescence syndrome in plants What impact does the short life span of Arabidopsis som
provide a clear example of the operation of processes that may tissues have on the longevity of the whole plant? In the
favor reproductive success through remobilization of nutrients the iterative nature of plant development could allow fo
but also lead to decreased fitness (i.e., senescence) of the tis? indefinite life span. In practice, this is true for Arabidopsis o
sues in which they are expressed. in cases where reproductive development is delayed. In
The SAG genes we have identified by differential hybridization case, the primary meristem continues to generate new ro
conform to the theoretical definition of antagonistic pleiotropy leaves for many months (Napp-Zinn, 1985). The transitio
if the encoded proteins function in the mobilization of nutrients, flowering, however, marks the beginning of the end for the
favoring reproduction, and also decrease the viability of the plant because all aerial meristems are converted over
tissues in which they operate. The SAG2 gene, which encodes reproductive development. While the tissues produced by
a cysteine proteinase, may provide a specific case. The de? inflorescence and flower meristems are capable of photo
rived amino acid sequence of this gene shares a high degree thesis, inflorescence meristems have a limited prolifer
of amino acid identity with oryzain y and aleurain, which are capacity; they cease meristematic activity after the pro
specifically expressed in germinating grain seeds and are tion of afew dozen flowers (Shannon and Meeks-Wagner, 1
thought to function in the mobilization of nitrogen from protein Alvarez et al., 1992). Thus, the combination of limited lon
reserves (Rogers et al., 1985; Watanabe et al., 1991). Thus, ity of somatic tissues and the limited proliferative capacit
a role for the SAG2 gene in nutrient mobilization seems likely. the inflorescence meristems ultimately limit the life sp
It is also plausible that the SAG2 gene, in concert with other the adult Arabidopsis plant.
SAG genes, contributes actively to the autolytic process of
senescence that leads to the death of the somatic tissues in
which these genes are expressed. Considering the evolutionary
METHODS
origin of senescence syndrome-associated salvage pathways,
most of the SAG genes we have identified are expressed at
detectable levels in the young photosynthetically active leaf
Strains
(Figure 6; V. Grbic, unpublished results), which is consistent
with a role for some of these genes in the maintenance-related
Seeds for Landsberg erecta (Ler) and the isogenic mutant lines co-2,
turnover of macromolecules (e.g., damaged proteins). This con-
ms1-1, and fca were obtained from A.R. Kranz (Kranz, 1978; Kirchheim
cept is supported by the high degree of amino acid sequenceand Kranz, 1981), Columbia seeds were obtained from Chris Somer-
identity between SAG2 and cathepsin H from animals, which ville (Michigan State University, East Lansing). tfH-2 seeds were
is thought to function in general protein turnover in animal cell
provided by D. Smyth (Monash University, Melbourne, Australia)
(Alvarez et al., 1992).
lysosomes (Takio et al., 1983). Sequence analysis of additional
SAG gene clones should provide information about how these
differentially expressed genes function in cellular maintenance
Plant Material and Measurements
and senescence.
Assuming that SAG genes are responsible for the progres-
All plants were grown at a density of approximately one plant per 25
sion of senescence in somatic tissues of plants, the regulatory
cm2, at 22?C, and 65 to 85% relative humidity under fluorescent illu?
pathways that govern their expression patterns may actually
mination supplemented with incandescent light (100 to 150 \iE rrr2
determine the longevity of these tissues. We have presented
sec~1) on a 2:1 mixture of Jiffy Mix (Jiffy Products of America, Bata-
an argument that in Arabidopsis, SAG genes are activated
via, IL) toas
perlite with a continuous wicking system of 10% Hoagland's
a consequence of age-related declines in photosynthesis.
solutionNot(Hoagland and Arnon, 1938). All plants were grown under

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562 The Plant Cell

continuous illumination unless otherwise stated. Seeds were surface were washed in 1 x SSPE (0.15 M NaCI, 10 mM sodium phosphate,
sterilized with a 30% bleach and 0.5% Triton solution and stratified 1 mM EDTA, pH 7.2), 0.1% SDS, and then in 0.2 x SSPE, 0.1% SDS,
at 4?C for 24 to 48 hr prior to planting. Leaf and internode longevityeach at 68?C for 45 min, followed by autoradiography overnight. Ra?
were measured as the time from visual emergence (1 mm) to moredioactivity was quantified with a Betagen Betascope 630.
than or equal to 50% degreening. Dry weight was determined from For quantification of rRNA per leaf, 10-fold serial dilutions (103 to
fully mature plants harvested 52 days after planting. Plants were dried106 of total RNA) were slot blotted on nylon membranes and probed
for 3 days at 65?C prior to weighing. with a pea rRNA gene, pHA2 (Polans et al., 1986). rRNA levels were
calculated from the linear range of signals on the slot blot and cor-
rected for losses during extraction using recovery of the 35S-labeled
tracer RNA.
Nucleic Acid Preparation

DNA for gel blot analysis was prepared from 5-week-old plants according
to the method of Shure et al. (1983). Total RNA was prepared as de?DNA Sequencing Analysis
scribed by Puissant and Houdebine (1990). Total RNA was isolated
from a pool of 20 adult leaves (the seventh and eighth leaves to emergeThe dideoxynucleotide chain termination method (Sanger et al., 1977)
was used for sequencing SAG2. Sequencing was done using the Se-
from the primary inflorescence meristem). All nucleic acid prepara?
tions were isolated from tissues of the Columbia strain, except the RNAquenase kit (U.S. Biochemicals) according to the manufacturer's
protocol. The deduced amino acid sequence of the SAG2 polypeptide
for Figure 5A, which was isolated from tissues of the Landsberg strain.
To correct for losses in total RNA during isolation, 35S-labeled RNAwas compared to the GenBank sequence data base using FASTA
was added as a tracer to leaf homogenates. The 35S-labeled tracer (Pearson and Lipman, 1988).
was made from the 1.15-kb Hindlll fragment of the 5' end of the TMK1
gene (Chang et al., 1992), which was cloned into the pGEM-7 plasmid
Biochemical Assays
(Promega). In vitro transcription from the SP6 promoter (Riboprobe
kit; Promega) produced 35S-labeled RNA of ~1 kb in length.
For protein, chlorophyll, and carboxylase activity, each data point
represents the average of three pools of three 0.5-cm2 leaf discs taken
from the middle of the leaf. The discs were frozen immediately in liq?
Screening the cDNA Library
uid nitrogen and stored at -80?C until measurements were made. Discs
used for total chlorophyll measurements were ground in 2 mL of 80%
The cDNA library made from RNA of senescing leaves was provided
acetone and quantified photometrically using the method of Arnon
by M. Michael (CalGene Pacific, Australia). Clones from the amplified
(1949). Initial carboxylase activity was determined by grinding the tis?
cDNA library were screened with 32P-labeled cDNA from green
sue in 2 mL of buffer (100 mM Bicine, pH 7.8, 5 mM MgCI2, 1 mM
(young) and yellow (old) leaves according to the method of Sambrook
EDTA, 5 mM DTT, 1.5% polyvinylpolypyrrolidone) and analyzed pho?
et al. (1989). The screening of 7000 recombinant cDNA clones resulted
tometrically using the method of Sharkey et al. (1991). Soluble protein
in identification of 40 differentially expressed clones belonging to nine
was quantified using 30 nL of the soluble extract from the carboxy?
families that do not cross-hybridize, designated senescence-associated
lase measurements by the method of Bradford (1976) using BSA as
(SAG)1 through SAG9.
the standard.

DNA and RNA Gel Blot Analysis Gas Exchange Measurements

For DNA gel blots, 3 ng of DNA was digested with EcoRI and Hindlll
C02 assimilation measurements were performed on the fifth leaf
(Promega), fractionated on an 0.8% agarose gel (05 x Tris-borate/EDTA
using the method of Loreto and Sharkey (1990). A 2.0-cm2 area of leaf
electrophoresis buffer), transferred to a nylon membrane (MSI-Magna
was clamped in a 1.59-cm3 aluminum cuvette with glass windows,
NT; Micron Separations Inc, Westborough, MA), and hybridized at 68?C
maintained at 24?C, and illuminated with 150 \iE nrr2 sec~1 white light.
with random primer 32P-labeled probes (Prime-A-Gene; Promega) ac?
Data points represent the average of measurements from three leaves.
cording to standard protocols (Sambrook et al., 1989). For RNA gel
blot analyses, 3 \ig of total RNA was denatured in 70% formamide,
20% formaldehyde, 10% 10 x running buffer (0.25 3-(A/-morpholino) ACKNOWLEDGMENTS
propanesulfonic acid, 50 mM sodium acetate, 10 mM EDTA, pH 7.0),
fractionated on an 0.8% agarose gel (1 x running buffer), transferred
to a nylon membrane (MSI-Magna NT; Micron Separations Inc.) withLinda L. Hensel and Vojislava Grbic contributed equally to this work
CE (10 mM sodium citrate, pH 7.0, 1 mM EDTA), and hybridized asWe appreciate the invaluable technical contributions of Sara Patters
was done for DNA gel blot analysis. The chlorophyll alb binding pro?
and the laboratory of Rick Amasino. We thank Michael Michael of C
tein (CAB) probe, pAB 140, was obtained from E. Tobin (University gene Pacific (Victoria, Australia) for the cDNA library from senesc
of California, Los Angeles) (Leutwiler et al., 1986). The ribulose Arabidopsis leaves. We thank Tony Cashmore and Elaine Tobin f
bisphosphate carboxylase small subunit (rbcS) probe, pATS-3, is thea rbcS gene and the CAB gene, respectively. We also thank To
genomic clone that spans the promoter and the 5' half of the coding Sharkey and Rick Amasino for critical reviews of this manuscript. T
sequence of the ATS3B member of the rbcS gene family (Krebberswork presented here was funded by grants from the National Scien
et al., 1988). The elongation factor (EF)-1a probe was made by poly?Foundation (No. DMB-9005164) and National Institute on Aging (N
merase chain reaction amplification with oligonucleotides designed5F32AG05542-02). V.G. and D.A.B. are graduate students in the Univ
to be unique to EF-1a-A1 (Curie et al., 1991), and the amplified frag?
sity of Wisconsin Genetics Program, which is supported by the Natio
ment was cloned into Bluescript KS+ (Stratagene). Hybridized filtersInstitutes of Health (No. GM07133-181).

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 Longevity and Senescence in Arabidopsis 563

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