Mechanism of phytohormone involvement in feedback regulation of cotton leaf senescence induced by potassium deficiency
absorb and accumulate metals from sediments (Sanchiz The wide adoption of Bt cotton cultivars, noted for greater sus-
Department of Soil Science, Faculty of Agriculture
, Forestry and Wildlife Resources Management,
University of Calabar
Chemical Control, China Agricultural University
Beijing 100193, China
State Key Laboratory of Plant Physiology and Biochemistry, Key Laboratory of Crop Cultivation and Farming System, Center of Crop
a metal bioindicator species (Maserti et al., 1988; Pergent cotton-producing areas (Dong et al., 2006; Tian et al., 2008)
For this reason, this seagrass is widely considered to be an increasing occurrence of premature senescence in Chinese
et al., 1990; Pergent-Martini, 1998; Maserti et al., 2005) thus ceptibility to potassium (K) deficiency (Zhang et al., 2007; Yang
through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, rTeosepleucctiidvaetlye. tThheepehxyptorehsosrmioonnoafl obnaesismoefmthbeerfeoefdtbhaecCkHreRgOuMlaOtioMnEoTfHlYeLaAfSseEn(eCsMceTn)cfea minidlyu, caedDNbyA pmoetathsysliturman(sKf e)rdaesfei-, interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd stage. K deficiency (0.03 mM for standard and Y grafting, and 0.01 mM for inverted Y grafting) increased the root absciperturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are sic acid (ABA) concentration by 1.6- to 3.1-fold and xylem ABA delivery rates by 1.8- to 4.6-fold. The K deficiency also linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. decreased the delivery rates of xylem cytokinins [CKs; including the zeatin riboside (ZR) and isopentenyl adenosine Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants. (iPA) type] by 29-65% and leaf CK concentration by 16-57%. The leaf ABA concentration and xylem ABA deliveries were consistently greater in CCRI41 (more sensitive to K deficiency) than in SCRC22 (less sensitive to K deficiency) DNA-methylation, Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile. spective of rootstock cultivar or grafting type, indicating that cotton shoot influences the levels of ABA and CKs in leaves and xylem sap. Because the scions had little influence on phytohormone levels in the roots (rootstocks) of all three types of grafts and rootstock xylem sap (collected below the graft union) of Y and inverted Y grafts, it appears that the site for basipetal feedback signal(s) involved in the regulation of xylem phytohormones is the hypocotyl of
metabolism, as well as a reduction in water and mineral
and faster fruiting cotton cultivars across the Cotton Belt of
Shukla et al., 2003; Sobkowiak and Deckert, 2003).
et al., 1995).
At the genetic level, in both animals and plants, Cd
can induce chromosomal aberrations, abnormalities in
It is generally considered that cytokinins (CKs) and abscisic
acid (ABA) are two major phytohormones involved in the
initiation and progression of plant senescence. The results of
measurement of endogenous CK levels before and during senescence
(van Staden et al., 1988; Singh et al., 1992; Gan and Amasino,
1995), external application of CKs (van Staden et al., 1988;
Zavaleta-Mancera et al., 1999), and manipulation of endogenous
production of CKs in transgenic plants (Gan and Amasino, 1995;
Rivero et al., 2007; Ghanem, et al., 2011a) showed that CKs can
inhibit leaf senescence. The gain-of-function analyses also
indicated that CK receptors AHK2 and AHK3 participate in
regulating the onset of leaf senescence in Arabidopsis (Kim et al., 2006;
Riefler et al., 2006). In contrast to CKs, the ABA level increases
in senescing leaves (Gepstein and Thimann, 1980; Samet and
Sinclair, 1980), and genes coding for proteins involved in ABA
synthesis and signalling are up-regulated during leaf senescence
(Buchanan-Wollaston et al., 2005; van der Graaff et al., 2006;
Jukanti et al., 2008). Furthermore, it has been known that
exogenous application of ABA promotes leaf abscission and senescence
(Zeevaart and Creelman, 1988), and exogenously applied ABA
induces the expression of several SAGs (senescence-associated
genes; Weaver et al., 1998), which is consistent with its
promotion of leaf senescence.
Nutrient deprivation (1% or 10% full-strength nutrient
solution) not only decreased the levels of CKs in shoot and root
(Kuiper et al., 1989; Vysotskaya et al., 2009), but also caused
an accumulation of ABA in the shoot (Vysotskaya et al., 2009).
It is well known that CK levels in roots, shoots, and xylem sap
are reduced in nitrogen-starved plants (Takei et al., 2001; Dodd
et al., 2004). With respect to K, although increased ABA levels
have been measured in grains and flag leaves of wheat (Triticum
aestivum) (Haeder and Beringer, 1981), roots, xylem, and
phloem sap of castor bean (Ricinus communis L.) (Jeschke et al.,
1997; Peuke et al., 2002), and leaves of Arabidopsis (Kim et al.,
2009), the physiological consequences of this increase in ABA
are less well known. One report (Salama and Wareing, 1979)
showed that K deficiency also caused a marked decrease in CK
levels in both roots and leaves.
A previous grafting study determined the roles of shoot and
root in the regulation of premature leaf senescence induced by K
deficiency in cotton (Li et al., 2012). The results showed that the
effect of rootstocks on leaf senescence was significant in some
cases, but the scion cultivars explained a higher percentage of
variation within grafting treatments. It was speculated that the
shoot-to-root feedback signal(s) that mediates xylem
phytohormone delivery was involved in the shoot regulation of premature
senescence of cotton (Li et al., 2012).
Several studies have shown that the root system dominates the
root export of CKs via the xylem (Sitton et al., 1967; McKenzie
et al., 1998; Dong et al., 2008; Albacete et al., 2009, 2010;
Ghanem et al., 2011b). However, results from studies on grafted
pea branching mutants and Arabidopsis branching mutants
suggested a feedback regulation of xylem sap CKs by some
longdistance signals that move from shoot to root (Beveridge et al.,
1997; Foo et al., 2007). Furthermore, the interaction of root and
shoot in terms of xylem phytohormone delivery may exist when
considering the recirculation of phytohormones between xylem
and phloem (Dodd, 2005).
Although premature senescence is a typical symptom of K
deficiency in cotton (Bednarz and Oosterhuis, 1995; Wright,
1999; Zhao et al., 2001), there has not been much attention
paid to the role of endogenous hormones in the leaf senescence
induced by K deficiency. Therefore, the objectives of this study
were to (i) examine the effects of K deficiency on the
endogenous ABA, zeatin riboside (ZR) + zeatin (Z), and isopentenyl
adenosine (iPA) + isopentenyladenine (iP) levels in roots, leaves,
and xylem sap of cotton seedlings; (ii) determine whether there
is a linkage between leaf senescence induced by K deficiency
and changes in endogenous CK and ABA levels in leaves; and
(iii) test the hypothesis that the leaf and xylem CK and ABA
levels are dependent on feedback signal(s) derived from the shoot
by using a grafting study as in previous work (Li et al., 2012).
The results should aid our understanding of mechanisms of
phytohormone involvement in the feedback regulation of leaf
senescence induced by K deficiency, and facilitate the development of
approaches to managing this problem.
Materials and methods
Two cotton cultivars contrasting in sensitivity to K deficiency were
used in the present study: CCRI41, developed by the Cotton Research
Institute, Chinese Academy of Agricultural Sciences, which is more
likely to senesce under K deficiency, and SCRC22, developed by the
Cotton Research Center, Shandong Academy of Agricultural Sciences,
which shows relatively late senescence under the same condition.
The experiment was performed in a growth chamber with 12 h
light/12 h dark at 30 2/22 2 C, 7080% humidity, and 600 mol m2
s1 photosynthetically active radiation. Seeds were surface-sterilized
by soaking in 9% H2O2 for 30 min, rinsed with tap water, and then
germinated in K-free sand medium for 4 d in the dark. After
germination, uniform seedlings were cultured hydroponically by transferring
them to 16 cm13 cm16 cm plastic pots filled with 2.2 litres of
halfstrength modified Hoaglands solution. The constituents of the
solution were (mM) 2.5 Ca (NO3)2, 1 MgSO4, 0.5 (NH4)H2PO4, 2 104
CuSO4, 1 103 ZnSO4, 0.1 Fe Na EDTA, 2 102 H3BO3, 5 106
(NH4)6Mo7O24, and 1 103 MnSO4. The concentration of K in the form
of potassium sulphate (K2SO4) in the solution was 0.1 mM (mild K
deficiency) before grafting and during graft recovery to ensure either
a higher survival rate of grafts or faster occurrence of leaf senescence
induced by severe K deficiency (0.03 mM or 0.01 mM) after graft
Standard, Y, and inverted Y grafting of cotton seedlings were
performed hypocotyl-to-hypocotyl at the cotyledonary or one-leaf stage as
described previously (Li et al., 2012). For each type of grafting, two
cultivars were self- and reciprocally grafted; standard grafts are denoted
as scion/rootstock, and Y and inverted Y grafts are denoted as
(scion+scion)/rootstock and scion/(rootstock+rootstock), respectively.
After establishment under high humidity and low light (80 mol m2
s1), surviving grafts were transferred to severe K-deficient (0.03 mM
for standard and Y grafts, and 0.01 mM for inverted Y grafts because
of its two rootstocks providing nutrients for only one scion) solutions
to induce premature senescence, with a 2.5 mM (K-sufficient) medium
as control. One week after establishment, the cotyledons and axillary
bud from cotyledonary nodes of rootstock (standard and Y grafts) were
Sampling of xylemsap
At the 89 leaf stage (~32 d after severe K deficiency treatment as
above), xylem sap was collected basically according to Noodn et al.
(1990). Ten grafts per treatment were decapitated 510 mm above the
graft union to collect scion xylem sap, and another 10 grafts per
treatment were cut below the graft union (~5 mm away) to collect rootstock
xylem sap. The cut surface was wiped with distilled water to remove
disrupted cells and residual cell elements, then a flexible silicon tube
(length 15 mm, internal diameter 2 mm) was placed ~510 mm over the
stump and tied tightly in place. The collection period started at 10:00 h
and lasted for 24 h in the dark each time. After collection, the sap
volume was quickly determined to calculate the sap flow rate over the
collection period, and then the sap was freeze-dried (SIM FD5-6, LA,
USA) and stored in the dark at 40 C. The xylem ABA and CK delivery
rates were calculated by multiplying phytohormone concentrations by
the xylem sap flow rates.
Extraction and purification of phytohormone in leaves androots
The fourth main-stem leaf from the apex and the whole roots were
harvested before and after xylem sap collection to determine CKs and
ABA. About 0.5 g of fresh samples was extracted and homogenized in
2 ml of 80% methanol (containing 40 mg ll butylated hydroxytoluene
as an antioxidant). The extract was incubated at 4 C for 48 h, and then
centrifuged at 4000 rpm for 15 min at 4 C. The supernatant was passed
through C18 Sep-Pak cartridges (Waters Corp., Millford, MA, USA),
and the phytohormone fraction was eluted with 10 ml of 100% (v/v)
methanol and then 10 ml of ether. The eluate was dried down by pure N2
at 20 C, and then stored at 40 C.
Quantification of ABA and CKs by enzyme-linked
immunosorbent assay (ELISA)
Freeze-dried xylem sap and N2-dried extracts of leaf and root
samples were dissolved in 2.0 ml of phosphate-buffered saline
(PBS) containing 0.1% (v/v) Tween-20 and 0.1% (w/v) gelatin (pH
7.5) to quantify free ABA, ZR, and iPA by ELISA following the
protocol described in Zhao et al. (2006). The mouse monoclonal
antigen and antibodies against free ABA, ZR, and iPA were
produced at the Center of Crop Chemical Control, China Agricultural
University, China, according to Weiler et al. (1981). As the anti-ZR
antibody also detects Z, the CKs quantified by this antibody are
described as ZR-type CKs. Similarly, the anti-iPA antibody detects
iP and iPR, hence the CKs quantified by this antibody are described
as iPA-type CKs. Calculations of the ELISA data were performed
as described in Weiler et al. (1981). The recovery percentage
obtained by using internal standards during extraction and analysis
was all >90%.
Leaf photosynthesis measurement
In a separate experiment with four replicates and four plants for each
replicate (Li et al., 2012), the photosynthesis rate (Pn) of the fourth
main-stem leaf from the apex (functional leaf) was measured by
a Li-6400 portable photosynthesis system (LI-COR, Lincoln, NE,
USA) with 1000 mol m2 s1 quantum flux and 500 mol mol1 CO2
Three replicates were used for each grafting treatment, and each
replicate consisted of 10 plants for either rootstock or scion xylem sap
samples and 20 plants for leaf and root samples. A similar trend of results
was found in three independent repeat experiments; thus data were
pooled across repeats. Analysis of variance (ANOVA) was performed
using SAS statistical software (V8, SAS Institute Inc., Cary, NC, USA)
to determine the significance of the effects of K supply, rootstock, and
scion, and their interaction under K deficiency. Treatment means were
compared using Duncans multiple range test.
Spatial pattern of ABA and CKs in cotton seedlings
As shown in Figs 1, 3, and 5, the ABA concentrations in
cotton leaves were much higher than those in roots. The iPA-type
concentrations in roots and leaves were higher than those of the
ZR-type, whereas the xylem iPA-type delivery rate was much
lower than that of the ZR-type (Figs 2, 4, 6), indicating that the
ZR-type is the dominant type of CKs in cotton xylem sap, as
in other plants (Singh et al., 1992; Beveridge et al., 1997; Foo
et al., 2007).
There were no significant differences in the xylem ABA and
CK delivery rates (the product of phytohormone concentration
in the xylem sap and sap flow rate) between rootstock (collected
below the graft union) and scion (collected above the graft union)
within a standard graft (Figs 1, 2), implying that the graft union
itself had little influence on phytohormone delivery as
previously demonstrated (Holbrook et al., 2002; Dodd et al., 2008). In
addition, the sum of the xylem phytohormone levels of the two
scions was more than that of the rootstock within a Y graft (Figs
3, 4), and the xylem phytohormone level in the scion was less
than that of the sum of the two rootstocks within an inverted Y
graft (Figs 5, 6), suggesting that the scion xylem phytohormone
deliveries were independent of the xylem of the rootstocks.
ABA and CK levels in standard grafts
Under K sufficiency, there were no significant differences
between SCRC22 and CCRI41 in either root (rootstocks) ABA
concentrations or leaf (scion) ABA concentrations (Fig. 1a).
Also, SCRC22 had similar xylem ABA delivery rates to CCRI41
both in rootstocks and in scions (Fig. 1a).
When submitted to K deficiency, the root ABA
concentrations and xylem ABA delivery rates across grafts significantly
(P < 0.001) increased by 1.6- and 4.6-fold, respectively, whereas
the leaf ABA concentrations changed little (Fig. 1b; Table 1).
Compared with SCRC22 self-grafts, CCRI41 self-grafts had
88, 90, and 28% greater ABA levels in roots, leaves, and xylem
sap, respectively. In addition, it was observed that although the
SCRC22 rootstock could reduce the ABA levels in leaves and
xylem sap of CCRI41 scions compared with CCRI41 self-grafts,
the corresponding values were greater than those of SCRC22
self-grafts. Similarly, the CCRI41 rootstock had a tendency to
enhance the ABA levels in leaves and xylem sap of SCRC22
scions compared with SCRC22 self-grafts, but the
corresponding values were lower than those of CCRI41 self-grafts (Fig. 1b).
These results suggest a feedback regulation of leaf ABA
concentrations and xylem ABA delivery rates by scion cultivars.
Furthermore, the scion did not affect the rootstock in terms of
root ABA concentrations under K deficiency (Fig. 1b; Table 1).
Under K sufficiency, the ZR- and iPA-type concentrations in
roots and leaves of SCRC22 tended to be higher than those of
CCRI41, but the differences were not significant in most cases.
In addition, the xylem ZR-type delivery rates in SCRS22 scions
were significantly greater than those in CCRI41 scions
regardless of rootstock cultivars. However, there were no significant
variations in the xylem iPA-type delivery rates between scions
of the two cultivars.
When exposed to K deficiency, the roots showed 73%
(P < 0.001) more ZR-type concentrations across grafts
compared with K sufficiency, and a slight but significant (P =
0.009) increase occurred in iPA-type concentrations (Fig. 2b;
Table 1). Nonetheless, the ZR-type levels in leaves and xylem
sap decreased by 32% and 29% (P < 0.001), and the iPA-type
levels decreased 48% and 63% (P < 0.001), respectively. The
ZR- and iPA-type levels in leaves and xylem sap of reciprocal
grafts were altered insignificantly compared with self-grafts with
the same scions as reciprocal grafts, suggesting a feedback
regulation by scion cultivars (Fig. 2b). The same cultivar rootstock
had similar root ZR- or iPA-type concentrations regardless of
scion cultivar, indicating little influence of scion on rootstock.
Considering genotypic variations, SCRC22 rootstocks across
self- and reciprocal grafts showed 26% and 87% greater ZR- and
iPA-type concentrations in roots (Fig. 2b). Also, SCRC22 scions
showed 61% and 60% greater ZR- and iPA-type concentrations
in leaves, and 26% and 42% greater ZR- and iPA-type delivery
rates in xylem sap (averaged across above and below the graft
union) than CCRI41 scions.
ABA and CK levels in Y grafts
Under K sufficiency, although there were no typical symptoms
of premature senescence, genotypic variations in root and xylem
ABA levels were observed between the two cultivars (Fig. 3a).
Compared with SCRC22, CCRI41 rootstocks had greater ABA
levels not only in roots but also in xylem sap (collected below the
graft union) regardless of scion cultivars. Also, the xylem ABA
delivery rates in CCRI41 scions were 93% significantly greater
than those of SCRC22 scions regardless of rootstock cultivars.
There were no significant differences in the leaf ABA
concentrations between CCRI41and SCRC22 scions.
Under K deficiency, the ABA levels significantly increased by
3.1-fold in roots, 1.9-fold in rootstock xylem sap, and 1.6-fold in
scion xylem sap compared with K sufficiency (Fig. 3b; Table 1).
However, there was no significant alteration in leaf ABA
concentrations. CCRI41 rootstocks showed 86% and 32% greater ABA
levels in roots and xylem sap than SCRC22 rootstocks; and its
scions had consistently greater ABA levels in leaves and xylem
sap than SCRC22 scions, even if they were grafted together onto
the same rootstock, clearly suggesting a feedback regulation by
scion cultivar. The scions did not affect root and xylem ABA
levels in rootstocks (Fig. 3b).
Under K sufficiency, there were no significant differences in root
ZR- and iPA-type concentrations between SCRC22 and CCRI41
rootstocks. However, the xylem ZR- and iPA-type delivery rates
of SCRC22 rootstocks were much greater than those of CCRI41
rootstocks in most cases, regardless of scion cultivars. Similarly,
SCRC22 scions showed greater xylem ZR- and iPA-type
levels than CCRI41 scions, even if they were grafted together onto
P-values are presented for each main effect or interaction.
Effect or interaction
Xylem sap below
the graft union
Xylem sap above
the graft union
the same rootstock (Fig. 4a). No significant differences in leaf
ZR- and iPA-type concentrations between the two cultivars were
When subjected to K deficiency, the ZR-type levels in roots
across grafts increased by 58% (P < 0.001) compared with K
sufficiency, and iPA-type levels changed slightly but significantly
(Fig. 4b; Table 1). However, the ZR-type levels in xylem sap of
rootstocks, xylem sap of scions, and leaves decreased by 39, 36,
and 41% (P < 0.001); and the iPA-type levels decreased by 38,
43 (P < 0.001), and 16% (P = 0.004), respectively. Compared
with CCRI41 self-graft, the rootstock of the SCRC22 self-graft
had 37% and 55% greater ZR-type, and 37% and 43% greater
iPA-type levels, in roots and xylem sap, respectively (Fig. 4b).
When one of two scions of a self-graft was replaced by the other
cultivar, the ZR- and iPA-type levels in both SCRC22 or CCRI41
rootstocks changed little. SCRC22 scions had more ZR- and
iPAtype CKs in leaves and xylem sap than CCRI41 scions, even if
they were grafted together onto the same rootstock (insignificant
for the ZR type; Fig. 4b). The mean values of leaf ZR- and
iPAtype concentrations and xylem ZR- and iPA-type delivery rates
across SCRC22 scions were 72% and 32%, and 41% and 60%
more, respectively, than those across CCRI41 scions.
ABA and CK levels in inverted Y grafts
Under K sufficiency, there were no significant differences in
root and xylem ABA levels between CCRI41 and SCRC22
rootstocks, and in xylem ABA delivery rates between CCRI41 and
SCRC22 scions. Nevertheless, CCRI41 scions showed greater
leaf ABA concentrations than SCRC22 scions regardless of
rootstock (Fig. 5a).
Under K deficiency, the ABA levels in roots and xylem
sap across rootstocks strongly increased by 2.3- and 1.9-fold
(P < 0.001) compared with K sufficiency; and those in xylem
sap across scions increased by 78% (P < 0.001); no significant
changes were observed in leaf ABA concentrations (Fig. 5b;
Table 1). CCRI41 rootstocks showed significantly greater ABA
levels in roots and xylem sap than SCRC22 rootstocks whether
in self- or reciprocal grafts (except xylem ABA delivery rates
in reciprocal grafts). In addition, the ABA levels in leaves and
xylem sap of CCRI41 scions were more than those of SCRC22
scions, even if they were separately grafted onto the same
combination of rootstocks, displaying the characteristic of feedback
regulation. The mean values of ABA in leaves and xylem sap of
CCRI41 scions were 53% and 31% more than those of SCRC22
Under K sufficiency, SCRC22 rootstocks had greater ZR- and
iPA-type levels in roots and xylem sap than CCRI41 rootstocks,
but not all differences were significant (Fig. 6a). Similarly, the
xylem ZR- and iPA-type delivery rates in SCRC22 scions tended
to be higher than those in CCRI41 scions. However, the leaf
ZRand iPA-type concentrations of SCRC22 scions were equivalent
to those of CCRI41 scions.
Under K deficiency, the ZR- and iPA-type levels in roots
across grafts increased by 90% and 47% (P < 0.001) but
decreased by 33% and 63% (P < 0.001) in rootstock xylem
sap, 42% and 73% (P < 0.001) in scion xylem sap, and 45%
and 57% (P < 0.001) in leaves (Fig. 6b). SCRC22 rootstocks
had more ZR- and iPA-type CKs in roots and xylem sap than
CCRI41 rootstocks, even if they were grafted together with the
same scion (Fig. 6b). In addition, SCRC22 scions showed more
ZR- and iPA-type CKs in leaves and xylem sap than CCRI41
scions, even if they were separately grafted onto the same
combination of rootstocks (Fig. 6b), suggesting a feedback
regulation by shoot cultivars. The mean ZR- and iPA-type
levels in leaves and xylem sap of SCRC22 scions were 64%
and 55% (leaves), and 1.2-fold and 61% (xylem sap) more
than those of CCRI41 scions.
Potassium deficiency increased ABA levels in roots
and xylem sap but decreased CK levels in xylem sap
and leaves of cotton seedlings
It was observed that the flow rates of xylem sap under K
deficiency were lower than those under K sufficiency in the
present study (data not shown) since K deficiency decreases root
hydraulic conductance (Cabanero and Carvajal, 2007), which
may result in the overestimation of the xylem phytohormone
concentration (Dodd, 2005). Therefore, the phytohormone
delivery rate (the product of xylem phytohormone concentration and
sap flow rate) was used to compare the effects of different K
supplies on xylem phytohormone levels.
In the present study, K deficiency (0.01 mM or 0.03 mM)
increased root ABA concentrations by 1.6- to 3.1-fold, and
xylem ABA flux by 1.8- to 4.6-fold across grafting types and
cultivars, as compared with adequate K supply (2.5 mM). These
results agreed with those of Peuke et al. (2002) showing that low
K supply increased the deposition of ABA in the roots (1.9-fold),
and root-to-shoot ABA signal in the xylem (4.6-fold). However,
there was no significant change in leaf ABA concentrations
across cultivars regardless of grafting type under K deficiency
(Table 1), possibly due to the high degradation of xylem-sourced
ABA in leaves (Zhang et al., 1997; Holbrook et al., 2002; Peuke
et al., 2002).
The responses of ZR- and iPA-type CKs to K deficiency were
different among parts of cotton seedlings. The levels of ZR- and
iPA-type CKs decreased in leaves and xylem sap under K
deficiency, but increased in the root tissue (albeit insignificantly for
the iPA type in Y and inverted Y grafts). Salama and Wareing
(1979) studied the effects of low supply of nitrogen (N),
phosphorus (P), and K on endogenous CKs in sunflower (Helianthus
annuus), and found that not only low N and low P but also low K
decreased the levels of CKs in the roots. The difference between
the present results and those of Salama and Wareing (1979) may
be due to interspecific differences (cotton versus sunflower) in
responses to K deficiency and the different intensities of K
deficiency applied (~1/100 versus 1/10).
Recent studies showed potential co-regulation of the ABA
and CK status in plants. Changes in ABA status can regulate
shoot CK concentrations via altering cytokinin oxidase activity
(Vysotskaya et al., 2009). Root overexpression of the ipt gene can
decrease ABA accumulation in roots, xylem sap, and the mature
leaf of salinized plants (Ghanem et al., 2011a). In the present
study, significant negative relationships of ABA with ZR- and
iPA-type CKs were also found in the organs determined (data not
shown). However, no causal relationship, or direct interaction,
between them can be discerned.
ABA and CKs are responsible for premature
senescence induced by K deficiency
Ghanem et al. (2008) and Albacete et al. (2010) have
demonstrated that CKs in tomato was the hormonal parameter best
related to photosystem II efficiency (Fv/Fm, indicative of leaf
senescence) under salinity. In the present study, a close negative
correlation of photosynthesis rate (Pn) in the fourth main-stem
leaf from the apex (Li et al., 2012), indicative of leaf senescence,
with leaf ABA concentrations, scion xylem ABA delivery rates,
and ratios of ABA/(ZR+iPA) type in leaves and xylem sap under
K deficiency were found; a close positive correlations was also
noted between Pn and CK levels in leaves and xylem sap (Figs
In previous work (Li et al., 2012), a close positive
relationship between leaf K content and senescence induced by K
deficiency was observed. In order to investigate whether leaf K
content influences leaf senescence via phytohormones, K
content in the fourth main-stem leaf from the apex (Li et al., 2012)
was correlated with ABA and CK concentrations in the same leaf
under K deficiency. There was a significant negative
relationship between K content and ABA concentration and a significant
positive relationship between K content and ZR-type
concentration (except standard grafting; Fig. 9). In addition, significant
relationships were found between K deliveries and
phytohormone levels in rootstock xylem sap of standard (except iPA-type
CKs) and inverted Y grafts and in scion xylem sap of Y grafts
(data not shown). However, there were no significant
relationships between root K content and ABA and CK concentrations
in root tissues.
Feedback regulation of ABA and CK levels in leaves
and scion xylem sap (collected above the graft union)
is guaranteed by the different graftingtypes
Grafting is a useful tool to test whether there was a feedback
regulation of some physiological responses, including leaf
senescence. For the seedlings exposed to K deficiency, the rootstocks
have a tendency to alter phytohormone levels in leaves and scion
xylem sap in some cases (Table 1), even significantly for the leaf
ABA concentration of a CCRI41 scion grafted onto a SCRC22
rootstock (compared with CCRI41 self-graft; Fig. 1b), but the
scion cultivars explained a higher percentage of variation within
grafting treatments under K deficiency irrespective of grafting
types. No interactions were found between rootstock and scion
in the standard grafting experiment regardless of plant organs
and phytohormone types (Table 1), indicating that rootstock and
scion are autonomous to a great extent in terms of ABA and CK
The levels of ZR- and iPA-type CKs were consistently lower
in leaves and xylem of CCRI41 scions than those of SCRC22
scions, and the leaf ABA concentrations and scion xylem ABA
flux were consistently greater in grafts with CCRI41 as scions
than those with SCRC22 as scions. These results revealed that
the cotton shoot can modify (i.e. feedback regulate) the import
of phytohormones from the root, as reported in Beveridge et al.
(1997) and Foo et al. (2007) for pea and Arabidopsis, and the
leaf phytohormone concentrations. Furthermore, it was noticed
that it is the xylem phytohormone concentration rather than sap
volume that was regulated by shoot cultivars. With respect to
seedlings grown under K sufficiency, although significant
genotypic differences in scion xylem CK flux were observed in some
cases, the leaf CK concentrations were similar between CCRI41
and SCRC22 scions (Figs 2a, 4a, 6a; Table 1).
Consistent with the present results, Gan and Amasino (1995)
and Faiss et al. (1997) found that the CK-overproducer rootstock
failed to delay leaf senescence via a grafting study. Due to the
absence of data for xylem CKs in grafts which they performed, it
remained unclear whether or not there was a feedback regulation
of CKs exported from roots by the shoots. However, the present
results are different from the classic notion that the roots play an
important role in regulation of leaf senescence by delivering
hormones to shoots (van Staden et al., 1988; McKenzie et al., 1998;
Dong et al., 2008; Albacete et al., 2009, 2010; Ghanem et al.,
2011b), reflecting the diversity and complexity of the
longdistance signalling system in higher plants. As for ABA,
studies with reciprocal grafts of wild-type plants and ABA-deficient
mutants indicated that the mutant rootstocks had no/little effect
on ABA concentrations in xylem sap (Dodd et al., 2009) and in
the leaves (Holbrook et al., 2002) of wild-type scions, which are
similar to those in the present study.
The site of feedback regulation of xylem ABA and CK
levels is the hypocotyl
Xylem sap was collected below and above the graft union, and
the phytohormone levels were measured separately. This permits
the site of feedback regulation to be deduced more precisely.
The three types of grafting were all performed
hypocotyl-tohypocotyl. Because scions did not influence the root (rootstock)
ABA and CK concentrations (Figs 1b, 2b, 3b, 4b, 5b, 6b), it is
postulated that the site of feedback regulation of xylem
phytohormones by scion lies beyond the root tissue. For standard
grafts, no significant differences in xylem ABA and CK delivery
between rootstock (collected below the graft union) and scion
(collected above the graft union) within a graft were found (Figs
1b, 2b), suggesting that the site of feedback regulation of xylem
phytohormones in standard grafts is most probably the rootstock
hypocotyl (from the rootshoot junction to the graft union). In
terms of Y and inverted Y grafts, the scion(s) did not significantly
affect the xylem ABA and CK flux in rootstock(s) (collected
below the graft union) (Figs 3b, 4b, 5b, 6b), thus implying that
the main site of feedback regulation of xylem phytohormones by
scion(s) is the scion hypocotyl (above the graft union), and the
target of feedback signal(s) is more likely to be the changes in
xylem phytohormone levels within hypocotyl tissues rather than
the export of phytohormones from the roots.
It is considered that when/after xylem phytohormoes passed
through the graft union from rootstock(s) to scion(s) in Y and
inverted Y grafts, they experienced great changes in
metabolism, and/or exchanges with xylem parenchyma cells via the
action of feedback signal(s). This presumption can at least partly
explain the imbalance between rootstock(s) and scion(s) within
either a Y graft or an inverted Y graft in terms of xylem
phytohormone delivery rates (Figs 36), and is supported by the
literature. Sauter and Hartung (2002) demonstrated that the lateral
transport of ABA in the stem (from the stem parenchyma to the
xylem, and vice versa) may contribute to modifying the xylem
ABA levels, and xylem sap can be enriched with ABA sourced
from xylem parenchyma cells by the higher xylem pH (Li et al.,
2011). Moreover, it is known that CKs may be modulated along
their transport pathway, for example by movement from xylem
to parenchyma cells in the stem or petioles (Singh et al., 1992).
Future studies need to confirm the site of feedback regulation of
xylem phytohormones further by using interstock grafting and
top grafting (on the main stem above the cotyledons, as opposed
to the hypocotyl in the present study), and the physiological and
molecular mechanisms underlying the changes in xylem
phytohormones levels within hypocotyl tissues.
The mechanism of feedback regulation of leaf ABA and
CK concentrations remains unclear
Theoretically, the level of phytohormones in leaf is regulated at
diverse steps, including de novo synthesis, activation,
conjugation, and degradation, as well as local and long-distance
transport. Since the differences in xylem CKs levels between CCRI41
and SCRC22 scions were similar to those differences in leaf CKs
concentrations under K deficiency, we cannot exclude the xylem
contribution to leaf phytohormone status by long-distance
transport in the present study.
The capacity of the leaf to biosynthesize ABA and CKs has
been well demonstrated (Miyawaki et al., 2004; Endo et al.,
2008). Furthermore, both ABA inactivation (the conjugation
with glucose by ABA glucosyltransferase to form ABA glucose
ester; Xu et al., 2002) and CK degradation by cytokinin oxidase
(Vysotskaya et al., 2009) can occur in the leaf. Therefore,
further metabolic studies such as on de novo synthesis, activation,
conjugation, and degradation, and local and long-distance
transport need to be undertaken to find the exact mechanism underlying
the feedback regulation of leaf phytohormone concentrations.
K deficiency strongly increased ABA levels in roots and xylem
sap of cotton seedlings and decreased those of ZR- and iPA-type
CKs in xylem sap and leaves. Correlation analysis indicated
that ABA, ZR-, and iPA-type levels in leaves and scion xylem
sap (collected above the graft union) under K deficiency were
closely associated with leaf senescence. The results of both
standard (one scion grafted onto one rootstock) and Y (two
scions grafted onto one rootstock) or inverted Y (one scion grafted
onto two rootstocks) grafting showed that the ABA and CK
levels in leaves and scion xylem sap were feedback regulated by
scion cultivars under K deficiency. The main action site of
basipetal feedback signal(s) involved in xylem phytohormones is the
Fig.9. Relationships between K content (x) and ABA, ZR-, and
iPA-type concentrations in the youngest fully expanded leaf
(the fourth leaf from the top of plant) grown under K deficiency
(0.03 mM for standard and Y grafts, and 0.01 mM for inverted
Y grafts). Open and filled circles denote CCR141 and SCRC22
scions of standard grafts, respectively; open and filled triangles
denote CCR141 and SCRC22 scions of Y grafts; and open and
filled inverted triangles denote CCR141 and SCRC22 scions of
inverted Y grafts. Each point represents the mean of a grafting
treatment averaged across three or four replicates, and each
replicate consisted of 420 plants. The linear regressions were
fitted with Sigmaplot 11.0.
rootstock hypocotyl (below the graft union) in standard grafting
and the scion hypocotyl (above the graft union) in Y and inverted
Y grafting. The target of this feedback signal(s) is more likely to
be the changes in xylem phytohormones within hypocotyl
tissues rather than the export of phytohormones from the roots. The
mechanism of feedback regulation of leaf ABA and CK
concentrations remains unclear. The results of this study have provided
an improved understanding of communication between shoot
and root, and are beneficial for the development of approaches to
managing this problem.
This research was supported by the NSFC (National Natural
Science Foundation of China, 30571118 and 30971708) and the
Program for New Century Excellent Talents in University of
China (NCET-08-0533). We thank the Cotton Research Institute,
Chinese Academy of Agricultural Sciences, Anyang, Henan, and
the Cotton Research Center, Shandong Academy of Agricultural
Sciences, Jinan, Shandong for providing cotton seeds.
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