Transcript Leaf senescence, cytokinoin level and cytokininoxidase

LEAF SENESCENCE, LEVELS OF CYTOKININS AND NITRATE AND ACTIVITY OF THEIR
DEGRADATION ENZYMES IN WILD TYPE AND TRANSGENIC PSAG 12 -IPT WHEAT CV. SCAMP
PLANTS.
1Blanka
ŠOLCOVÁ, 2Gabriela JANDOVÁ, 1Václav MOTYKA, 2Marie TRČKOVÁ, 3Malcolm C. ELLIOTT, 1Miroslav KAMÍNEK
1Institute
of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 135, Prague 6, 165 02, Czech Republic, phone: +42020390436, fax: +42020390446, Email: [email protected]
2Research Institute of Crop Production, Drnovská 505, Prague 6, 161 00, Czech Republic
3The Norman Borlaug Institute for Plant Science Research, De Montfort University, Scraptoft, Leicester LE7 9SU, U.K.
The aim
Introduction
COMPARISON OF
• THE RATE OF NITRATE UPTAKE
Leaf senescence is an integral part of plant development. It is accelerated by different external stimuli including
mineral deficiency, especially deficiency of nitrogen. As programmed process it is under control of internal
mechanisms which include hormonal regulations. Cytokinins (CKs) are known to slow-down the progress of
senescence. Expression of most genes is down-regulated during the progressing leaf senescence but expression
of distinct sets of genes, so called SAGs (senescence-associated genes), is increased (for review see Yoshida
2003).
IN THE 1ST AND 2ND LEAF OF THE WILD-TYPE AND PSAG 12 – IPT TRANSGENIC WHEAT CV. SCAMP PLANTS
AT OPTIMAL ( 1158 mM) AND SUBOPTIMAL (773 mM) NITRATE LEVEL.
We compare here the progress of senescence, changes in CK levels and in uptake and assimilation of nitrogen in
transgenic wheat plants transformed with PSAG 12 -IPT and untransformed controls.
* THE PROGRESS OF SENESCENCE
• CYTOKININ CONTENT AND CYTOKININ OXIDASE/DEHYDROGENASE ACTIVITY
• NITRATE CONTENT AND NITRATE REDUCTASE ACTIVITY
CHLOROPHYLL CONTENT IN WILD TYPE AND
TRANSGENIC P SAG 12-IPT WHEAT CV. SCAMP
PSAG 12 -IPT
140
3
2
1
0
1st leaf
2nd leaf
1st leaf
HN
100
80
60
40
20
0
2nd leaf
1st leaf
2nd leaf
Leaf position
HN
100
80
60
40
20
1st leaf
2nd leaf
1st leaf
2nd leaf
Leaf position
control
PSAG 12-ipt
200
150
[mmol]
LN
Nitrate content in medium
PSAG 12 -IPT
-
[nmol NO3 / g FW / min]
Nitrate reductase activity
NITRATE UPTAKE OF WILD TYPE AND
TRANSGENIC PSAG 12-IPT WHEAT CV. SCAMP
120
0
2nd leaf
Figure 2: control = wild type wheat, PSAG 12 – IPT = ipt transformed plants
with senescence inducible promotor, 1stleaf = flag leaf, LN (low nitrate) =
suboptimal level of NO3- (773 mM) in medium, HN (high nitrate) = optimal level
of NO3- (1158 mM) in medium. Columns characterize nitrate content in mmol
NO3- / g leaf fresh weight.
NITRATEREDUCTASE ACTIVITY IN WILD TYPE AND
TRANSGENIC PSAG 12-IPT WHEAT CV. SCAMP
140
1st leaf
Leaf position
Figure 1: control = wild type wheat, PSAG 12 – IPT = ipt transformed plants
with senescence inducible promotor, 1stleaf = flag leaf, LN (low nitrate) =
suboptimal level of NO3- (773 mM) in medium, HN (high nitrate) = optimal
level of NO3- (1158 mM) in medium. Columns characterize the chlorophyll
(a+b) content in mg / g leaf fresh weight.
control
PSAG 12 -IPT
LN
120
-
HN
Nitrate content
LN
control
[mmol NO3 / g FW]
4
[mg / g FW]
Chlorophyll content
control
NITRATE CONTENT IN WILD TYPE AND TRANSGENIC
PSAG 12-IPT WHEAT CV. SCAMP
100
50
0
0
40 72 104 134 167 197 127 157
Time [min]
Figure 4. control = wild type wheat, PSAG 12 – IPT = ipt
transformed plants with senescence inducible promotor,
Points characterize nitrate content in medium in mmol et
time.
Figure 3: control = wild type wheat, PSAG 12 – IPT = ipt transformed plants
with senescence inducible promotor, 1stleaf = flag leaf, LN (low nitrate) =
suboptimal level of NO3- (773 mM) in medium, HN (high nitrate) = optimal level
of NO3- (1158 mM) in medium. Columns characterize nitrate reductase activity
in nmol NO3- / g leaf fresh weight / min.
CYTOKININ CONTENT IN WILD TYPE AND TRANSGENIC PSAG 12-IPT WHEAT CV. SCAMP
[pmol / g FW]
control
PSAG 12 -IPT
Results
Chlorophyll content decreased with the leaf insertion and age and the senescence was delayed in leaves of
transgenic PSAG 12 –IPT plants as compared to control ones (fig. 1). Optimal nitrate level in medium a line the
differences in chlorophyll content in flag leaf between controls and ipt transformed plants. The physiologically
active CKs in flag leaves of transgenic PSAG 12 –IPT plants were to large extent converted to storage O-glucosides
and inactive N-glucosides. Transgenic P SAG 12 –IPT plants also contained much higher amount of inactive
derivates of cis-zeatin (table 1) High level of active cytokinin forms remain in control plants. Optimal nitrate level
shift cytokinins in wild type plants towards to inactive N-glucosides. Cytokinin oxidase/dehydrogenase (CKX) was
more active in transgenic P SAG 12 –IPT plants compared to controls but the optimal nitrate level decreased the
CKX activity (table 2). The nitrate reductase (NR) activity was higher in flag leaves than in older leaves in both
control and ipt transformed plants (fig. 2). Leaves of transgenic P SAG 12 -IPT plants displayed significantly lower
NR activity than leaves of corresponding controls and levels of nitrate positively correlated with NR activity (fig. 1).
These results indicate that accumulation of active CKs in transgenic PSAG 12 -IPT plants is accompanied with their
inactivation and degradation to maintain cytokinin homeostasis. Lowering of nitrate levels and activity of NR in
leaves of transgenic PSAG 12 –IPT plants implies that nitrate uptake was either decreased or its assimilation was
enhanced in ipt transformed plants.
B+R
O-G
N-G
ribotides
cis
derivates
Σ
LN
142,8
19,8
94,0
11,6
820,9
1089,1
HN
3,4
33,2
129,1
10,5
1520,4
1696,6
LN
10,2
39,1
32,1
12,0
1602,5
1695,9
• THE SENESCENCE OF TRANSGENIC PSAG 12 -IPT PLANTS WAS DELAYED, MAINLY IN THE FLAG LEAF
HN
6,5
15,9
8,3
8,9
1370,0
1409,6
(FIG. 1).
CYTOKININ OXIDASE/DEHYDROGENASE ACTIVITY IN WILD TYPE AND TRANSGENIC PSAG 12-IPT
WHEAT CV. SCAMP
nitrate level
PSAG 12 - IPT
Plant material: Wheat (Triticum aestivum L., cv. Scamp) wild type and the ipt transformed plants with senescence
inducible promotor (PSAG 12 – IPT).
Cultivation: Both PSAg 12 –IPT transgenic and wild type wheat were cultivated in hydroponic conditions (82,5 mM
MgSO4, 190 mM KH2PO4, 105 mM KCl) at 16 / 8 h and 20 / 15°C (day / night) under optimum (1158 mM) and
suboptimum (773 mM) NO3- supply.
Transformation: The wheat immature embryos were co-bombarded by plasmids pSG516 and pDB1. Plasmid
pSG516 contained PSAG 12-ipt as the target construct while plasmid pDB1 contained gusA and bar as reporter
and selectable marker, respectively.
Selection of transformants was performed on MS medium without plant growth regulators but supplemented with 5
mg/l bialaphos.
Presence of ipt in DNA was determined using PCR.
Senescence progress was detected on the chlorophyll loss basis.
Chlorophyll was extracted using 85% acetone acording to AOAC Official Methods of Analysis.
Endogenous cytokinins were isolated and quantified using HPLC/MS according to Dobrev and Kaminek (2002).
Nitrate content was estimated spectrophotometrically using a Skalar San plus analyzer.
Nitrate uptake was determined by depletion from nutrient solution.
Cytokinin oxidase/dehydrogenase activity was estimated by in vitro conversion of [2,8-3H] isopentenyladenine
to [2,8-3H] adenine according to Motyka et al (1996).
Nitrate reductase activity was determined by an in vitro assay using azo dye.
Nitrate level
Table 1: control = wild-type wheat, PSAG 12 –IPT= ipt transformed plants with senescence inducible promotor, B+R = bases
and ribosides (cis and trans zeatin, isopentenyladenine and dihydrozeatin and their ribosides), O-G = O-glucosides of
zeatin, zeatin riboside and dihydrozeatin, N-G = N7- and N9 –glucosides of zeatin, dihydrozeatin and isopentenyladenine,
ribotides – of trans and cis zeatin riboside, dihydrozeatin riboside and isopentenyladenine riboside, cis derivates of zeatin
N7- and N9–glucosides, zeatin O-glucoside and zeatine riboside O-glucoside, Σ = total cytokinin content, LN (low nitrate) =
suboptimal level of NO3- (773 mM) in medium, HN (high nitrate) = optimal level of NO3- (1158 mM) in medium.
control
Material and Methods
activity
specific activity
[pmol Ade / h x g FW]
[nmol Ade / mg prot. x h]
LN
0,0306
0,0086
HN
0,0146
0,0020
LN
0,0057
0,0086
HN
0,0147
0,0082
Table 2: control = wild type wheat, PSAG 12 –IPT = ipt transformed plants with senescence inducible promotor, LN (low nitrate) =
suboptimal level of NO3- (773 mM) in medium, HN (high nitrate) = optimal level of NO3- (1158 mM) in medium, Ade = adenine, FW = fresh
weight.
Knowledges
This research was supported by the Grant Agency of the Czech Republic (grant No. 522/02/0530).
Conclusions
• MAJORITY OF PHYSIOLOGICALLY ACTIVE CYTOKININS IN TRANSGENIC PSAG 12 -IPT PLANTS WAS
CONVERTED TO STORAGE AND INACTIVE DERIVATES INCLUDING O- AND N-GLUCOSIDES AND CIS
DERIVATES OF ZEATIN. OPTIMAL LEVEL OF NITRATES IN MEDIUM INCREASED THE CONVERSION OF
ACTIVE CYTOKININS TO INACTIVE N-GLUCOSIDES IN CONTROLS (TABLE 1).
• CYTOKININ OXIDASE/DEHYDROGENASE ACTIVITY AND TOTAL PROTEIN CONTENT WERE HIGHER IN
CONTROLS COMPARE TO TRANSGENIC PSAG 12 -IPT PLANTS; OPTIMAL LEVEL OF NITRATES
DECREASED CKX ACTIVITY IN CONTROLS (TABLE 2).
•LEAVES OF CONTROL PLANTS SHOWED HIGHER LEVEL OF NITRATE AND SIMILARY HIGHER ACTIVITY
OF NITRATE REDUCTASE COMPARED TO TRANSGENIC PSAG 12 –IPT WHEAT (FIG. 2 AND 3).
• NITRATE UPTAKE ET ANTHESIS WAS HIGHER AND MORE EXTENDED IN TRANSGENIC PSAG 12 -IPT
PLANTS COMPARE TO CONTOLS (FIG. 4).
References
AOAC Official Methods of Analysis: Chlorofhyll in plants. Spectrophotometric metod for total chlorophyl and the a and b components, pp 62 - 63
(1990)
Dobrev P., Kaminek M. (2002). J. Chromat. A 950:21-29
Motyka V., Faiss M., Strnad M., Kamínek M., Schmülling T. (1996). Plant. Physiol. 112: 1035-1043.
Yoshida A. S. (2003). Curent Opinion in Plant Biology 6, 79-84