Lectures L6.1 Session 6. New Vistas in Plant Molecular Biology L6.2

Transkrypt

Session 6. New Vistas in Plant Molecular Biology
Lectures
L6.2
L6.1
Phytohormone-binding
proteins in legume plants
Signal transduction in plant
immunity response
Justin Lee1, Gerit Bethke2, Lennart Eschen-Lippold1,
Ines Lassowskat1, Siska Herklotz1, Luis Maldonado
Bonilla1, Kai Naumann1, Mieder Palm-Forster1,
Pascal Pecher1, Stefanie Ranf1, Dierk Scheel1
1Leibniz
Institute of Plant Biochemistry, Department of Stress &
Developmental Biology, Halle, Germany; 2Microbial and Plant Genetics
Institute, University of Minnesota, St. Paul, MN, USA
e-mail: Justin Lee <[email protected]>
Plant immunity responses are initiated upon recognition of conserved microbe-associated molecular patterns
(MAMPs) that trigger a network of cellular signalling
events. Our research emphasis is on two aspects of the cellular signal transduction, viz. calcium and mitogen-activated protein kinase (MAPK) signalling.
Calcium flux is one of the earliest events following MAMP
perception. Several known signalling components acting
concomitantly with cellular Ca2+ elevation were tested for
an influence on calcium signalling. Among the investigated
components, BAK1, a receptor-like kinase associated with
several receptors, appears to differentially amplify the signals to attain stimuli-specific calcium amplitudes. Additionally, the overall “calcium signature” is shaped by reactive
oxygen species accumulation. To further search for novel
components regulating this crucial signalling step, a genetic
screen was performed to isolate mutants with altered calcium signature after application of the MAMP, flg22. This
led to several novel alleles of the flg22 receptor, FLS2 and
its associated partner, BAK1, as well as several new mutants with changed calcium elevation (cce).
Downstream of calcium, mitogen-activated protein kinase
(MAPK) cascades control signal transduction pathways
in eukaryotes through protein phosphorylation. As several MAPK components are shared in multiple distinct
pathways, specificity must somehow be maintained to
prevent erroneous signalling. We propose that signal fidelity of MAPK pathways is, in part, maintained through
the repertoire and combination of diverse MAPK substrates. Towards this end, we have identified a number
of MAPK-interacting proteins and putative substrates by
yeast-two-hybrid analysis or a protein-array-based kinase
screen. These potential MAPK substrate targets include
potential transcription factors, RNA-binding proteins, enzymes or proteins of yet unknown functions. We are in the
process to verify how some of the isolated MAPK substrates are involved in plant immunity or other processes.
Michal M. Sikorski
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań,
Poland
e-mail: Michal Sikorski <[email protected]>
Plant pathogenesis-related proteins of class 10 show an
ability to bind phytohormones. They are cytosolic proteins
present in various plant tissues. Some their homologues are
developmentally regulated. These proteins are also involved
in the plant defense reactions. Among them, two major protein subclasses can be distinguished: very abundant and not
abundant showing less than 20% sequence identity to the
first subclass. The low abundant protein subclass specifically
binds cytokinins (CSBP, cytokinin-specific binding protein),
which evolved from very abundant PR-10 protein class.
In order to recognize physiological function of the MtCSBP,
we have applied RNAi-mediated gene silencing strategy in a
transgenic Medicago truncatula. Two Mtcsbp coding DNA
fragments (1 - 443nt and 1-130nt) were subcloned into pHellsgate 12 vector, introduced into Agrobacterium tumefaciens
GV3101, and used for plant transformation. The transgenic
plants were obtained via direct shoot organogenesis.
We have analyzed the expression pattern of csbp gene and
other genes of the cytokinin transduction pathway in transgenic plants. As a consequence of csbp gene silencing, a
significant drawdown expression of the gene coding for
cytokinin receptor cre1 was pointed out. A visible decrease
of the pr-10.1 gene was also observed.
The csbp gene silencing causing visible changes in the cre1
and pr-10.1 genes expression level shows that cytokinin
perception in the cell is weaker and plant becomes less sensitive to phytohormone stimulus.
The proteome analysis of the transgenic plants by 2DPAGE revealed elevated level of OEE1 protein (response
to the imbalance of hormonal level). A similar expression pattern was obtained for glycoside hydrolase GH17
that belongs to the PR-2 class. This protein overtakes the
function of the PR-10.1 defense protein the expression
of which in transgenic plants was reduced. Minor changes
in accumulation of protein of the 14-3-3A protein family
(binds a wide range spectrum of small ligands and plays a
function of dense proteins) were also observed.
We have recently co-crystalized the Medicago truncatula
CSBP with gibberellic acid and it has been found in the
hydrophobic cavity of the protein crystal. It showed that
the previously described protein can bind a higher spectrum of plant hormones. Therefore, we have postulated
to rename the MtCSBP to MtPhBP (Medicago truncatula
phytohormone-binding protein).
Taking into account the above results, we checked the expression of genes involved in gibberellin pathway in transgenic
plants. For all tested plants we observed a decrease of the transcript level of the gibberellin oxidase Mtga2ox gene responsible for degradation of the biologically active gibberellins. In
transgenic plants it provides to accumulation of gibberellins
at higher level. Therefore, the hormonal equilibrium remains
shifted towards the gibberellin transduction pathway.
47th Congress of the Polish Biochemical Society, 2012
Session 6. New Vistas in Plant Molecular Biology155
L6.3
L6.4
TIME FOR COFFEE mediates between
the circadian clock and JA signaling by
repressing MYC2 protein accumulation
Are plants intelligent? Exponentially
integrated quantum-molecular
overall regulation of growth,
photosynthesis, defence and
acclimatory responses in Arabidopsis
Seth J. Davis
Department of Plant Developmental Biology, Max-Planck-Institute for
Plant Breeding Research, Köln, Germany
e-mail: Seth J. Davis <[email protected]>
Plants undergo physiological and developmental changes
in harmony with the day/night cycle. Perception of various
rhythmic environmental cues, such as light and temperature,
set the phase of the clock to allow for signaling systems
to be maximal at the appropriate time of the day. TIME
FOR COFFEE (TIC) was identified as a clock regulator
important to maintain circadian period and amplitude. TIC
protein has been shown to localize in the nucleus, however,
it is a pioneer protein whose biochemical activities cannot
be inferred from its amino-acid sequence. To investigate
the underlying molecular mechanism of TIC by finding its
interacting partners, we performed yeast-two-hybrid screen
using the N-terminus of TIC as bait, and found MYC2 as
a TIC interacting protein. MYC2 encodes a bHLH protein,
which positively regulates the jasmonic acid (JA) signaling.
Through molecular genetic and biochemical approaches,
we found that TIC negatively regulates MYC2 protein accumulation, and tic mutants are hypersensitive to JA in a
MYC2-dependent manner. Further, we defined that JA responses are time specifically gated by the clock, with higher
response at dawn and lower response at dusk. Notably, tic
mutants showed hypersensitivity to JA at all times of the
day. Therefore, TIC is necessary for the clock gating of
JA responses. Collectively, we identified an interconnection
between the circadian clock and JA signaling, and TIC plays
a key role in clock-gated JA responses.
Stanisław M. Karpiński
Warsaw University of Life Sciences-SGGW, Faculty of Horticulture and
Landscape Architecture, Department of Genetics, Breading and Plant
Biotechnology, Warsaw, Poland
e-mail: Stanislaw Karpinski <[email protected]>
In a simplified model of photosynthesis, light energy absorbed by chlorophylls of photosystem II is distributed
between photochemistry, fluorescence, and heat. Spectrally
and time-resolved fluorescence combined with foliar heat
dynamics measurements demonstrates that higher plants
evolved genetic and physiological overall regulatory system, which optimizes photosystem II quantum-molecular
functions and the fate of photons absorbed in excess [1,
2]. This in turn specifically influences overall electrochemical signalling [3] that regulate growth, acclimatory and
defence responses in Arabidopsis [4-6]. Moreover, changes
in photochemistry, water use efficiency, hormonal and reactive oxygen species cellular homeostasis, and seed yield
of Arabidopsis can be defined by the exponential function
and simple equation with natural logarithm (y = y0*e-Kx),
that depends on molecular regulators: LESION SIMULATING DISEASE 1 (LSD1), ENHANCED DISEASE
SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4) (4-6). The LSD1 recessive null mutant
(lsd1) regardless of permissive laboratory or non-permissive laboratory and field conditions demonstrates constant
seed yield, but significant variation in photochemistry and
water use efficiencies, and in foliar transcriptomes that
depend on EDS1 and PAD4. Obtained results suggest
that LSD1/EDS1/PAD4 constitute at least tree-component -molecular machinery regulating plant Darwinian fitness. This intelligent processing allows to reach the best
possible seed yield and Darwinian fitness in multivariable
natural environment.
References
1. Kulasek M et al. (2012) submitted.
2. Karpiński S et al. (1999) Science 284: 654-657.
3. Szechyńska-Hebda M et al. (2010) Plant Cell 22: 2201-2218.
4. Mateo A et al. (2004) Plant Physiol 136: 2818-2830.
5. Mühlenbock P et al. (2008) Plant Cell 20: 2339-2356.
6. Wituszynska W et al. (2012) submitted.
The First Polish-German Biochemical Societies Joint Meeting, 2012
156
L6.5
L6.6
Ab initio identification of the substrate
of an orphan enzyme via non-targeted
metabolomics — the glucosyltransferase
UGT76B1 and its substrate isoleucic
acid modulate plant defense
Crosstalk between the chaperone
network and heat stress
transcription factors in plants
Veronica von Saint Paul1, Wei Zhang1, Rafał Maksym1,
Basem Kanawati2, Philippe Schmitt-Kopplin2,
Theresa Faus-Kessler3, Anton R. Schäffner1
Goethe-University Frankfurt, Frankfurt am Main, Germany
1Institute
of Biochemical Plant Pathology, Helmholtz Zentrum
München, Neuherberg, Germany; 2Analytical Biogeochemistry,
Helmholtz Zentrum München, Neuherberg, Germany; 3Institute of
Developmental Genetics, Helmholtz Zentrum München, Neuherberg,
Germany
e-mail: Anton Schäffner <[email protected]>
Plants tightly regulate defense against biotrophic and necrotrophic pathogens by the mostly antagonistic salicylate(SA)- and jasmonate-(JA)-mediated signaling pathways.
The Arabidopsis thaliana small-molecule glucosyltransferase
UGT76B1 and its newly identified substrate isoleucic
acid (ILA) are novel modulators of this SA-JA crosstalk.
UGT76B1 is a member of a large, 122-numbered gene
family in A. thaliana. Most of these glycosyltransferases are
orphan enzymes without known physiological roles. However, public expression data indicated that UGT76B1 was
the top stress-induced isoform responsive to many biotic
and abiotic cues. Loss of UGT76B1 function led to enhanced resistance to the biotrophic bacterium Pseudomonas
syringae, yet higher susceptibility towards the necrotrophic
fungus Alternaria brassicicola. This is accompanied by constitutively elevated SA levels and SA-related marker gene
expression, whereas JA-dependent markers are repressed.
The opposite was found in lines constitutively overexpressing UGT76B1. Both induction of SA pathway and suppression of JA pathway in the ugt76b1 mutant are dependent on
SA and abolished in NahG lines completely degrading SA.
Based on a non-targeted metabolome analysis of mutant,
overexpression and wild-tpye extracts using FT-ICR mass
spectrometry, we succeeded in identifying a substrate of
UGT76B1 without any prior clue on its chemical nature.
ILA (2-hydroxy-3-methyl pentanoic acid) was also glucosylated in vitro by recombinant UGT76B1. Importantly,
exogenous application of ILA induced the SA marker gene
PR1 and led to an enhanced resistance to P. syringae. This
action of ILA required at least basal SA level, since NahG
lines, efficiently degrading internal SA, disabled PR1 induction. In contrast, ILA was still relatively effective in a sid2
line leading to the loss of defense-induced SA, though PR1
expression was not restored to the wild-type level. Thus,
ILA effects are only partially dependent on SID2-related
SA. In conclusion, these findings indicate (1) UGT76B1 as
a novel player in plant defense attenuating SA-dependent
defense and promoting JA response and (2) a novel link of
amino acid-derived molecules to plant defense via smallmolecule glucosylation. Currently, we explore whether ILA
is also effective on defense systems in other plant species.
Alexander Hahn, Sascha Röth, Daniela Bublak,
Klaus-Dieter Scharf, Enrico Schleiff
e-mail: Enrico Schleiff <[email protected]>
Heat stress transcription factors (Hsfs) are involved in the
control of gene expression in all eukaryotes as regulatory
components of the heat stress response, but also as regulator of other types of stress and developmental processes.
In plants, at least 20 Hsfs are involved in adaptation of
transcription to changes in environmental conditions. The
function of Hsfs is controlled on the transcriptional level,
but also by a tight crosstalk with the chaperone network
and protein degradation network of the cell. This interaction of the two functional distinct networks targeting either
transcriptional regulation or protein folding is consistent
with the accumulation of both, Hsfs and heat stress proteins (Hsps) in cells during stress.
Our data will demonstrate that in tomato (Lycopersicon esculentum) the functional crosstalk between the two networks is
manifested by physical interactions of the three major Hsfs,
i.e. HsfA1a, HsfA2, and HsfB1, with Hsp70 and Hsp90. At
the same time the functional consequences of these interactions are factor-specific. On the one hand, Hsp70 was
found to repress the activity of HsfA1a including its binding to the target DNA. On the other hand, HsfB1 is tightly
controlled by both Hsp70 and Hsp90. The latter is involved
in the targeting of HsfB1 for proteasomal degradation,
however, at the same time Hsp90 enforces the DNA-binding of this Hsf. In contrast to HsfA1a and HsfB1, which
are both regulated at the protein level, the crosstalk between HsfA2 and Hsp90 is manifested by the influence of
Hsp90 on the degradation of hsfA2 transcripts.
Summarizing these and recent data it becomes obvious
that during repeated cycles of HS and recovery the activity
and composition of the cellular Hsf network is dynamically controlled by factor-specific physical and functional
interactions with distinct chaperones from the Hsp70,
Hsp90 and small Hsp families. Our findings implicate the
existence of a versatile regulatory system based on mutual
feedback mechanisms between the two central networks required for the efficient adaptation of protein homeostasis
under permanently changing temperature conditions.
Reference
von Saint Paul V et al. (2011) Plant Cell 23: 4124–4145.
L6.7
Dinucleoside polyphosphates and
some other uncommon nucleotides
function as alarmones in plants
Andrzej Guranowski1, Katarzyna Nuc1, Łukasz Czekała2,
Małgorzata Zielezińska2, Małgorzata Pietrowska-Borek2
1Department
of Biochemistry and Biotechnology and 2Department of
Plant Physiology of Poznań University of Life Sciences, Poznań, Poland
e-mail: Andrzej Guranowski <[email protected]>
Diadenosine polyphosphates, such as ApppA and AppppA can be synthesized by some ligases or transferases [1]. These enzymes are able to transfer adenyl- moiety from
mixed anhydride, e.g. aminoacyl-AMP [2] or coumaroylAMP [3], onto an acceptor which contains pyrophosphate
residue, such as ADP, ATP or any NDP and NTP. In early
1980s, Np3N′s or Np4N′s were demonstrated to occur in
eucaryotic and procaryotic cells [4, 5] and their levels increased from submicromolar (when the cells were under
normal conditions) to nearly millimolar concentration under various types of stress, such as temperature, ethanol or
heavy metals [6]. Because of that, these compounds have
been called alarmones. However, molecular “addressee” of
the postulated “alarm” has not been experimentally found.
It is known that in plants all kinds of biotic or abiotic stress
induce the synthesis of different aromatic compounds
(flavonoids, anthocyanins, salicylic acid or lignins) that
are the products of the phenylpropanoid pathway [7]. We
have studied the effects induced in Arabidopsis thaliana by
cadmium (Cd2+) and found that the cation dramatically increased the specific activities of enzymes that initiate the
phenylpropanoid pathway [8]. Knowing that Cd2+causes
accumulation of Np3-4N′-alarmones in various cells [5,
6], we wondered whether those compounds would induce
the aforementioned rescue action in Arabidopsis seedlings
when exogenously administrated? And in fact, we demonstrated that submicromolar concentration of either
ApppA or AppppA triggered in the Arabidopsis seedlings
the expression of the genes for and specific activities of
phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA
ligase (4CL) [9]. The response was quick (could be noticed
in 10 min), dramatic (the PAL2 expression was up to 70fold bigger than in the untreated plants), and specific (none
of the potential ApppA or AppppA degradation products: AMP, ADP, ATP or adenosine) evoked the effects. Also our studies of cyclic nucleotides, cAMP and cGMP,
showed that Cd2+ stimulates the expression and activity of
cyclic adenylate- and guanylate cyclases [10] and increases
the level of cAMP in A. thaliana (unpublished). Our recent
experiments demonstrated that both 5 µM cAMP or cGMP
administrated to the Arabidopsis growth medium induced
the specific activities of PAL and 4CL in the seedling extracts [11]. Finally, we have recently observed that the same
effects could be evoked by another type of nucleotides:
adenosine 5′-phosphoramidate (NH2-pA) or adenosine
5′-phosphofluoride (F-pA). These compounds are good
substrates of the ubiquitously occurring nucleoside phosphoramidase, the Hint1 protein which is the member of
the conservative HIT-protein family [12, 13]. The former
compound proved to be a naturally occurring metabolite
[14]. The biochemistry of NH2-pA and biological role of
the compound are practically unknown. It is synthesized by
adenylylsulfate:ammonia adenylyltransferase (EC 2.7.7.51)
that catalyzes the replacement of sulfate with amino group
[15].
We put forwards the hypothesis that all the aforementioned
uncommon nucleotides can function as signal molecules
and their action is possible due to reactions catalyzed by
specific enzymes that hydrolytically or phosphorolytically
release adenyl- moiety and energy which is used for activation of appropriate transcription factors controlling the
expression of corresponding genes. The hypothesis needs
experimental verification and our findings only open the
promising avenue.
Reference
1. Fraga H, Fontes R (2011) Biochim Biophys Acta 1810: 1195-1204.
2. Zamecnik PC, Stephenson ML, Janeway CM, Randerath K (1966) Biochem Biophys Res Commun 24: 91-97.
3. Pietrowska-Borek M, Stuible HP, Kombrink E, Guranowski A (2003)
Plant Physiol 131: 1401-1410.
4. Lee PC, Barry R, Ames BN (1983) J Biol Chem 258: 6827-6834.
5. Brevet A, Plateau P, Best-Belpomme M, Blanquet S (1985) J Biol Chem
260: 15566-15570.
6. Coste H, Brevet A, Plateau P, Blanquet S (1987) J Biol Chem 262: 1209612103.
7. Dixon RA, Paiva NL (1995) Plant Cell 7: 1085-1097.
8. Pietrowska-Borek M, Nuc K (2010) Acta Soc Bot Pol 79 (S1): 90.
9. Pietrowska-Borek M, Nuc K, Zielezińska M, Guranowski A (2011)
FEBS OpenBio 1: 1-6.
10. Pietrowska-Borek M, Nuc K, Stroiński A, Zielezińska M (2009) Acta
Biol Crac 51 (S2): 64.
11. Pietrowska-Borek M, Nuc K (2012) Plant Cell Physiol (submitted).
12. Guranowski A, Wojdyła AM, Zimny J, Wypijewska A, Kowalska J
Łukaszewicz M, Jemielity J, Darżynkiewicz E, Jagiełło A, Bieganowski P
(2010) New J Chem 34: 888-893.
13. Guranowski A, Wojdyła AM, Rydzik AM, Stępiński J, Jemielity J
(2011) Acta Biochim Pol 58: 131-136.
14. Frankhauser H, Berkowitz GA, Schiff JA (1981) Biochem Biophys Res
Commun 101: 524-532.
15. Frankhauser H, Schiff JA, Graber LJ (1981) Biochem J 195: 545-560.
158
Oral presentations
O6.2
O6.1
Small RNAs in plant nutrient
signaling and homeostasis
Chromatin mechanisms and
hormone signaling in plants
Rafal Archacki
University of Warsaw, Faculty of Biology, Department of Plant Molecular
Biology, Warsaw, Poland
e-mail: Rafal Archacki <[email protected]>
Przemyslaw Nuc1,2, Patrick May3, Magdalena
Musialak-Lange1, Bikram Pant4, Wolf Scheible4
1Max Planck Institute of Molecular Plant Physiology, Germany;
2Adam Mickiewicz University, Institute of Molecular Biology
and Biotechnology, Poznań, Poland; 3University of Luxembourg,
Luxembourg Centre for Systems Biomedicine, Luxembourg; 4The
Samuel Roberts Noble Foundation, Plant Biology Division, USA
e-mail: Przemyslaw Nuc <[email protected]>
In eukaryotes, the activation and repression of genes largely
depends on their chromatin state. The mechanisms underlying modulation of chromatin structure, including posttranslational modifications of histones, DNA methylation,
histone variants exchange, and ATP-dependent chromatin
remodeling are the main components of the epigenetic system that serve to integrate genetic programs with environmental signals and enables inheritance of gene expression
patterns. Chromatin remodeling depends on the activity of
evolutionarily conserved multimeric assemblages, of which
SWI/SNF complexes have been the most thoroughly studied. During regulation of gene expression, SWI/SNF complexes have both global and highly specific functions. One
example is their central role in steroid hormone signaling
through protein-protein interactions with hormone nuclear
receptors. Our recent results indicate that similarly to animals, SWI/SNF complexes may be also directly involved in
hormonal signaling in plants. Possible mechanisms underpinning this role and links to other components of epigenetic system will be discussed.
In sequenced genomes of land plants about 200 miRNA
genes can be found considering strict annotation rules [1].
Far more different small RNAs can be detected in RISC
complexes and considered as potential facilitators of targeted mRNA degradation [2]. Several ancient miRNAs
participate in the maintenance of macronutrient homeostasis. MiR399 regulates phosphate uptake and allocation
[3, 4]. Sulphur starvation induced miR395 regulates S assimilation and allocation [5]. MiR398 and 408 accommodate plant’s copper resources to its availability and miR393
participates in nitrate sensing [6, 7].
Several reports show more or less dramatic changes in
small RNA accumulation in response to nutrient stress. The
number of A. thaliana miRNAs which potentially participate in sensing and adaptation to P limitation extends for
several others, like miR156, miR 827, miR 2111, miR828,
miR778 and miR408 [8, 9]. MiRNA expression response to
nutrient stress is tissue specific and opposite changes can
be observed in different tissues upon the same treatment.
This response extends to small RNAs of different origin
which can be co-immunoprecipitated with argonautes. The
naïve approach of miRNA expression profiling allows for
selection of biomarker molecules which can be easily detected and quantitated in plants under nutrient stress. If a
change in miR accumulation is of a biological relevance,
the interaction with its target mRNA is the simplest way
to follow the signaling pathway [10]. Transcriptome-wide
analysis of plant mRNA degradation shows which target
predictions can be functional. Finally deregulation of miRNA/mRNA interactions can give a more detailed picture
of miRNA driven signaling cascades and plant’s benefits
from evolutionary conserved complementarities between
mRNAs and their imperfect inverted repeat copies [10].
References
1. Nozawa M et al. (2011) Genome Biol Evol 4: 230-239.
2. Mi S et al. (2008) Cell 133: 116-127.
3. Aung K et al. (2006) Plant Phys 141: 1000-1011.
4. Bari R et al. (2006) Plant Phys 141: 988-999.
5. Kawashima C et al. (2011) Plant J 66: 863-76.
6. Yamasaki H et al. (2009) Plant Cell 21: 374-361.
7. Vidal E et al. (2010) Proc Natl Acad Sci 107: 4477-4482.
8. Pant B et al. (2009) Plant Phys 150: 1541-1555.
9. Hsieh L et al. (2009) Plant Phys 151: 2120-2132.
10. Nuc P et al. (2011) GABI Status Seminar.
11. Todesco M et al. (2010) PLOS 6: 1-10.
O6.3
O6.4
Regulation of gene expression during
the sexual reproduction in liverworts
— the oldest living land plants
Cyclic nucleotide signal
transduction in plants
Izabela Sierocka, Halina Pietrykowska,
Zofia Szweykowska-Kulinska
Department of Gene Expression, Institute of Molecular Biology and
Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań,
Poland
e-mail: Izabela Sierocka <[email protected]>
In lower plants, like bryophytes, sex determination is manifested in the gametophyte generation by production of
egg- and sperm-forming gametangia, in many species on
separate male and female individuals. RDA-cDNA was
performed to search for genes specifically expressed in the
male and female thalli producing sex organs of dioecious
liverwort Pellia endiviifolia species B.
Four genes were selected as specifically expressed in the
male thalli. These are PenB_TUA1 coding for an α-tubulin
family protein, PenB_Raba1/11 coding for a Rab family
protein, PenB_HMGbox coding for an HMGbox family
protein and PenB_MT coding for an unknown transcript
containing an ORF of 295 amino acid residues in length.
PenB_TUA1 and PenB_Raba1/11 transcripts are expressed in the male thalli, regardless of whether they develop antheridia or not. PenB_HMGbox and PenB_MT are
exclusively expressed in the male thalli producing antheridia. Moreover, two genes PenB_TUA1 and PenB_Raba1/11
are encoded only in the male genome of P.endiviifolia sp B.
Five RDA-cDNA fragments were selected as specifically
expressed in the female thalli. Molecular characterisation
of the genes and their transcripts need to be further investigated.
Our studies show for the first time the specific contribution
of identified genes in the liverwort male or female gametophyte development.
In higher plants properly regulated specific types of
α-tubulin and Rab family proteins activity are essential
for tip-focused membrane trafficking and growth of the
male gametophyte. Thus they are pivotal to reproductive
success of these plants. HMGbox family plant proteins
display a binding preference toward DNA what increases
the structural flexibility of DNA, promoting the assembly
of nucleoprotein complexes that control DNA-dependent
processes including transcription. Our results show that
genes connected with the gametogenesis processes in higher plants already have their potential counterpart genes in
liverworts — the oldest living land plants.
Adriana Szmidt-Jaworska
Nicholaus Copernicus University, Chair of Plant Physiology and
Biotechnology, Torun, Poland
e-mail: Adriana Szmidt-Jaworska <[email protected]>
Cyclic nucleotides: 3’:5’-cyclic adenyl monophosphate and
3’:5’-cyclic guanyl monophosphate, commonly known as
cAMP and cGMP, are key second messengers in living organisms ranging from bacteria to Homo sapiens.
Cyclic AMP and cyclic GMP are formed from ATP and
GTP by adenylyl cyclase (AC) and guanylyl cylase (GC),
respectively and hydrolyzed to AMP and GMP by the cyclic
nucleotide phosphodiesterase.
Cyclic nucleotides (cNMP) are involved in the transduction of many environmental and developmental stimuli
in bacteria, fungi, animals and algae. In the last decade a
growing number of reports have described both the occurrence and function of cNMP also in vascular plants. It
is evident that cNMPs are playing significant role in some
morphological processes including germination, flowering, xylogenesis and are taking part in regulation of multiple plant responses toward a variety of abiotic and biotic
stresses such as pathogen infection, UV light, drought and
mechanical wounding.
Despite the progress in understanding the diverse biological function of cNMP, it is only recently that the first plant
enzymes, of the unique structure, with AC and GC activity
have been identified and functionally characterized. Moreover, several reports have implicated the existence in plants
of cNMP effectors such as cyclic nucleotide regulated kinases and cyclic nucleotide-gated ion channels.
In this lecture, the mechanisms of plant cyclic nucleotide
signaling will be evaluated and discussed.
160
O6.5
O6.6
Down-regulation of CBP80 gene
expression as a strategy to engineer
drought-tolerant potato Sumoylation of phototropin2,
Arabidopsis blue light receptor
Marcin Pieczynski1, Waldemar Marczewski2,
Jacek Hennig3, Jakub Dolata1, Dawid Bielewicz1,
Paulina Piontek1, Anna Wyrzykowska1, Dominika
Krusiewicz2, Danuta Strzelczyk-Zyta2, Dorota
Konopka-Postupolska3, Magdalena Krzeslowska4,
Artur Jarmolowski1, Zofia Szweykowska-Kulinska1
1Department
of Gene Expression, Faculty of Biology, Adam Mickiewicz
University, Poznań, Poland; 2Plant Breeding and Acclimatization
Institute, National Research Institute, Młochów, Poland; 3Institute of
Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa,
Poland; 4Department of General Botany, Faculty of Biology, Adam
Mickiewicz University, Poznań, Poland
e-mail: Marcin Pieczynski <[email protected]>
The threat of drought is the major climate change problem
recognized worldwide. Innovative plant breeding requires
good quality information on the molecular strategies developed by plants to adapt and resist to drought stress. In
our previous investigations we have shown that A. thaliana
mutants with silenced CBP20 or CBP80 genes are more
drought tolerant in comparison to wild type plants. Based
on these results we carried out experiments with the aim
to obtain transgenic potato lines with higher drought tolerance. We amplified and sequenced CBP20 and CBP80
cDNAs for all four alleles of two potato cultivars Sante
and Desiree. Conservative fragments from each cDNA sequences were used as targets for gene silencing. We prepared
two types of silencing constructs. The first one generates
siRNAs and targets mRNA of CBP20 gene. The second
one contains artificial microRNA gene targeting mRNA of
CBP80 gene. Transgenic potato lines were obtained using
Agrobacterium tumefaciens mediated transformation and
tested for the expression level of CBP20 and CBP80 genes
using PCR real-time method. Western analysis showed the
lack of CBP80 protein in selected transgenic lines transformed with the construct generating artificial microRNA.
Obtained transgenic potato plants revealed higher tolerance to drought, ABA-hypersensitive stomatal closing, the
increase in leaf stomata and trichome density, compact
cuticle structure with the lower number of microchannels
in comparison to Desiree plants. All these findings correspond to higher tolerance to water stress.
According to literature date, it is known that cbp20 and
cbp80 Arabidopsis seedlings present lower level of microRNA 159 in response to ABA, in comparison to wild type
plants. In our research we found that the level of miR159
is decreased, and its targets MYB33, MYB101 mRNAs
are increased in the transgenic potato plants subjected
to drought. Similar changes were observed in Arabidopsis
cbp80 mutant after drought treatment.
Selected transgenic potato lines with silenced CBP20 or
CBP80 genes were grown in greenhouse and plastic tunnel in optimal and drought conditions. Plants with silenced
CBP80 gene showed much more improved water tolerance,
in both grown experiments, in comparison to Desiree potato plants.
The evolutionary conservation of the CBP80 gene role in
plant response to drought nominates it as a good candidate
for genetic manipulation to obtain improved water deficit
tolerant crop plants. Our research has proved that artificial
microRNAs represent a useful tool for gene silencing in
polyploid species like potato.
Olga Sztatelman1, Wojciech Strzałka1, Sylwia KędrackaKrok2, Weronika Krzeszowiec1, Halina Gabryś1
1Department
of Plant Biotechnology, Faculty of Biochemistry,
Biophysics and Biotechnology, Kraków, Poland; 2Department of
Physical Biochemistry, Faculty of Biochemistry, Biophysics and
Biotechnology, Kraków, Poland
e-mail: Olga Sztatelman <[email protected]>
SUMO (Small Ubiquitin-related Modifier) is a small protein
that can post-translationally and reversibly modify other
proteins. Modification by SUMO leads to changes in protein-protein interactions, activity, localization and stability
of the targets. Sumoylation has been implied in many processes in plants, from regulation of development, disease
resistance and flowering to hormone signaling. So far, no
links between sumoylation and light perception has been
reported. Phototropins are blue light receptors responsible for numerous adaptive responses, including chloroplast
relocations. The aim of the present work was to examine
whether phototropin2 can be a target of sumoylation and
if this process has a function in chloroplast movements. N-terminal part of phototropin2 underwent sumoylation
when expressed in E. coli together with SUMO and its
activating and conjugating enzymes. The process was observed both without E3 ligase or enhanced in the presence
of MMS21 ligase. Four examined SUMO isoforms gave
different modification patterns: SUM1 and SUM2 formed
poly- or multi-sumoylated products, in the case of SUM3
most of the modified protein was monosumoylated, and
no modification was detected with SUM5. Analysis of
shorter phot2 fragments showed that SUMO conjugation
can occur in different areas of the protein, consistent with
in silico analysis. MS/MS analysis of SUM3 adduct revealed
numerous potential sumoylation sites.
The interaction between phot2 and sumoylation machinery was additionally confirmed in planta using bimolecular
fluorescence complementation in Nicotiana benthamiana
cells. We tested two SUMO isoformes and two E3 ligases:
MMS21 and SIZ1. Phot2 interacted with SUM1, SUM3
and MMS21, but not with SIZ1. This result was consistent
with subcellular localization of analyzed proteins.
To elucidate physiological significance of phototropin2 sumoylation, chloroplast movements of several Arabidopsis
knock-out mutants in sumoylation-related genes were analyzed. sum1, sum2 and sum3 exhibited wild-type chloroplast relocations in response to continuous blue light and
light pulses. By contrast, siz1 mutants showed normal chloroplast responses to continuous light, but increased sensitivity to short pulses of strong light, with higher accumulation amplitude, but no changes in reaction velocities.
Taken together, we show that phototropin2 is a potential
sumoylation target and this process can be involved in signaling from this photoreceptor.
O6.7
Posters
High-resolution crystal structures
of complexes of plant S-adenosyl-Lhomocysteine hydrolase (Lupinus luteus)
P6.1
Krzysztof Brzezinski1, Mariusz Jaskolski2,3
1Institute
of Chemistry, University of Bialystok, Bialystok, Poland; 2Center
for Biocrystallographic Research, Institute of Bioorganic Chemistry,
Polish Academy of Sciences, Poznań, Poland; 3Department of
Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznań,
Poland
e-mail: Krzysztof Brzezinski <[email protected]>
S-Adenosyl-L-methionine (SAM) is the most common donor of methyl group in cellular methylation of a wide range
of substrates, from small-molecule compounds to macromolecules, including proteins, nucleic acids and polysaccharides. SAM-Dependent methylation generates equimolar
amounts of S-adenosyl-L-homocysteine (SAH), which is
a strong inhibitor of SAM-dependent methyltransferases.
S-Adenosyl-L-homocysteine hydrolase (SAHase) catalyzes
the reversible breakdown of SAH to adenosine and L-homocysteine. By removing the SAH byproduct, SAHase is
an essential enzyme involved in the regulation of methylation reactions.
We present the first crystal structure of SAHase of plant
origin, from the legume yellow lupine. The structures have
been determined for three complexes of the enzyme, with
a reaction byproduct/substrate (adenosine), its nonoxidizable analog (cordycepin), and a product of inhibitor cleavage
(adenine). In all three complexes, the enzyme has a closed
conformation. In addition to the adenosine, adenine or
cordycepin ligands found in the substrate-binding domain,
each subunit contains a tightly bound NAD+ molecule in
the cofactor-binding domain. A sodium cation is found
near the active site, coordinated by residues from a conserved loop that hinges domain movement upon reactant
binding. An insertion segment present in all plant SAHases
is located near an substrate-pocket access channel and participates in its formation. In contrast to bacterial and mammalian SAHases, the channel is open when adenosine or
cordycepin is bound, and is closed in the adenine complex.
Contrary to SAHases from other organisms, which form
tetrames, the plant enzyme is active as a homodimer.
Participation of Lhcb4, Lhcb5 and Lhcb6
— apoproteins of minor, peripheral,
energetic antennae in excitation energy
transfer in higher plant photosystem II
Małgorzata Adamiec1, Anna Pera1, Robert
Luciński1, Krzysztof Gibasiewicz2, Wojciech
Giera2, Edyta Głów2, Grzegorz Jackowski1
1University
of Adam Mickiewicz in Poznań, Faculty of Biology, Institute
of Experimental Biology, Plant Physiology Department, Poznań,
Poland; 2University of Adam Mickiewicz in Poznań, Faculty of Physics,
Department of Molecular Biophysics, Poznań, Poland
e-mail: Małgorzata Adamiec <[email protected]>
Photosystem II (PSII) minor, peripheral energetic antennae
(CP29, CP26 and CP24) may serve as a bridge mediating
excitation energy transfer from LHCII to the core of PSIILHCII supercomplex. Still, fundamental questions relating
to dynamics of excitation energy transfer in PSII minor
antennae remain unanswered. In order to gain more knowledge regarding this issue stationary and time-resolved fluorescence measurements were carried out on stacked thylakoid membrane fragments (BBY particles) isolated from
wild type and T-DNA insertion lhcb4-6 Arabidopsis thaliana mutants. The level of Lhcb1-6 apoproteins as well as
contamination by PSI core apoproteins was determined
for wild type plants and mutants. The dynamics of excitation energy transfer in PSII-LHCII complexes was studied
by time-resolved fluorescence. There are two main bands
in steady-state fluorescence spectra at 77 K: a smaller one
at ~687 nm and a stronger one at ~702 nm, the latter one
being absent at room temperature. The relative amplitudes
of these two bands are slightly different for all the preparations under study. The band at ~702 nm was ascribed
to low-energy chlorophylls in PSII-LHCII complex. The
kinetics of fluorescence decay at 682 nm (fluorescence
maximum at room temperature) is similar for all the preparations, including wild type, except for Lhcb6 mutant, for
which the decay is significantly slower. This observation indicates an important role of CP24 apoprotein in excitation
energy transfer from LHCII to PSII core. Detailed global
analysis in the whole spectral region yielded two lifetimes
of fluorescence decay for all the preparations: 46-59 ps and
137-174 ps. The exact lifetimes values and their relative
contributions depends on preparations.
162
P6.2
P6.3
SERRATE: an important factor of Arabidopsis
thaliana miRNA biogenesis machinery
Lead stress accelerates cell death of
Lupinus angustifolius sp. root meristems
Mateusz Bajczyk, Agata Stępień, Katarzyna
Skorupa, Dawid Bielewicz, Jakub Dolata, Zofia
Szweykowska-Kulińska, Artur Jarmołowski
Łucja Balcerzak, Agnieszka Kobylińska, Mirosław Godlewski
Department of Gene Expression, Institute of Molecular Biology and
Biotechnology, Adam Mickiewicz University, Poznań, Poland
e-mail: Łucja Balcerzak <[email protected]>
e-mail: Mateusz Bajczyk <[email protected]>
MicroRNAs are small non-coding RNAs which are involved
in post transcriptional gene silencing. They take part in the
regulation of many physiological and cellular processes. In
plants, miRNA genes are transcribed by RNA polymerase
II that synthesizes long miRNA primary precursors (primiRNAs), which are processed by DCL1 (an RNase III
type ribonuclease) into pre-miRNAs, and subsequently
cleaved into miRNA/miRNA* duplexes. The miRNA/
miRNA* duplexes are exported to the cytoplasm, where
mature miRNAs are loaded into RNA silencing complex
(RISC), and can pair with target mRNAs, inducing mRNA
cleavage or translational inhibition. Although the conversion of pri-miRNAs into mature miRNAs is catalyzed in
the plant cell nucleus by one enzyme, DCL1, other proteins
are also involved in the process. These are: CBC (a nuclear
cap-binding complex, which is composed of two subunits, CBP20 and CBP80), a dsRNA binding protein HYL1
(HYPONASTIC LEAVES1), and a zinc-finger containing
protein SE (SERRATE). It has been reported that all these
factors are required for the efficient and correct excision
of miRNAs from pri-miRNAs, however, their direct roles
in miRNA biogenesis are still not clear. Moreover, in addition to miRNA biogenesis, SERRATE and CBC take part
in splicing of pre-mRNA. Thus, the involvement of SE
and CBC in processing of pri-miRNA can be connected
with both, splicing of introns from pri-miRNA precursors,
and/or excision of miRNA from its precursor. Therefore,
we compared the level of pri-miRNAs in cbc, hyl1-2 and se-1
mutants with the level observed in wt Arabidopsis plants.
The results showed that the level of over 50% A. thaliana
pri-miRNAs was changed in the se-1 and cbc mutants. These
changes concerned largely the same precursors and were
similar, while in hyl1-2 mutant plants, the expression level
of 40% different pri-miRNAs was altered. In aim to understand the role of SE in plant RNA metabolism, and propose the mechanism of its action, we decided to search for
RNA molecules and proteins that are SE binding partners.
To this end, we have constructed the Arabidopsis thaliana
transgenic plant, in which, in the genetic background of
the se-1 mutant, the FLAG-tagged version of the SE gene
is integrated into the genome. The FLAG-tagged version
of SE is fully active, since it complements all phenotypic disturbances of the se-1 mutant, and the transformed
plants look just like wt. We chose two stable lines with the
highest expression level of FLAG-SE, and performed immunoprecipitation with antiFLAG antibodies, in order to
find proteins interacting with SE. Performing Western blot
analyses, we confirmed the presence of CBP80 and HYL1
in the immunoprecipitated protein complex. The interaction between CBP80 and SE has been previously detected
by us in transfected Arabidopsis protoplasts using the BiFC
method. Identification of novel protein binding partners
of SE will be performed by mass spectrometry. In addition, we will also sequence RNA molecules present in the
complexes co-immunoprecipitated with SE.
University of Łódź, Department of Plant Cytology and Cytochemistry,
Łódź, Poland
Programmed cell death (PCD) is a conserved suicide program which is an integral part of plant development and
of responses to biotic and abiotic stress. Although no homologes of mammalian caspases and Bcl-2 family members have been identified there is some evidence for the
existence of evolutionarily conserved PCD in both plants
and animals. It has been demonstrated the proapoptotic
Bax-like protein may exists in cells, mammalian Bax can
initiate plant cell death, Bax-induced cell death is similar to
hypersensitive response and lamine-like protein is cleaved
by caspase-6- like proteases.
As it was previously described lead may induce many
disturbances in various plant species probably leading to
death. However, mechanisms by which it affects and damages plants as well as morphology of lead-induced cell
death are still unclear and not characterized. Therefore,
studies on Lupinus angustifolius sp. seedling roots in hydroponic cultures using 100 μM concentration of lead nitrate
were undertaken.
The obtained results revealed progressive cell vacuolization during lead treatment. In consequence after 48-hour
incubation of roots in the metal solution vacuoles occupied
the greater part of a cell. The process of cell vacuolization
was accompanied by a gradual decrease in the volume of
cytoplasm and destruction of cell organelles. It suggested
so called vacuolar cell death, therefore the vigour tests using tetrazolium chloride or indigo carmine were made. Our
investigations showed that root tips were dead already after
24 hours of lead incubation. Moreover, an increase in proteolytic activity in lupine root tissue and root exudates after
metal treatment was observed. This result was accompanied by increase in Bax-like protein expression level. Additionally, enhanced level of the 16 kDa polypeptides which
were identified as a member of PR-10 pathogenesis related
proteins in root tips exposed to lead was found.
From these findings we can conclude that lead-induced
lupine root meristem cell death is connected with the increase in proapoptotic protein expression.
P6.4
P6.5
Comparative analysis of phenotypes of
two deg5 mutants of Arabidopsis thaliana
Arginase activity in Arabidopsis plants
infect with cyst-forming nematode
Małgorzata Baranek, Grzegorz Jackowski
Monika Borowska-Komenda1, Elżbieta Różańska2,
Mirosław Sobczak2, Jolanta M. Dzik1
Institute of Experimental Biology, Department of Plant Physiology,
Poznań, Poland
e-mail: Małgorzata Baranek <[email protected]>
Deg5 is a serine-type protease peripherally bound to the
lumen side of thylakoid membrane. Very little is known
about the physiological substrates of this enzyme. There
are indications that Deg5 participates in degradation of
some proteins of photosystem II in responses to different stresses. Much less is known about the role of Deg5 in
plant growth and development under comfortable conditions.
This communication contains information about selected
elements of phenotype of two homozygous Arabidopsis thaliana mutants in which Deg5 was repressed to various levels, namely to 0% (deg5-1 mutant) and to 80%
(deg5-2 mutant) of the value found for wild type plants.
The phenotypic analysis included: leaf blade morphology
(length, width, perimeter, area and shape coefficient), inflorescence appearance and seed morphology. Electron
microscopy was used to analyze the average cross-sectional
area of chloroplasts, cross-sectional area of plastoglobules, number of plastoglobules per cross-sectional area of
chloroplast, length and width of grana stacks, number of
thylakoids stacked per granum and the presence of “undulations” of thylakoids (they highlight entering of chloroplasts into early senescence phase). Furthermore, the level
of accumulation of Lhcb1-6 apoproteins and in organello
degradation rate of chloroplast proteins in mutants vs wild
type plants were studied as well. The results indicate that
Deg5 is required for normal leaf morphology as well as for
chlorlast life cycle, the thylakoid membranes biogenesis and
their final ultrastructure. 1Department
of Biochemistry, 2Department of Botany, Warsaw
University of Life Sciences, Warsaw, Poland
e-mail: Monika Borowska-Komenda <[email protected]>
Arginase (EC. 3.5.3.1), commonly found in plants, is an
important enzyme in nitrogen metabolism. It hydrolyses
arginine to urea and ornithine, which is used as a precursor
for proline and polyamines biosynthesis. Increased levels
of these compounds and induction of arginase activity are
typical responses to mechanical wounding. Arginase plays
significant role also in plant defense responses to bacterial
and fungal infections. However, very little is known about
its function during parasitism of cyst-forming nematodes.
Heterodera schachtii, a representative of this group of nematodes, is a sedentary endoparasite of sugar beet and many
Brassicaceae plants roots. Its second-stage juveniles penetrate
roots of the host plant and induce permanent feeding site,
called a syncytium. This specific feeding structure constitutes the sole source of nutrients for the developing nematode. Very high energy and nutritional demands of the
nematode strongly change plant metabolism, as well.
The aim of presented study was examination of changes
in plant arginase activity during syncytium development.
Arabidopsis thaliana plants were inoculated with juveniles of
H. schachtii 11 days after germination and collected 7 and
15 days post inoculation (dpi). Uninfected plants were collected at the same time points. In extracts obtained from
roots and schoots of infected and control plants arginase
activity and protein content were measured.
In shoots of infected plants, arginase activity was 3- and
1.5-fold higher in plants collected at 7 and 15 dpi, respectively, than in appropriate controls. Protein content in
shoots of infected plants was approximately 2-fold lower
than in uninfected plants. On the contrary, higher protein
content was found in infected roots than in uninfected
ones.
164
P6.6
P6.7
Purification and biochemical
characterization of serine endopeptidase
from developing triticale (X
Triticosecale Wittm.) seeds
Cloning, molecular characterization
and expression analysis of glutamine
synthetase type I gene from triticale
Justyna Fidler, Beata Prabucka
Warsaw University of Life Sciences, Department of Biochemistry,
Warsaw, Poland
e-mail: Justyna Fidler <[email protected]>
Plant serine peptidases are a large group of enzymes detected in many species. These enzymes occur in almost all
tissues and organs and are involved in a lot of processes
related to plant physiology and development. Plant serine
peptidases participate in i.a. microsporogenesis, differentiation of specialized plant tissues, signal transduction or
hypersensitive response after pathogen attack which leads
to cell death, preventing the spread of pathogens. In seeds
serine peptidases might be involved in mobilization of
storage protein during germination, however knowledge
of the role that this enzymes play in developing seeds
is still poor. According to recent studies serine peptidase
might affect formation of epidermal surface on embryos
and seedlings and can play role in the endosperm development. The results of our earlier experiments indicate a
predominant contribution of this group of endopeptidases
in total proteolytic activity in extracts of developing triticale caryopses. In this paper serine endopeptidase able to
hydrolysis gliadin (main storage protein of triticale caryopses) active up to 35 days after pollination was partially
purified. The enzyme was obtained after a four-step purification procedure, consisting homogenization and extraction, ammonium sulphate precipitation, ion exchange
chromatography and gel filtration. The activity of partially
purified endopeptidase was completely inhibited by DFP
(serine peptidase inhibitor) and PMSF (serine and cysteine
peptidase inhibitor) while pepstatine A (aspartic peptidase
inhibitor) and E-64 (cysteine peptidase inhibitor) had no
effect on enzyme activity. Molecular weight of this enzyme
was estimated at 62.5 kDa by gel filtration and this result
was confirmed by SDS-PAGE. The enzyme was active over
a wide range of temperature from 0 to 50ºC and loses stability at 70°C. Partially purified endopeptidase was able to
hydrolyze wheat gliadin in a very wide range of pH from
3.6 to 5.6 in acetate buffer and from 7.2 to 9.2 in Tris-HCl.
Agnieszka Grabowska, Joanna Kwinta
Warsaw University of Life Sciences, Faculty of Agriculture and Biology,
Department of Biochemistry, Warsaw, Poland
e-mail: Agnieszka Grabowska <[email protected]>
Nitrogen is a crucial nutrient both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS, EC 6.3.1.2) is a key enzyme in plant nitrogen
assimilation that catalyzes the ATP-dependent condensation of ammonium with glutamate to produce glutamine.
Three GS types, GSI, GSII and GSIII, have been described
in living organisms, based on molecular mass, quaternary
structure and gene sequences. GSI and GSII are both present in eukaryotes and prokaryotes, with GSI being more
abundant in prokaryotes, GSII in eukaryotes and GSIII
has been described in prokaryotes. In plants, GS (type II)
can exist as distinct isoforms that are classified into groups
according to their cellular localization. These isoforms
include a cytosolic form, GS1, and a chloroplastic form,
GS2. In higher plants, the GSII type is the most abundant,
although some recent studies have shown the presence
of GSI-encoding genes in Hordeum vulgare, Oryza sativa or
Brachypodium distachyon.
In the molecular approach the aim of the present project
was cloning of glutamine synthetase type I (GSI) gene from
triticale. The first part of sequence GSI based on reverse
transcriptase polymerase chain reaction (RT-PCR) was
cloned. The cloned cDNA was designated as TsGSI. Complete TsGSI cDNA was obtained by 3’ and 5’ RACE (Rapid
Amplification of cDNA Ends). The full-length of TsGSI
is 2797 bp and contains a 2529 bp open reading frame, 5’
untranslated region (5’ UTR) of 135 bp and 3’ UTR 133
bp. TsGSI encodes a protein of 842 amino acids with a calculated molecular mass of 93 kDa and an isoelectric point
of 5.80. TsGSI protein contains the glutamine synthetase
catalytic domain towards the C-terminal end of the peptide, additional TsGSI includes an amidohydro domain. All
conserved regions found in TsGSI show a high level sequence similarity to other glutamine synthetase type I. The
level of TsGSI expression was studied by semi-quantitative
RT-PCR analysis. The results showed that TsGSI has high
expression level in: roots, leaves, kernels of triticale. 47th Congress of the Polish Biochemical Society, 2012
P6.8
P6.9
Exposure of Arabidopsis thaliana
plants to low irradiance leads
to oxidative modifications and
aggregation of LS Rubicso
Cyclic nucleotides are involved
in the defense response against
mechanical wounding and
pathogen attack in Hippeastrum
Magda Grabsztunowicz, Grzegorz Jackowski
Weronika Grzegorzewska, Brygida Świeżawska,
Piotr Szewczuk, Adriana Szmidt-Jaworska
Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz
University, Poznań, Poland
e-mail: Magda Grabsztunowicz <[email protected]>
In terrestrial plants Rubisco exists as a holoenzyme composed of eight large (LS) and eight small subunits (SS). Its
abundance varies during a leaf lifecycle and under stressful conditions. It has been demonstrated that aggregation
and/or degradation is an important regulatory factor influencing LS Rubisco level in leaves. Reactive oxygen species
generated under stressful conditions may induce oxidative
modifications of LS Rubisco molecules rendering them
more susceptible to aggregation/degradation.
To study the influence of exposure to low irradiance on LS
Rubisco level A. thaliana plants were grown under moderate irradiance (250 µmol quanta × m-2 × s-1) and then acclimated to low irradiance (25 or 50 µmol quanta × m-2 ×
s-1) for 5min-96h. Acclimation to low irradiance was found
to be accompanied by a clear decrease in LS Rubisco abundance, maximally to about 60% of its initial level in leaves
of plants acclimated for 24 h to 50 µmol quanta × m-2 ×
s-1. The decrease was not accompanied by an increase in
the non-chloroplast pool of LS Rubisco and this finding
suggests that intrachloroplastic phenomena are responsible
for the decline in Rubisco LS level.
We have shown that a massive accumulation of LS Rubisco homo and heteroaggregtaes takes place during the
acclimation with no sings of a degradation of aggregated
molecules. The aggregates were found to arise due to both
disulphide bridges non-disulphide bonds. As there are reports suggesting the exposure to light of low intensity can
generate an oxidative stress by promoting 1O2 release and,
possibly other reactive oxygen species we were following
the level of O2•- and H2O2 in leaves of plants acclimated
to low irradiance. No clear signs of H2O2 or O2•- burst
preceding the maximal decrease in LS Rubisco abundance
were found yet a prominent rise in MDA was identified
after 6h of acclimation to low irradiance and this suggests
that in fact the acclimation to low irradiance was accompanied by the rapid accumulation of reactive oxygen species
other than O2 •- and H2O2, probably 1O2 which may be the
effective promoter of LS Rubisco oxidative modifications
resulting in a massive aggregation.
Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus
University, Toruń, Poland
e-mail: Weronika Grzegorzewska <[email protected]>
Plants have developed various mechanisms that act on
local and systemic levels, and their role is to adapt plant
organisms to environmental factors. Physical, chemical or
biological factors may lead to wounding, which opens entrances for pathogens. Defense responses have been studied in the aspects of their development mechanisms and
signaling pathways that transmit the signal to distant plant
tissues and organs. However, very little is known regarding
the components of wounding and pathogen attack signaling, starting from perception, through transduction, to the
activation of stress-responsive genes.
Adenosine 3’:5’-cyclic monophosphate (cAMP) and guanosine 3’:5’-cyclic monophosphate (cGMP) are universal
second messengers, playing key roles in the many diverse
physiological responses and processes in prokaryotes and
eukaryotes. Recently, cyclic nucleotides have been shown to
play an important role in abiotic stress responses, particularly in osmotic stress and pathogen defense.
The aim of our study was to investigate the role of cAMP
and cGMP in defense responses, taking place in wounded
Hippeastrum x hybr scales. It has been revealed that the mechanical injury of Hippeastrum organs leads to the formation of phytoalexins (PA), antimicrobial substances, which
make the damaged tissue red. The application of adenylyl
cyclase (the enzymes responsible for cAMP synthesis) inhibitors reduced the level of PA, whereas the application
of adenylyl cyclases activators or pure cyclic AMP has accelerated phytoalexin synthesis. In the case of cGMP, the
observed effect was exactly opposite. As the consequence
of bulb wounding, an increase in the cAMP level has been
observed, reaching the maximum in the 48th hour. A significant increase in the cGMP concentration, preceding the
death of a tissue, appeared in the 96th hour after wounding. A simultaneous treatment (wounding and pathogen)
has slightly increased the level of cyclic nucleotides. However independent infection had a greater influence on the
cNMP level, than the wounding itself. These results show
that the cNMP accumulation was restricted to the wounded
and infected tissue and the cAMP accretion was necessary
to phytoalexin synthesis, whereas the increase in the cGMP
level could be a signal to programmed cell-death.
The results presented herein enable a better understanding
of the role of both cAMP and cGMP as second messengers in plant responses to environmental stimuli, particularly to stress responses.
166
P6.10
P6.11
Activity and subcellular localization
of IAA-amido synthetase in pea
(Pisum sativum) seedlings
The impact of protein carbonylation
on beech (Fagus sylvatica L.)
seeds during storage
Maciej Ostrowski1, Michał Świdziński2, Elżbieta
Bednarska-Kozakiewicz2, Anna Jakubowska1
Ewa Kalemba, Ewelina Ratajczak, Stanisława Pukacka
1Institute
of General and Molecular Biology, Department of
Biochemistry; 2Institute of General and Molecular Biology, Department
of Cell Biology Nicolaus Copernicus University, Toruń, Poland
e-mail: Anna Jakubowska <[email protected]>
Plant acyl acid amido synthetases encoded by early-auxin
responsive genes GH3 conjugate amino acids to diverse
acyl acid substrates through a two-step mechanism involving adenylation and transferase activities. The GH3 proteins are wide-spread in plants and contribute to the active
levels of plant hormones necessary for regulating distinct
physiological responses. Immature seeds of pea contain
acyl acid amido synthetase that catalyzes the formation of
indole-3-acetyl-aspartate (IAA-Asp). The prevalence of
GH3 genes in genomes of mosses, gymnosperms and angiosperms as well as IAA-Asp in most plants analyzed so
far shows that IAA-amido synthetases are important for
the regulation of free auxin levels. In this study, we identified pea IAA-Asp synthetase as a member of the GH3
family proteins based on amino acid sequence determined
by tandem mass spectrometry (LC-MS/MS). IAA-Asp was
highly homologous (54–84% amino acid identity) to the
GH3 proteins from Populus trichocarpa, Glycine max, Riccinus
communis, Nicotiana tabacum, and Arabidopsis thaliana. Moreover, we studied the subcellular distribution and activity of
IAA amido synthetase in developing pea seedlings. The
cytoplasmic localization of GH3 IAA amido synthetase
was indicated in 10-day old pea seedlings by immunofluorescence method using A. thaliana anti-GH3.5 antibodies
and laser confocal microscopy technique. This finding was
confirmed by the measurement of the enzyme activity in
the subcellular fractions collected by differential centrifugation. The most of IAA-Asp synthetase activity (22.6 nmol
[14C]-IAA-Asp h-1 mg-1 of protein, i.e. 88 % of the total activity) was detected in the 100 000 × g supernatant fraction
containing soluble cytoplasmic proteins. 3-day and 6-day
old pea seedlings showed a weak IAA-Asp synthetase activity and GH3 expression levels. These results were supported by reverse transcription — quantitative PCR (qRT-PCR)
assay using specific primer for PsGH3-5. We hypothesized
that IAA-amido synthetase expression and activity depend
on developmental stage of vegetative tissues and this is associated with changes in free IAA level.
Laboratory of Seed Biochemistry, Institute of Dendrology, Polish
Academy of Sciences, Kórnik, Poland
e-mail: Ewa Kalemba <[email protected]>
Proteins can become modified by a large number of reactions involving reactive oxygen species. Direct oxidation of
amino acids produces 2,4-dinitrophenylhydrazine detectable protein products. Carbonylation is irreversible and
because of its unrepairable nature carbonylated proteins
are marked for proteolysis by the proteasome and the Lon
protease or can escape degradation and form high-molecular-weight aggregates that accumulate with age. Quantitative and proteomic analyses of carbonylated proteins using
two-dimensional gel electrophoresis, followed by immunoblotting and mass spectrometry were performed. This
redox proteomics approach allowed for the identification
of carbonylated proteins in embryonic axes of beech (Fagus
sylvatica L.) seeds that have been stored at optimal condition
through different periods from 1 to 13 years. Regardless of
their orthodox or suborthodox categorization, long-term
storage of beech seeds is intricate. What is more a good
cropping year for those forest tree seeds occurs in long intervals – up to 10 years. Therefore protein carbonylation as
a potential cause of vitality lost of beech seeds was tested
and characteristic spots of the longest stored seeds or seeds
with the lowest germination capacity were identified. Considering the fact that oxidation process may also play roles
in cell signal transduction a correlation between protein
carbonylation level and reactive oxygen species production
and distribution was made. Here we present and discuss
the role of carbonylation in beech seeds proteome in two
aspects: as the source of viability lost and seed aging basis
and potential participation in signal transduction pathway.
P6.12
P6.13
Excitation transfer from phycobilisome
to photosystem I changes upon
photosystem I trimerization
Negatively regulated transcription
profiling reveals changes in photosynthetic
apparatus of Brassica oleracea var. capitata
f. alba during black spot disease
Kinga Kłodawska1, Przemysław Malec1, Mihály
Kis2, Zoltán Gombos2, Kazimierz Strzałka1
1Jagiellonian
University, Faculty of Biochemistry, Biophysics and
Biotechnology, Department of Plant Physiology and Biochemistry, Kraków, Poland; 2Hungarian Academy of Sciences, Biological Research
Center, Institute of Plant Biology, Szeged, Hungary
e-mail: Kinga Klodawska <[email protected]>
Cyanobacteria conduct photosynthesis in the way that
very much resembles plant photosynthesis. Photosynthetic
machinery is organized in plant-like manner and consists
of the two photosystems connected by a chain of electron carriers. Polypeptide composition of photosystem I
and photosystem II is similar but not identical as in higher
plants. Photosystem I of cyanobacteria exhibits a unique
capability of oligomerization into trimeric supercomplexes.
In wild type cells trimerization level increases with the rise
of growth temperature. Capability of photosystem I oligomerization depends on the presence of PsaL subunit of
photosystem I. With the use of wild type Synechocystis
PCC6803 and trimerless psaL- mutant we attempt to unravel the mystery of physiological significance of the photosystem I trimer formation.
Room temperature and low temperature fluorescence data
are presented. Based on these results we conclude that the
energy transfer from outer antenna - phycobilisome, to
photosystem reaction center is altered by the oligomerization state of photosystem I.
We propose that the structural changes upon PS I trimerization cause differences in phocobilisome–photosystem
interactions. This phenomenon may be considered a part
of the photoprotective mechanisms network in cyanobacterial cells.
Violetta K. Macioszek1, Agnieszka Żmieńko,2 Magdalena
Gapińska3, Karolina Rousseau1, Andrzej Kiejstut Kononowicz1
1University
of Lodz, Department of Genetics, Plant Molecular Biology
and Biotechnology, Łódź, Poland, 2Institute of Bioorganic Chemistry,
PAS, Poznań, Poland, 3University of Lodz, Laboratory of Electron
Microscopy, Łódź, Poland
e-mail: Andrzej K. Kononowicz <[email protected]>
Foliar plant pathogen Alternaria brassicicola causes one of the
most destructive leaf spot diseases of cultivated crops of
Brassica genus worldwide. Spreading necrotic lesions lead
to the death of infected seedlings or foliar tissues of mature plants and consequently to the host plant decay. Brassica’s foliar tissues contain many phenolic compounds and
can produce broad spectrum of antimicrobial secondary
metabolites e.g. phenolics and flavonoids, during pathogen
attack, but such defense is insufficient against A. brassicicola. From the agricultural point of view, disadvantageous
changes in photosynthetic potential appear to be the most
important one for plant cultivation. Chloroplasts are the
target organelles for A. brassicicola and damage of chloroplast structure such as degradation of chloroplast envelope
and disturbances of thylakoids during disease development
in Brassica oleracea var. capitata f. alba leaf`s cells have been
confirmed by electron microscopy analyses. Changes in
negatively regulated transcription profiling of infected B.
oleracea tissues revealed gradual increase (from 12 to 48
hpi) of number of suppressed chloroplast and photosynthesis-related genes. Decreased of chlorophyll a, b and increased carotenoids concentrations were observed as well. Acknowledgements
This work was supported by grant of Polish Ministry of Science and
Higher Education N302 318833 and in part by grant of National Center
of Science 2011/01/B/NZ1/04315.
168
P6.14
P6.15
Osmopriming — old method new aspects
— improvingrape seeds germination
Leaf age-dependent changes in
photosynthetic dyes content during
black spot disease development
of Brassica oleracea var. capitata
f. alba and Brassica juncea
S. Kubala1, Ł. Wojtyla1, A. Kosmala2, M. Garnczarska1
1Adam
Mickiewicz University, Institute of Experimental Biology,
Departmentof Plant Physiology, Poznań, Poland, 2Polish Academy of
Sciences, Institute of Plant Genetics, Poland
e-mail: Szymon Kubala <[email protected]>
Seed germination is a very important stage during plant
ontogenetic development. Several physiological and biochemical changes take place during germination. Seed germination is also influenced by many abiotic factors such as
temperature, humidity, salinity, which restrict or inhibit this
process. One known way to improve seeds germination
performance as well as stress tolerance and seedlings vigor
is osmopriming. Osmopriming is a pre-sowing treatment
that exposes seeds to a low external water potential that
allows partial hydration but prevents germination. However, the mechanism of seeds priming remains unknown.
In this work, rape (Brassica napus L. cv. Libomir) seeds were
osmoprimed with –1.2 MPa polyethylene glycol (PEG
6000) for 7 days and then germinated on water for 48h.
The results of this work showed that seed coat rapture of
more than half of osmoprimed seeds population occurred
at 8–12h of imbibition, while in not conditioned seeds
at 20–24h of imbibition. The germination percentage as
well as dynamic of germination and time to reach 50% of
germination of osmoprimed seeds were greater than that
of non-treated seeds. The differences in kinetic of water
uptake were also observed between primed and unprimed
seeds. Proteins of dry osmoprimed and unprimed seeds
were analysed by two-dimensional electrophoresis 2DIEF-SDS-PAGE in order to detect osmpriming induced
changes in protein profile. Approximately 236 protein
spots ranged from 10 to 145 kDa and pI from 4 to 10 were
detected. During osmopriming, a total of 89 protein spots
changed more than 20% in abundance. From these 89 proteins expression of 49 was up regulated and expression
of 40 was down regulated. The identification of selected
proteins should gain more insight into the molecular and
physiological processes underlying seeds priming.
Violetta Katarzyna Macioszek, Izabela Kołodziejczyk,
Karolina Rousseau, Paweł Walczak, Małgorzata
Rożniata, Andrzej Kiejstut Kononowicz
University of Lodz, Department of Genetics, Plant Molecular Biology
and Biotechnology, Łódź, Poland
e-mail: Violetta Macioszek <[email protected]>
Brassica oleracea and Brassica juncea are vegetable crops susceptible to Alternaria brassicicola infection. On their foliar
tissues develop spreading necrotic lesions that can cause
even whole plant decay. However, it has been observed that
the younger leaf of the plant the smaller lesion has developed. Alternaria brassicicola development on plants with
five mature leaves has shown leaf age-dependent tendency.
Number of germination tubes and appressoria decreased
on younger leaves from early hours of infection to 24 hpi.
Spreading lesions especially on B. juncea leaves have been
surrounded by chlorotic “halo” and after 5 days of infection older leaves (first and second) yellowed. Decreased
contents of chlorophyll a and b have been observed from
the younger to the oldest leaves compared to the uninfected
samples. The same tendency was observed for carotenoids,
although up to 72 hpi three oldest leaves showed higher
carotenoids concentrations then control ones.
Basal defense response of infected Brassicas, evaluated as
transcriptional activity of genes encoding PR proteins,
PDF1.2 and PR-1, has been suppressed in the oldest leaves
after 5 dpi and strong activity of PR genes was found in the
youngest leaves, but also with decreasing tendency at 5 dpi.
Acknowledgements
This work was supported by grant of National Science Centre 2011/01/B/
NZ1/04315.
P6.16
P6.17
The role of genes encoding enzymes
involved in the gibberellin deactivation
(InGA2ox) in the growth and
development of Ipomoea nil seedlings
Two cucumber Metal Transport Proteins,
MTP8 and MTP9, are involved in the
cellular manganese homeostasis
Katarzyna Marciniak, Jacek Kęsy, Jan Kopcewicz
Nicolaus Copernicus University, Chair of Plant Physiology and
Biotechnology, Toruń, Poland
e-mail: Katarzyna Marciniak <[email protected]>
Gibberellins (GAs) are one of the seven classical phytohormones involved in the plant growth and development
because they regulate many important processes such as
seed germination, stem elongation and floral induction.
The studies on GA metabolism pathway in a model, short
day plant Ipomoea nil have led to the identification of two
genes encoding 2-gibberellin oxidases (InGA2oxs). These
enzymes oxidize the second carbon atom in the active
GAs and convert them to the catabolites.
Obtaining positive results at the identification of nucleotide sequences (GeneBank no GU189414 — InGA2ox1,
GU911364 — InGA2ox2) was the starting point to determine the transcriptional activity of known genes in all
vegetative organs during the five days of I. nil growth and
development in different light and photoperiodic conditions and after ethylene application — a hormone, which is
regarded as a major flower inhibitor. To achieve the objective used a sensitive and reliable Real Time PCR method.
The results indicate that the gene sequences encoding InGA2oxs are homologous to genes found in other plant
species. Their transcriptional activity is not equal in time
and space. This means that the concentration of GAs in
different cells and tissues is subject to complex and multidirectional control, which underlies the correct growth and
development of I. nil. The results of this study allow also
suppose that GAs do not play a significant role in the photoperiodic induction of flowering that occurs in the cotyledons of I. nil seedlings or even inhibit this process in a long
day. Only the removal of the hormone molecules during
the second half of the inductive night can produce flower
inducer, which after moving to the shoot apex initiates a
series transformations leading to changes in the differentiation pattern. The situation is different at the stage of
floral evocation and morphogenesis, where GAs is likely
to accelerate these processes. In turn, ethylene contributing
positively to the accumulation of mRNA InGA2ox genes,
mainly in the tops of shoot growth, contributes to the inhibition of flowering due to decreased of GAs levels. Therefore, gibberellins action takes place in close cooperation
with other phytohormones.
Magdalena Migocka1, Piotr Poździk1, Tadeusz
Marchewka1, Anna Papierniak1, Anna Kosieradzka1,
Ewelina Posyniak1, Arnold Garbiec2
1Institute
of Experimental Biology, Department of Plant Physiology,
University of Wrocław, Wrocław, Poland; 2Institute of Experimental
Biology, Department of Animal Developmental Biology, University of
Wrocław, Wrocław, Poland
e-mail: Magdalena Migocka <[email protected]>
Members of ubiquitous cation facilitator (CDF) family are
integral membrane transporters that are involved in the
transport of heavy metal ions across cells and cellular organelles. Through the regulation of cellular divalent heavy
metal homeostasis, CDF transporters influence metal accumulation, metal tolerance or oxidative stress resistance.
In comparison to bacterial, yeast or mammal CDFs, only a
few representative members of this family have been studied in plants. Plant CDFs are designated as Metal Transport
Proteins (MTPs) and divide into two separate groups based
on the sequence similarity. Here, we reveal the functional
properties of two cucumber MTPs, CsMTP8 and CsMTP9,
homologous to the MTP8-MTP11 cluster of Arabidopsis thaliana MTPs. The two full cDNAs of CsMTP8 and
CsMTP9 have been isolated from the cDNA library prepared from cucumber roots. Heterologous expression of
cucumber genes in yeast mutants showed that CsMTP8
and CsMTP9 were able to complement the mutant strains'
hypersensitivity to Mn and partially to Ni, but not other
metals including Co, Cd and Zn. CsMTP8 and CsMTP9
fused to green fluorescent protein were localized in yeast
endomembranes, consistent with the function of both proteins in heavy metal transporting. The expression of genes
encoding CsMTP8 and CsMTP9 was differentially upregulated by Cd, Cu or Ni, and significantly reduced under manganese deficiency in cucumber roots and leaves. The results
indicate that the two MTP transporters participate in the
intracellular Mn sequestration and thus, maintain manganese homeostasis in cucumber cells.
Acknowledgments
This work was financially supported by the Ministry of Science and Higher Education (grant no. IP2010 026470).
Acknowledgements
This work was supported by the National Science Centre (Poland) grant
No NN303811240.
170
P6.18
P6.19
Indole-3-acetic acid glucosyltransferase
is involved in modification of
glycoproteins from pea seeds
Cucumber CAX4 is a tonoplast exchanger
involved in the Mn2+, Ni2+ and Ca2+
sequestration within vacuoles
Maciej Ostrowski, Iwona Sławkowska,
Anna Hetmann, Anna Jakubowska
Anna Papierniak, Magdalena Migocka, Grażyna Kłobus
Institute
of General and Molecular Biology, Department of
Biochemistry, Nicolaus Copernicus University, Toruń, Poland
e-mail: Maciej Ostrowski <[email protected]>
UDP-glucose: indole-3-acetic acid glucosyltransferase
(1-O-IA-Glc synthase) forms the 1-O-indole acetyl glucose
ester (1-O-IA-Glc), the glucosylated conjugate of indole3-acetic acid (IAA), according to the equation:
IAA + UDPG ↔ 1-O-IA-Glc + UDP
In monocotyledonous plants this catalytic activity is required for the biosynthesis of the lower molecular mass
as well as the higher molecular mass conjugates of IAA.
These conjugates are generally considered to represent the
storage forms of the phytohormone and are involved in a
homeostatic mechanism for control of IAA levels.
Recently, we have identified the 1-O-IA-Glc synthase activity in pea seeds where IAA-amide conjugates are major
forms of auxin. However, the significance of 1-O-IA-Glc
synthase in dicotyledonous plants remains elusive. We now
report the IAA-modification of pea seeds glycoproteins by
UDP-glucosyltransferase of IAA. The enzyme was purified
from immature seeds of pea during our previously studies. The specific activity of the final enzyme preparation
was 30.3 nmol min–1 mg-1 of protein. Coomassie Brilliant
Blue R-250 staining after SDS-PAGE of this preparation
revealed a single protein band corresponding to the 56
kDa molecular mass. Our preliminary investigations suggested that the product of the UDP-glucosyltransferase
activity, 1-O-IA-Glc, is an intermediate in the transfer of
indolylacetyl moiety from 1-O-IA-Glc to glycoproteins.
To examine the modification of glycoproteins by IAA, we
used the protein fraction from the extract of immature pea
seeds eluted by 15% (w/v) α-methylmannopyranoside during Concanavalin A-affinity chromatography. We observed
more than 100-fold incorporation of [14C]-IAA labeled to
pea glycoproteins after addition of the 1-O-IA-Glc synthase preparation to the glycoprotein fraction. The highest
radioactivity of [14C]-IAA labeled proteins was detected for
glycoprotein band corresponding to the molecular mass of
28 kDa (RF 0.22).
In addition, a detailed steady-state kinetic characterization
of the 1-O-IA-Glc synthase was performed. Determination of the substrate specificity revealed the highest UDPglucose-transferring activity on IAA, whereas activities on
other phytohormones were not significant or not detected.
KmIAA = 5.2×10-4 M and VmaxIAA=160.7 nmol min-1 parameters were determined by Lineweaver-Burk doublereciprocal plot and verified by Michaelis-Menten equation.
The inhibition kinetic was analyzed by graphical methods
using Cornish-Bowden and Dixon plots. Two natural auxins, indole-3-butyric acid (IBA) and indole-3-propionic acid
(IPA) which were poorly acceptors of the glucose moiety,
act as competitive inhibitors of 1-O-IA-Glc synthase activity (KiIBA=10×10-5 M, Kmapp=8×10-3 M, KiIPA=1.5×10-4
M, Kmapp=5.5×10-3M ). Another plant hormone, gibberellin, was also a competitive inhibitor (Ki=1.5×10-4 M,
Kmapp=5.5×10-3M) of the enzyme. In contrast, salicylic acid
(SA) non-competitively inhibited of the 1-O-IA-Glc synthase (Ki=1.5×10-4 M, Vmaxapp=14.6 nmol min-1). Based on
these results, it has been supposed that UDP-glucosyltransferase of IAA is a part of the mechanism that controls of
phytohormone homeostasis in pea tissues. Institute of Experimental Biology, Department of Plant Physiology,
Wroclaw University, Wrocław, Poland
e-mail: Anna Papierniak <[email protected]>
Plant cell vacuoles play an important role in the homeostasis of metals within the cell. The presence of a tonoplast
metal/proton antiporters (Cd2+/H+, Ca2+/H+, Zn2+/H+)
involved in vacuolar metals sequestration has been well
documented in several plant species, including cucumber.
Within the past few years, a great effort has been made to
identify and characterize these proteins. Among multiple
families of metal transporters in plants, that members of
CAX (CAtion eXchangers) family appear to be the key proteins responsible for metals sequestration within vacuole.
Although part of them CAXs (AtCAX1 to AtCAX6) have
been characterized in Arabidopsis thaliana, no information
concerning other species are available.
Here, we present the first molecular and functional characterization of cucumber protein homologous to AtCAX4.
The gene encoding CsCAX4 and the remaining five cucumber CAXs were identified through the screening of cucumber genome (ACHR01000000) using AtCAX1-6 as the
query sequences. Among six cucumber CAXs, CsCAX4
clusters into the type IA of plant CAXs together with CsCAX3 and CsCAX1. According to hydropathy analysis, the
open reading frame of 1218-bp encodes a protein transporter of 406 amino acids with 11 putative transmembrane
domains. The CsCAX4 mRNA was abundant in all tissues.
In roots the expression of CsCAX4 was significantly stimulated upon Cd2+ and slightly enhanced by Zn2+ and Ca2+,
whereas in shoots Ni2+, Mn2+ and Cd2+ markedly increased
the level of CsCAX4 transcript. In addition, the presence
of the CsCAX4 gene on a multicopy plasmid increased
tolerance to Mn2+ and Ni2+ in the Δmnr mutant lacking
the vacuolar manganese transporter. The Ca2+ sensitivity
phenotype of another yeast mutant (Δvcx1pmc1) was also
fully complemented by CsCAX4-pYES-DEST52 plasmid.
In protoplasts isolated from Arabidopsis cell suspension
culture, the N-terminal GFP-fusion CsCAX4 protein localized clearly to the vacuolar membrane. Our results provide
the first evidence for the contribution of CAX4-like protein in manganese, nickel and calcium sequestration within
plant cell vacuoles.
Acknowledgments
This work was financially supported by the Ministry of Science and Higher Education (grant no. N N303 544639).
P6.20
P6.21
Involvement of protein kinases, MAPKs
and CDPKs, in the response of Solanum
genotypes to Phytophthora infestans
Alteration of GWD genes expression during
low temperature exposure in potato leaves
Lidia Polkowska-Kowalczyk, Krzysztof Olszak,
Justyna Tarwacka, Grażyna Muszyńska,
Bernard Wielgat, Jadwiga Szczegielniak
Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Department of Plant Biochemistry, Warsaw, Poland
e-mail: Lidia Polkowska-Kowalczyk <[email protected]>
Plants are constantly exposed to stress factors. Due to sedentary life, in order to meet the changing environmental
conditions, plants have evolved a complex defence strategy
providing a protection against stresses. Mitogen-activated
protein kinases (MAPKs) cascades and calcium-dependent
protein kinases (CDPKs) are a crucial components of
plants signalling network to defend against numerous potential pathogens. The proteins phosphorylated by protein
kinases are involved in gene expression, signalling pathways, ion and water transport through membranes, metabolism and function of cytoskeleton (DeFalco et al., 2010).
Changes in MAPKs and CDPKs activities as well as their
expression profiles were investigated in leaves of Solanum
tuberosum cv Bzura, S. tuberosum clone H-8105 and S. nigrum
var. gigantea that exhibited field resistance, susceptibility
and non-host resistance, respectively, in response to the
Phytophthora infestans, the pathogenic oomycete that causes
late blight, the most destructive potato disease. Leaves of
Solanum genotypes were treated with elicitor (culture filtrate
of P. infestans, CF). Activities of protein kinases were determined using “in gel kinase assay”. The expression levels
of MAPKs and CDPKs were measured by method of RTPCR.
We found that MAPKs and CDPKs activities increased
in response to CF treatment in all studied genotypes, but
varied with respect to intensity and timing. The highest increase in all enzymes activities was noted in S. nigrum var.
gigantea, completely resistant to P. infestans, and the lowest
in the susceptible H-8105. It means that these enzymatic
activities were positively correlated with the level of plant
resistance in response to CF treatment. Moreover, we have
demonstrated that transcripts of MAP kinases and CDPKs
are present in all studied Solanum genotypes, although only
in the case of CDPKs the increments in transcript levels
in elicitor-treated leaves in comparison with control were
observed.
The obtained results indicated that MAPKs and CDPKs
are involved in signalling pathways occurring in Solanum
genotypes after treatment with elicitor from P. infestans.
Reference
DeFalco TA et al. (2010) Biochem J 425: 27-40.
Acknowledgements
This work was supported by the MNiSW, Project 0329/B/P01/2008/34.
Dorota Sitnicka, Joanna Simińska, Sławomir Orzechowski
Warsaw University of Life Sciences, Faculty of Agriculture and Biology,
Department of Biochemistry, Warsaw, Poland
e-mail: Joanna Simińska <[email protected]>
Starch occurs in nature as insoluble granules synthesized in
plastids. The phosphorylation of amylopectin by the glucan, water dikinases (GWDs: GWD- EC 2.7.9.4; PWD/
GWD3- EC 2.7.9.5) is an essential step within starch metabolism. Starch phosphorylation together with activities
of β-amylase (BAM- EC 3.2.1.2) and isoamylase (ISA3- EC
3.2.1.68) are required for normal starch breakdown in A.
thaliana and S. tuberosum leaves (Delatte T et al., 2006, J Biol
Chem 281: 12050-12059).
Recent studies on the cold acclimation capacity of A. thaliana have shown that the first carbohydrate that concentration increases after cold exposure was maltose which
is a direct product of starch degradation catalyzed by
β-amylase. It is considered that sugar accumulation at low
temperatures may partly contribute to the enhancement
of freezing tolerance in various cold-hardy plants during
cold acclimation. Hence, along with photosynthesis, starch
degradation might play a significant role in cold induced
sugar accumulation and increased tolerance to cold stress
(Kaplan F et al., 2007, Plant J 50: 967-981). Starch related
glucan water dikinases are involved in regulation of starch
degradation in plastids through phosphorylating the surface of starch granules, thereby ensuring better accessibility
to starch hydrolyzing enzymes (Hejazi M et al., 2008, Plant
J 55: 323-334). Taken together, it is expected that starch
phosphorylation as the initial step in starch degradation
might play a significant role in sugar accumulation in response to low temperature.
The aim of presented project was to define changes in expression profiles of glucan water dikinases under low temperature conditions in potato leaves. Real-time RT-PCR
data analysis has shown that a few hours after transferring
of the plants to the chilling temperature the amount of
transcripts of both dikinases increased. Our results confirmed those of previous studies which demonstrated that
the GWD transcript level in Arabidopsis increase during an
early phase of cold acclimation (Yano R et al., 2005, Plant
Physiol 138: 837-846). Additionally, we have shown that
the expression of second homolog of GWD in potato –
GWD3 also increased in response to low temperature. The
obtained results suggest that the enzymes encoded by this
genes are probably involved in cold induced starch degradation in potato leaves. Hypothetically, starch hydrolysis
may likely be an important adaptation mechanism in potato
leaves to cold conditions, which often occurs in Polish climate after these plant emerge.
172
P6.22
P6.23
HpAC1 gene from Hippeastrum encodes
active plant adenylyl cyclase
Generating of small RNAs from
ribosomal RNAs as a response
to herbicide stress in plants
Brygida Świeżawska, Piotr Szewczuk, Weronika
Grzegorzewska, Krzysztof Jaworski, Adriana Szmidt-Jaworska
Nicolaus Copernicus University, Chair of Plant Physiology and
Biotechnology, Toruń, Poland
e-mail: Brygida Swiezawska <[email protected]>
The structure, regulation of activity and role of adenylyl
cyclases (ACs) in the animal kingdom is well know. These
enzymes catalyse the conversion of ATP to cyclic AMP
and pyrophosphate. cAMP is involved in many and diverse
physiological responses and processes. In contrast to animal cells, since the mid-1980s the presence of cAMP and
ACs in plants was questioned. At present, there is no doubt
about it. ACs and cAMP are involved in various physiological and biochemical mechanisms, but the knowledge about
it remains obscure.
As a results of recent studies we have cloned cDNA that
represents a putative member of the ACs gene family in
plants. A full length cDNA, designated as HpAC1, was isolated using the RACE-PCR method. The HpAC1 cDNA
clone (HM991704.1) had a total length of 979 bp with 621
bp — long ORF which encoded 206 amino acid peptide
(ADM83595.1) with a molecular mass of 23.07 kDa and
an isoelectric point of 5.07. HpAC1 contains conservative
CYTH like Pase domain characteristic for bacterial class IV
adenylyl cyclase.
Level of HpAC1 mRNA was investigated in leaves, stems,
roots, bulbs, petals, stamens (anthers and filaments), carpels (stigma and style), ovaries and pollen grains. The highest amount of HpAC1 transcript was observed in bulbs,
leaves, stems, respectively and the lowest in pollen grains.
We also investigated the changes in HpAC1 transcript level
in response to abiotic and biotic stress conditions. Our
studies revealed increase in HpAC1 transcript level after
mechanical wounding and Phoma narcissi infection in several minutes and 8–10 hours after stress.
Next we obtained recombinant HpAC1 protein with using E.coli BL21 strain and pGEX-6P-2 expression vector
system. The molecular mass of protein was 25.9 kDa and
corresponded to in silico prediction.
We observed formation of cAMP from ATP catalysed by
HpAC1 in the presence of Mg 2+ and Mn 2+.
Subsequently, we cloned ORF to E. coli SP850, which
was deficient in AC and cannot ferment lactose, in effect
produces colorless colonies on MacConkey agar. After the
complementation test a bright red colonies were observed.
To sum up, we isolated and identified HpAC1 gene from
Hippeastrum and confirmed that it encodes an active adenylyl cyclase enzyme.
Aleksandra Szopa1, Aleksander Tworak2,
Marek Figlerowicz2, Tomasz Twardowski2
1Institute of Technical Biochemistry, Technical University of Łódź, Łódź,
Poland; 2Institute of Bioorganic Chemistry, Polish Academy of Sciences,
Poznań, Poland
e-mail: Aleksandra Szopa <[email protected]>
Glyphosate is one of the most widely used nonselective herbicide in crops. Recently it has been proven that
glyphosate can cause oxidative stress in plant cell. It was
also shown that oxidative stress in plants can cause degradation of constitutive RNA molecules, giving rise to small
RNAs that can act as regulatory RNAs. In our research we
try to find out if such RNAs can be generated from ribosomal RNAs by Dicer-like 1 ribonuclease (DCL1), which
is involved in miRNA and siRNA biogenesis, as a plant
response to herbicide stress caused by using of glyphosate
in plants cultivation.
Our plant model is Zea mays. We are testing two maize lines,
displaying natural sensitivity or resistance to glyphosate. We
are using plants treated with herbicide and control plants.
Plant material was collected in two time points: on the day
of herbicide application and a week after glyphosate treatment.
We isolated total RNA and subsequently sequestered the
ribosomal RNA by extraction from agarose gel. The next
step will be the digestion of rRNA by DCL1. Obtained
molecules will be analyzed by sequencing. At the same time
we are carrying out the in vitro selection of small RNAs
that could be involved in herbicide stress plant response.
We will make the comparison of molecules obtained from
both projects. Our aim is to identify RNA molecules that
play regulatory role in translation process during herbicide
stress.
P6.24
Different antioxidant responses
of glicophytic and halophytic
plants subjected to salinity
Mrah S et al. (2006) Effects of NaCl on the growth ion accumulation
and photosynthetic parameters of Thellungiella halophila. J Plant Physiol 163:
1022-1031.
Monika Wiciarz1, Jerzy Kruk1, Ewa Niewiadomska2,3
1Department
of Plant Physiology and Biochemistry, Faculty of
Biochemistry, Biophysics and Biotechnology, Jagiellonian University,
Kraków, Poland; 2Institute of Biology, The Jan Kochanowski University of
Humanities and Science, Kielce, Poland; 3Institute of Plant Physiology,
PolishAcademy of Sciences, Kraków, Poland
e-mail: Monika Wiciarz <[email protected]>
Antioxidants are reducing agents inhibiting oxidation of
other molecules and therefore protect cells against reactive oxygen species (ROS). Under unstressed conditions,
removal and formation of ROS is well balanced but under
stress, an imbalance occurs and efficiency of antioxidant
system may determine survival of cells. To overcome this
hazardous situation, signal transduction mechanisms activate genes related to antioxidant activity and metabolic
adjustments (Møller & Sweetlove, 2010).
The aim of this study was to investigate the response of
different antioxidants to salinity treatment in glicophytic and halophytic plants. To elucidate this, we used two
closely related species: glicophytic Arabidopsis thaliana and
halophytic Thellungiella halophila. T. halophila is a model plant,
tolerating high salinity. During stress conditions, this plant
accumulates NaCl at controlled rates without producing
salt glances and other morphological structures. Mrah et
al. (2006) postulate that leaves remained intact from oxidative damage and the process of photosynthesis remains
unchanged, in contrary to glicophytic plants.
A. thaliana and T. halophila plants were irrigated for 7 days
with 0.15 and 0.3 M NaCl, respectively. Analysis of total
antioxidant capacity showed their significant decline after
salinity treatment in A.t.. On the other hand, the level of
antioxidants increased in T.h.. It is noteworthy, that this level was different already under control conditions and was
higher in A.t. than in T.h.. We compared the level of two
antioxidant enzymes – superoxide dismutase (SOD, EC
1.15.1.1.) and catalase (EC. 1.11.1.6.) which are known to
be the first line of defense against ROS. The level of SOD
increased significantly in T.h. (MnSOD and CuZnSOD isoforms) and slightly decreased in A.t. (FeSOD and CuZnSOD isoforms) after salinity treatment when compared to
control conditions. Whilst, the activity of CAT increased in
T.h. and declined in A.t. after salinity.
In the next step we investigated low molecular mass antioxidants: ascorbic acid, glutathione, α-tocopherol and plastochromanol (PC) with its oxidation product - hydroxy-plastochromanol (PC-OH). The level of ascorbic acid significantly
decreased in A.t. and remained unchanged in T.h. after salinity
treatment, although, under control conditions, the level was
higher in A.t.. After the salt treatment, the content of glutathione was significantly higher in T.h. in comparison to A.t.
The analysis of hydrophobic antioxidants (α-tocopherol, PC
and its derivative PC-OH) demonstrated a different pattern in
these species after salinity. In A.t., thelevel of these molecules
increased significantly while remained unchanged in T.h..
Altogether, our data suggest that tolerance of salinity in
T.h. is linked with a significant activation of antioxidant
defense. In contrast, lack of such activation in A.t. may
determine a high sensitivity to salinity of this species. An
exception are hydrophobic antioxidants which indicate a
high production of singlet oxygen in A.t..
References
Møller IM, Sweetlove LJ (2010) ROS signaling — specificity is required.
Trends Plant Sci 15: 370-374.
174
P6.25
Molecular characteristics, subcellular
and tissue localization of PsAOγ — an
aldehyde oxidase isoform oxidizing
abscisic aldehyde to ABA in pea
Edyta Zdunek-Zastocka1, Mirosław
Sobczak2, Małgorzata Dudkiewicz3
1Department
of Biochemistry, Faculty of Agriculture and Biology,
Warsaw University of Life Sciences-SGGW, Warsaw, Poland;
2Department of Botany, Faculty of Agriculture and Biology, Warsaw
University of Life Sciences-SGGW, Warsaw, Poland; 3Department of
Experimental Design and Bioinformatics, Faculty of Agriculture and
Biology, Warsaw University of Life Sciences-SGGW, Warsaw, Poland
e-mail: Edyta Zdunek-Zastocka <[email protected]>
Aldehyde oxidase (AO) catalyzes the final step of the abscisic acid (ABA) biosynthesis, the oxidation of abscisic
aldehyde to ABA. Its isoforms effectively using abscisic
aldehyde as a substrate were detected, however, only in
Arabidopsis and pea (PsAOγ). Expression analysis suggests
that the homodimerc PsAOγ isoform is encoded by PsAO3
gene. To confirm this hypothesis, the heterologous expression of the pea PsAO3 was performed in Pichia pastoris
cells (strain KM71H). The recombinant PsAOγ protein
was purified on DEAE-cellulose and Ni-IDA resin, and its
biochemical characteristics was carried out. As revealed by
activity staining following native PAGE, the recombined
enzyme prefers indole-3-aldehyde and naphthaldehyde as
substrates, although a high activity for abscisic aldehyde
and geranialdehyde was also observed. Acetaldehyde and
heptaldehyde were not oxidized by the recombinant protein while cinnamaldehyde and benzaldehyde were oxidized
with a very low efficiency. The Km values of recombinant
PsAOγ against naphthaldehyde and abscisic aldehyde (4.6
and 5.1 μM, respectively) were the lowest among the substrates tested. The activity of recombinant PsAOγ was almost totally inhibited by potassium cyanide that removes
sulphur from the molybdenum cofactor of AO. Significant
inhibition was also observed in the presence of p-hydroxymercuribenzoate, which modifies sulfhydryl groups, and
menadione that blocks the electron transfer from FAD
center. Mass spectrometry analysis confirmed that the peptides derived from purified recombinat PsAOγ are encoded
indeed by PsAO3 gene.
A polyclonal antibodies were raised against PsAOγ-specific
15-amino acid peptide (VVYQLSLRPTPGKV) and used
for subcellular and tissue localization of PsAOγ protein.
Immunofluorescence microscopy revealed the presence of
the PsAOγ protein in the phloem companion cells and in
some stomatal cells in pea leaves. In stem and root, PsAOγ
protein was found in the youngest phloem cells located
close to the xylem vessels. Immunogold transmission electron microscopy indicated that PsAOγ protein was present
only in the cytoplasm of vascular cells. These results point
the vascular tissue cells as potential sites of ABA synthesis.

Lectures L6.1 Session 6. New Vistas in Plant Molecular Biology L6.2

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