TRANSCRIPTIONAL RESPONSE TO HYDROGEN PEROXIDE IN GRAPEVINE BERRY SKIN
AT THE BEGINNING OF RIPENING
Stefania Pilati*, Giorgia Bagagli, Daniele Brazzale, Paolo Sonego, Marco Moretto, Kristof Engelen and Claudio Moser.
Fondazione E. Mach, Research and Innovation Center, via E. Mach, 1 - 38010 San Michele all'Adige (TN)- Italy
*stefania.pilati@fmach.it
Fleshy fruit ripening represents the final stage of fruit development, when pulp and skin undergo many metabolic and morphological transformations to become attractive for seed
dispersing animals. In grapevine, the beginning of ripening can be identified by the softening and coloring of the berries (véraison). The transition from mature green to ripening
berries is controlled by many internal signals, such as hormones, transcription factors and metabolites, tuned to external stimuli, mainly light, temperature and water availability.
Ripening onset is characterized by the gradual loss of photosynthetic activity and a transient shift to an aerobic fermentative metabolism (grapevine is a non climacteric plant)
which are likely to favor an oxidative stress. We reported the transient accumulation of hydrogen peroxide (H2O2) and singlet oxygen (1O2) respectively in the cytosol and
plastids of Pinot noir berry skin at the beginning of ripening (Pilati et al, BMC Plant Biology 2014). In order to ascertain the signaling function of H2O2, we collected pre-véraison
berries and treated them ex-vivo for 20 hours either with 1 mM H2O2, 0.2 mM abscisic acid (ABA), known to be an important signal for ripening transition, a cocktail of ROS
scavengers or a combination of them. RNA extracted from berry skin has been anayzed to study transcriptional response at the genome-wide level by RNA-seq for the H2O2
and ABA treatment and at the gene level by Real-time PCR considering all the conditions. Main results are described here.
Experimental setting
RNA-seq analysis results
Comparison between ABA e H
2O
2responses in pre-véraison berry skin
Future work: H
2O
2visualization with hyper
E-L 33
mature hard green berries
(approx. two weeks before véraison)
Introduction
Treatments (in biological triplicates): - control
- H2O2 1mM
- ABA 0,2 mM
- ABA 0,2 mM + anti-oxidants (pyridoxal-phosphate 1mM, Na/Ascorbate 1mM, tocopherol 0,25 mM)
- ABA 0,2 mM + H2O2 1 mM
- anti-oxidants (pyridoxal-phosphate 1mM, Na/Ascorbate 1mM, tocopherol 0,25 mM)
20 hours in mild agitation in the dark
Gene expression analysis (gene and genome-wide level)
196 down-regulated genes by H2O2 122 up-regulated genes by H2O2 berry skin flower young leaf berry ripening grafting treatments with JA and salicylic acid
berry skin flower
berry ripening
After reads quality check, mapping and normalization, differentially expressed
genes have been selected by t-test analysis imposing thresholds on p-value (<0.05) and on fold change (|FC| > 1.5).
318 differentially expressed genes were obtained for berries treated with H2O2 and 871 for berries treated with ABA.
Here, we focus on results concerning H2O2 response and the comparison with ABA treatment.
H2O2 response is slightly unbalanced towards gene repression (62%).
24% of the genes are unknown and 65% of the annotated genes have been assigned to a metabolic pathway (https://www.sdstate.edu/ps/research/vitis/pathways.cfm).
The more represented metabolisms are:
- lipid metabolism - response to stress - photosynthesis
- secondary metabolism
- plant-pathogen interaction - cell wall metabolism
- transport
- regulation of transcription
MarcoPaolo: a gene expression compendium for
Vitis vinifera
MarcoPaolo is an atlas based on COLOMBOS technology which
incorporates 1883 samples coming from 64 microarray and RNA-seq experiments publicly available at GEO, ArrayExpress, PlexDB and SDRA.
Here, MarcoPaolo has been used to select and visualize conditions in which genes responding to H2O2 in pre-véraison berry skin are highly modulated and then to cluster genes according to their co-expression behaviour.
It becomes evident that the genes we find up- and down-regulated by H2O2 are co-expressed not only in other experiments on berries
(focused on development or tissues) but even in other conditions, like flower and leaf development, grafting and response to jasmonic and salicylic acid.
Moreover, we can distinguish blocks of co-expressed genes, suggesting a modular organization of gene expression.
This analysis supports the role of H2O2 in signaling in Vitis physiology and provides candidates for gene expression regulation.
logFC_H2O2 logFC_ABA20 VIT_15s0024g01760 VIT_12s0057g00800 VIT_18s0001g06170 VIT_06s0004g06820 VIT_09s0018g01670 VIT_12s0134g00560 VIT_11s0016g02800 VIT_10s0116g00520 VIT_11s0016g05840 VIT_18s0001g06020 VIT_18s0001g12610 VIT_02s0154g00300 VIT_02s0154g00310 VIT_11s0052g01200 VIT_01s0011g05110 VIT_01s0146g00260 VIT_11s0016g05830 VIT_14s0066g01670 VIT_06s0004g07910 VIT_13s0067g02910 VIT_14s0068g00920 VIT_05s0077g01670 VIT_19s0014g01180 VIT_15s0046g03290 VIT_13s0067g00330 VIT_06s0004g07680 VIT_05s0029g01540 VIT_07s0129g00320 VIT_15s0046g03240 VIT_13s0019g05340 VIT_19s0014g01770 VIT_15s0048g01000 VIT_01s0010g02850 VIT_03s0063g00380 VIT_06s0004g00960 VIT_17s0000g01290 VIT_03s0091g00420 VIT_18s0086g00180 VIT_08s0007g08030 VIT_03s0091g00310 VIT_07s0191g00220 VIT_05s0049g00040 VIT_18s0089g01270 VIT_00s0246g00010 VIT_04s0008g01280 VIT_14s0060g01980 VIT_02s0025g04330 VIT_17s0000g08080 VIT_06s0004g01510 VIT_13s0067g02360 VIT_00s0347g00030 VIT_09s0002g06770 VIT_00s0203g00210 VIT_14s0066g00300 VIT_00s0429g00040 VIT_15s0046g00490 VIT_12s0059g00110 VIT_00s1206g00010 −2 0 2 Value Color Key
ABA
H
2O
2H
2O
2ABA
18% of the genes modulated by H2O2 treatment are modulated also by ABA, 50% of them with a coherent trend. This overlap suggests that H2O2 could act as secondary signal down-stream of ABA at ripening onset.
Lipoxygenase (PnLOXA), a gene modulated by ABA and H2O2,
and 9-cis-epoxycarotenoid dioxygenase (NCED), modulated only by ABA, were analyzed by RT-PCR in all the conditions tested. This analysis confirms that PnLOXA requires not only ABA but also H2O2 for its full induction, and suggests that H2O2
accumulation is a consequence of the presence of ABA.
This hypothesis will be further investigated.
Hyper, a recently developed cell probe for H2O2-specific and quantitative measurements in living cells, has been stably introduced in a dwarf mutant of Vitis vinifera. Two constructs have been employed, for cytosolic and plastidial probe localization.
So far, we can see the probe in the leaves of acclimated plants. This probe will allow to monitor oxidative stress in berries at ripening onset. Then, the same treatments describe above
(i.e. with ABA, H2O2 and anti-oxidants) will be repeated in order to test the hypothesis of a cause-effect relationship between ABA increase and H2O2 accumulation.
Hyper Clorophyll Merge
cytosolic pro
be
chloro plastc prob
